RU2778405C2 - Methods for protection of pig fetuses from virus infection - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a method for the protection of pig fetus from the infection with the porcine reproductive and respiratory syndrome virus (hereinafter – PRRSV), including breeding of a female pig with a male pig. At the same time, the female pig contains modified chromosome sequences in both alleles of gene CD163, wherein the modified chromosome sequence is an insert in a gene encoding protein CD163, deletion in the gene encoding protein CD163, or a combination thereof.

EFFECT: invention is effective for the reduction in sensitivity of a female pig to infection with PRRSV.

31 cl, 24 dwg, 18 tbl, 4 ex

Description

LINK TO ELECTRONIC SEQUENCE LISTING

[0001] The official copy of the sequence listing is provided electronically via EFS-Web as an ASCII sequence listing file named "SEQ LISTING 18054WO", created on March 27, 2018 and having a size of 175.5 kilobytes, and submitted simultaneously with the description . The sequence listing contained in this document in ASCII format is part of the specification and is incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY

[0002] The present invention relates to methods for protecting fetal pigs from infection with the porcine reproductive and respiratory syndrome virus (PRRSV).

BACKGROUND OF THE INVENTION

[0003] Porcine reproductive and respiratory syndrome (PRRS) is the most economically important swine disease in North America, Europe and Asia, costing North American producers approximately $600 million annually (Holtkamp et al., 2013). Clinical disease syndromes caused by infection with the porcine reproductive and respiratory syndrome virus (PRRSV) were first reported in the US in 1987 (Keffaber, 1989) and then in Europe in 1990 (Wensvoort et al., 1991). PRRSV infection leads to respiratory problems, including cough and fever, reproductive failure in late pregnancy, and decreased growth. The virus is also involved in multiple interactions of the polymicrobial disease syndrome while maintaining subclinical infection throughout life (Rowland et al., 2012). Losses are the result of respiratory diseases in young pigs, poor growth performance, reproductive failure and intrauterine infection (Keffaber, 1989).

[0004] The reproductive form of the disease accounts for about 45% of losses due to abortion, dead fetuses and respiratory diseases in newborns. In its most severe form, reproductive PRRS can result in 90% fetal/newborn mortality as well as increased sow mortality. The reproductive form of PRRS occurs after infection in pregnant pigs or sows approximately 90 days out of a 114-day gestation period (Christianson et al., 1993; Rowland, 2010). After an initial phase of replication in maternal macrophages, the virus crosses the placenta and begins to productively infect the fetus. Initially, the virus infects only a small number of fetuses, after which there is horizontal transmission of the virus from fetus to fetus (Wiltdnson et al., 2016). The exact mechanism of how the virus crosses the placenta remains unknown, but may be similar to macrophage infection acting as a Trojan horse, a mechanism previously described for lactate dehydrogenase (LDV) elevating virus (Cafruny, 1996). Unlike alveolar macrophages in adult animals, the main site of PRRSV replication in the fetus is the thymus (Rowland, 2003). Because the pig fetus becomes immunocompetent around day 70 of gestation, PRRSV infection occurs in the fetal immune environment containing functional B and T cells (Rowland, 2003; Rowland, 2010).

[0005] Pigs that survive intrauterine infection become persistent sources of virus in later production stages, resulting in endemically infected herds (Rowland, et al., 2003). The most severe form of reproductive disease is associated with a group of highly virulent isolates called atypical PRRSVs (Halbur et al., 1997; Mengeling et al., 1998). Interestingly, many of the atypical PRRSV isolates have been raised on farms vaccinated against the PRRS virus (Key et al., 2001). In 2006, an atypical virus named Highly Pathogenic PRRSV (HP-PRRSV) emerged in China and continues to devastate pig populations in that country (Tian et al., 2007). With about 5,000 sows in a standard commercial breeding facility, an outbreak of reproductive PRRS with high mortality can be devastating. To ensure the sustainability of pork production and food security, reproductive PRRS control solutions remain a priority. Vaccines have failed to control the disease, mainly due to the genetic diversity of the structural proteins of the virus (Shi et al., 2010). In practice, intensive biosecurity measures are the only means of protecting the reproductive herd.

[0006] The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) belongs to the Arterividae family, together with the murine lactate dehydrogenase-increasing virus, simian hemorrhagic fever virus, and equine arteritis virus. Structurally, arteriviruses resemble togaviruses but are similar to coronaviruses, replicating through a nested 3'-terminal set of subgenomic mRNAs that share a common leader and poly-A tail. Arteriviruses have important properties associated with viral pathogenesis, including tropism for macrophages and the ability to cause severe disease and persistent infection (Plagemann, 1996). Molecular comparisons between North American and European viruses assign all PRRSV virus isolates to one of two genotypes, respectively. Type 2 or Type 1. Although the two genotypes share only about 70% identity at the nucleotide level (Nelsen et al., 1999), both have tropism for CD163-positive cells, cause long-term infections, and cause similar clinical signs.

[0007] CD163 is a 130 kDa type 1 membrane protein consisting of nine cysteine-rich phagocyte receptor (SRCR) domains and two spacer domains, as well as a transmembrane domain and a short cytoplasmic tail (Fabriek et al., 2005). Porcine CD163 contains 17 exons that encode a peptide signal sequence followed by nine SRCR domains, two linker domains (also called proline-serine-threonine (PST) domains located after SRCR 6 and SRCR 9) and a cytoplasmic domain followed by short cytoplasmic tail. Surface expression of CD163 is limited to cells of monocyte-macrophage origin. In addition to functioning as a viral receptor, CD163 performs several important functions related to maintaining normal homeostasis. For example, after infection or tissue damage, CD163 acts as a scavenger molecule, removing haptoglobin-hemoglobin complexes from the blood (Kristiansen et al., 2001). The resulting heme degradation products regulate the associated inflammatory response (Fabriek et al., 2005). HbHp utilization is the main function of CD163 and is located in SRCR 3 (Madsen et al., 2004). Metabolites released by macrophages after HbHp degradation include bilirubin, CO, and free iron. One important function of CD163 is to prevent oxidative toxicity caused by free hemoglobin (Kristiansen et al., 2001; Scares et al., 2009).

[0008] Other important C163 functions include erythroblast adhesion (SRCR2), TWEAK receptor function (weak inducer of apoptosis similar to tumor necrosis factor) (SRCR 1-4 and 6-9), bacterial receptor (SRCR5), and African porcine virus receptor (Sanchez -Torres et al. 2003). CD163 may also play an immunomodulatory role (discussed in Van Gorp et al. 2010).

[0009] CD163 as a PRRSV receptor was first described by Calvert et. al. (2007). Transfection of non-permissive CD163 cDNA cell lines from various species, including monkeys, humans, dogs, and mice, can render cells susceptible to PRRSV infection (Calvert et al., 2007). In addition to CD163, a second receptor protein, CD169 (also known as sialoadhesin or SIGLEC1), has been identified as the primary PRRSV receptor involved in the initial interaction with the CP5 matrix (M) heterodimer, the major protein on the surface of the virion (Delputte et al., 2002 ). In this model, the subsequent interaction between CD163 and the GP2, 3, 4 heterotrimer in the endosomal compartment mediates deenvelopment and release of the viral genome into the cytoplasm (Van Breedam et al., 2010, Allende et al., 1999). A previous model describing PRRSV infection of alveolar macrophages identified SIGLEC1 (CD169) as the primary viral receptor on the surface of macrophages; however, previous work using SIGLEC1-/- pigs showed no difference in viral replication compared to wild-type pigs (Prather et al., 2013). These results support previous in vitro studies demonstrating that PRRSV-resistant cell lines lacking surface CD169 and CD163 support viral replication after transfection with the CD163 plasmid (Welch et al., 2010).

[0010] Many characteristics of both PRRSV pathogenesis (especially at the molecular level) and epidemiology are poorly understood, making it difficult to control. It is now common practice for manufacturers to vaccinate pigs against PRRSV with modified live attenuated strains or killed virus vaccines, but existing vaccines often do not provide satisfactory protection. This is due both to strain variability and to insufficient stimulation of the immune system. In addition to concerns about the effectiveness of current PRRSV vaccines, there is strong evidence that the modified live vaccine currently used can persist in individual pigs and pig herds and accumulate mutations (Mengeling et al. 1999), as demonstrated with virulent field isolates after experimental infection of pigs (Rowland et al., 1999). In addition, vaccine virus has been shown to be shed in the semen of vaccinated boars (Christopher-Hennings et al., 1997). As an alternative to vaccination, some experts recommend a "test and discard" strategy in breeding herds (Dee et al., 1998). Successful use of this strategy depends on the removal of all pigs that are acutely or persistently infected with PRRSV, followed by strict controls to prevent re-introduction of the virus. The difficulty, and much of the cost associated with this strategy, is that little is known about the pathogenesis of persistent PRRSV infection, and therefore there are no reliable methods to detect persistently infected pigs.

[0011] As can be seen, there is a need in the art to develop strategies for inducing resistance to the PRRSV virus in animals. There is also a particular need for methods to protect the fetus from PRRSV infection in utero and to prevent transmission of PRRSV from mother to fetus.

DISCLOSURE OF THE INVENTION

[0012] A method for protecting a porcine fetus from porcine reproductive and respiratory syndrome virus (PRRSV) infection is provided. The method includes breeding a female pig with a male pig. The female pig contains modified chromosomal sequences in both alleles of its CD163 gene, with the modified chromosomal sequences reducing the susceptibility of the female pig to PRRSV infection compared to the susceptibility to PRRSV infection of a female pig that does not contain any modified chromosomal sequences in the alleles of its CD163 gene. The male pig contains at least one wild-type CD163 allele.

[0013] Other objects and features will be in part obvious, and in part indicated below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1. Targeted vectors and CRISPR are used to modify CD163. Panel A shows wild-type exons 7, 8 and 9 of the CD163 gene that has been modified using CRISPR. Panel B shows a targeted vector designed to replace porcine exon 7 (porcine CD163 SRCR5 domain) with DNA encoding human CD163L SRCR8. This targeted vector was used in G418 susceptibility selection transfections. PCR primers for right arm and left arm long fragment analysis are arrowed for 1230, 3752, 8791, 7765, and 7775. Panel C shows a targeted vector identical to that shown in panel B, but from which the Neo cassette has been removed. . This targeted vector was used to target CD163 in cells that were already resistant to neomycin. Primers used in small deletion assays are indicated by arrows and labeled GCD163F and GCD163R. Panel D highlights the exons targeted by CRISPR. The location of CRISPR 10, 131, 256, and 282 is shown by downward arrows on exon 7. The CRISPR numbers represent the number of base pairs from the intron-exon junction of intron 6 and exon 7.

[0015] FIG. 2. Targeted vector and CRISPR were used to modify CD1D. Panel A shows exons 3, 4, 5, 6 and 7 of the wild-type CD1D gene that has been modified by CRISPR. Panel B shows a targeted vector designed to replace exon 3 with the Neo selectable marker. This targeted vector was used in conjunction with CRISPR to modify CD1D. PCR primers for analysis of long fragments on the left and right arms for 3991, 4363, 7373 and 12806 are indicated by arrows. Panel C shows exons; targeted by CRISPR. The location of CRISPR 4800, 5350, 5620 and 5626 is shown with down arrows on exon 3. Primers used in small deletion assays are shown with arrows and labeled GCD1DF and GCD1DR.

[0016] FIG. 3. Creation of CD163 and CD1D knockout pigs using CRISPR/Cas9 and SCNT. A) Targeted deletion of CD163 in somatic cells after transfection with CRISPR/Cas9 and donor DNA. The wild-type genotype gives a band at 6545 base pairs (bp). Lanes 16 represent six distinct colonies from a single transfection of CRISPR 10 with Cas9 and donor DNA containing Neo. Lanes 1, 4 and 5 show a large homozygous deletion of 1500-2000 bp. Lane 2 represents the smaller homozygous deletion. Lanes 3 and 6 represent either the wild-type allele and a small deletion, or a biallelic modification of both alleles. The exact modifications of each colony were determined only by sequencing the colonies used for somatic cell nuclear transfer (SCNT). A faint wild-type band in some lanes may indicate cross-contamination of fetal fibroblasts from a neighboring wild-type colony. NTC - matrixless control. B) Targeted deletion of CD1D in somatic cells after transfection with CRISPR/Cas9 and donor DNA. The wild-type genotype produces the 8729 bp band. Lanes 14 are colonies with a 500-2000 bp CD1D deletion. Lane 4 appears to be a wild-type colony. NTC = matrixless control. B) Image of a CD163 knockout pig obtained by SCNT during the course of the study. This male pig contains a homozygous CD163 deletion of 1506 bp. D) Image of CD1D pigs obtained during the study. These piglets contain a 1653 bp CD1D deletion. E) The genotype of two SCNT litters containing a 1506 bp CD163 deletion. Lanes 1-3 (litter 63) and lanes 1-4 (litter 64) represent the genotype of each piglet from each litter. Sow indicates the female recipient of the SCNT embryos and wild type is the wild type control. NTC - matrixless control. E) Genotype of two SCNT litters containing a 1653 bp CD1D deletion. Lanes 1-7 (litter 158) and lanes 1-4 (litter 159) represent the genotype of each piglet.

[0017] FIG. 4. Effect of the CRISPR/Cas9 system on pig embryos. A) Frequency of blastocyst formation after injection of various concentrations of the CRISPR/Cas9 system into zygotes. The toxicity of the CRISPR/Cas9 system was lowest at 10 ng/µl. B) The CRISPR/Cas9 system can successfully disrupt eGFP expression in blastocysts when injected into zygotes. Original magnification X4. C) Types of eGFV mutations generated by the CRISPR/Cas9 system: wild-type genotype (SEQ ID NO:16), #1 (SEQ ID NO:17), #2 (SEQ ID NO: 18) and #3 (SEQ ID NO: 19).

[0018] FIG. Fig. 5. Effect of the CRISPR/Cas9 system upon targeted action on CD163 in porcine embryos. A) Examples of mutations generated in CD163 by the CRISPR/Cas9 system: wild-type genotype (SEQ ID NO:20), #1-1 (SEQ ID NO:21), #1-4 (SEQ ID NO:22) and #2 -2 (SEQ ID NO:23). All embryos examined by DNA sequencing showed mutations in CD163 (18/18). CRISPR 131 is in bold. B) Sequencing readout of a homozygous deletion caused by the CRISPR/Cas9 system. The image shows #1-4 of panel A with a 2 bp deletion. CD163.

[0019] FIG. 6. Impact of the CRISPR/Cas9 system when administered with two types of CRISPR. A) PCR amplification of CD163 in blastocysts injected with CRISPR/Cas9 as zygotes. Lanes 1, 3, 6 and 12 show targeted deletion between two different CRISPRs. B) PCR amplification of CD/D in blastocysts injected with CRISPR/Cas9 as zygotes. According to gel electrophoresis, CD/D has a lower deletion frequency compared to CD163 (3/23); lanes 1, 8 and 15 show obvious CD/D deletions. B) The CRISPR/Cas9 system successfully targeted two genes when the system was provided with two CRISPRs targeting CD163 and eGFP. Modifications of CD163 and eGFP are shown: wild-type CD163 (SEQ ID NO:24), CD163 #1 (SEQ ID NO:25), CD163 #2 (SEQ ID NO:26), CD163 #3 (SEQ ID NO:27), wild-type eGFP (SEQ ID NO:28), eGFP #1-1 (SEQ ID NO:29), eGFP #1-2 (SEQ ID NO:30), eGFP #2 (SEQ ID NO:31), and eGFP No. 3 (SEQ ID NO:32).

[0020] FIG. 7. CD163 knockout pigs generated with the CRISPR/Cas9 system introduced into zygotes. A) PCR amplification of CD163 from knockout pigs; a clear sign of a deletion was found in litters 67-2 and 67-4. B) Image of CD163 knockout pigs with a surrogate mother. All animals are healthy and show no signs of abnormalities. C) The genotype of CD163 knockout pigs. The wild-type sequence is shown as SEQ ID NO: 33. Two animals (from litters 67-1 (SEQ ID NO: 34) and 67-3 (SEQ ID NO: 37)) carry a homozygous deletion or insertion in CD163. Two other animals (from litters 67-2 and 67-4) carry the biallelic modification of CD163: #67-2 Al (SEQ ID NO:35), #67-2 A2 (SEQ ID NO:36), #67-4 Al (SEQ ID NO:38) and No. 67-4 a2 (SEQ ID NO:39). The deletion was caused by the introduction of two different CRISPRs with the Cas9 system. None of the animals injected with CD163 into the zygote showed a mosaic genotype.

[0021] FIG. 8. CD1D knockout pigs generated with the CRISPR/Cas9 system introduced into zygotes. A) PCR amplification of CD1D from knockout pigs; 166-1 shows the mosaic genotype of CD1D. 166-2, 166-3 and 166-4 show no change in amplicon size, however amplicon sequencing revealed modifications. Wild type FF - wild type embryonic fibroblasts. B) PCR amplification of the long fragment assay showed a clear deletion of one allele in piglets 166-1 and 166-2. B) Image of CD1D knockout pigs with a surrogate mother. D) Sequence data of CD1D knockout pigs; wild-type (SEQ ID NO:40), #166-1.1 (SEQ ID NO: 41), #166-1.2 (SEQ ID NO:42), #166-2 (SEQ ID NO:43), #166-3.1 (SEQ ID NO:44), #166-3.2 (SEQ ID NO:45) and #166-4 (SEQ ID NO:46). The atg start codon in exon 3 is shown in bold and also in lowercase.

[0022] FIG. 9. Clinical signs of acute PRRSV infection. Results of daily assessment for respiratory signs and fever for CD163+/+(n=6) HCD163-/-(n=3).

[0023] FIG. 10. Lung histopathology in acute PRRSV infection. Representative photomicrographs of hematoxylin-eosin-stained tissue from wild-type and knockout pigs. The left panel shows edema and mononuclear cell infiltration. The right panel from the knockout pig shows the pulmonary structure of a normal lung.

[0024] FIG. 11. Viremia in different genotypes. Note that the data for CD163-/- piglets are located along the x-axis.

[0025] FIG. 12. Antibody production in pigs with a null allele, a wild-type allele and an uncharacterized allele.

[0026] FIG. 13. Cell surface expression of CD163 in selected pigs. The lines on the right in panels A of uncharacterized alleles, panels B of uncharacterized alleles, and panels CD163+/+, represent the CD163 antibody, while the lines on the left side of these panels are control elements without antibodies (background). Note that in CD163-/- animals, CD163 staining overlaps with background control, and that CD163 staining in uncharacterized alleles is roughly in the range between wild-type and background levels (also note that this is a logarithmic scale, so less than ~10%) .

[0027] FIG. 14. Level of CD169 on alveolar macrophages from three representative pigs and control without antibodies (anti-CD169 labeled with FITC).

[0028] FIG. 15. Viremia in different genotypes. Note that the data for piglets with amino acid D43 lie along the x-axis.

[0029] FIG. 16. Genomic sequence of wild-type exons 7-10 of CD163 used as control sequence (SEQ ID NO: 47). The sequence includes 3000 bp. above exon 7 to the last base of exon 10. The underlined areas show the location of exons 7, 8, 9, and 10, respectively.

[0030] FIG. 17. Schematic of CD163 modifications illustrating several CD163 chromosomal modifications, predicted protein product for each modification, and relative macrophage expression for each modification as measured by surface CD163 levels on porcine alveolar macrophages (AMCs). Black areas indicate introns and white areas indicate exons. . The shaded area indicates hCD163L1 exon 11 mimetic, a porcine exon 7 homologue. The gray area indicates the synthesized intron with the PGK Neo construct.

[0031] FIG. 18. Sequence diagram of the porcine CD163 protein and gene. A) SRCR (ovals) and PST (squares) domains of the CD163 protein along with the corresponding gene exons. B) Comparison of porcine CD163 SRCR 5 (SEQ ID NO: 120) with the human homologue CD163L1 SRCR 8 (SEQ ID NO: 121).

[0032] FIG. 19. Representative results of surface expression of CD163 and CD169 on AMC from wild-type and CD163-modified pigs. Panels A E show results for CD163 modifications as shown in FIG. 17. Combined data for d7(1467) and d7(1280) are shown in panel D.

[0033] FIG. 20. Serum levels of haptoglobin in wild-type and CD163-modified pigs.

[0034] FIG. 21. Relative permissivity of wild-type AMS and HL11m to infection with type 2 PRRSV isolates.

[0035] FIG. 22. Infection of CD 163 modified pigs with PRRSV type 1 and type 2 isolates.

[0036] FIG. 23. Viral load for wild-type and modified CD 163 pigs infected with type 2 viruses.

[0037] FIG. 24. Fetal outcomes after maternal infection with PRRSV. The numbers on the left identify each sow (“sow #”; see Table 16 below). Below each sow number in brackets is the PCR result for serum PRRS, measured as log10 number of templates per reaction. "N" means no PRRSV nucleic acid (Ct>39). Fruits are identified by number and relative position in each uterine horn. Asterisks denote fetal PCR samples obtained from abdominal fluid. The number below each fetus represents the fetal serum PRRS PCR result (logio number of templates per reaction). The number in each circle indicates the presence of anatomical pathology: 1) normal fetus; 2) a small fetus; 3) placental changes such as placental abruption and/or necrosis; 4) a fetus stained with meconium; 5) the fetus is dead and necrotic. Lower case letters identify the genotype of individual fetuses (see Table 16). Key: a, A/A; b, C/A; s, V/A; d, E/A; e, B/C; f, B/D; g, D/C; h, D/D; i, E/C; j, E/D; HO - not determined, as the fetus was necrotic; but the genotype has not been determined.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention is directed to methods for protecting fetal pigs from infection with the porcine reproductive and respiratory syndrome virus (PRRSV). Pigs with inactivating mutations in both alleles of the CD 163 gene are resistant to PRRSV infection. It has now surprisingly been found that CD163-positive fetuses (eg, fetuses that have one or two wild-type CD163 alleles) can be protected from PRRSV infection in utero as long as the mother has inactivating mutations in both alleles of her CD163 gene. For example, females with inactivating mutations in both alleles of the CD163 gene can be bred to males with two wild-type CD163 alleles and the resulting heterozygous fetuses will be protected from PRRSV infection.

Definitions

[0039] When presenting elements of the present invention or its preferred embodiments, the designations in the singular and "specified" are intended to indicate that there is one or more elements.

[0040] The term "and/or" means any of the elements, any combination of elements, or all of the elements with which the term is associated.

[0041] As used herein, the term "breeding" refers to the union of male and female gametes such that fertilization occurs. Such a connection can be carried out by mating (copulation) or by artificial methods in vitro or in vivo. Such artificial methods include, without limitation, artificial insemination, artificial insemination by surgical intervention, in vitro fertilization, intracytoplasmic sperm injection, vitelline preparation, culture of fertilized oocytes in vitro, ovarian transfer, and ovarian separation. As used herein, the term "breeding" also includes the transfer of a fertilized oocyte into the reproductive tract of the female.

[0042] The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than those listed.

[0043] The term "CRISPR" means "clustered regular intermediate short palindromic repeats". CRISPR systems include Type I, Type II, and Type III CRISPR systems.

[0044] The term "Cas" refers to "protein associated with CRISPR". Cas proteins include, without limitation, Cas9 family members, Cas6 family members (eg, Csy4 and Cas6), and Cas5 family members.

[0045] The term "Cas9" can generally refer to a polypeptide containing at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild-type Cas9 polypeptide (eg, Cas9 from S. pyogenes). Exemplary Cas9 sequences are provided by SEQ ID NNs 1-256 and 795-1346 in US Patent Publication No. 2016/0046963. SEQ ID NN 1-256 and 795-1346 US Patent Publications No. 2016/0046963 are incorporated herein by reference. "Cas9" may refer to a polypeptide with no more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or similarity sequences with a wild-type Cas9 polypeptide (eg, from S. pyogenes). "Cas9" may refer to a wild-type or modified form of the Cas9 protein, which may include an amino acid substitution such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

[0046] The term "Cas5" can generally refer to a polypeptide containing at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to an exemplary wild-type Cas5 polypeptide (eg, Cas5 from D. vulgaris). Exemplary Cas5 sequences are shown in Figure 42 of US Patent Publication No. 2016/0046963. Fig. 42 US Patent Publication No. 2016/0046963 is incorporated here by reference. "Cas5" can generally refer to a polypeptide with a maximum of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity with a wild-type Cas5 polypeptide (eg, Cas5 from D. vulgaris). "Cas5" may refer to a wild-type or modified form of the Cas5 protein, which may include an amino acid substitution such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

[0047] The term "Cas6" may generally refer to a polypeptide containing at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100 % sequence identity and/or sequence similarity to an exemplary wild-type Cas6 polypeptide (eg, Cas6 from T. thermophilus). Exemplary Cas6 sequences are shown in Figure 41 in US Patent Publication No. 2016/0046963. Figure 41 of US Patent Publication No. 2016/0046963 is incorporated here by reference. "Cas6" as a whole may refer to a polypeptide with a maximum of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or similarity sequences with a wild-type Cas6 polypeptide (eg, from T. thermophilus). "Cas6" may refer to a wild-type or modified form of the Cas6 protein, which may include an amino acid substitution such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

[0048] The terms "CRISPR/Cas9" or "CRISPR/Cas9 system" refer to a programmable genetic engineering nuclease system that includes the Cas9 protein or a derivative thereof, as well as one or more non-coding RNAs that can provide CRISPR-PHK (crRNA) function. ), and trans-activating RNA (tracrRNA) for Cas9. CrRNA and tracrRNA can be used alone, or they can be combined to produce "guide RNA" (gRNA). CrRNA or gRNA provide a sequence that is complementary to the genomic target.

[0049] References herein to a deletion in the nucleotide sequence from nucleotide x to nucleotide y means that all nucleotides in the range have been deleted, including x and y. For example, the phrase "an 11 base pair division from nucleotide 3137 to nucleotide 3147 compared to SEQ ID NO: 47" means that each of nucleotides 3317 to 3147 has been deleted, including nucleotides 3317 and 3147.

[0050] "Animal resistance to disease" is a characteristic of an animal in which an animal avoids disease symptoms that result from interactions between animals and pathogens, such as interactions between a pig and PRRSV. That is, the ability of pathogens to cause animal diseases and associated disease symptoms is prevented, or, alternatively, a reduction in the frequency and/or severity of clinical signs or a reduction in clinical symptoms. One of skill in the art will appreciate that the methods disclosed herein may be used with other compositions and methods available in the art to protect animals from attack by a pathogen.

[0051] As used herein, the terms "gene editing", "edited gene", "genetically edited/modified" and "gene editing effectors" refer to the use of homing technology with natural or artificially created nucleases, also referred to as "molecular scissors", " homing endonucleases" or "targeted endonucleases". Nucleases create specific double-stranded chromosomal breaks (DSBs) at desired locations in the genome, which in some cases use endogenous cellular mechanisms to repair the break induced by natural processes of homologous recombination (GR) and/or nonhomologous end-joining. - NHEJ). Gene editing effectors include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), clustered regular short palindromic repeat (CRISPR) systems (e.g., the CRISPR/Cas9 system), and meganucleases (e.g., modified meganucleases like homing- nuclease end). The terms also include the use of transgenic procedures and methods, including, for example, where the change is a deletion or a relatively small insertion (typically less than 20 nucleotides) and/or does not introduce DNA from an alien species. The term also includes offspring animals, such as animals created by sexual interbreeding or asexual reproduction from an animal with an edited original gene.

[0052] The terms "genomic engineering", "genetically engineered", "genetically engineered", "genetically altered", "genetic alteration", "genome modification", "genome modification" and "genetically modified" may refer to alteration of the genome by deletion, insertion, mutation or substitution of certain nucleic acid sequences. The change may be specific to a gene or place. Genetic engineering can use nucleases to cut the nucleic acid, thereby creating a site for change. The development of a non-genomic nucleic acid is also being considered. A protein containing a nuclease domain can bind and cleave a target nucleic acid to form a complex with a nucleic acid that targets the nucleic acid. In one example, cleavage may result in double-strand breaks in the target nucleic acid. The nucleic acid can be repaired, for example, using the endogenous non-homologous end joining (NHEJ) mechanism. In the following example, a nucleic acid fragment may be inserted. Modifications to nucleic acids that target nucleic acids and site-directed polypeptides can introduce new functions that will be used for genomic engineering.

[0053] As used herein, the terms "DNA homing technology", "homing technology", and "homing endonuclease" include any mechanisms that allow targeting of a specified molecule to a specified DNA sequence, including zinc finger (ZF) proteins, effectors like activators transcription (TALE) meganuclease; and CRISPR systems (eg, CRISPR/Cas9 system).

[0054] The terms "increased resistance" and "reduced susceptibility" as used herein means, without limitation, a statistically significant reduction in the frequency and/or severity of clinical signs or clinical symptoms that are associated with infection by a pathogen. For example, "increased resistance" or "reduced sensitivity" may refer to a statistically significant reduction in the frequency and/or severity of clinical signs or clinical symptoms that are associated with PRRSV infection in an animal containing a modified chromosomal sequence in the CD163 gene protein compared to a control animal, having an unmodified chromosomal sequence. The term "statistically significant reduction in clinical symptoms" means, without limitation, the frequency of occurrence of at least one clinical symptom in a group of modified subjects, which, after infection with an infectious agent, is at least 10%, preferably at least 20%, more preferably at least at least 30%, even more preferably at least 50% and even more preferably at least 70% lower than the unmodified control group.

[0055] "Knockout" means a violation of the structure or regulatory mechanism of a gene. Knockouts can be generated by homologous recombination of targeted vectors, replacement vectors, or hit-and-run vectors, or random insertion of a gene decoy vector resulting in complete, partial, or conditional loss of gene function.

[0056] As used herein, the term "mutation" includes changes in the nucleotide sequence of a polynucleotide, for example, in a gene or DNA coding sequence (CDS), compared to the wild-type sequence. The term includes, without limitation, substitutions, insertions, frameshifts, deletions, inversions, translocations, duplications, splice-donor site mutations, point mutations, and the like.

[0057] Here, “reducing the frequency and/or severity of clinical signs” or “reducing clinical symptoms” means, without limitation, reducing the number of infected subjects in a group, reducing or eliminating the number of subjects with clinical signs of infection, or reducing the severity of any clinical signs that are present in one or more subjects compared to wild-type infection. For example, these terms encompass any clinical signs of infection, lung disease, viremia, antibody production, reduction in pathogen load, pathogen isolation, reduction in pathogen transmission, or reduction in any clinical signs symptomatic of PRRSV. Preferably, these clinical signs are reduced in one or more animals of the present invention by at least 10% compared to infected subjects without the modification in the CD163 gene. More preferably, clinical signs are reduced in subjects of the invention by at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%.

[0058] A "TALE DNA binding domain" or "TALE" is a polypeptide containing one or more repeating TALE domains/units. The repeat domains are involved in the binding of TALE to its cognate target DNA sequence. A single "repeating unit" (also referred to as a "repeat") is typically 33-35 amino acids long and shows at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. Zinc finger and TALE binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example, by constructing (altering one or more amino acids) the helix recognition region of naturally occurring zinc finger or TALE proteins. Therefore, engineered DNA binding proteins (zinc fingers or TALE) are proteins not found in nature. Non-limiting examples of methods for constructing DNA-binding proteins are design and selection. An engineered DNA binding protein is a non-naturally occurring protein whose design/composition is based primarily on rational criteria. Rational design criteria include the use of replacement rules and computerized algorithms to process information in a database that stores information about existing ZFP and/or TALE designs, as well as binding data. See, for example. US Patents No. 6140081; 6453242 and 6534261; see also WO 98/53058; W098/53059; W098/53060; WO 02/016536 and WO 03/016496 and US Publication No. 20110301073.

[0059] A "zinc finger DNA-binding protein" (or binding domain) is a protein or domain within a larger protein that binds DNA in a sequence-specific manner via one or more zinc fingers, which are sections of an amino acid sequence in the binding domain, structure which is stabilized by the coordination of the zinc ion. The term zinc finger protein, which binds DNA, is often abbreviated as "zinc finger protein" or ZFP.

[0060] A "selected" zinc finger or TALE protein is a non-naturally occurring protein that is produced primarily by an empirical process such as phage display, interaction trap, or hybrid selection. See for example. US Patent No. 5789538; US Patent No. 5925523; US Patent No. 6007988; US Patent No. 6013453; US Patent No. 6200759; WO 95/19431; W096/06166; W098/53057; W098/54311; WO00/27878; WO 01/60970; WO 01/88197; WO 02/099084 and US Publication No. 20110301073.

[0061] Various other terms are defined below.

Ways to protect fetal pigs from PRRSV infection

[0062] A method for protecting a porcine fetus from porcine reproductive and respiratory syndrome virus (PRRSV) infection is provided. The method includes breeding a female pig with a male pig. The female pig contains modified chromosomal sequences in both alleles of its CD163 gene, and the modified chromosomal sequences reduce the susceptibility of the female pig to PRRSV infection compared to the susceptibility to PRRSV infection of the female pig, which does not contain any modified chromosomal sequences in the alleles of the CD163 gene. The male pig contains at least one wild-type CD163 allele.

[0063] In the methods described herein, modified chromosomal sequences can be sequences that are altered such that the function of the CD163 protein associated with PRRSV infection is impaired, reduced, or abolished. Thus, female pigs used in the methods described herein may be referred to as "knockout" animals.

[0064] A male pig may contain two wild-type CD163 alleles.

[0065] The term "wild-type CD163 allele" as used herein means that the sequence of the CD163 allele is a sequence found in nature, or that the sequence of the CD163 allele contains one or more mutations (e.g., insertions, deletions, or substitutions) that significantly do not impair CD163 activity. Thus, wild-type CD163 alleles may contain polymorphisms and/or mutations, provided that these polymorphisms or mutations do not significantly reduce CD163 activity.

[0066] Using the methods described herein, the fetus will be protected from both type 1 and type 2 PRRSV viruses, including various type 1 and type 2 PRRSV isolates.

[0067] Thus, in the methods described herein, modified chromosomal sequences can desensitize female pigs to PRRSV type 1, PRRSV type 2, or PRRSV type 1 and type 2.

[0068] Modified chromosomal sequences can reduce the sensitivity of female pigs to a PRRSV isolate selected from the group consisting of NVSL 97-7895, KS06-72109, P129, VR2332, C090, AZ25, MLV-ResPRRS, KS62-06274, KS483 (SD23983) , C084, SD13-15, Lelystad, 03-1059, 03-1060, SD01-08, 4353PZ and combinations thereof.

[0069] The female pig may include a genetically modified female pig.

[0070] The genetically modified female pig may be an animal modified with a homing endonuclease. The homing endonuclease may be a naturally occurring endonuclease, but is preferably a rationally engineered, non-naturally occurring homing endonuclease that has a DNA recognition sequence designed such that the endonuclease targets a chromosomal sequence in the gene encoding the CD163 protein.

[0071] Thus, the homing endonuclease may be a constructed homing endonuclease. The homing endonuclease may include, for example, a regularly spaced clustered short palindromic repeat (CRISPR) system, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a recombinase fusion protein, a meganuclease, or a combination of any of these.

[0072] Homing nuclease preferably includes a CRISPR system. Examples of CRISPR systems that can be used to create female pigs for use in the methods described herein include, without limitation, CRISPR/Cas9, CRISPR/Cas5, and CRISPR/Cas6. The use of CRISPR systems to create genetically modified animals is discussed below.

[0073] In any of the methods described herein, the female pig may contain the same modified chromosomal sequence in both alleles of the CD163 gene.

[0074] Alternatively, the female pig may contain a first modified chromosomal sequence in the first allele of the CD163 gene and a second modified chromosomal sequence in the second allele of the CD163 gene, wherein the first and second modified chromosomal sequences are different from each other.

[0075] In any of the methods described herein, each allele of the female porcine CD163 gene may contain an insertion, a deletion, or a combination thereof.

[0076] For example, at least one allele of the female porcine CD163 gene may contain a deletion.

[0077] At least one allele of the CD163 gene may contain an in-frame deletion.

[0078] At least one allele of the female porcine CD163 gene may contain an insert.

[0079] In the methods described herein, the modified chromosomal sequences preferably cause a decrease in the production or activity of the CD163 protein compared to the production or activity of the CD163 protein in female pigs that lack the modified chromosomal sequences.

[0080] Preferably, the modified chromosomal sequences result in substantially no functional CD163 protein in the female pig. By "substantially free of functional CD163 protein" is meant that the level of CD163 protein in the animal, progeny, or cell is not detectable or, if detectable, is at least about 90% lower, preferably at least about 95% lower, more preferably at least about 98%, lower and even more preferably at least about 99% lower than the level observed in an animal, progeny or cell that does not contain the modified chromosomal sequences.

[0081] For example, in any of the methods described here, the female pig does not produce the CD163 protein.

[0082] In any of the methods described herein, each allele of the female porcine CD163 gene may include an exon 7 modification, an exon 8 modification, an intron modification adjacent to exon 7 or exon 8, or a combination of any of these.

[0083] For example, one or both alleles of the CD163 gene in a female pig may include a modification of exon 7 of the CD163 gene.

[0084] The modification in exon 7 may include a deletion.

[0085] In any of the methods described herein, where the allele of the female porcine CD163 gene contains a deletion, the deletion may include an in-frame deletion.

[0086] The modification in exon 7 may include an insert.

[0087] In any of the methods described herein, modified chromosomal sequences in one or both alleles of the female pig CD163 gene can lead to miscoding.

[0088] If the modified chromosomal sequence results in incorrect encoding of the CD163 gene allele, the incorrect encoding may result in a premature stop codon below the incorrect encoding in the CD163 gene allele.

[0089] In any of the methods described herein, at least one of the alleles of the CD163 gene in a female pig contains a modification selected from the group consisting of: deletion of 11 base pairs from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47; insertions of 2 base pairs between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a deletion of 377 base pairs from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47 on the same allele; deletions of 124 base pairs from nucleotide 3024 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47; deletions of 123 base pairs from nucleotide 3024 to nucleotide 3146 compared to the reference sequence of SEQ ID NO: 47; 1 bp inserts between nucleotides 3147 and 3148 compared to the reference sequence of SEQ ID NO: 47; deletions of 130 base pairs from nucleotide 3030 to nucleotide 3159 compared to the reference sequence of SEQ ID NO: 47; deletions of 132 base pairs from nucleotide 3030 to nucleotide 3161 compared to the reference sequence of SEQ ID NO: 47; deletions of 1506 base pairs from nucleotide 1525 to nucleotide 3030 compared to the reference sequence of SEQ ID NO: 47; insert 7 base pairs between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47; deletions of 1280 base pairs from nucleotide 2818 to nucleotide 4097 compared to the reference sequence of SEQ ID NO: 47; deletions of 1373 base pairs from nucleotide 2724 to nucleotide 4096 compared to the reference sequence of SEQ ID NO: 47; deletions of 1467 base pairs from nucleotide 2431 to nucleotide 3897 compared to the reference sequence of SEQ ID NO: 47; a 1930 bp deletion from nt 488 to nt 2,417 compared to the reference sequence of SEQ ID NO: 47 where the deleted sequence is replaced by a 12 bp insert starting at nt 488 and where there is an additional 129 bp deletion in exon 7 from nt 3044 up to nucleotide 3172 compared to the reference sequence of SEQ ID NO: 47; deletions of 28 base pairs from nucleotide 3145 to nucleotide 3172 compared to the reference sequence of SEQ ID NO: 47; deletions of 1387 base pairs from nucleotide 3145 to nucleotide 4531 compared to the reference sequence of SEQ ID NO: 47; deletions of 1382 base pairs from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113; deletions of 1720 base pairs from nucleotide 2440 to nucleotide 4160 compared to the reference sequence of SEQ ID NO: 47; deletions of 452 base pairs from nucleotide 3015 to nucleotide 3466 compared to the reference sequence of SEQ ID NO: 47; and combinations of any of them.

[0090] At least one of the alleles of the CD163 gene in the female pig may contain an 11 base pair deletion from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47.

[0091] At least one of the alleles of the CD163 gene in a female pig may contain an insertion of 2 base pairs between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a deletion of 377 base pairs from nucleotide 2573 to nucleotide 2949 compared to reference sequence SEQ ID NO: 47 on the same allele.

[0092] If the modification includes an insertion of 2 base pairs between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a deletion of 377 base pairs from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47 on the same allele, and a 2 base pair insert may contain an AG dinucleotide.

[0093] At least one of the alleles of the CD163 gene in the female pig may contain a 124 base pair deletion from nucleotide 3024 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47.

[0094] At least one of the alleles of the CD163 gene in the female pig may contain a 123 base pair deletion from nucleotide 3024 to nucleotide 3146 compared to the reference sequence of SEQ ID NO: 47.

[0095] At least one of the alleles of the CD163 gene in the female pig may contain an insertion of 1 base pair between nucleotides 3147 and 3148 compared to the reference sequence of SEQ ID NO: 47.

[0096] If the modification includes a 1 base pair insert between nucleotides 3147 and 3148 compared to the reference sequence of SEQ ID NO: 47, the 1 base pair insert may contain one adenine residue.

[0097] At least one of the alleles of the CD163 gene in the female pig may contain a 130 bp deletion from nucleotide 3030 to nucleotide 3159 compared to the reference sequence of SEQ ID NO: 47.

[0098] At least one of the alleles of the CD163 gene in the female pig may contain a 132 base pair deletion from nucleotide 3030 to nucleotide 3161 compared to the reference sequence of SEQ ID NO: 47.

[0099] At least one of the alleles of the CD163 gene in the female pig may contain a deletion of 1506 base pairs from nucleotide 1525 to nucleotide 3030 compared to the reference sequence of SEQ ID NO: 47.

[00100] At least one of the alleles of the CD163 gene in the female pig may contain a 7 bp insertion between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47.

[00101] If the modification includes a 7 base pair insert between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47, the 7 base pair insert may contain the sequence TASTAST (SEQ ID NO: 115).

[00102] At least one of the alleles of the CD163 gene in the female pig may contain a 1280 bp deletion from nucleotide 2818 to nucleotide 4097 compared to the reference sequence of SEQ ID NO: 47.

[00103] At least one of the alleles of the CD163 gene in the female pig may contain a deletion of 1373 base pairs from nucleotide 2724 to nucleotide 4096 compared to the reference sequence of SEQ ID NO: 47.

[00104] At least one of the alleles of the CD163 gene in the female pig may contain a 1467 base pair deletion from nucleotide 2431 to nucleotide 3897 compared to the reference sequence of SEQ ID NO: 47.

[00105] At least one of the alleles of the CD163 gene in a female pig may contain a deletion of 1930 base pairs from nucleotide 488 to nucleotide 2417 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 12 base pairs; starting at nucleotide 488, and there is an additional 129 bp deletion in exon 7 from nucleotide 3044 to nucleotide 3172 compared to the reference sequence of SEQ ID NO: 47.

[00106] If the modification includes a 1930 base pair deletion from nucleotide 488 to nucleotide 2417 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by a 12 base pair insert starting at nucleotide 488, and there is an additional 129 base pair deletion bases in exon 7 from nucleotide 3044 to nucleotide 3172 compared to the reference sequence of SEQ ID NO: 47, a 12 base pair insert may contain the sequence TGTGGAGAATTC (SEQ ID NO: 116).

[00107] At least one of the alleles of the CD163 gene in the female pig may contain a 28 base pair deletion from nucleotide 3145 to nucleotide 3172 compared to the reference sequence of SEQ ID NO: 47.

[00108] At least one of the alleles of the CD163 gene in the female pig may contain a 1387 base pair deletion from nucleotide 3145 to nucleotide 4531 compared to the reference sequence of SEQ ID NO: 47.

[00109] At least one of the alleles of the CD163 gene in a female pig may contain a 1382 base pair deletion from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113.

[00110] If the modification includes a deletion of 1382 base pairs from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an 11 base pair insert starting at nucleotide 3113, the 11 base pair insert may contain the sequence AGCCAGCGTGC (SEQ ID NO: 117).

[00111] At least one of the alleles of the CD163 gene in the female pig may contain a 1720 base pair deletion from nucleotide 2440 to nucleotide 4160 compared to the reference sequence of SEQ ID NO: 47.

[00112] At least one of the alleles of the CD163 gene in the female pig may contain a 452 base pair deletion from nucleotide 3015 to nucleotide 3466 compared to the reference sequence of SEQ ID NO: 47.

[00113] For example, at least one of the alleles of the CD163 gene in a female pig contains a modification selected from the group consisting of a 7 base pair insert between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47; insertions of 2 base pairs between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a deletion of 377 base pairs from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47 on the same allele; deletions of 11 base pairs from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47; deletions of 1382 base pairs from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113; and combinations of any of them.

[00114] The CD163 gene in a female pig may contain any combination of the modified chromosomal sequences described herein.

[00115] For example, a female pig may contain an insertion of 7 base pairs between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47 in one allele of the CD163 gene; and insertion of 2 base pairs between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a deletion of 377 base pairs from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47, in the other allele of the CD163 gene.

[00116] The female pig may contain a 1382 base pair deletion from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113 in one allele of the CD163 gene; and a 2 bp insertion between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a 377 base pair deletion from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47, in the other allele of the CD163 gene.

[00117] The female pig may contain an insertion of 7 base pairs between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47 in one allele of the CD163 gene; and deletion of 11 base pairs from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47 in another allele of the CD163 gene.

[00118] The female pig may contain a 1382 base pair deletion from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113 in one allele of the CD163 gene; and deletion of 11 base pairs from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47 in another allele of the CD163 gene.

[00119] In any of the methods described herein, alleles of the female pig CD163 gene may contain a chromosomal sequence having at least 80% sequence identity with SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion.

[00120] Alleles of the female pig CD163 gene may contain a chromosomal sequence having at least 85% sequence identity with SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion.

[00121] Alleles of the female pig CD163 gene may contain a chromosomal sequence having at least 90% sequence identity with SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion.

[00122] Alleles of the female porcine CD163 gene may contain a chromosomal sequence having at least 95% sequence identity with SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion.

[00123] Alleles of the female pig CD163 gene may contain a chromosomal sequence having at least 98% sequence identity with SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion.

[00124] Alleles of the female pig CD163 gene may contain a chromosomal sequence having at least 99% sequence identity with SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion.

[00125] Alleles of the female porcine CD163 gene may contain a chromosomal sequence having at least 99.9% sequence identity with SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion.

[00126] Alleles of the female pig CD163 gene may contain a chromosomal sequence having 100% sequence identity with SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion.

[00127] In any of the methods described herein, the female pig may comprise a chromosomal sequence comprising SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 , 113, 114 or 119 in one or both alleles of the CD163 gene.

[00128] For example, a female pig may contain a chromosomal sequence comprising SEQ ID NO: 99, 102, 103 or 113 in one or both alleles of the CD163 gene.

[00129] Alleles of the CD163 gene in a female pig may contain any combination of chromosomal sequences, including SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 , 113, 114, or 119. Thus, a female pig may comprise a chromosomal sequence comprising any of SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 119 a water allele of the CD163 gene and a chromosome sequence comprising any of SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 119 in another allele of the CD163 gene.

[00130] For example, a female pig may contain a chromosomal sequence comprising SEQ ID NO: 99 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 103 in another allele of the CD163 gene.

[00131] The female pig may contain a chromosomal sequence comprising SEQ ID NO: 113 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 99 in another allele of the CD163 gene.

[00132] The female pig may contain a chromosomal sequence comprising SEQ ID NO: 99 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 102 in another allele of the CD163 gene.

[00133] The female pig may contain a chromosomal sequence comprising SEQ ID NO: 113 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 102 in another allele of the CD163 gene.

[00134] In any of the methods described here, breeding will produce one or more fetuses that contain a modified chromosomal sequence in a single allele of the CD163 gene. Because the female pig used in the methods described herein contains modified chromosomal sequences in both alleles of its CD163 gene, breeding a female pig with a male pig containing at least one wild-type CD163 allele will produce fetuses that inherit the CD163 allele. containing a modified chromosomal sequence from a female pig and a wild-type CD163 allele from a male pig. Thus, as a result of breeding, fruits that are heterozygous for the modified CD163 chromosomal sequence will be obtained. If a male contains two wild-type CD163 alleles, all breeding fetuses will be heterozygous for the modified CD163 chromosomal sequence.

[00135] Breeding fetuses will have a reduced susceptibility to PRRSV infection in utero compared to fetuses in the womb of a wild-type pig.

[00136] In any of the methods described herein, breeding may include mating (copulation) of a female pig with a male pig.

[00137] In any of the methods described herein, breeding may include artificial insemination of a female animal with sperm obtained from a male animal.

[00138] In any of the methods described herein, breeding may include the transfer of a fertilized oocyte into the reproductive tract of a female pig.

[00139] In methods in which breeding involves transferring the fertilized oocyte into the reproductive tract of a female pig, the fertilized oocyte can be obtained by fertilizing the oocyte in vitro with sperm obtained from a male pig.

[00140] In vitro fertilization may include intracytoplasmic injection of an oocyte with sperm obtained from a male pig.

[00141] When breeding involves the transfer of a fertilized oocyte into the reproductive tract of a female pig, the oocyte may be an oocyte derived from a female pig such that the oocyte contains modified chromosomal sequences in both alleles of the CD163 gene. Alternatively, the oocyte may be an oocyte derived from another female pig containing modified chromosomal sequences in both alleles of its CD163 gene, wherein the modified chromosomal sequences reduce the animal's susceptibility to PRRSV infection compared to the susceptibility to PRRSV infection of a female pig that does not contain the modified chromosomal sequences in the alleles of their CD163 gene. Thus, for example, any oocyte having modified chromosomal sequences in both alleles of its CD163 gene (eg, knockouts of both alleles of the C163 gene) can be used.

[00142] However, when breeding involves the transfer of a fertilized oocyte into the reproductive tract of a female pig, the oocyte need not contain any modified chromosomal sequences in the CD163 gene alleles. An oocyte containing two wild type CD163 alleles can be used. An oocyte containing two wild-type CD163 alleles can be fertilized with sperm obtained from a male pig, where the sperm contains two wild-type CD163 alleles, to create a fertilized oocyte containing two wild-type CD163 alleles. If such a fertilized oocyte (containing two wild-type CD163 alleles) is transferred into the reproductive tract of a female pig containing modified chromosomal sequences of both alleles of its CD163 gene, the resulting fetus will be protected from PRRSV infection.

[00143] Alternatively, where breeding involves transfer of a fertilized oocyte into the reproductive tract of a female pig, the fertilized oocyte may contain a modified chromosomal sequence in the single allele of its CD163 gene. Such fertilized oocytes will also produce fetuses that are protected from PRRSV infection upon transfer of the fertilized oocyte into the reproductive tract of a female pig containing modified chromosomal sequences in both alleles of its CD163 gene.

[00144] Thus, when breeding involves the transfer of a fertilized oocyte into the reproductive tract of a female pig, the fertilized oocyte may contain modified chromosomal sequences in both alleles of its CD163 gene (for example, knockouts of both alleles of the CD163 gene), may contain a modified chromosomal sequence in only one allele its CD163 gene or may contain only wild-type CD163 alleles.

Affine labels

[00145] An "affinity tag" can be either a peptide affinity tag or a nucleic acid affinity tag. The term "affinity tag" generally refers to a protein or nucleic acid sequence that can be linked to a molecule (eg, linked to a small molecule, protein, or linked by a covalent bond). The affine label may be a non-native sequence. The peptide affinity tag may include a peptide. The peptide affinity tag may be part of a split system (eg, two inactive peptide fragments may be combined together in trans form to form an active affinity tag). A nucleic acid affinity tag may comprise a nucleic acid. A nucleic acid affinity tag may be a sequence that can selectively bind to a known nucleic acid sequence (eg, via hybridization). A nucleic acid affinity tag can be a sequence that can selectively bind to a protein. The affinity tag can be fused to the native protein. An affinity tag may be fused to a nucleotide sequence.

[00146] Sometimes one, two or more affinity tags can be fused to a native protein or nucleotide sequence. An affinity tag can be introduced into a nucleic acid that targets the nucleic acid using in vitro or in vivo transcription methods. Nucleic acid affinity tags may include, for example, a chemical tag, an RNA binding protein binding sequence, a DNA binding protein binding sequence, a sequence hybridizable to an affinity tagged polynucleotide, a synthetic RNA aptamer, or a synthetic DNA aptamer. Examples of chemical affinity labels for nucleic acids may include, without limitation, ribonucleotriphosphates containing biotin, fluorescent dyes, and digoxeginin. Examples of protein-binding nucleic acid affinity tags may include, without limitation, MS2 binding sequence, U1A binding sequence, stem-loop binding protein sequences, boxB sequence, eIF4A sequence, or any sequence recognized by the RNA-binding protein. Examples of nucleic acid affinity tagged oligonucleotides may include, without limitation, biotinylated oligonucleotides, 2,4-dinitrophenyl oligonucleotides, fluorescein oligonucleotides, and primary amine conjugated oligonucleotides.

[00147] The nucleic acid affinity tag can be an RNA aptamer. Aptamers may include aptamers that bind to theophylline, streptavidin, dextran B512, adenosine, guanosine, guanine/xanthine, 7-methyl-GTP, amino acid aptamers such as aptamers that bind to arginine, citrulline, valine, tryptophan, cyanocobalamin, N β-methylmesoporphyrin IX, flavin, NAD, and antibiotic aptamers such as aptamers that bind to tobramycin, neomycin, lividomycin, kanamycin, streptomycin, viomycin, and chloramphenicol.

[00148] A nucleic acid affinity tag may contain an RNA sequence that can be linked to a site-directed polypeptide. The site-directed polypeptide may be conditionally enzymatically inactive. The RNA sequence may contain a sequence that may be associated with a member of the type I, type II and/or type III CRISPR systems. The RNA sequence can be associated with a protein - a member of the RAMP family. The RNA sequence may be associated with a protein - a member of the Cas9 family, a protein - a member of the Cas6 family (for example, Csy4, Cas6). The RNA sequence may be associated with a protein - a member of the Cas5 family (for example, Cas5). For example, Csy4 can bind with high affinity to a specific RNA hairpin sequence (Kd ~ 50 pM) and can cleave RNA at the 3' hairpin region.

[00149] A nucleic acid affinity tag may contain a DNA sequence that can be linked to a site-directed polypeptide. The site-directed polypeptide may be conditionally enzymatically inactive. The DNA sequence may include a sequence that may be associated with a member of the type I, type II and/or type III CRISPR systems. The DNA sequence can be linked to the argonaut protein. The DNA sequence can be linked to a protein containing a zinc finger domain, a TALE domain, or any other DNA binding domain.

[00150] A nucleic acid affinity tag may contain a ribozyme sequence. Suitable ribozymes may include 23 SrRNA peptidyltransferase, RNase P, group I introns, group II introns, GIR1 branched ribozyme, Leadzyme, hairpin ribozymes, hammerhead ribozymes, HDV ribozymes, CPEB3 ribozymes, VS ribozymes, glmS ribozyme, CoTC ribozyme, and synthetic ribozymes.

[00151] Peptide affinity tags may contain tags that can be used for tracking or purification (e.g., fluorescent protein such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato; His tag, (e.g., 6XHis tag ); hemagglutinin (HA) tag; FLAG tag; Myc tag; GST tag; MBP tag; chitin binding protein tag; calmodulin tag; V5 tag; streptavidin binding tag; etc.).

[00152] Both nucleic acids and peptide affinity tags can contain small molecule tags such as biotin or digitoxin and fluorescent tags such as, for example, fluorescein, rhodamine, Alexa fluorescent dyes, Cyanine3 dye, Cyanine5 dye.

[00153] Nucleic acid affinity tags can be located at the 5' end of the nucleic acid (eg, a nucleic acid that targets a nucleic acid). Nucleic acid affinity tags can be located at the 3' end of the nucleic acid. Nucleic acid affinity tags can be located at the 5' and 3' end of the nucleic acid. Nucleic acid affinity tags can be located within the nucleic acid. Peptide affinity tags may be located at the N-terminus of the polypeptide sequence. Peptide affinity tags may be located at the C-terminus of a polypeptide sequence. Peptide affinity tags can be located at the N-terminus and C-terminus of the polypeptide sequence. A plurality of affinity tags may be fused to a nucleic acid and/or polypeptide sequence.

Capture Agents

[00154] As used herein, the term "capture agent" can generally refer to an agent that can purify a polypeptide and/or nucleic acid. The capture agent can be a biologically active molecule or material (for example, any biological substance, naturally occurring or synthetic, and includes, without limitation, cells, viruses, subcellular particles, proteins, in particular antibodies, immunoglobulins, antigens, lipoproteins, glycoproteins, peptides, polypeptides, protein complexes, (strept)avidin-biotin complexes, ligands, receptors or small molecules, aptamers, nucleic acids, DNA, RNA, peptide nucleic acids, oligosaccharides, polysaccharides, lipopolysaccharides, cellular metabolites, haptens, pharmacologically active substances, alkaloids, steroids, vitamins, amino acids and sugars). In some embodiments, the capture agent may contain an affinity tag. In some embodiments, the capture agent may preferentially bind to the target polypeptide or nucleic acid of interest. Capture agents are free to float in the mixture. Capture agents may be associated with a particle (eg, bead, microsphere, nanoparticle). Capture agents may be associated with a solid or semi-liquid surface. In some cases, capture agents are irreversibly bound to the target. In other cases, the capture agents are reversibly bound to the target (for example, if the target can be eluted, or with a chemical such as imidazole).

DNA binding polypeptides

[00155] Site-specific integration can be achieved using factors that are able to recognize and bind to specific nucleotide sequences, for example, in the genome of the host organism. For example, many proteins contain polypeptide domains that are able to recognize and bind to DNA in a site-specific manner. A DNA sequence that is recognized by a DNA binding polypeptide may be referred to as a "target" sequence. Polypeptide domains that are capable of recognizing and binding to DNA in a site-specific manner usually fold correctly and function independently, binding DNA in a site-specific manner, even when expressed in a different polypeptide from the protein from which the domain was originally isolated. Similarly, target sequences for recognition and binding by DNA-binding polypeptides can generally be recognized and bound by such polypeptides even if they are present in large DNA structures (eg, a chromosome), especially when the site where the target sequence is located is at a location which is known to be accessible to soluble cellular proteins (eg, in a gene).

[00156] Although DNA-binding polypeptides identified from naturally occurring proteins are typically associated with a specific nucleotide sequence or motif (e.g., a consensus recognition sequence), methods exist and are known in the art to modify many such DNA-binding polypeptides. for the purpose of recognizing another nucleotide sequence or motif. DNA binding polypeptides include, for example, and without limitation: zinc finger DNA binding domains; leucine zippers; DNA-binding domains of UFA; GAL4; TAL; LexA; Tet repressors; LacI; and steroid hormone receptors.

[00157] For example, the DNA binding polypeptide can be a zinc finger. Individual zinc finger motifs can be designed to target and specifically bind to any of a wide range of DNA regions. Canonical Cys2His2 (as well as non-canonical Cys3His) zinc finger polypeptides bind DNA by inserting an α-helix into the major groove of the target DNA double helix. Zinc finger DNA recognition is modular; each finger contacts primarily three consecutive base pairs in the target, and several key residues in the polypeptide mediate recognition. By incorporating several zinc finger DNA-binding domains into the homing endonuclease, the DNA-binding specificity of the homing endonuclease can be further increased (and therefore the specificity of any gene regulatory effects associated therewith can also be increased). See, for example, Umov et al. (2005) Nature 435:646-51. Thus, one or more zinc finger DNA-binding polypeptides can be designed and used such that a homing endonuclease introduced into a host cell interacts with a DNA sequence that is unique within the host cell's genome.

[00158] Preferably, the zinc finger protein is not naturally occurring because it is designed to bind to a chosen target site. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; US Patents No. 6453242; 6534261; 6599692; 6503717; 6689558; 7030215; 6794136; 7067317; 7262054; 7070934; 7361635; 7253273; and US Patent Publication No. 2005/0064474; 2007/0218528; 2005/0267061.

[00159] The engineered zinc finger binding domain may have novel binding specificity compared to natural zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, the use of databases containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more zinc finger amino acid sequences that link a particular triplet or quadruplet sequence. . See, for example, US Patent Nos. 6,453,242 and 6,534,261.

[00160] Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in US Pat. Nos. 5,789,538; 5925523; 6007988; 6013453; 6410248; 6140466; 6200759; and 6242568; as well as WO 98/37186; W098/53057; WO00/27878; WO 01/88197 and GB 2338237. In addition, increased binding specificity for zinc finger binding domains is described, for example, in WO 02/077227.

[00161] In addition, as described in these and other references, zinc finger domains and/or multifinger zinc finger proteins can be linked together using any suitable linker sequences, including, for example, linkers of 5 or more amino acids in length. For exemplary linker sequences of 6 or more amino acids in length, see also US Pat. Nos. 6,479,626; 6903185; and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

[00162] Selection of target sites: ZFPs and methods for designing and constructing fusion proteins (and polynucleotides encoding them) are known to those skilled in the art and are detailed in US Pat. Nos. 6,1400,815; 789538; 6453242; 6534261; 5925523; 6007988; 6013453; 6200759; WO 95/19431; W096/06166; W098/53057; W098/54311; WO00/27878; WO 01/60970; WO 01/88197; WO 02/099084; W098/53058; W098/53059; W098/53060; WO 02/016536 and WO 03/016496.

[00163] In addition, as disclosed in these and other references, zinc finger domains and/or multi-finger zinc finger proteins can be linked together using any suitable linker sequences, including, for example, linkers of 5 or more amino acids in length. See also US Patent Nos. 6479626; 6903185; and 7153949 for exemplary linker sequences of 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

[00164] If a female pig for use in the methods described herein has been genetically modified using a zinc finger nuclease, the female animal can be generated using a process comprising introducing into an embryo or cell at least one RNA molecule encoding the target nuclease a zinc finger; and optionally at least one helper polynucleotide. The method further includes incubating the embryo or cell to allow expression of the zinc finger nuclease, wherein the double strand break introduced into the target chromosomal sequence by the zinc finger nuclease is repaired by an error-prone non-homologous end-joining DNA repair process or a homology-directed DNA repair process. The method for editing chromosomal sequences encoding a germline developmental protein using targeted zinc finger nuclease technology is fast, accurate and highly efficient.

[00165] Alternatively, the DNA binding polypeptide is a DNA binding domain from GAL4. GAL4 is a modular transactivator in Saccharomyces cerevisiae, but it also acts as a transactivator in many other organisms. See, for example, Sadowski et al. (1988) Nature 335:563-4. In this regulatory system, the expression of genes encoding enzymes of the galactose metabolic pathway in S. cerevisiae is tightly regulated by the available carbon source. Johnston (1987) Microbiol. Rev. 51:458-76. Transcriptional control of these metabolic enzymes is mediated by an interaction between a positive regulatory protein, GAL4, and a 17 bp symmetrical DNA sequence to which GAL4 specifically binds (upstream activation sequence (UAS)).

[00166] Native GAL4 consists of 881 amino acid residues with a molecular weight of 99 kDa. GAL4 includes functionally autonomous domains, the cumulative activity of which determines the activity of GAL4 in vivo. Ma and Ptashne (1987) Cell 48:847-53); Brent and Ptashne (1985) Cell 43(3 Pt 2):729-36. The 65 N-terminal amino acids of GAL4 make up the DNA-binding domain of GAL4. Keegan et al. (1986) Science 231:699-704; Johnston (1987) Nature 328:353-5. Sequence-specific binding requires the presence of a divalent cation coordinated by the six Cys residues present in the DNA binding domain. The coordinated cation-containing domain interacts with a conserved CCG triplet at each end of the 17 bp UAS. and recognizes it through direct contact with the major groove of the DNA helix. Marmorstein et al. (1992) Nature 356:408-14. The DNA-binding function of proteins positions the C-terminal transcription activating domains near the promoter so that the activating domains can direct transcription.

[00167] Additional DNA binding polypeptides that can be used include, for example, but not limited to, a binding sequence from a gene inducible by AVRBS3; a consensus binding sequence from an AVRBS3 inducible gene or a synthetic binding sequence constructed therefrom (eg, a UPA DNA binding domain); TAL; LexA (see, for example, Brent & Ptashne (1985), supra); LacR (see e.g. Labow et al. (1990) Mol. Cell. Biol. 10:3343-56; Bairn et al. (1991) Proc. Natl. Acad. Sci. USA 88(12):5072-6 ); steroid hormone receptor (Elliston et al. (1990) J. Biol. Chern. 265:11517-121); penpeccop Tet (US Patent No. 6271341) and a mutant Tet repressor that binds to the tet operator sequence in the presence but not in the absence of tetracycline (Tc); DNA-binding domain of NF-kappaB; and regulatory system components described in Wang et al. (1994) Proc. Natl. Acad. sci. USA 91(17):8180-4 which uses a fusion of GAL4, hormone receptor and VP16.

[00168] The DNA binding domain of one or more nucleases used in the methods and compositions described herein may include a natural or engineered (non-naturally occurring) effector TAL DNA binding domain. See, for example. US Patent Publication No. 2011/0301073.

[00169] Alternatively, the nuclease may include a CRISPR system. For example, the nuclease may comprise the CRISPR/Cas system.

[00170] The system (associated with CRISPR) evolved in bacteria and archaea as an adaptive immune system to protect against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. The RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. This RNA, which contains a complementary sequence to the viral genome, mediates targeting of a Cas protein (eg, Cas9 protein) to a sequence in the viral genome. The Cas protein cleaves and thereby disables the viral target. Recently, the CRISPR/Cas system has been adapted for genome editing in eukaryotic cells. The introduction of site-specific double strand breaks (DSBs) allows the target sequence to be altered using one of two endogenous DNA repair mechanisms, either non-homologous end-joining (NHEJ) or homologous-directed DNA repair. reparations (homology-directed repair - HDR). The CRISPR/Cas system has also been used for gene regulation, including transcriptional repression and activation without altering the target sequence. Targeted gene regulation based on the CRISPR/Cas system can, for example, use enzymatically inactive Cas9 (also known as catalytically dead Cas9).

[00171] CRISPR/Cas systems include the CRISPR locus (grouped at regular intervals, short palindromic repeats), which encodes components of the RNA system, and the Cas (CRISPR-associated) locus, which encodes proteins. (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput Biol 1: e60). CRISPR loci in microbial hosts contain a combination of Cas genes as well as non-coding RNA elements capable of programming the specificity of CRISPR-mediated nucleic acid cleavage.

[00172] Type II CRISPR is one of the most well-studied systems and performs targeted DNA double-strand breaks in nature in four successive steps. In the first step, two non-coding RNAs, template pre-crRNA and tracrRNA, are transcribed from the CRISPR locus. In the second step, tracrRNA hybridizes to repetitive regions of pre-crRNA and mediates the processing of pre-crRNA into mature crRNA containing individual spacer sequences. In the third step, the mature crRNA:tracrRNA complex guides Cas9 to the target DNA via Watson-Crick base pairing between a spacer on the crRNA and a protospacer on the target DNA next to the adjacent protospacer motif (AMS), which is an additional requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA to form a double-strand break within the protospacer.

[00173] To use the CRISPR/Cas system to create targeted insertions and deletions, two non-coding RNAs (crRNA and TracrRNA) can be replaced by a single RNA called guide RNA (gRNA). The activity of the CRISPR/Cas system consists of three steps: (i) insertion of exogenous DNA sequences to prevent future attacks into the CRISPR template in a process called "adaptation", (ii) expression of the appropriate proteins, and expression and processing of the template, followed by (iii) RNA-mediated interference with a foreign nucleic acid. In a bacterial cell, several Cas proteins are involved in the natural function of the CRISPR/Cas system and perform functions such as the insertion of foreign DNA, etc.

[00174] The Cas protein may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound that has a qualitative biological property in common with the native sequence polypeptide. "Functional derivatives" include, without limitation, native sequence fragments and derivatives of a native sequence polypeptide and fragments thereof, provided they share biological activity with the corresponding native sequence polypeptide. The biological activity considered here is the ability of a functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both variants of the amino acid sequence of a polypeptide and their covalent modifications and fusions. Suitable derivatives of a Cas polypeptide or fragment thereof include, without limitation, mutants, fusions, covalent modifications of the Cas protein or fragment thereof. The Cas protein, which includes the Cas protein or a fragment thereof, as well as derivatives of the Cas protein or a fragment thereof, can be obtained from a cell or synthesized chemically or by a combination of the two. The cell may be a cell that naturally produces a Cas protein or a cell that naturally produces a Cas protein and is genetically engineered to produce an endogenous Cas protein with a higher level of expression, or to produce a Cas protein from an exogenously introduced nucleic acid that encodes a Cas that is same or different from endogenous Cas. In some cases, the cell does not naturally produce the Cas protein and is genetically engineered to produce the Cas protein.

[00175] If a female pig for use in the methods described herein has been genetically modified using a CRISPR system, the CRISPR/Cas9 system can be used to create a female pig. To use Cas9 to edit genomic sequences, the protein can be delivered directly into the cell. Alternatively, the mRNA encoding Cas9 can be delivered to the cell, or the gene that allows expression of the mRNA encoding Cas9 can be delivered to the cell. In addition, either the target-specific crRNA or tracrRNA can be delivered directly to the cell, or the target-specific gRNA(s) can be delivered to the cell (these RNAs can alternatively be produced by a gene engineered to express these RNAs). The choice of target sites and construction of crRNA/gRNA is well known in the art. A discussion of gRNA construction and cloning can be found at http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509.pdf.

[00176] A DNA-binding polypeptide can specifically recognize and bind to a target nucleotide sequence contained in the host's genomic nucleic acid. In some examples, any number of discrete examples of the target nucleotide sequence can be found in the host genome. The target nucleotide sequence may be rare in an organism's genome (e.g., less than about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 copy(s) may exist in the genome. ) of the target sequence). For example, the target nucleotide sequence may be located at a unique site in the organism's genome. Target nucleotide sequences, for example and without limitation, can be randomly distributed throughout the genome relative to each other; located in different linkage groups in the genome; be in the same link group; are located on different chromosomes; located on the same chromosome located in the genome at sites that are expressed under similar conditions in the body (eg, under the control of the same or essentially functionally identical regulatory factors); and are located close to each other in the genome (for example, target sequences may be contained in nucleic acids integrated as concatemers at genomic loci).

Homing endonucleases

[00177] A DNA binding polypeptide that specifically recognizes and binds to a target nucleotide sequence may be contained in a chimeric polypeptide so as to allow specific binding to the target sequence of the chimeric polypeptide. In examples, such a chimeric polypeptide may comprise, for example, but not limited to, nuclease, recombinase and/or ligase polypeptides according to the polypeptides described above. Chimeric polypeptides containing a DNA binding polypeptide and a nuclease, recombinase and/or ligase polypeptide may also include other functional polypeptide motifs and/or domains, such as, for example, and without limitation: a spacer sequence located between the functional polypeptides in the chimeric protein; leader peptide; a peptide that targets the fusion protein to an organelle (eg, nucleus); polypeptides that are cleaved by a cellular enzyme; peptide labels (eg, Myc, His, etc.); and other amino acid sequences that do not affect the function of the chimeric polypeptide.

[00178] Functional polypeptides (eg, DNA binding polypeptides and nuclease polypeptides) in a chimeric polypeptide may be operably linked. Functional polypeptides of a chimeric polypeptide can be operably linked by expression from a single polynucleotide encoding at least functional polypeptides ligated to each other in frame to create a chimeric gene encoding a chimeric protein. Alternatively, the functional polypeptides of the chimeric polypeptide may be operably linked in other ways, such as by cross-linking independent expressed polypeptides.

[00179] A DNA-binding polypeptide or guide RNA that specifically recognizes and binds to a target nucleotide sequence may be contained within a naturally occurring isolated protein (or a mutant thereof), wherein the naturally occurring isolated protein or a mutant thereof also contains a nuclease polypeptide (and may also contain recombinase and/or ligase polypeptide). Examples of such isolated proteins include TALEN, recombinases (eg, Cre, Hin, Tre, and FLP recombinases), CRISPR/Cas9, guided RNA, and meganucleases.

[00180] In the context of the present description, the term "homing endonuclease" refers to natural or engineered isolated proteins and their mutants that contain a DNA-binding polypeptide or guide RNA and a nuclease polypeptide, as well as chimeric polypeptides containing a DNA-binding polypeptide or guide RNA and nuclease. Any homing endonuclease containing a DNA-binding polypeptide or guide RNA that specifically recognizes and binds to a target nucleotide sequence contained in the CD 163 locus may be used (for example, either because the target sequence is contained in a native sequence at the locus, or because that the target sequence has been introduced into the locus, eg by recombination).

[00181] Some examples of suitable chimeric polypeptides include, without limitation, combinations of the following polypeptides: Zinc fingers DNA-binding polypeptides; a FokI nuclease polypeptide; TALE domains; leucine zippers; DNA-binding transcription factor motifs; and DNA recognition and/or cleavage domains isolated from, for example, but not limited to, TALEN, recombinase (eg, Cre, Hin, RecA, Tre and FLP recombinase), CRISPR/Cas9 guided RNA, meganuclease; and other polypeptides known to those skilled in the art. Specific examples include a chimeric protein containing a site-specific DNA-binding polypeptide and a nuclease polypeptide. Chimeric polypeptides can be designed by methods known to those skilled in the art to alter the recognition sequence of a DNA binding polypeptide contained in a chimeric polypeptide so as to target the chimeric polypeptide to a particular nucleotide sequence of interest.

[00182] The chimeric polypeptide may contain a DNA binding domain (eg, zinc finger, TAL effector domain, etc.) and a nuclease (cleavage) domain. The cleavage domain may be heterologous to a DNA binding domain, such as a zinc finger DNA binding domain and a cleavage domain from a nuclease, or a TALEN DNA binding domain and a cleavage domain, or a meganuclease DNA binding domain and a cleavage domain from another nuclease. Heterologous cleavage domains can be derived from any endonuclease or exonuclease. Examples of endonucleases from which a cleavage domain can be derived include, without limitation, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional DNA-cleaving enzymes are known (eg, nuclease 51; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast endonuclease HO; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.

[00183] Similarly, a cleavage half-domain can be derived as above from any nuclease or portion thereof that requires dimerization for cleavage activity. In general, if fusion proteins contain cleavage half-domains, two fusion proteins are required for cleavage. Alternatively, a single protein containing two cleavage half-domains can be used. The two cleavage half-domains may be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain may be derived from a different endonuclease (or functional fragments thereof). In addition, the target sites for the two fusion proteins are located preferentially with respect to each other such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in spatial orientation to each other, which allows the cleavage half-domains to form a functional cleavage domain, e.g. , by dimerization. Thus, the near edges of the target sites can be separated by 5-8 nucleotides or 15-18 nucleotides. However, any integer number of nucleotides or base pairs (eg, 2 to 50 base pairs or more) can be between two target sites. Typically, the cleavage site is between the target sites.

[00184] Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequentially binding to DNA (at the recognition site) and cleaving DNA at or near the binding site, for example, so that one or more exogenous sequences (donors/transgenes) integrated into binding sites (targets) or near them. Some restriction enzymes (eg type IIS) cleave DNA at sites remote from the recognition site and have separate binding and cleavage domains. For example, the US Fok I type enzyme catalyzes double-stranded DNA cleavage 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, for example, US Patent Nos. 5356802; 5436150 and 5487994; and Li et al. (1992) Proc. Natl. Acad. sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. sci. USA 90:2764-2768; Kirn et al. (1994a) Proc. Natl. Acad. sci. USA 91:883-887; Kirn et al. (1994b) J. Biol. Chem. 269:31.978-31.982. Thus, fusion proteins may include a cleavage domain (or cleavage half-domain) of at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.

[00185] An example of a type IIS restriction enzyme whose cleavage domain is separated from the binding domain is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. sci. USA 95: 10570-10575. Accordingly, for the purposes of the present invention, the portion of the Fok I enzyme used in the described fusion proteins is considered to be the cleavage half-domain. In this way; For targeted double-stranded cleavage and/or targeted replacement of cellular sequences using a zinc finger-Fok I fusion, two fusion proteins each containing a FokI cleavage half-domain can be used to restore the catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a DNA binding domain and two FokI cleavage half-domains can also be used.

[00186] A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity or that retains the ability to multimerize (eg, dimerize) to form a functional cleavage domain.

[00187] Exemplary type IIS restriction enzymes are described in US Patent Publication No. 2007/0134796. Additional restriction enzymes also contain shared binding and cleavage domains, and these are discussed in the present description. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

[00188] A cleavage domain may contain one or more engineered cleavage half-domains (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, as described, for example, in US Patent Publication Nos. 2005/0064474; 2006/0188987 and 2008/0131962.

[00189] Alternatively, nucleases can be assembled in vivo at the target site of a nucleic acid using so-called "split enzyme" technology (see, for example, US Patent Publication No. 20090068164). The components of such cleaving enzymes may be expressed either in separate expression constructs, or may be linked in a single open reading frame where the individual components are separated, for example, by a 2A self-cleaving peptide or an IRES sequence. The components can be individual zinc finger binding domains or meganuclease and nucleic acid binding domain domains.

Nucleases "zinc fingers"

[00190] The chimeric polypeptide may comprise a specially designed zinc finger nuclease (ZFN) that can be engineered to deliver a target site-specific double-stranded DNA break into which exogenous nucleic acid or donor DNA can be integrated (see US Patent Publication 2010/0257638 ). ZFNs are chimeric polypeptides containing a non-specific restriction endonuclease cleavage domain (eg, FokI) and a zinc finger DNA-binding domain polypeptide. See, for example, Huang et al. (1996) J. Protein Chem. 15:481-9; Kirn et al. (1997a) Proc. Natl. Acad. sci. USA 94:3616-20; Kirn et al. (1996) Proc. Natl. Acad. sci. USA 93:1156-60; Kirn et al. (1994) Proc Natl. Acad. sci. USA 91:883-7; Kirn et al. (1997b) Proc. Natl. Acad. sci. USA 94:12875-9; Kirn et al. (1997 c) Gene 203:43-9; Kirn et al. (1998) Biol. Chem. 379:489-95; Nahon and Raveh (1998) Nucleic Acids Res. 26:1233-9; Smith et al. (1999) Nucleic Acids Res. 27:674-81. ZFNs may contain non-canonical zinc finger DNA binding domains (see US Patent Publication 2008/0182332). The restriction endonuclease Fokl must dimerize through the nuclease domain in order to cleave the DNA and introduce a double-strand break. Therefore, ZFNs containing a nuclease domain from such an endonuclease also require dimerization of the nuclease domain in order to cleave the target DNA. Mani et al. (2005) Biochem. Biophys. Res. commun. 334:1191-7; Smith et al. (2000) Nucleic Acids Res. 28:3361-9. ZFN dimerization can be facilitated by two adjacent, oppositely oriented DNA-binding sites. There.

[00191] A method for site-specific integration of an exogenous nucleic acid into at least one CD163 locus of a host may include introducing a ZFN into the host cell, wherein the ZFN recognizes and binds to a target nucleotide sequence, wherein the target nucleotide sequence is contained in at least one CD163 locus of the host . In some examples, the target nucleotide sequence is not contained in the host genome at any position other than at least one CD163 locus. For example, a ZFN DNA binding polypeptide can be designed to recognize and bind to a target nucleotide sequence identified at at least one CD163 locus (eg, by sequencing the CD163 locus). A method for site-specific integration of an exogenous nucleic acid into at least one functional CD163 host locus, which comprises introducing a ZFN into the host cell, may also include introducing the exogenous nucleic acid into the cell, wherein the exogenous nucleic acid is recombined into a host nucleic acid containing at least at least one CD163 locus, promotes site-specific recognition and binding of ZFN to the target sequence (and subsequent cleavage of the nucleic acid comprising the CD163 locus).

Optional exogenous nucleic acids for integration into the CD163 locus

[00192] Exogenous nucleic acids for integration into the CD163 locus include: exogenous nucleic acid for site-specific integration at at least one CD163 locus, such as, but not limited to, ORF; a nucleic acid containing a nucleotide sequence encoding a homing endonuclease; and a vector containing at least one of any or both of the above. Thus, specific nucleic acids include polypeptide-encoding nucleotide sequences, structural nucleotide sequences, and/or DNA-binding polypeptide recognition and binding sites.

Optional exogenous nucleic acid molecules for site-specific integration

[00193] As noted above, the insertion of an exogenous sequence (also referred to as a "donor sequence", "donor" or "transgene") is provided, for example, for the expression of a polypeptide, the correction of a mutant gene, or for increased expression of a wild-type gene. It is clear that the donor sequence is usually not identical to the genomic sequence in which it is placed. The donor sequence may contain a non-homologous sequence flanked by two regions of homology to allow efficient homology targeted repair (HDR) at the site of interest. In addition, donor sequences may include a vector molecule containing sequences that are not homologous to the region of interest in the cell chromatin. The donor molecule may contain several discontinuous regions of homology with cellular chromatin. For example, for targeted insertion of sequences not normally present in the region of interest, said sequences may be present in the donor nucleic acid molecule and flanked by regions of homology to a sequence in the region of interest.

[00194] The donor polynucleotide may be DNA or RNA, single or double stranded, and may be introduced into the cell in linear or circular form. See, for example, US Patent Publication Nos. 2010/0047805, 2011/0281361, 2011/0207221 and 2013/0326645. When introduced in linear form, the ends of the donor sequence can be protected (eg, from exonucleolytic degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methylibose or deoxyribose residues.

[00195] A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, origins of replication, promoters, and genes encoding antibiotic resistance. Moreover, the donor polynucleotides can be administered as a naked nucleic acid, as a complex of a nucleic acid with an agent such as a liposome or a poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus, and lentivirus, defective by integrase (IDLV)).

[00196] The donor is usually integrated such that its expression is driven by an endogenous promoter at the integration site, namely a promoter that drives the expression of the endogenous gene into which the donor is integrated (eg, CD163). However, it will be appreciated that the donor may include a promoter and/or enhancer, such as a constitutive promoter or an inducible or tissue-specific promoter.

[00197] In addition, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides, and/or polyadenylation signals.

[00198] Exogenous nucleic acids that can be integrated in a site-specific manner into at least one CD163 locus to modify the CD163 locus include, for example, but not limited to, nucleic acids comprising a nucleotide sequence encoding a polypeptide of interest; nucleic acids containing an agronomic gene; nucleic acids containing a nucleotide sequence encoding an RNAi molecule; or nucleic acids that disrupt the CD163 gene.

[00199] An exogenous nucleic acid can be integrated into the CD163 locus so as to modify the CD163 locus, wherein the nucleic acid contains a nucleotide sequence encoding a polypeptide of interest such that the nucleotide sequence is expressed in the host from the CD163 locus. In some examples, the polypeptide of interest (eg, a foreign protein) is expressed from a nucleotide sequence encoding the polypeptide of interest in commercial amounts. In such examples, the polypeptide of interest may be extracted from a host cell, tissue, or biomass.

Nucleic acid molecules containing a nucleotide sequence encoding a homing endonuclease

[00200] A nucleotide sequence encoding a homing endonuclease can be constructed by manipulating (eg, ligating) native nucleotide sequences encoding polypeptides contained in the homing endonuclease. For example, the nucleotide sequence of a gene encoding a protein containing a DNA binding polypeptide can be examined to identify the nucleotide sequence of a gene that corresponds to a DNA binding polypeptide, and this nucleotide sequence can be used as an element of a nucleotide sequence encoding a homing endonuclease containing DNA binding polypeptide. Alternatively, the amino acid sequence of the homing endonuclease can be used to deduce the nucleotide sequence encoding the homing endonuclease, for example, according to the degeneracy of the genetic code.

[00201] In exemplary nucleic acid molecules containing a nucleotide sequence encoding a homing endonuclease, the last codon of a first polynucleotide sequence encoding a nuclease polypeptide and the first codon of a second polynucleotide sequence encoding a DNA binding polypeptide may be separated by any number of nucleotide triplets, e.g. , without intron encoding or "STOP". Similarly, the last codon of a nucleotide sequence encoding a first polynucleotide sequence encoding a DNA binding polypeptide and the first codon of a second polynucleotide sequence encoding a nuclease polypeptide can be separated by any number of nucleotide triplets. The last codon (ie, the 3'-most in the nucleic acid sequence) of the first polynucleotide sequence; polypeptide-encoding nuclease and a second polynucleotide sequence encoding a DNA-binding polypeptide may be fused in a phase register with the first codon of an additional polynucleotide coding sequence immediately adjacent to it, or separated from it by no more than a short peptide sequence, such as the sequence encoded by a synthetic nucleotide linker (eg, a nucleotide linker that could be used to achieve fusion). Examples of such additional polynucleotide sequences include, for example, but not limited to, tags targeting peptides and enzymatic cleavage sites. Similarly, the first codon from the 5'-most end (in the nucleic acid sequence) of the first and second polynucleotide sequences may be fused in the phase register to the last codon of the additional polynucleotide coding sequence immediately adjacent to it, or separated from it by no more than a short peptide sequence. sequence.

[00202] A sequence separating polynucleotide sequences encoding functional polypeptides in a homing endonuclease (e.g., a DNA-binding polypeptide and a nuclease polypeptide) may, for example, consist of any sequence such that the encoded amino acid sequence is unlikely to significantly alter translation of the target endonuclease . Due to the autonomous nature of known nuclease polypeptides and known DNA-binding polypeptides, intermediate sequences will not affect the respective functions of these structures.

Other knockout methods

[00203] Various other methods known in the art can be used to inactivate genes to produce knockout animals and/or to introduce nucleic acid constructs into animals to produce founder animals and to create animal strains in which the construct with a knockout or nucleic acid is integrated into the genome. Such methods include, but are not limited to, pronucleus microinjection (U.S. Patent No. 4,873,191), retrovirus mediated gene transfer to germ lines (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6148-1652), gene targeting in embryonic stem cells (Thompson et al. (1989) Cell 56, 313-321), embryo electroporation (Lo (1983) Mol. Cell. Biol. 3, 1803-1814), sperm-mediated gene transfer (Lavitrano et al (2002) Proc. Natl. Acad. Sci. USA 99, 14230-14235; Lavitrano et al. (2006) Reprod. Fert. Develop. 18, 19-23) and in vitro transformation of somatic cells such as cumulus cells or breast cells, or adult, fetal or embryonic stem cells, followed by nuclear transplantation (Wilmut et al. (1997) Nature 385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374). Particularly useful methods are pronuclear microinjection, sperm-mediated gene transfer, and somatic cell nuclear transfer. A genetically modified animal is an animal in which all cells have been modified, including germline cells. When methods are used to produce an animal that is mosaic in its modification, the animals can be inbreeded and offspring selected that are genetically modified. Cloning, for example, can be used to create a mosaic animal if its cells are modified in a blastocyst state, or genomic modification can take place when a single cell is modified. Animals modified so that they do not reach sexual maturity may be homozygous or heterozygous for the modification, depending on the particular approach used. If a particular gene is inactivated by a knockout modification, homozygosity is usually required. If a particular gene is inactivated by RNAi or a dominant-negative strategy, then heterozygosity is often sufficient.

[00204] Typically, in embryo/zygote microinjection, a nucleic acid construct or mRNA is introduced into a fertilized egg; one or two fertilized eggs are used as a nuclear structure containing the genetic material from the sperm head and egg and are visible inside the protoplasm. Fertilized eggs in the pronuclear stage can be obtained in vitro or in vivo (i.e. surgically removed from the oviduct of donor animals). Oocytes fertilized in vitro can be obtained as follows. For example, pig ovaries can be harvested from a slaughterhouse and maintained at 22-28°C during transport. The ovaries can be washed and isolated for follicle aspiration, and 4-8 mm follicles can be aspirated under vacuum into 50 ml conical centrifuge tubes using 18 gauge needles. Follicular fluid and aspirated oocytes can be washed through pre-filters with commercial TL-HEPES (Minitube, Verona, Wis.). Oocytes surrounded by a compact cumulus mass can be selected and placed in TCM-199 OOCYTE MATURATION MEDIUM (Minitube, Verona, Wis.) supplemented with 0.1 mg/ml cysteine, 10 ng/ml epidermal growth factor, 10% porcine follicular fluid , 50 μM 2-mercaptoethanol, 0.5 mg / ml cFMP, 10 IU / ml of gonadotropin in the blood serum of pregnant mares (pregnant mare serum gonadotropin-PMSG) and human chorionic gonadotropin (hCG) for approximately 22 hours in humidified air at 38, 7 °C and 5% CO ₂ . Subsequently, oocytes can be transferred to fresh TCM-199 maturation medium, which will not contain cAMP, PMSG, or hCG, and incubated for an additional 22 hours. Mature oocytes can be separated from cumulus cells by shaking in 0.1% hyaluronidase for 1 minute.

[00205] For pigs, mature oocytes can be fertilized in 500 µl Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, Wis.) microtubes in Minitube 5-well fertilization plates. In preparation for in vitro fertilization (IVF), freshly harvested or frozen boar semen can be washed and resuspended in PORCPRO IVF medium for up to 400,000 spermatozoa. Semen concentration can be analyzed using computer semen analysis (SPERMVISION, Minitube, Verona, Wis.). Final in vitro fertilization can be performed in a volume of 10 µl at a final concentration of approximately 40 motile spermatozoa/oocyte, depending on the boar. All fertilizing oocytes can be incubated at 38.7°C in an atmosphere with 5.0% CO ₂ for six hours. Six hours after insemination, putative zygotes can be washed twice in NCSU-23 and transferred to 0.5 ml of the same medium. This system can regularly produce 20-30% blastocysts in most boars with a 10-30% polyspermic rate.

[00206] The linearized nucleic acid or mRNA constructs can be introduced into one of the pronuclei or into the cytoplasm. The injected eggs can then be transferred into the recipient female (eg, into the recipient female's oviducts) and allowed to develop in the recipient female to produce transgenic or gene-edited animals. In particular, in vitro fertilized embryos can be centrifuged at 15,000 x g for 5 minutes to precipitate lipids, allowing visualization of the pronucleus. Embryos can be injected with the Eppendorf FEMTOJET injector and cultured to form a blastocyst. It is possible to record the rate of embryo cleavage, the formation and quality of the blastocyst.

[00207] Embryos can be surgically transferred into the uterus of asynchronous recipients. Typically, 100-200 (eg, 150-200) embryos can be placed in the ampulla-isthmus junction using a 5.5-inch TOMSAT® catheter. After the operation, a real-time ultrasound examination of pregnancy can be performed.

[00208] In somatic cell nuclear transfer, a transgenic or genome-edited cell, such as an embryonic blastomere, fetal fibroblast, adult ear fibroblast, or granulosa cell, which includes the nucleic acid construct described above, can be introduced into the enucleated oocyte to form a combined cells. Oocytes can be enucleated by partial dissection of the zone near the polar body, followed by extrusion of the cytoplasm in the area of dissection. Typically, a sharp, beveled injection pipette is used to inject a transgenic or genome-edited cell into an enucleated meiosis 2-arrested oocyte. In some conventions, oocytes retained in meiosis-2 are called oocytes. After obtaining a porcine or bovine embryo (for example, by fusion and activation of an oocyte), the embryo is transferred into the oviducts of the recipient female approximately 20-24 hours after activation. See, for example, Cibelli et al. (1998) Science 280, 1256-1258 and US Patent Nos. 6548741, 7547816, 7989657 or 6211429. Recipient female pigs can be tested for pregnancy approximately 20-21 days after embryo transfer.

[00209] To create animals that are homozygous for the inactivated gene, you can use standard breeding methods from the original heterozygous founding animals. However, homozygosity may not be required. The genetically modified pigs described herein can be crossed with other pigs of interest.

[00210] Once gene-edited animals have been created, endogenous nucleic acid inactivation can be assessed using standard techniques. An initial screening can be done with a Southern blot to determine if inactivation has occurred. For a description of Southern blotting, see sections 9.37-9.52 in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, second edition. Cold Spring Harbor Press, Plainview; N.Y. Polymerase chain reaction (PCR) techniques may also be used in initial screening. PCR refers to a procedure or technique in which target nucleic acids are amplified. In general, sequence information from or beyond the ends of the region of interest is used to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific DNA sequences as well as RNA, including total genomic DNA or total cellular RNA sequences. Primers are typically 14 to 40 nucleotides in length, but may be 10 nucleotides to hundreds of nucleotides in length. PCR is described, for example, in the PCR primer example: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Nucleic acids can also be amplified by ligase chain reaction, strand displacement amplification, self-sustaining sequence replication, or nucleic acid sequence-based amplification. See, for example, Lewis (1992) Genetic Engineering News 12.1; Guatelli et al. (1990) Proc. Natl. Acad. sci. USA 87:1874; and Weiss (1991) Science 254:1292. At the blastocyst stage, embryos can be individually processed for analysis by PCR, Southern hybridization, and PCR using sprinklers (see, for example, Dupuy et al. Proc Natl Acad Sci USA (2002) 99:4495).

Interfering RNAs

[00211] Many interfering RNA (RNAi) systems are known. Double-stranded RNA (dsRNA) induces sequential degradation of transcripts of homologous genes. The RNA-induced silencing complex (RISC) metabolizes dsRNA to small 21-23 nucleotide small interfering RNAs (siRNAs). RISC contains a double-stranded RNase (dsRNase, eg Dicer) and ssRNase (eg Argonaut 2 or Ago2). RISC uses the antisense strand as a guide to find a cleavable target. Both miRNAs and microRNAs (miRNAs) are known. A method for inactivating a gene in a genetically modified animal includes inducing RNA interference against a target gene and/or nucleic acid such that expression of the target gene and/or nucleic acid is reduced.

[00212] For example, an exogenous nucleic acid sequence can induce RNA interference with a nucleic acid encoding a polypeptide. For example, double-stranded small interfering RNA (siRNA) or short hairpin RNA (shRNA) homologous to the target DNA can be used to reduce the expression of this DNA. The siRNA constructs can be prepared as described, for example, in Fire et al. (1998) Nature 391:806; Romano and Masino (1992) Mol. microbiol. 6:3343; Cogoni et al. (1996) EMBO J. 15:3153; Cogoni and Masino (1999) Nature 399:166; Misquitta and Paterson (1999) Proc. Natl. Acad. sci. USA 96:1451; and Kennerdell and Carthew (1998) Cell 95:1017. ShRNA constructs can be prepared as described by Mchityre and Fanning (2006) BMC Biotechnology 6:1. In general, shRNAs are transcribed as single-stranded RNA molecules containing complementary regions that can anneal and form short hairpins.

[00213] The probability of finding a single individual functional shRNA or siRNA directed to a particular gene is high. For example, the predictability of a particular siRNA sequence is about 50%, but a range of interfering RNAs can be made with good confidence that at least one of them will be effective.

[00214] An in vitro cell, an in vivo cell, or a genetically modified animal such as a farm animal that expresses an RNAi directed against the gene encoding CD 163 can be used. The RNAi can be, for example, selected from the group consisting of siRNA, shRNA, dsRNA , RISC and miRNA.

Inducible systems

[00215] An inducible system can be used to inactivate the CD163 gene. Various inducible systems are known that make it possible to control gene inactivation in space and time. Some of them have been shown to function in vivo in pigs.

[00216] An example of an inducible system is the tetracycline (tet)-OH promoter system, which can be used to regulate nucleic acid transcription. In this system, the mutated Tet repressor (TetR) is fused to the activation domain of the herpes simplex virus VP 16 transactivator protein to create a tetracycline-controlled transcriptional activator (tTA) that is regulated by tet or doxycycline (dox). In the absence of an antibiotic, transcription is minimal, while in the presence of tet or dox, transcription is induced. Alternative inducible systems include ecdysone or rapamycin systems. Ecdysone is an insect moulting hormone whose production is controlled by the ecdysone receptor heterodimer and the product of the ultraspiracle (USP) gene. Expression is induced by treatment with ecdysone or an ecdysone analog such as muristerone A. An agent that is administered to an animal to trigger an inducible system is referred to as an induction agent.

[00217] The tetracycline inducible system and the Cre/loxP recombinase system (constitutive or inducible) are among the most commonly used inducible systems. The tetracycline inducible system includes the tetracycline controlled (tTA)/reverse tTA (rtTA) transactivator. The method of using these systems in vivo involves the creation of two lines of genetically modified animals. One animal line expresses an activator (tTA, rtTA or Cre recombinase) under the control of a selected promoter. Another line of animals expresses an acceptor in which the expression of the gene of interest (or the gene to be changed) is under the control of the target sequence for tTA/rtTA transactivators (or flanked by loxP sequences). Mating two animals provides control of gene expression.

[00218] Tetracycline-dependent regulatory systems (tet-systems) are based on two components, namely, a tetracycline-controlled transactivator (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of the downstream cDNA in a tetracycline-dependent manner. In the absence of tetracycline or its derivatives (such as doxycycline), tTA binds to tetO sequences, allowing transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target and transcription does not occur. The tet system that uses tTA is called tet-OFF because tetracycline or doxycycline allows transcriptional repression. The introduction of tetracycline or its derivatives allows you to control the expression of the transgene in vivo in time. rtTA is a tTA variant that does not function in the absence of doxycycline, but requires the presence of a ligand for transactivation. This tet system is therefore called tet-ON. The tet systems have been used in vivo for the inducible expression of several transgenes encoding, for example, reporter genes, oncogenes, or proteins involved in the signaling cascade.

[00219] The Cre/lox system uses a Cre recombinase that catalyzes site-specific recombination by crossover between two distant Cre recognition sequences, i.e. loxP sites. A DNA sequence introduced between two loxP sequences (called loxP flanked DNA) is excised by Cre-mediated recombination. Controlling Cre expression in a transgenic and/or gene-edited animal using either spatial control (with a tissue or cell specific promoter) or temporal control (with an inducible system) results in control of DNA excision between the two loxP sites. One application is for conditional gene inactivation (conditional knockout). Another approach is to overexpress the protein when a loxP flanked stop codon is inserted between the promoter sequence and the DNA of interest. Genetically modified animals do not express the transgene until Cre is expressed, which will remove the loxP flanked stop codon. This system has been applied for tissue-specific oncogenesis and controlled expression of antigen receptors in B-lymphocytes. Inducible Cre recombinases have also been developed. The inducible recombinase Cre is activated only upon the introduction of an exogenous ligand. Inducible Cre recombinases are fusion proteins containing the original Cre recombinase and a specific ligand-binding domain. The functional activity of the Cre recombinase depends on an external ligand that is able to bind to this specific domain in the fusion protein.

[00220] An in vitro cell, an in vivo cell, or a genetically modified animal such as a farm animal that contains the CD163 gene under the control of an inducible system can be used. An animal's chromosome modification can be genomic or mosaic. The inducible system may, for example, be selected from the group consisting of Tet-On, Tet-Off, Cre-lox and Hif1 alpha.

Vectors and nucleic acids

[00221] Various nucleic acids can be introduced into cells for purposes of knockout, to inactivate a gene, to obtain gene expression, or for other purposes. As used herein, the term "nucleic acid" includes DNA, RNA, and nucleic acid analogs, as well as nucleic acids that are double-stranded or single-stranded (ie, sense or antisense single-stranded). Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, the stability, hybridization, or solubility of the nucleic acid. Modifications to the core moiety include deoxyuridine for deoxythymidine and 5-methyl-2'-deoxycytidine and 5-bromo-2'-doxycytidine for deoxycytidine. Sugar moiety modifications include modification of the ribose sugar 2'-hydroxyl to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholinonucleic acids in which each core moiety is linked to a six-membered morpholino ring or peptide nucleic acids in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and four bases are retained. See Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7(3): 187; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5. In addition, the deoxyphosphate backbone can be replaced by, for example, a phosphorothioate or phosphorodithioate backbone, phosphoramidite, or an alkylphosphotriester backbone.

[00222] The target nucleic acid sequence may be operably linked to a regulatory region, such as a promoter. The regulatory regions may be pig regulatory regions or may be derived from other species. In the context of the present description, the term "operably linked" refers to the location of the regulatory region relative to the nucleic acid sequence in such a way as to allow or facilitate transcription of the target nucleic acid.

[00223] Any type of promoter may be operably linked to a target nucleic acid sequence. Examples of promoters include, without limitation, tissue-specific promoters, constitutive promoters, inducible promoters, and promoters that are responsive or non-responsive to a particular stimulus. Suitable tissue-specific promoters can result in preferential expression of the nucleic acid transcript in beta cells and include, for example, the human insulin promoter. Other tissue-specific promoters may result in preferential expression, for example, in hepatocytes or heart tissue, and may include albumin or alpha-myosin heavy chain promoters, respectively. A promoter can be used that promotes the expression of the nucleic acid molecule without significant tissue or temporal specificity (ie, a constitutive promoter). For example, a beta-actin promoter such as the chicken beta-actin gene promoter, ubiquitin promoter, miniCAGs promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or 3-phosphoglycerate kinase (PGK) promoter, as well as viral promoters such as the herpes simplex virus thymidine kinase (HSV-TK), SV40 promoter or cytomegalovirus (CMV) promoter. For example, a hybrid of the chicken beta actin gene promoter and the CMV enhancer can be used as a promoter. See, for example, Xu et al. (2001) Hum. Gene Ther. 12:563; and Kiwaki et al. (1996) Hum. Gene Ther. 7:821.

[00224] Additional regulatory regions that may be useful in nucleic acid constructs include, without limitation, polyadenylation sequences, translation control sequences (eg, internal ribosome entry segment, IRES), enhancers, inducible elements, or introns. Such regulatory regions may not be necessary, although they may increase expression by affecting transcription, mRNA stability, translation efficiency, and the like. ). However, it is sometimes possible to obtain sufficient expression without such additional elements.

[00225] A nucleic acid construct that encodes signal peptides or selectable markers can be used. Signal peptides can be used such that the encoded polypeptide is directed to a specific location on the cell (eg, the surface of the cell). Non-limiting examples of selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin B phosphotransferase, thymidine kinase (TK), and xanthine guanine phosphoribosyl transferase (XGPRT). Such markers are useful for selecting stable transformants in culture. Other selectable markers include fluorescent polypeptides such as green fluorescent protein or yellow fluorescent protein.

[00226] A sequence encoding a selectable marker may be flanked by recognition sequences for a recombinase such as, for example, Cre or Flp. For example, a selectable marker may be flanked by loxP recognition sites (34 bp recognition sites recognized by Cre recombinase) or FRT recognition sites so that a selectable marker can be excised from the construct. See Orban, et al., Proc. Natl. Acad. sci. (1992) 89:6861 review of Cre/lox technology and Brand and Dymecki, Dev. Cell (2004) 6:7. A transposon containing a Cre- or Flp-activated transgene interrupted by a selectable marker gene can also be used to produce animals with conditional expression of the transgene. For example, the promoter driving the expression of the marker/transgene may be either ubiquitous or tissue-specific, resulting in ubiquitous or tissue-specific expression of the marker in F0 animals (eg, pigs). Tissue-specific activation of a transgene can be achieved, for example, by crossing a pig that ubiquitously expresses a marker-interrupted transgene with a pig that expresses Cre or Flp in a tissue-specific manner, or by crossing a pig that expresses a marker-interrupted transgene in a tissue-specific manner with a pig that ubiquitously expresses the Cre recombinase. or FLP. Controlled expression of the transgene or controlled deletion of the marker allows the expression of the transgene.

[00227] An exogenous nucleic acid may encode a polypeptide. The nucleic acid sequence encoding the polypeptide may include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation of the encoded polypeptide (eg, to facilitate localization or detection). Label sequences can be inserted into a nucleic acid sequence encoding a polypeptide such that the encoded label is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include glutathione S-transferase (GST) and the FLAG™ tag (Kodak, New Haven, Conn.).

[00228] Nucleic acid constructs can be methylated using SssI CpG methylase (New England Biolabs, Ipswich, Mass.). In general, the nucleic acid construct can be incubated with S-adenosylmethionine and SssI CpG methylase in buffer at 37°C. Hypermethylation can be confirmed by incubating the construct with one unit of HinP1I endonuclease for 1 hour at 37° C. and analyzing by agarose gel electrophoresis.

[00229] Nucleic acid constructs can be introduced into any type of embryonic, fetal, or adult animal cell using various techniques, including, for example, germ cells such as an oocyte or egg, a progenitor cell, an adult or embryonic stem cell, a primordial germ cell , a kidney cell such as a PK-15 cell, an islet cell, a beta cell, a liver cell, or a fibroblast such as a dermal fibroblast. Non-limiting examples of methods include the use of transposon systems, recombinant viruses that can infect cells, or liposomes, or other methods, such as electroporation, microinjection, or calcium phosphate precipitation, which are capable of delivering nucleic acids to cells.

[00230] In transposon systems, the transcriptional unit of nucleic acid construct, i. a regulatory region operably linked to an exogenous nucleic acid sequence is flanked by an inverted transposon repeat. Several transposon systems, including, for example, Sleeping Beauty (see US Pat. No. 6,613,752 and US Patent Publication No. 2005/0003542); Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31:6873); Tol2 (Kawakami (2007) Genome Biology 8(Suppl.l):S7; Minos (Pavlopoulos et al. (2007) Genome Biology 8(Suppl.l):S2)) Hsmarl (Miskey et al. (2007)) Mol Cell Biol 27:4589); and Passport were designed to introduce nucleic acids into cells, including those of mice, humans, and pigs. The Sleeping Beauty transposon is especially useful. The transposase can be delivered as a protein encoded by the same nucleic acid construct as the exogenous nucleic acid, can be introduced into a separate nucleic acid construct, or provided as an mRNA (eg, in vitro transcribed and capped mRNA).

[00231] Insulator elements may also be included in the nucleic acid construct to maintain expression of the exogenous nucleic acid and inhibit unwanted transcription of host genes. See, for example, US Patent Publication No. 2004/0203158. Typically, the insulator element flanks each side of the transcription unit and is internal to the inverted repeat of the transposon. Non-limiting examples of insulator elements include matrix attachment region (MAR) type insulators and border type insulator elements. See, for example, US Pat.

[00232] Nucleic acids can be included in vectors. Vector is a broad term that includes any specific segment of DNA that is intended to pass from host to target DNA. A vector may be referred to as an expression vector or a vector system, which is a set of components necessary to insert DNA into a genome or other target DNA sequence, such as an episome, plasmid, or even a virus/phage DNA segment. Vector systems such as viral vectors (for example, retroviruses, adeno-associated virus, and integrating phage viruses) and non-viral vectors (for example, transposons) used to deliver genes to animals have two main components: 1) a vector consisting of DNA (or RNA, which is reverse transcribed into cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and the target DNA sequence and inserts the vector into the target DNA sequence. The vectors most often contain one or more expression cassettes that contain one or more expression control sequences, where the expression control sequence is a DNA sequence that controls and regulates the transcription and/or translation of another sequence, respectively DNA or mRNA.

[00233] Many different types of vectors are known. For example, plasmids and viral vectors, such as retroviral vectors, are known. Mammalian expression plasmids typically have an origin of replication, a suitable promoter and an optional enhancer, the necessary ribosome binding sites, a polyadenylation site, splicing donor and acceptor sites, transcription termination sequences, and 5' flanking non-transcribed sequences. Examples of vectors include: plasmids (which may also carry another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (eg, modified HIV-1, SIV, or FIV), retrovirus (eg, ASV, ALV, or MoMLV), and transposons (e.g. Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac).

[00234] As used herein, the term nucleic acid refers to both RNA and DNA, including, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, and naturally occurring and chemically modified nucleic acids, e.g., synthetic bases or alternative backbones. The nucleic acid molecule can be double-stranded or single-stranded (ie, sense or antisense single-stranded).

[00235] After a detailed description of the invention, it will become apparent that modifications and changes are possible without departing from the scope of the invention as defined in the appended claims.

EXAMPLES

[00236] To further illustrate the present invention, the following non-limiting examples are provided.

Example 1 Use of the CRISPR/Cas9 System to Produce Genetically Engineered Pigs from In Vitro Oocytes and Embryos

[00237] Recent reports describing homing endonucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and components of the Clustered Regular Intermediate Short Palindromic Repeat (CRSCR)/CRISPR-associated system (Cas9 ) suggest that genetic engineering (GE) in pigs may now be more efficient. Targeted homing endonucleases can induce double-strand breaks (DSBs) at specific locations in the genome and cause either random mutations due to non-homologous end joining (NHEJ) or, if donor DNA is available, stimulation of homologous recombination (HR). Targeted genome modification by GR can be achieved with homing endonucleases if donor DNA is provided along with a targeted nuclease. After making specific modifications to the somatic cells, these cells were used to obtain pig GI for various purposes using SCNT. Thus, homing endonucleases are a useful tool for creating GIs in pigs. Among various homing endonucleases, the CRISPR/Cas9 system adapted from prokaryotes, where it is used as a defense mechanism, provides an effective approach. In nature, the Cas9 system requires three components: an RNA (~20 bp) that contains a region complementary to the target sequence (cis-repressed RNA [crRNA]), an RNA that contains a region complementary to crRNA (transactivating crRNA [tracrRNA]), and Cas9 , the enzymatic protein component in this complex. A single guide RNA (gRNA) can be designed to perform the functions of base-paired crRNA and tracrRNA. The gRNA/protein complex can scan the genome and catalyze DSBs in regions that are complementary to crRNA/gRNA. Unlike other developed nucleases, only a short oligomer needs to be developed to create the reagents needed to target a gene of interest, while a series of cloning steps is required to assemble ZFN and TALEN.

[00238] In contrast to existing standard gene disruption methods, the use of engineered nucleases allows the use of zygotes as starting material for GI. Standard methods of disrupting genes in farm animals include GH in cultured cells and subsequent embryo reconstruction by somatic cell nuclear transfer (SCNT). Because SCNT-derived cloned animals sometimes show signs of developmental defects, progeny of SCNT/GI founders is usually used for research in order to avoid confounding SCNT and phenotype abnormalities that can occur when founder animals are used in experiments. Given the longer gestation period for pigs and the higher cost of keeping pigs compared to rodents, reducing the need for breeding offers time and cost advantages. A recent report has demonstrated that direct injection of ZFN and TALEN into pig zygotes can disrupt the endogenous gene and produce piglets with desired mutations. However, only about 10% of the piglets showed a biallelic modification of the target gene, and some presented mosaic genotypes. A recent article has demonstrated that the CRISPR/Cas9 system can induce mutations in developing embryos and produce GI pigs more efficiently than either ZFN or TALEN. However, GI pigs derived from the CRISPR/Cas9 system also had mosaic genotypes. In addition, all of the above studies used in vivo zygotes for experimentation, which require intensive labor and a large number of sows to obtain a sufficient number of zygotes.

[00239] This example describes an efficient approach to use the CRISPR/Cas9 system to generate GI pigs by injecting in vitro derived zygotes and modifying somatic cells followed by SCNT. Two endogenous genes (CD163 and CD1D) and one transgene (eGFP) were targeted, and only in vitro derived oocytes or zygotes, respectively, were used for SCNT or RNA injections. CD163 appears to be required for productive infection with the porcine reproductive and respiratory syndrome virus, a virus known to cause significant economic damage to swine production. CD1D is considered a non-classical major histocompatibility complex protein and is involved in the presentation of lipid antigens to invariant natural killer T cells. Pigs deficient in these genes have been created as models for agriculture and biomedicine. The eGFP transgene was used as a target for preliminary proof-of-concept and method optimization experiments.

MATERIALS AND METHODS

[00240] Chemicals and reagents. Unless otherwise noted, all chemicals used in this study were purchased from Sigma.

Design of gRNA to obtain specific CRISPR

[00241] Guide RNAs were designed for regions in exon 7 of CD163 that were unique to wild-type CD163 and were not present in the targeting vector for domain exchange (described below), such that CRISPR would result in DSB in the wild-type CD163 vector, but not in the scope of the directed vector to implement the domain exchange. There were only four locations where the targeting vector could introduce a single nucleotide polymorphism (SNP) that would alter the adjacent S. pyogenes (AMC) protospacer (Spy) motif. All four targets were chosen, including:

(SEQ ID NO:1) GGAAACCCAGGCTGGTTGGAgGG (CRISPR 10),

(SEQ ID NO:2) GGAACTACAGTGCGGCACTGtGG (CRISPR 131),

(SEQ ID NO:3) CAGTAGCACCCCGCCCTGACgGG (CRISPR 256) and

(SEQ ID NO:4) TGTAGCCACAGCAGGGACGTcGG (CRISPR 282).

The AMCs in each gRNA can be identified by the bold type.

[00242] For CD1D mutations, the search for CRISPR targets was arbitrarily limited to the coding strand within the first 1000 bp of the primary transcript. However, RepeatMasker [26] (Pig's repeat library) identified a repeat element starting at base 943 of the primary transcript. The search for CRISPR targets was then limited to the first 942 bp. primary transcript. Further, the search was limited to the first 873 bp. primary transcript, since the last Spy AMC is located at base 873. The first target (CRISPR 4800) was chosen because it overlapped with the start codon located at base 42 in the primary transcript (CCAGCCTCGCCCAGCGACATgGG (SEQ ID NO: 5)). Two additional targets (CRISPR 5620 and 5626) were selected because they were furthest away from the first selection (CTTTCATTTATCTGAACTCAgGG (SEQ ID NO: 6)) and TTATCTGAACTCAGGGTCCCcGG (SEQ ID NO: 7) in the randomly selected region. These targets overlap. With respect to the start codon, the most proximal Spy AMCs were located in a simple sequence containing a largely homopolymeric sequence, as determined by visual assessment. The fourth target (CRISPR 5350) was chosen because, compared to the selected first target, it was the most proximal target that did not contain extensive homopolymer regions (CAGCTGCAGCATATATTTAAgGG (SEQ ID NO: 8)). The specificity of the constructed crRNAs was confirmed by searching for similar porcine sequences in GenBank. Oligonucleotides (Table 1) were annealed and cloned into a p330X vector containing two expression cassettes, human codon-optimized S.pyogenes Cas9 (hSpy), and a chimeric guide RNA. P330X was digested with ith BbsI (New England Biolabs) followed by Zhang's laboratory protocol (http://www.addgene.org/crispr/zhang/).

[00243] To target eGFP, two specific gRNAs were designed to target the eGFP coding sequence in the first 60 bp. eGFP start codon. Both gRNAs, both eGFP1 and eGFP2, were on the antisense strand, and eGFP1 was directed directly to the start codon. The eGFP1 gRNA sequence was CTCCTCGCCCTTGCTCACCAtGG (SEQ ID NO: 9) and the eGFP2 gRNA sequence was GACCAGGATGGGCACCACCCcGG (SEQ ID NO: 10).

Synthesis of donor DNA for CD163 and CD1D genes

[00244] Both porcine GD163 and CD1D were amplified by PCR from DNA isolated from fetal fibroblasts to be used for subsequent transfections in order to ensure an isogenic match between the targeting vector and the transfected cell line. Briefly, to amplify the 9538 bp CD163 fragment. LA tag (Clontech), forward primer STTSSSTSASTSTAACSTATT (SEQ ID NO: 11) and reverse primer TATTTCTCTCACATGGCCAGTC (SEQ ID NO: 12) were used. The fragment was a verified DNA sequence and was used to construct a targeting vector with a domain change (FIG. 1). This vector included 33 point mutations in exon 7 such that it encoded the same amino acid sequence as human CD163L from exon 11. The substituted exon was 315 bp. In addition, the subsequent intron was replaced with a modified myostatin B intron that contained a selectable marker gene that could be removed by Cre recombinase (Cre) and that previously showed normal splicing while maintaining the loxP site (Wells, unpublished results). The long arm of the construct was 3469 bp. and included the DS exon with a domain change. The short arm was 1578 bp. and included exons 7 and 8 (Fig. 1, panel B). This plasmid was used to attempt to replace the exon 7 coding region in the first transfection experiments and allowed the selection of targeted events using a selectable marker (G418). If targeting had occurred, the marker could be removed by Cre recombinase. The vector was then modified to target DS CD 163 for use with cell lines that already contained the Neo disrupted SIGLEC1 gene that could not be removed by Cre. In this targeting vector, the Neo cassette, loxP and myostatin B intron were removed, leaving only the DS exon in the long and short arms of the wild type (FIG. 1, panel C).

[00245] The porcine CD1D genomic sequence was amplified with LA taq using the forward primer STTSSSSTCASTSTAACSTATT (SEQ ID NO: 13) and the reverse primer GACTGGCCATGTGAGAGAAATA (SEQ ID NO: 14), resulting in a fragment of 8729 bp. The DNA fragment was sequenced and used to construct the targeting vector shown in FIG. 2. The Neo cassette is under the control of a phosphoglycerol kinase (PGK) promoter and is flanked by loxP sequences that were introduced for selection. The long arm of the construct was 4832 bp, and the short arm was 3563 bp. and included exons 6 and 7. If successful GR occurs, exons 3,4 and 5 should have been removed and replaced with a Neo cassette. If NHEJ repair did not occur correctly, exon 3 will be broken.

Obtaining embryonic fibroblasts

[00246] Pig fetal tissue was obtained on day 35 of gestation to establish cell lines. Two male and female wild-type fetal fibroblast cell lines were derived from a hybrid of a local pig and a Large White pig. These studies also used male and female fetal fibroblasts that had previously been modified to contain the Neo cassette (SIGLEC1-/- genetics). Embryonic fibroblasts were harvested as described with minor modifications; chopped tissue from each fetus was digested in 20 ml of digestion medium (Dulbecco's Modified Eagle's Medium [DMEM] containing L-glutamine and 1 g/l D-glucose [Cellgro] supplemented with 200 units/ml collagenase and 25 Kunitz units/ml DNA -zy I) for 5 hours at 38.5°C. After digestion, fetal fibroblast cells were washed and cultured with DMEM, 15% fetal bovine serum (FBS) and 40 μg/ml gentamicin. After culturing overnight, the cells were trypsinized and aliquoted at -80° C. in FBS with 10% dimethyl sulfoxide and stored in liquid nitrogen.

Transfection and genotyping of cells

[00247] The transfection conditions were essentially as previously reported. Donor DNA was always used at a constant amount of 1 μg with varying amounts of CRISPR/Cas9 plasmid (listed below). Donor DNA was linearized with MLUI (CD163) (NEB) or AFLII (CD1D) (NEB) before transfection. The generated cell lines were gendered by PCR prior to transfection as described previously. Both male and female cell lines were transfected and these genome modifications were analyzed together between transfections. Cell lines of embryonic fibroblasts with the same number of passages (2-4) were cultured for 2 days and grown to 75-85% confluency in DMEM containing L-glutamine and 1 g/l D-glucose (Cellgro) supplemented with 15% FBS, 2.5 ng/ml basic fibroblast growth factor and 10 mg/ml gentamicin. Fibroblast cells were washed with phosphate buffered saline (PBS) (Life Technologies) and treated with trypsin. Once cells were detached, cells were washed with electro-operation medium (75% cell salts [120 mM KCl, 0.15 mM CaCl ₂ , 10 mM K ₂ HPO ₄ , pH 7.6, 5 mM MgCl ₂ ]) and 25% Opti-MEM (Life Technologies). The cell concentration was quantified using a hemocytometer. Cells were pelleted at 600 x g for 5 minutes and resuspended at a concentration of 1×10 ⁶ in electroporation medium. Each electroporation used 200 µl of cells in 2 mm gap cuvettes with three (1 ms) rectangular pulses applied through a BTX ECM 2001 at 250 V. After electroporation, the cells were resuspended in DMEM as described above. For selection 24 hours after transfection, 600 μg/ml G418 (Life Technologies) was added and the medium was changed on day 7. Colonies were collected on the 14th day after transfection. Embryonic fibroblasts were plated at 10,000 cells/plate if G418 selection was used and at 50 cells/plate if G418 selection was not used. Embryonic fibroblast colonies were harvested using 10 mm autoclaved cloning cylinders, sealing around each colony with autoclaved vacuum grease. Colonies were washed with PBS and harvested with trypsin; then resuspended in DMEM culture medium. Part (1/3) of the resuspended colony was transferred to a 96-well PCR plate, and the remaining (2/3) cells were cultured in the wells of a 24-well plate. Cell pellets were resuspended in 6 µl lysis buffer (40 mM Tris, pH 8.9, 0.9% Triton X-100, 0.4 mg/ml proteinase K [NEB]), incubated at 65°C for 30 minutes for cell lysis followed by incubation at 85°C for 10 minutes to inactivate proteinase K.

PCR squeaks for DS, large and small deletions

[00248] Identification of repair directed to homologous recombination (GR). Long fragment PCR was used to identify mutations in CD163 or CD1D. Three different PCR analyzes were used to identify GR events: PCR amplification of regions from CD163 or CD1D sequences in donor DNA to endogenous CD163 or CD1D sequences on the right or left side, and PCR of long fragments, which amplified large regions of CD163 or CD1D containing engineered donor DNA. An increase in the size of the PCR product by 1.8 kb. (CD1D) or 3.5 kb. (CD163), resulting from the addition of exogenous Neo sequences, was considered evidence of GH-directed gene repair. All PCR conditions included initial denaturation at 95°C for 2 minutes, followed by 33 cycles of 30 seconds at 94°C, 30 seconds at 50°C and 7-10 minutes at 68°C. LA taq was used for all assays as recommended by the manufacturers. Primers are presented in Table 2.

[00249] Small Deletion Analysis (NHEJ). Small deletions were determined by PCR amplification of CD163 or CD1D flanking the putative incision site introduced by the CRISPR/Cas9 system. The size of the amplicons was 435 bp. and 1244 p. for, respectively, CD163 and CD1D. Embryo and fetal fibroblast lysates were amplified by PCR with LA taq. Conditions for PCR assays were initial denaturation at 95°C for 2 minutes followed by 33 cycles of 30 seconds at 94°C, 30 seconds at 56°C and 1 minute at 72°C. For genotyping of transfected cells, insertions and deletions (INDELs) were identified by separating PCR amplicons by agarose gel electrophoresis. For embryo genotyping, the resulting PCR products were then subjected to DNA sequencing to detect small deletions using the forward primers used in PCR. Primer information is presented in Table 3.

Somatic cell nuclear transfer (SCNT)

[00250] Either sow oocytes (ART, Inc.) or pig origin oocytes from a local slaughterhouse were used to obtain embryos by SCNT. Sow-derived oocytes were sent overnight to maturation medium (TCM-199 with 2.9 mM Hepes, 5 µg/mL insulin, 10 ng/mL epidermal growth factor [EGF], 0.5 µg/mL porcine follicle-stimulating hormone [p-FSH], 0.91 mM pyruvate, 0.5 mM cysteine, 10% porcine follicular fluid and 25 ng/ml gentamicin) and transferred to fresh medium 24 hours later. After 40-2 hours of maturation, cumulus cells were removed from oocytes by shaking in the presence of 0.1% hyaluronidase. Oocytes obtained from young pigs were matured as described below for in vitro fertilization (IVF). During manipulation, oocytes were placed in manipulation medium (TCM-199 [Life Technologies] with 0.6 mM NaHCO ₃ , 2.9 mM Hepes, 30 mM NaCl, 10 ng/ml gentamicin, and 3 mg/ml BSA with an osmolarity of 305 mOsm ) with the addition of 7.0 μg/ml cytochalasin B. The polar body, together with a portion of the adjacent cytoplasm, presumably containing the metaphase II plate, was removed, and the donor cell was placed in the perivitelline space using a thin glass capillary. The reconstructed embryos were then fused in fusion medium (0.3 M mannitol, 0.1 mM CaCl ₂ , 0.1 mM MgCl ₂ and 0.5 mM Hepes) with two DC pulses (1 second interval) at 1.2 kV/cm for 30 seconds using a BTX Electro Cell Manipulator (Harvard Apparatus). After fusion, fused embryos were fully activated in the dark with 200 μM thimerosal for 10 minutes and 8 mM dithiothreitol for 30 minutes. The embryos were then incubated in PZM3-MU1 modified porcine zygote medium with 0.5 μM Scriptaid (S7817; Sigma-Aldrich) , a histone deacetylase inhibitor, for 14-16 hours as previously described.

In vitro fertilization (IVF)

[00251] For IVF, prepubescent pig ovaries were obtained from a slaughterhouse (Farmland Foods Inc.). Immature oocytes were aspirated from medium-sized (36 mm) follicles using a #18 hypodermic needle attached to a 10 ml syringe. Then, oocytes with uniformly dark cytoplasm and intact surrounding cumulus cells were selected for maturation. Approximately 50 cumulus oocyte complexes were placed in a well containing 500 µl of maturation medium, TCM-199 (Invitrogen) with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/ml EGF, 0.5 µg/ml luteinizing hormone (LH), 0.5 µg/ml FSH, 10 ng/ml gentamicin (APP Pharm) and 0.1% polyvinyl alcohol, for 42-44 hours at 38.5°C, 5% CO ₂ and humidified air. At the end of maturation, the surrounding cumulus cells were removed from the oocytes by shaking for 3 minutes in the presence of 0.1% hyaluronidase. Then, in vitro matured oocytes were placed in drops of 50 µl of IVF medium (modified Tris buffer medium containing 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2, 11 mM glucose, 20 mM Tris, 2 mM caffeine, 5 mM sodium pyruvate and 2 mg/ml bovine serum albumin (BSA)) in groups of 25-30 oocytes. One 100 µl frozen semen pellet was thawed in 3 ml of Dulbecco PBS supplemented with 0.1% BSA. Frozen wild-type or fresh eGFP sperm were washed by centrifugation in 60% percoll for 20 minutes at 650 3 g and in modified medium with Tris buffer for 10 minutes. In some cases, freshly harvested sperm heterozygous for the previously described eGFP transgene were washed three times in PBS. The sperm pellet was then resuspended in IVF medium to 0.5×10 ⁶ cells/ml. Fifty microliters of sperm suspension were injected into droplets with oocytes. The gametes were co-incubated in air for 5 hours at 38.5°C in an atmosphere with 5% CO ₂ . After fertilization, the embryos were incubated in air in PZM3-MU1 at 38.5°C and 5% CO ₂ .

Embryo transfer

[00252] Embryos designed to produce genetically engineered CD163 or CD ID pigs were transferred to surrogate mothers either day 1 (SCNT) or day 6 (zygote injected) after the first active estrus. For transfer to day 6, zygotes were cultured for five additional days in PZM3-MU1 in the presence of 10 ng/ml ps48 (Stemgent, Inc.). The embryos were surgically transferred into the ampulla-isthmus junction of the oviduct of the surrogate mother.

Synthesis of RNA. in vitro for CRISPR/Cas9 system

[00253] Template DNA for in vitro transcription was amplified by PCR (Table 4). The PCR template was the CRISPR/Cas9 plasmid used for cell transfection experiments. In order to express Cas9 in zygotes, the mMESSAGE mMACHINE Ultra Kit (Ambion) was used to obtain Cas9 mRNA. The poly-A signal was then added to the Cas9 mRNA using the Poly (A) tailing kit (Ambion). CRISPR guide RNAs were generated using MEGAshortscript (Ambion). The quality of the synthesized RNAs was visualized on a 1.5% agarose gel and then diluted to a final concentration of 10 ng/µl (both gRNA and Cas9) and distributed into 3 µl aliquots.

Microinjection of the engineered CRISPR/Cas9 system into zygotes

[00254] Messenger RNA encoding Cas9 and gRNA was injected into the cytoplasm of fertilized oocytes 14 hours after fertilization (putative zygotes) using a FemtoJet microinjector (Eppendorf). Microinjection was performed in manipulation medium on a heated stage of a Nikon inverted microscope (Nikon Corporation; Tokyo, Japan). Then the injected zygotes were transferred to PZM3-MU1 with 10 ng/ml ps48 until further use.

Statistical analysis

[00255] The number of genome-modified colonies was classified as 1 and colonies without genome modification were classified as 0. Differences were determined using PROC GLM (SAS) with a P-value of 0.05, which is considered significant. Means were calculated as least squares means. Data are presented as numerical means ± SEM (standard error of the mean).

RESULTS

CD163 and CD1D knockout in somatic cells mediated by CRISPR/Cas9

[00256] The performance of four different CRISPR plasmids (guides 10, 131, 256 and 282) targeting CD163 was tested at 2 μg/μl of donor DNA (Table 5). CRISPR 282 resulted in significantly greater mean colony formation than CRISPR 10 and 256 treatments (P<0.05). From the long fragment PCR analysis described above, instead of DS, large deletions ranging from 503 bp to 1506 bp were detected by GR, as originally expected (Fig. 3, panel A). This was unexpected as previous reports with other DNA editing systems have shown much smaller deletions of 6333 bp. using ZFN in pigs. CRISPR 10 and a mixture of all four CRISPRs resulted in more genome-modified colonies than CRISPR 256 and 282 (Table 5, P<0.002). Transfection with CRISPR 10 and a plasmid containing Neo but not homologous to CD163 did not result in large deletion colonies. Interestingly, one monoallelic deletion was also found when the donor DNA was introduced without any CRISPR. This analysis is likely an underestimate of the mutation rate because the sequencing was not screened for any potential small divisions that could not be detected on agarose gel in transfected somatic cells.

* Mix of 4 + Donor DNA is mixing equal amounts of 0.5 µg of each CRISPR with 1 µg of Donor DNA. Donor DNA treatment served as a non-CRISPR control, and 10 + Neo treatment illustrates that the large divisions seen with CRISPR treatment were only present when CD163 of the donor DNA was also present.

ANOVA был проведен для сравнения среднего количества колоний на чашку для оценки токсичности CRISPR и процента колоний с модифицированным геномом. Р-значения составили, соответственно, 0,025 и 0,0002. н/п = для этой обработки не было повторов, поэтому статистический анализ не проводился.

ANOVA was performed to compare the mean number of colonies per plate to assess CRISPR toxicity and the percentage of genome-modified colonies. P-values were 0.025 and 0.0002, respectively. n/a = there were no repeats for this treatment, so no statistical analysis was performed.

Единственная колония с ГР представляет собой событие частичной ГР.

A single colony with GR represents a partial GR event.

^{ac Superscript} letters indicate significant difference between treatments for both the average number of colonies per plate and the percentage of genome-modified colonies (P<0.05)

[00257] The original goal was to have a domain swap (DS) targeting event by GR for CD163, but CRISPR did not increase the effectiveness of CD163 targeting. Of note, various combinations of this targeting vector have been used to modify CD163 by conventional transfections with GH and resulted in 0 target events after screening 3399 colonies (Whitworth and Prather, unpublished results). Two pigs were obtained with complete DS as a result of GR, which contained all 33 mutations that were tried to be introduced by transfection with CRISPR 10 and a DS-targeted vector as donor DNA.

[00258] The effectiveness of CRISPR/Cas9-induced mutations was then tested without drug selection; the embryonic fibroblast cell line used in this study already had Neo stable cassette integration and SIGLEC1 knockout. It was also tested whether the ratio of CRISPR/Cas9 and donor DNA would increase genome modification or cause toxic effects at high concentrations. CRISPR 131 was chosen for this testing because in the previous experiment it resulted in the total number of colonies and an increase in the percentage of colonies with an altered genome. Increasing the amount of CRISPR 131 DNA from 3:1 to 20:1 did not significantly affect the survival of fetal fibroblasts. The percentage of colonies with the NHEJ modified genome did not differ significantly at different concentrations of CRISPR, however, at a ratio of 10:1, the highest number of NHEJs was observed (Table 6, P=0.33). Even at the highest ratio of CRISPR DNA to donor DNA (20:1), no GH was noted.

^a Significant difference between treatments for the percentage of colonies with NHEJ repair (P>0.05).

^b No significant difference was observed in the number of genome-modified colonies with increasing RISPR concentration (P>0.33).

[00259] Based on this experience, an attempt was made to target the destruction of CD1D in somatic cells. Four different CRISPRs have been developed and tested on both male and female cells. CD1D modifications could be detected from the three CRISPRs applied, but the use of CRISPR 5350 did not result in a CD1D modification with a deletion large enough to be detected by agarose gel electrophoresis (Table 7). Interestingly, no genetic changes were obtained as a result of GH, despite the fact that donor DNA was provided. However, large deletions similar to those found in CD163 knockout experiments were observed (FIG. 3, panel B). If CRISPR/Cas9 was not used with donor DNA, no targeted modification of large deletion CD1D was detected. Modification of CD1D as a result of targeted exposure under the control of CRISPR/Cas9 was 4/121 and 3/28 in male and female cell colonies, respectively. Only INDELs detected by agarose gel electrophoresis were included in the transfection data.

Obtaining pigs CD163 and CD1D by SCNT using genetically edited cells

[00260] CD163 or CD1D modified cells were used for SCNT to generate CD163 and CD1D knockout pigs (FIG. 3). Seven embryo transfers (CD163, Table 8), six embryo transfers (CD163-no Neo), and five embryo transfers (CD1D) were performed in young pig recipients with SCNT embryos from male and female fibroblasts transfected with CRISPR/Cas9 systems. Six (CD163), two (CD163-no Neo), and four (CD1D) (Table 9) young recipient pigs were delivered and delivered at term, resulting in pregnancy rates of 85.7%, 33.3%, respectively. and 80%. Of the CD163 recipients, five gave birth to healthy piglets by caesarean section. One (O044) farrowed naturally. Litter size ranged from one to eight. Four pigs were euthanized due to the fact that after birth they stopped developing. One piglet was euthanized due to severe cleft palate. All remaining piglets appeared healthy (Fig. 3, panel C). Two litters of male piglets derived from fetal fibroblasts transfected with CRISPR 10 and donor DNA, described in FIG. 3, panel B had a 30 bp deletion. in exon 7 adjacent to CRISPR 10 and an additional 1476 bp deletion. the previous intron, thus removing the CD163 nitron 6/exon 7 junction (FIG. 3, panel E). Genotypes and predicted translations are shown in Table 10. One male piglet and one female litter (4 piglets) were obtained by transfection of CD163-no Neo cells previously modified with SIGLEC1. All five piglets were knocked out twice for SIGLEC1 and CD163. The male piglet had a biallelic modification of CD163 with a 28 bp deletion. in exon 7 on one allele and a deletion of 1387 bp. on the other allele, which included a partial deletion of exon 7 and a complete deletion of exon 8 and the subsequent intron, thus removing intron exon junctions. The female piglets had a biallelic CD163 mutation, including a 1382 bp deletion. with insertion 11 b.p. on one allele and a 1720 bp deletion. CD163 on a different allele. A summary of CD163 modifications and predicted translations can be found in Table 10. A summary of CD1D modifications and predicted translations by CRISPR modification can be found in Table 11. Briefly, one female litter and two male litters were born, resulting in 13 piglets. One pig died immediately after birth. Twelve of 13 piglets had either a biallelic or homozygous CD1D deletion (FIG. 3, panel F). One piglet was wild type.

*CD163 CRISPR NT line are embryos created by nuclear transfer (NT) with a line of embryonic fibroblasts modified by transfection. Embryos injected with CRISPR were IVF embryos injected at cell stage 1 with CD163 guide RNA with CAS9 RNA. The CD163 CRISPR NT-no Neo embryonic line is nuclear transfer embryos with pre-modified embryonic fibroblasts that were already a kAb resistant line modified by transfection without the use of a selectable marker.

MU относится к ооцитам молодых свиней, которые были аспирированы и созревали в Университете Миссури, как описано в разделе ЭКО Материалов и способов. ART относится к ооцитам свиней, которые были приобретены и созревали, как описано в разделе SCNT Материалов и способов.

MU refers to oocytes from young pigs that have been aspirated and matured at the University of Missouri as described in the IVF Materials and Methods section. ART refers to porcine oocytes that have been purchased and matured as described in the Materials and Methods section of the SCNT.

* The CD1D CRISPR NT line is an embryo created by nuclear transfer (NT) with a line of embryonic fibroblasts modified by transfection. Embryos injected with CRISPR were IVF embryos injected at cell stage 1 with CD1D guide RNA with CAS9 RNA.

MU относится к ооцитам молодых свиней, которые были аспирированы и созревали в Университете Миссури, как описано в разделе ЭКО Материалов и способов. ART относится к ооцитам свиней, которые были приобретены и созревали, как описано в разделе SCNT Материалов и способов.

MU refers to oocytes from young pigs that have been aspirated and matured at the University of Missouri as described in the IVF Materials and Methods section. ART refers to porcine oocytes that have been purchased and matured as described in the Materials and Methods section of the SCNT.

*KO, knockout

**Not included as pigs were euthanized.

SEQ ID NOs. в этой колонке относится к SEQ ID NOs. для последовательностей, демонстрирующих INDEL по сравнению с SEQ ID NO:47.

SEQID NOs. in this column refers to SEQ ID NOs. for sequences showing INDEL compared to SEQ ID NO:47.

^a The sequence entered was TASTAST (SEQ ID NO:115)

^b The sequence entered was AG.

^c The sequence introduced was a single adenine residue (A),

^d The sequence entered was TGTGGAGAATTC (SEQ ID NO:116).

^e The sequence introduced was AGCCAGCGTGC (SEQ ID NO:117).

Эффективность системы CRISPR/Cas9 в зиготах свиньи

Efficiency of the CRISPR/Cas9 System in Pig Zygotes

[00261] This approach, based on the targeted destruction of CD163 and CD1D in somatic cells using the CRISPR/Cas9 system, has been applied to porcine embryogenesis. First, the effectiveness of the CRISPR/Cas9 system was tested in developing embryos. The eGFP-targeted CRISPR/Cas9 system was introduced into zygotes fertilized with boar semen heterozygous for the eGFP transgene. Following the injection, subsequent embryos expressing eGFP were monitored. Various concentrations of the CRISPR/Cas9 system were tested and the cytotoxicity of the delivered CRISPR/Cas9 system was examined (Figure 4, panel A); embryo development after CRISPR/Cas9 injection was reduced compared to control. However, all concentrations of CRISPR/Cas9 tested were effective in producing the eGFP modification because no eGFP-expressing embryos were found in the CRISPR/Cas9 injection group (FIG. 4, panel B); 67.7% of uninjected control embryos stained green, indicating eGFP expression. When individual blastocysts were genotyped, small mutations near the CRISPR binding sites could be identified (Figure 4, panel C). Based on toxicity and efficacy, 10 ng/μl of gRNA and Cas9 mRNA were used for the following experiments.

[00262] When CRISPR/Cas9 components designed to target CD163 were introduced into putative zygotes, targeted gene editing was observed in subsequent blastocysts. When individual blastocysts were genotyped for the CD163 mutation, specific mutations were found in all embryos (100% gene editing efficiency). More importantly, while embryos could be found with homozygous or biallelic modifications (respectively 8/18 and 3/18) (Fig. 5), mosaic (monoallelic modifications) genotypes were also found (4/18 embryos ). Some of the pooled embryos (8/10) were injected with 2 ng/µl Cas9 and 10 ng/µl CRISPR and no difference in mutagenesis efficiency was found. Then, based on in vitro results, two CRISPRs representing different gRNAs were introduced to disrupt CD163 or CD1D during embryogenesis in order to induce a specific deletion of target genes. As a result, it was possible to successfully induce the planned deletion of CD163 and CD1D by introducing two guides. A planned deletion is defined as a deletion that removes a genomic sequence between two introduced guides. Among the embryos that received two CD165-targeted CRISPRs, all but one embryo had a targeted CD163 modification. In addition, 5/13 embryos were found to have an engineered deletion in CD163 (FIG. 6, panel A), and 10/13 embryos appeared to have CD163 modification either in a homozygous or biallelic fashion. Targeting CD1D with two CRISPRs was also effective as all embryos (23/23) showed CD1D modification. However, the engineered deletion of CD1D could only be detected in two embryos (2/23) (Fig. 6, panel B). Five of the twenty-three embryos were also found to have mosaic genotypes, but the remaining embryos were either homozygous or biallelic for CD1D. Finally, we tested whether the CRISPR Cas9 system could target multiple genes in the same embryo. For this purpose, targeting both CD163 and eGFP was carried out in zygotes fertilized with heterozygous eGFP sperm. When blastocysts from injected embryos were genotyped for CD163 and eGFP, it was found that CD163 and eGFP were successfully attacked during embryogenesis. The sequencing results demonstrated that multiple genes can be affected by introducing multiple CRISPRs with Cas9 (FIG. 6, panel C).

Generation of CD163 and CD1D Mutants from CRISPR/Cas9 Injected Zygotes

[00263] Based on the success of the previous in vitro study, several CRISPR/Cas9 injected zygotes were obtained and 46-55 blastocysts were transferred to the recipient (because this number has been shown to be effective in producing pigs from in vitro derived embryos). Four embryo transfers were performed, two each for CD163 and CD1D, and a pregnancy was obtained for each modification. Four healthy piglets carrying CD163 modifications were obtained (Table 8). All piglets in litter 67 from recipient sow ID O083 showed either homozygous or biallelic modification of CD163 (FIG. 7). Two piglets were found to have an engineered CD163 deletion using two delivered CRISPRs. All piglets were healthy. For CD1D, one pregnancy also produced four piglets (litter 166 from recipient sow ID 0165): one female and three males (Table 9). One pig (166-1) did carry a mosaic CD1D mutation, including a 362 bp deletion that completely deleted exon 3 containing the start codon (FIG. 8). One pig contained a 6 bp insert. with a pairing error of 2 b.p. on one allele, with a large deletion on the other allele. The other two piglets had a biallelic insertion of one bp. No mosaic mutations were found for CD163.

DISCUSSION

[00264] Increasing the efficiency of obtaining genome-modified pigs can be of great importance by increasing the number of genome-edited pigs for agriculture and biomedicine. The data described above show that genome-edited pigs carrying specific mutations can be produced with high efficiency using the CRISPR/Cas9 system. The CRISPR/Cas9 system has been successfully used for gene editing in both somatic cells and preimplantation embryos.

[00265] When the CRISPR/Cas9 system was introduced into somatic cells, it successfully induced targeted disruption of target genes by NHEJ, but did not increase targeting capability by gene editing. The targeting efficiency of individual CRISPR/Cas9 in somatic cells varied, indicating that targeting efficiency may be affected by guide design. In particular, when CRISPR 5350 and Cas9 were introduced into somatic cells, the target modification of CD1D could not be obtained. This indicates that it would be useful to develop several gRNAs and test their effectiveness before obtaining pigs. The reason for the lack of gene-editing-directed repair in the presence of donor DNA is still unclear. After screening 886 colonies (both CD163 and CD1D) transfected with CRISPR and donor DNA, only one colony had evidence of a partial gene editing event. The results demonstrated that the CRISPR/Cas9 system worked with the introduced donor DNA, causing unexpected large deletions in the target genes, but did not increase gene editing efficiency for these two specific targeting vectors. However, the specific mechanism of why large deletions were observed is unknown. Previous reports by our group suggested that donor DNA could be effectively used with ZFN to induce gene-editing repair. Similarly, an increase in targeting efficiency was observed when donor DNA was used with the CRISPR/Cas9 system, but complete repair aimed at gene editing was not observed. In a previous study using ZFN, it was noted that targeted modification could occur through a combination of gene editing and NHEJ, based on the fact that partial recombination of the introduced donor DNA was found after ZFN-induced DSBs. One explanation could be that gene editing and NHEJ pathways are not independent, but may act together to complete the repair process after DSBs induced by homing endonucleases. Higher concentrations of CRISPR may increase the effectiveness of somatic cell targeting, although no statistical difference was found in these experimental results. This may indicate that CRISPR is a limiting factor in the CRISPR/Cas9 system, but further verification is needed for this assumption. The target cells have been successfully used to produce gene-edited pigs by SCNT, indicating that the use of CRISPR/Cas9 does not affect the ability of the cells to clone. Several piglets were euthanized due to health problems; however, this is not uncommon in piglets produced by SCNT.

[00266] When the CRISPR/Cas9 system was introduced into developing embryos by zygote injection, almost 100% of embryos and pigs contained INDEL in the target gene, demonstrating that this technology is highly effective during embryogenesis. The efficacy observed during this study exceeds the rates reported in other studies using homing endonucleases during embryogenesis. The decrease in the number of embryos reaching the blastocyst stage suggests that the concentration of CRISPR/Cas9 administered in this study may be toxic to the embryos. Further optimization of the delivery system can improve embryo survival and thus improve the overall efficiency of the process. The almost 100% mutagenesis rate observed here was different from the previous report of CRISPR/Cas9-mediated knockout in pigs; however, the difference in performance between studies may be related to the chosen combination of guide and target. In the present study, lower concentrations of CRISPR/Cas9 (10 ng/µl each) were effective in generating mutations in developing embryos and producing gene-edited pigs. The concentration in porcine zygotes was lower than previously reported (125 ng/µl Cas9 and 12.5 ng/µl CRISPR). The lower concentration of CRISPR/Cas9 components may be beneficial for developing embryos, since the introduction of excessive amounts of nucleic acid into developing embryos can be toxic. In in vitro assays, some mosaic genotypes were observed in embryos injected with CRISPR/Cas9; however, only one pig produced by this approach had a mosaic genotype. Potentially, injection of CRISPR/Cas9 components may be more efficient than injection of other homing endonucleases, since the mosaic genotype has been considered a major obstacle to the use of the CRISPR/Cas9 system in zygotes. Another benefit of using the CRISPR/Cas9 system demonstrated by the present results is that no CD163 knockout pigs raised from IVF zygotes injected with the CRISPR/Cas9 system were lost, while several obtained from SCNT were euthanized after a few days. This suggests that the technology could not only bypass the need for SCNT to create knockout pigs, but could also solve common health problems associated with SCNT. Now that the injection of CRISPR/Cas9 mRNA into zygotes is optimized, future experiments will also include co-injection of donor DNA.

[00267] The present study demonstrates that the introduction of two Cas9 CRISPRs into zygotes can induce chromosomal deletions in developing embryos and produce pigs with a target deletion, ie a specific deletion between two CRISPR guides. This engineered deletion can be useful because you can specify the size of the deletion rather than relying on random events triggered by NHEJ. In particular, in the case where there is an insertion/deletion of nucleotides in multiples of three caused by homing endonuclease, the mutation is more likely to lead to a hypomorphic mutation because no frameshift will occur. However, by introducing two CRISPRs, larger deletions can be induced, which will have a higher chance of producing a non-functional protein. Interestingly, CRISPR CDIDs have been engineered in more of the genome than CD 163; the distance between CD163 CRISPR 10 and 131 was 124 bp, while the distance between CRISPR 4800 and 5350 for CD1D was 550 bp. As shown in the study, greater distance between CRISPRs is not very efficient in creating a deletion. However, since the present study included only a limited number of observations and it was not necessary to investigate the efficacy of individual CRISPRs, which are not discussed here, further studies are needed to test the relationship between CRISPR distance and the likelihood of putative deletions occurring.

[00268] The CRISPR/Cas9 system was also effective in targeting two genes simultaneously in the same embryo, with the only additional step being the introduction of one additional CRISPR with crRNA. This illustrates the ease of disruption of multiple genes compared to other homing endonucleases. These results suggest that this technology can be used to target gene clusters or gene families that may have a compensatory effect, making it difficult to determine the role of individual genes unless all genes are affected. The results demonstrate that CRISPR/Cas9 technology can be applied to create genome-edited pigs by increasing the efficiency of gene targeting in somatic cells and by direct injection into the zygote.

Example 2: Increased resistance to PRRSV in pigs having a modified chromosomal sequence in the gene encoding the CD163 protein

[00269] Over the past quarter century, the porcine reproductive and respiratory syndrome virus (PRRSV) has devastated the swine industry. Suggestions about the mode of entry of the virus include both SIGLEC1 and CD163. Although SIGLEC1 knockout did not affect response to viral infection, this example shows that CD163 null animals do not show the clinical signs of infection, lung disease, viremia, or antibody production that are hallmarks of PRRSV infection. Not only was the PRRSV proxy at the input confirmed; but if animals created in the same way were allowed to become a food resource, then a strategy could be described to prevent significant economic loss and animal suffering.

MATERIALS AND METHODS

Genotyping

[00270] Genotyping was based on both DNA sequencing and mRNA sequencing. There was an 11 bp deletion in the genotype of the male sire. in one allele, which when translated provided 45 amino acids in domain 5, resulting in a premature stop codon at amino acid 64. In the other allele, there was a 2 bp addition. in exon 7 and a 377 bp deletion. in the intron before exon 7, which, when translated, provided the first 49 amino acids of domain 5, leading to a premature stop of the code at amino acid 85. In one sow, 7 bp was added. in one allele that, when translated, provided for the first 48 amino acids of domain 5, resulting in a premature stop codon at amino acid 70. The other allele was not characterized (A) because there was no band from exon 7 obtained by both PCR and using PCR long fragments of 6.3 kb. The remaining 3 sows were clones and had a 129 bp deletion. in exon 7, which is predicted to result in a 43 amino acid deletion from domain 5. The other allele has not been characterized (B).

Growing PRRSV in culture and obtaining a viral inoculum for infecting pigs is described in accordance with the application 973, approved by the IBC

[00271] The PRRSV type strain, isolate NVSL 97-7895 (GenBank No. AF325691 2001-02-11), was grown as described in IBC approved protocol 973. This laboratory isolate has been used in experimental studies for about 20 years (Ladinig et al., 2015). The second isolate, KS06-72109, was used for the 2nd trial as described previously (Prather et al., 2013).

Infection of pigs with PRRSV

[00272] A standardized PRRSV infection protocol was used to infect pigs. Three week old piglets were inoculated with approximately 10 ⁴ TCID ₅₀ PRRS virus, which was administered by the intramuscular (IM) and intranasal (IN) routes. Pigs were observed daily and those showing symptoms of the disease were treated as recommended by CMG veterinarians. Pigs that were in serious condition and at risk of infection were humanely killed and their samples were collected. Staff and veterinarians were not aware of the genetic status of the pigs in order to avoid bias in evaluation or treatment. PRRSV is present in body fluids during infection; therefore, blood samples were collected and stored at -80° C. until measurements were taken to determine the amount or degree of viremia in each pig. At the end of the experiment, pigs were weighed and humanely euthanized, tissues collected and fixed in 10% buffered formalin, embedded in paraffin and processed for histopathological examination by a certified pathologist.

Assessment of the phenotype of infected pigs

[00273] Pig phenotype was blinded daily as follows: What is the feeding behavior of the pig? Behavior rating: 0: best, 1: questionable, 2: slightly depressed, 3: depressed, 4: dying animal. What is the body condition of the pig? Body condition score: 1: wasted, 2: lean, 3: perfect, 4: fat, 5: overweight/obese. What is the rectal temperature of a pig? Normal body temperature: 101.6-103.6°F (a fever is considered to be >104°F). Is there lameness (degree)? What limb? Examine the limbs for joint swelling and hoof damage (check the bottom and sides of the hoof). Lameness score: 1: no lameness, 2: slightly uneven when walking, feels stiff in some joints, but no lameness, 3: mild lameness, slight lameness when walking, 4: moderate lameness, pronounced lameness, including lameness from touching the toes , 5: severe lameness, limbs not supported by weight, need encouragement to stand/walk. Is there difficulty breathing (degree)? Is there open mouth breathing? Is there nasal discharge (discharge of color, amount of discharge: light/medium/heavy)? Have you noticed that the animal is coughing? Is there discharge from the eyes? Respiratory score: 0: normal, 1: mild dyspnea and/or tachypnea with stress (touch), 2: mild dyspnea and/or tachypnea at rest, 3: moderate dyspnoea and/or tachypnea with stress (touch), 4: moderate dyspnea and/or tachypnea at rest, 5: severe dyspnea and/or tachypnea during stress (on manipulation), 6: severe dyspnea and/or tachypnea at rest. Are there signs of diarrhea (severity) or vomiting? Is there blood or mucus? Diarrhea score: 0: no feces noted, 1: stools normal, 2: soft but formed stools (soft yogurt consistency, like cow pie), 3: brown/yellow-brown liquid diarrhea with small fecal particles, 4: liquid diarrhea brown/yellow brown without fecal solids, 5: watery, liquid diarrhea.

[00274] This scoring system was developed by Dr. Megan Niederwerder at KSU and is based on the following publications (Halbur et al., 1995; Merck; Miao et al., 2009; Patience and Thacker. 1989; Winckler and Willen. 2001). Scores and temperatures were analyzed using genotype-based split ANOVA as treatments.

Measurement of PRRSV Viremia

[00275] Viremia was determined using two approaches. Virus titration was performed by adding 1:10 serial dilutions of serum to confluent MARC-145 cells in a 96-well plate. Serum was diluted in Eagle's Minimum Essential Medium supplemented with 8% fetal bovine serum, penicillin, streptomycin, and amphotericin B as described previously (Prather et al., 2013). The cells were examined after 4 days of incubation for the presence of a cytopathic effect using a microscope. As the endpoint of the titration, the highest dilution showing a cytopathic effect was evaluated. Total RNA was isolated from serum using the Life Technologies MagMAX-96 Viral RNA Isolation Kit to measure viral nucleic acid. Reverse transcription polymerase chain reaction was performed using the EZ-PRRSV MPX 4.0 kit from Tetracore on a CFX-96 real-time PCR system (Bio-Rad) according to the manufacturer's instructions. Each reaction (25 µl) contained RNA from 5.8 µl of serum. A standard curve was generated by preparing serial dilutions of the control RNA supplied in the kit (Tetracore). The results are presented as the number of templates per PCR.

AMC cell staining for SIGLEC1 and CD163

[00276] Porcine alveolar macrophages (AMS) were harvested by excising the lungs and filling them with ~100 ml of cold phosphate-buffered saline. After retrieval, phosphate-buffered wash cells were pelleted and resuspended in 5 ml of cold phosphate-buffered saline and stored on ice. Approximately 10 ⁷ AMS were incubated in 5 ml of various antibodies (against porcine CD169 (clone 3B11/11; AbD Serotec); against porcine CD163 (clone 2A10/11; AbD Serotec)), diluted in phosphate buffered saline with 5% fetal bovine serum and 0.1% sodium azide, for 30 minutes on ice. Cells were washed and resuspended at a 1/100 dilution in fluorescein isothiocyanate (FITC) conjugated to anti-mouse IgG (life Technologies) diluted in staining buffer and incubated for 30 minutes on ice. At least 10 ⁴ cells were analyzed using a FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson).

Measurement of PRRSV-specific Ig

[00277] To measure PRRSV-specific Ig, recombinant PRRSV protein N was expressed in bacteria (Trible et al., 2012) and conjugated to Luminex magnetic beads using a kit (Luminex Corporation). N-protein bound beads were diluted in phosphate buffered saline containing 10% goat serum to 2500 beads/50 μl and placed in wells of a 96 well round bottom polystyrene plate. Serum was diluted 1:400 in PBS containing 10% goat serum and 50 µl was added to the wells in duplicate and incubated for 30 minutes with gentle shaking at room temperature. The plate was then washed (3 times) with phosphate buffered saline containing 10% goat serum and 50 µl of SP-biotin-labeled, affinity-purified goat anti-pig secondary antibody (IgG, Jackson ImmunoResearch) or biotin-labelled, affinity-purified goat anti-pig IgM. (KPL) diluted to 2 µg/ml in phosphate buffered saline containing 10% goat serum. After 30 minutes of incubation, the plates were washed (3 times) and then 50 μl streptavidin-conjugated phycoerythrin (2 μg/ml (Moss, Inc.) in phosphate-buffered saline containing 10% goat serum) was added. The plates were washed after 30 minutes and microspheres were resuspended in 100 µl phosphate buffered saline containing 10% goat serum and analyzed using MAGPIX and Luminex xPONENT 4.2 software Results are reported as mean fluorescence intensity MFI.

RESULTS

[00278] Mutations in CD163 were created using CRISPR/Cas9 technology as described above in Example 1. Several founding animals were generated by zygote injection and somatic cell nuclear transfer. Some of these founders have been bred while producing offspring for study. A single founding male was mated to females with two genotypes. The founding male (67-1) had an 11 bp deletion. in exon 7 on one allele and the addition of 2 b.p. in exon 7 (and a 377 bp deletion in the previous intron) of another allele and was determined to be a null animal (CD163 ^-/- ). One founding female (65-1) had a 7 bp addition. in exon 7 at one allele and an uncharacterized corresponding allele, and thus heterozygosity was predetermined for knockout. (CD163 ^-/? ). The second genotype of the founding female (3 animals that were clones) contained an as yet uncharacterized allele and an allele with a 129 bp deletion. in exon 7. This deletion is expected to result in a 43 amino acid deletion in domain 5. As a result of mating between them, all piglets inherited the null allele from the boar and either the 43 amino acid deletion or one of the uncharacterized alleles from the sows. In addition to the wild-type piglets that served as positive controls for viral challenge, this resulted in 4 additional genotypes (Table 12).

[00279] At weaning, age-matched modified and wild-type piglets were brought to Kansas State University for PRRSV challenge. PRRSV infection was performed as previously described (Prather et al., 2013). Three-week-old piglets were placed in the challenge room and kept as one group. All experiments were initiated after the approval of the institutional animal use and biosafety committees. After acclimation, pigs were challenged with PRRSV NVSL 97-7895 isolate (Ladinig et al., 2015) propagated on MARC-145 cells (Kim et al., 1993). Pigs were infected with approximately 10 ⁵ TCID ₅₀ virus. Half of the inoculum was administered intramuscularly and the remainder intranasally. All infected pigs were housed as one group, allowing continuous exposure to the virus from infected pen mates. Blood samples were collected on various days up to 35 days post infection and finally on day 35. Pigs were dissected, tissues fixed in 10% buffered formalin, embedded in paraffin and processed for histopathology. PRRSV-associated clinical signs reported during infection included respiratory distress, anorexia, lethargy, and fever. Results by clinical signs over the study period are presented in Fig. 9. As expected, wild-type pigs (CD163 ^+/+ ) showed early signs of PRRSV infection, which peaked between days 5 and 14 and persisted in the group during the remainder of the study. The percentage of pigs with fever peaked around day 10. In contrast, null (CD163 ^-/- ) piglets showed no clinical signs during the entire study period. Respiratory signs during acute PRRSV infection are reflected in significant histopathological changes in the lungs (Table 9). WT pig infection showed histopathology consistent with PRRS, including interstitial edema with mononuclear cell infiltration (FIG. 10). In contrast, there was no evidence of pulmonary changes in null (CD163 ^-/- ) pigs. The sample size for different genotypes is small; however, mean scores were 3.85 (n=7) for wild type, 1.75 (n=4) for uncharacterized A, 1.33 (n=3) for uncharacterized B, and 0 (n=3) and for null (CD 163 ^-/- ) genotype.

[00280] Peak clinical signs correlated with blood levels of PRRSV. Measurement of viral nucleic acid was performed by isolating total RNA from serum followed by amplification of PRRSV RNA using a commercial reverse transcriptase PRRSV real-time PCR assay (Tetracore, Rockville, MD). A standard curve was generated by preparing serial dilutions of the PRRSV control RNA supplied with the real-time PCR kit and the results were standardized as the number of templates per 50 µl PCR reaction. The PRRSV isolate was consistent with the course of PRRSV viremia in wild-type pigs CD163 ^+/+ (Fig. 11). Viremia was pronounced on the fourth day, peaked on day 11, and declined until the end of the study. In contrast, no viral RNA was detected in CD163 ^-/- pigs at any time during the study period. In accordance with viremia, the production of antibodies by pigs with a null and uncharacterized allele was detected by day 14 and increased by day 28. Null animals showed no antibody production (FIG. 12). Together, these data indicate that wild-type pigs maintain PRRSV replication with clinical signs consistent with PRRSV. In contrast, knockout pigs showed neither viremia nor clinical signs, although the pigs were inoculated and in constant contact with infected pen mates.

[00281] At the end of the study, porcine alveolar macrophages were isolated by lung lavage and stained for surface expression of SIGLEC1 (CD169, clone 3B11/11) and CD163 (clone 2A10/11) as previously described (Prather et al., 2013). Relatively high levels of CD163 expression were found in wild-type animals CD 163 ^+/+ (Fig. 13). In contrast, CD163 ^-/- pigs showed only baseline levels of anti-CD163 staining, confirming the knockout phenotype. Expression levels of another macrophage marker, CD169, were similar in both wild-type and knockout pigs (Fig. 14). Other macrophage surface markers, including MHC II and CD 172, were similar for both genotypes (data not shown).

[00282] Although the sample size was small, wild-type pigs tended to gain less weight during the experiment (mean daily weight gain 0.81 kg ± 0.33, n=7) compared to pigs of the other three genotypes (uncharacterized A 1.32 kg±0.17, n=4; uncharacterized B 1.20 kg±0.16, n=3; null 1.21 kg±0.16, n=3).

[00283] In the second study, 6 wild-type pigs, 6 pigs with Δ43 amino acids, and 6 pigs with an uncharacterized allele (B) were challenged as described above, except that KS06-72109 was used to inoculate piglets. Similar to the NVSL data, wild-type piglets and uncharacterized B piglets developed viremia. However, in pigs with Δ43 amino acids, KS06 did not cause viremia (Fig. 15; Table 7).

REASONING AND CONCLUSION

[00284] The most clinically significant disease in pig production is PRRS. While vaccination programs have been successful in preventing or attenuating most swine pathogens, PRRCV has proven to be more of a challenge. Here, CD163 is identified as the mediator of entry for this virus strain. The founder boar was created by injecting CRISPR/Cas9 into zygotes (Whitworth et al., 2014), and as a result, the transgene is missing. In addition, one of the sow's alleles (also generated with CRISPR/Cas9) does not contain the transgene. Thus, pig #40 carries an addition of 7 bp. in one allele and a deletion of 11 bp. in a different allele, but does not have the transgene. These CD163 virus resistance alleles represent minor genome edits given the fact that the pig genome is about 2.8 billion base pairs (Groenen et al., 2012). If animals created in a similar way were introduced as a food resource, significant economic losses could be averted.

Example 3: Increased resistance to porcine reproductive viruses genotype 1 and PRRS viruses in pigs with the replacement of CD163 SRCR domain 5 with SRCR domain 8 - human CD163-like homologue

[00285] CD163 is considered the major receptor for the porcine reproductive and respiratory syndrome virus (PRRSV). In this study, pigs were genetically modified (GE) to have one of the following genotypes: complete knockout (KO) of CD163, deletions in CD163 domain 5 of the cysteine-rich phagocyte receptor (SRCR), or replacement (domain swap) of the SRCR 5 domain with synthesized an exon encoding a homologue of the human CD163-like (hCD163Ll) domain of SRCR 8. Immunophenotyping of porcine alveolar macrophages (AMCs) showed that pigs with SRCR domain 5 knockouts or deletions did not express CD163, and AMCs did not support PRRSV infection. AMS from pigs that possessed the hCD163Ll 8 domain homologue expressed CD163 and supported replication of genotype 2 viruses but not type 1. Infection of CD163 modified pigs with typical type 1 and type 2 viruses produced similar results. Although type 1 and type 2 viruses are considered to be genetically and phenotypically similar on several levels, including the requirement for CD163 as a receptor, the results demonstrate a clear difference between the PRRSV genotypes in recognition of the CD163 molecule.

MATERIALS AND METHODS

Genomic modifications of the pig CD163 gene

[00286] Animal and virus experiments were conducted in accordance with the Federation of Animal Societies Guidelines for the Care and Use of Farm Animals in Research and Education, the USDA Animal Welfare Act, and the Animal Welfare Regulations, and were approved by the Institutional Care Committees and Use of Animals and the Institutional Biosafety Committees of the University of Kansas and the University of Missouri. Mutations in CD163 used in this study were created using CRISPR/Cas9 technology, as described above in the previous examples. Mutations are shown schematically in FIG. 17. Schematic of the genomic region shown in FIG. 17 covers the sequence from intron 6 to intron 8 of the porcine CD163 gene. Introns and exons are shown schematically in Fig. 17 is not to scale. The predicted protein product is shown to the right of each genomic structure. Relative macrophage expression, as measured by the level of surface CD163 on AMC, is shown at the far right of FIG. 17. Black areas indicate introns; white areas denote exons; the shaded area indicates hCD163Ll exon 11 mimic, a porcine exon 7 homologue; and the gray area indicates the synthesized intron with the PGK Neo construct, as shown in FIG. 17.

[00287] The KO-d7(11) CD163 gene construct shown in FIG. 17, has an 11 bp deletion in exon 7 from nucleotide 3137 to nucleotide 3147. The CD163 KO-i7(2) gene construct has a 2 bp insertion in exon 7 between nucleotides 3149 and 3150, and a 377 bp deletion in the intron before exon 7, nucleotide 2573 to nucleotide 2949. These changes are expected to cause frameshift mutations and premature stop codons, leading to only partial translation of SRCR 5 and the KO phenotype. Three other mutations cause deletions in exon 7. The first, d7(129), has a 129 bp deletion in exon 7 from nucleotide 3044 to nucleotide 3172. Construct d7(129) also has a deletion from nucleotide 488 to nucleotide 2,417 in exon 6, at the deleted sequence is replaced by a 12 bp insert. Two other deletion constructs, d7(1467) and d7(1280), have complete deletions of exons 7 and 8, as shown in FIG. 17. d7(1467) has a 1467 bp deletion from nt 2431 to nt 3897, and d7(1280) has a 1280 bp deletion from nt 2818 to nt 4097. For these deletion constructs, the remaining exons of CD 163 were left intact.

[00288] The last design shown in FIG. 17, HL11m, was generated using a targeting event that deleted exon 7 and replaced it with a synthesized exon encoding the SRCR 8 homologue of human CD163-like protein 1 (hCD163Ll domain 8 encoded by hCD163L1 exon 11). The peptide sequence of SRCR 8 was created by changing 33 nucleotides in the sequence of porcine exon 7. The neomycin cassette was included in the synthesized exon to enable modification screening. SEQ ID NO: 118 is the nucleotide sequence for the HL11m construct in the region corresponding to the same region in the reference sequence of SEQ ID NO: 47.

[00289] A schematic of the porcine CD 163 protein and its gene is shown in FIG. 18. The CD163 SCRC protein domains (ovals) and PST (squares) along with the corresponding gene exons are shown in panel A of FIG. 18. Comparison of the peptide sequences of porcine CD163 SRCR 5 (SEQ ID NO: 120) and human CD163 SRCR 8 homologue (SEQ ID NO: 121) is shown in panel B of FIG. 18. Figure based on GenBank accession numbers AJ311716 (porcine CD163) and GQ397482 (hCD163-Ll).

Viruses

[00290] The panel of viruses used in this example is shown in Table 14.

Isolates were expanded and titrated on MARC-145 cells (Kim et al., 1993). For titration, each virus was serially diluted 1:10 in MEM with 7% FBS, Pen-Strep (80 units/ml and 80 µg/ml, respectively), 3 µg ml FUNGIZONE (amphotericin B) and 25 mM HEPES. Diluted samples were added in quadruplicate to confluent MARC-145 cells in a 96-well plate to a final volume of 200 μl per well and incubated for four days at 37° C. with 5% CO ₂ . The end point of the titration was defined as the last cytopathic well (CPE). The tissue culture infectious dose of 50% (TMD50/ML) was calculated using the method described previously (Reed and Muench 1938).

**NA, no data available.

Infection of alveolar macrophages

[00291] Preparation and infection of macrophages was performed as previously described (Gaudreault, et al., 2009 and Patton, et al., 2008). Lungs were removed from euthanized pigs and washed by pouring 100 ml of cold phosphate-buffered saline (PBS) into the trachea. The tracheae were clamped and the lungs gently massaged. The alveolar contents were poured into 50 ml centrifuge tubes and stored on ice. Porcine alveolar macrophages (AMS) were pelleted by centrifugation at 1200×g for 10 minutes at 4°C. The pellets were resuspended and washed once with cold sterile PBS. Cell pellets were resuspended in freezing medium containing 45% RPMI 1640, 45% fetal calf serum (FBS) and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until use. Frozen cells were thawed on ice, counted and adjusted to 5×10 ⁵ cells/ml in medium (RPMI 1640 supplemented with 10% FBS, PenStrep and FUNGIZONE; RPMI-FBS). Approximately 10 ³ AMS per well were added to 96-well plates and incubated overnight at 37° C. in 5% CO ₂ . Cells were gently washed to remove non-adherent cells. Serial 1:10 dilutions of the virus were added to the wells in triplicate. After incubation overnight, the cells were washed with PBS and fixed for 10 minutes with 80% acetone. After drying, the wells were stained with SDOW-17 monoclonal antibody specific for the PRRSV N protein (Rural Technologies Inc.) diluted 1:1000 in PBS with 1% fish gelatin (PBS-FG; Sigma Aldrich). After a 30 minute incubation at 37° C., the cells were washed with PBS and stained with ALEXAFLUOR 488 labeled anti-mouse IgG (Thermofisher Scientific) diluted 1:200 in PBS-FG. The plates were incubated for 30 minutes in the dark at 37°C, washed with PBS and viewed under a fluorescent microscope. The infectious dose in tissue culture of 50% (TCID ₅₀ )/ml was calculated according to the method described previously (Reed and Muench 1938).

Measurement of surface expression of CD 169 and CD 163 on AMS

[00292] Staining for surface expression of CD 169 and CD 163 was performed as previously described (Prather et al., 2013). Approximately 1×10 ⁶ AMC were placed in 12 mm×75 mm polystyrene flow cytometry (FACS) tubes and incubated for 15 minutes at room temperature in 1 ml PBS with 10% normal mouse serum to block Fc receptors. Cells were pelleted by centrifugation and resuspended in 5 µl FITC-conjugated mouse anti-porcine CD169 mAbs (clone 3B11/11; AbD Serotec) and 5 µl PE-conjugated mouse anti-porcine CD163 mAbs (clone: 2A10/11, AbD Serotec). After 30 min incubation, cells were washed twice with PBS containing 1% bovine serum albumin (BSA Fraction V; Hyclone) and immediately analyzed on a BD LSR Fortessa flow cytometer (BD Biosciences) with FCS Express 5 software (De Novo Software). For each sample, at least 10,000 cells were analyzed.

Measurement of PRRS viremia

[00293] RNA was isolated from 50 μl of serum using the MagMAX 96 Ambion virus isolation kit (Applied Biosystems) according to the manufacturer's instructions. PRRSV RNA was quantified using Target-Specific Reagents (Tetracore), real-time RT-PCR EZ-PRRSV MPX 4.0 according to the manufacturer's instructions. Each plate contained Tetracore quantitation standards and control kits for use with RT-PCR reagents. PCR was performed on a CFX96 Touch Real Time PCR Detection System (Bio-Rad) in a 96-well format using the recommended run parameters. PCR results were reported as logio copy number of PRRSV RNA per 50 µl of reaction volume, which is approximately the number of copies per ml of serum. Area under the curve (AUC) for viremia over time was calculated using GraphPad Prism version 6.00 for Windows.

Measurement of antibodies to PRRSV

[00294] Microsphere immunofluorescence assay (FMIA) for detection of antibodies against PRRSV nucleocapsid (N) protein was performed as previously described (Stephenson et al., 2015). Recombinant PRRSV protein N was coupled to Luminex MAGPLEX carboxylated polystyrene microsphere beads according to the manufacturer's instructions. For FMIA, approximately 2500 antigen coated beads suspended in 50 μl of PBS with 10% goat serum (PBS-GS) were placed in each well of a 96-well polystyrene round bottom plate. Serums were diluted 1:400 in PBS-GS and 50 μl were added to each well. The tablet was wrapped in foil and incubated for 30 minutes at room temperature with gentle shaking. The plate was placed on a magnet and the beads were washed three times with 190 μl PBS-GS. For IgG detection, 50 µl biotin-SP conjugated affinity purified goat anti-pig secondary antibody (IgG, Jackson ImmunoResearch) was diluted to 2 µg/ml in PBS-GS and added 100 µl to each well. The plate was incubated at room temperature for 30 minutes and washed three times followed by the addition of 50 μl streptavidin-conjugated phycoerythrin (2 μg/ml in PBS-GS; SAPE). After 30 minutes, the microspheres were washed, resuspended in 100 μl PBS-GS and analyzed using the MAGPIX instrument (LUMINEX) and LUMINEX xPONENT 4.2 software. The mean fluorescence intensity (MFI) was calculated for at least 100 microspheres.

Measurement of haptoglobin (HP)

[00295] Serum Hp was measured using a porcine Hp ELISA kit (Genway Biotech Inc.) and steps were performed according to the manufacturer's instructions. Serum samples were diluted 1:10,000 in 1x diluent solution and pipetted in duplicate into a 96-well ELISA plate pre-coated with porcine Hp antibodies, incubated at room temperature for 15 minutes, then washed three times. Anti-Hp horseradish peroxidase (HRP) conjugate was added to each well and incubated in the dark at room temperature for 15 minutes. The plate was washed and 100 μl of chromogen substrate solution was added to each well. After incubation in the dark for 10 minutes, 100 μl of stop solution was added to each well. The plate was read at 450 nm on a microplate reader based on a Fluostar Omega filter (BMG Labtech).

RESULTS

Phenotypic properties of AMS from CD163-modified pigs

[00296] Forward and side scatter properties of the cells were used to capture the mononuclear subpopulation of cells in the lung lavage material. Representative staining results for CD169 and CD163 for various chromosomal modifications shown in FIG. 17 are shown in FIG. 19. In the representative example shown in panel A of FIG. 19, more than 91% of AMS from WT pigs were positive for both CD169 and CD163. Results for the 12 WT pigs used in this study showed an average of 85+/- 8% double positive cells. As shown in panel B of FIG. 19, AMS from CD163 knockout pigs showed no evidence of CD163 but retained normal surface levels of CD169. Although CD163 polypeptides derived from deletion genotypes (17(1467) and (17(1280)) were predicted to produce modified CD163 polypeptides attached to the AMC surface, immunostaining results did not show surface expression of CD163 (see Fig. 19, panel D) Because MAb 2A10 recognizes an epitope located in the first three domains of the SRCR, the lack of detection was not the result of a deletion of the immunoreactive epitope.The d7(129) genotype is expected to have a 43 amino acid deletion in SRCR 5 (see Fig. 17). In the example shown in panel C of Fig. 19, only 2.4% of the cells fell into the double positive quadrant.AMS analysis from the nine pigs (17(129) used in this study showed a percentage of double positive cells ranging from 0% to 3.6% (mean = 0.9%) Surface expression of CD 169 remained similar to wild-type AMS For the purposes of this study, all pigs with knockout genotypes, d7(1467), d7(1280) and d7(129) , were categorized as having a CD 163 null phenotype.

[00297] The CD163 modification containing the hCD163Ll domain 8 HL11m peptide sequence showed dual expression of CD163+ and CD169+ on AMC (panel E of Fig. 19). However, in all HL11m pigs analyzed in this study, CD163 surface expression was markedly reduced compared to wild-type AMS. CD163 levels fell on an expression continuum ranging from no detectable CD163 to pigs with moderate levels of CD163. In the example shown in panel E of FIG. 19, approximately 60% of the cells were in the double positive quadrant, while 40% of the cells stained for CD169 only. Analysis of AMC from 24 HL11m pigs showed that 38+/- 12% of AMC cells were positive for CD169 alone and 54+/- 14% were double positive (CD169 ⁺ CD163 ⁺ ).

Circulating Haptoglobin Levels in WT and CD 163 Modified Pigs

[00298] As an uptake molecule, CD163 is responsible for removing HbHp complexes from the blood (Fabriek, et al., 2005; Kristiansen et al., 2001; and Madsen et al., 2004). Serum Hp level provides a convenient way to determine the overall functional properties of macrophages expressing CD163. Serum Hp levels from WT, HLllm and CD 163 null pigs were measured at three to four weeks of age, just prior to PRRSV infection. The results shown in FIG. 20 showed that sera from WT pigs had the least amount of Hb (mean A450=23+/-0.18, n=10). The mean and standard deviation for each group was for wild type - 0.23+/- 0.18, n=10; for HL11m - 1.63+/-0.8, n=11; and for the zero group - 2.06+/- 0.57, n=9. The null group consisted of genotypes that did not express CD163 (pigs with a null CD 163 phenotype). Hp measurements were performed on a single ELISA plate. Groups with the same letter were not significantly different (p>0.05, one-way Kruskal-Wallis ANOVA followed by Dunnett's test). The mean A450 value for WT pigs was significantly different from that for HL11m and CD163 null pigs (p<0.05). Although the mean A450 was lower for the HLllm group compared to the non-CD163 group (A450=1.6+/-0.8 vs 2.1+/-0.6), the difference was not statistically significant. Since the interaction between HbHp and CD163 occurs via SRCR 3 (Madsen et al., 2004), the increased amount of circulating Hp in HLllm pigs compared to wild-type pigs was probably not due to a decrease in the affinity of CD 163 for Hb/Hp, but was the result of a decrease in the number of CD163 ⁺ macrophages along with reduced expression of CD 163 on the remaining macrophages (see panel E Fig. 19).

AMS infection with type 1 and type 2 viruses

[00299] The permissivity of CD165-modified pigs to PRRSV was initially assessed by infecting AMC cells in vitro with a panel of six PRRSV type 1 isolates and nine PRRSV type 2 isolates (see Table 14 for a list of viruses). The viruses in the panel represent different genotypes, as well as differences in nucleotide and peptide sequences, pathogenesis, and year of isolation. The data presented in Table 15 shows the results of experiments using AMS from three pigs for each group of the CD163 genotype. The listed viruses correspond to the PRRSV isolates shown in Table 14. The results are presented as the mean +/- standard deviation of the percentage of AMS infected. CD163-null AMCs were generated from pigs expressing the d7(129) allele (see Figures 17 and 19 for CD163 gene constructs and CD163 expression on AMCs, respectively).

[00300] As expected, wild-type AMS were infected with all viruses. In contrast, pigs with a CD163-null phenotype were negative for infection with all viruses. A marked difference was observed in AMC responses from HL11m pigs. None of the type 1 viruses were able to infect AMC HL11m; while all viruses in the type 2 panel infected HL11m AMS, albeit at a much lower percentage compared to wild-type AMS.

[00301] Permissivity was also assessed by comparing virus endpoint titration between WT and HL11m for the same type 2 viruses. Results are shown for two WT and two HL11m pigs (FIG. 21). Log ₁₀ TCID ₅₀ values were calculated based on infection of macrophage cultures with the same virus sample. The infection results represent two different pigs of each genotype. The viruses used for infection are listed in Table 14. The log ₁₀ TCID ₅₀ values for AMC from HL11m pigs were 1-3 log lower compared to wild-type AMC infected with the same virus. The only exception was infection with a modified live virus vaccine strain. Taken together, the results indicate that AMS from HL11m pigs have reduced susceptibility or permissivity to challenge with type 2 viruses.

Infection of CD 163-modified pigs with Type 1 and Type 2 viruses

[00302] WT (circles), HL11m (squares) and CD163-null (triangles) pigs were infected with representative type 1 (SD13-15) (Fig. 22, panel A, left graph) and type 2 (NVSL 97- 7895) (Fig. 22, panel A, right graph). Pigs with a null phenotype are descended from alleles with knockout and d(1567) (see Fig. 17). Pigs of three genotypes inoculated with the same virus were mixed in the same pen, allowing continuous exposure of CD163-modified pigs to viruses isolated from wild-type pen. The number of pigs infected with a representative type 1 virus were: wild type (n=4), HL11m (n=5) and null (n=3); and type 2 virus: wild type (n=4), HL11m (n=4) and null (n=3). As shown in FIG. 22, CD163 null pigs infected with type 1 or 2 virus were viraemic negative at all time points and did not seroconvert. As expected, wild-type pigs were productively infected, having an average level of viremia approaching 10 ⁶ templates per 50 μl PCR reaction 7 days after infection with both viruses. By day 14, all wild-type pigs had seroconverted (see Fig. 22, panel B). According to the results of AMC infection (Table 15), five HL11m pigs infected with Type 1 virus showed no signs of viremia or antibodies to PRRSV. All HL11m pigs infected with the Type 2 isolate, NVSL, maintained the infection and seroconverted (FIG. 22, panel B). The presence of reduced permissivity in HL11m pigs is unclear. The mean viremia for three out of four HL11m pigs was similar to wild-type pigs. However, in one HL11m pig, #101 (open squares in Fig. 22, panel A, right graph), viremia was significantly reduced compared to other pigs in the HL11m genotype group. The explanation for the 3-4-log reduction in viremia in pig #101 was not clear, but suggested that some HL11m pigs may be less susceptible to PRRSV, an observation supporting the results of in vitro AMC infection (Table 15). Since all pigs were infected with the same amount of virus and remained mixed with wild-type pigs, the lower viremia in pig #101 was not the result of receiving less virus or less exposure to the virus. Macrophage flow cytometry showed that CD163 expression in pig #101 was comparable to that of other HL11t pigs (data not shown). No differences were observed in the sequence of the exon 11 mimetic sequence.

[00303] Additional viral challenge studies were conducted using two viruses, NVSL 97-7895 and KS06-72109. The results are shown in FIG. 23. Pigs were observed for 35 days post challenge and data were reported as area under the curve (AUC) for viremia measurements taken at 3, 7, 11, 14, 21, 28 and 35 days post challenge. As shown in FIG. 23, for NVSL, the mean AUC for seven NVSL-infected WT pigs was 168+/-8 versus 165+/-15 for seven HL11m pigs. For KS06, the mean AUCs for six WT pigs and six HL11m pigs were 156+/-9 and 163+/-13, respectively. For both viruses, there was no statistically significant difference between WT and HL11m pigs (p>0.05 ). Overall, the results showed that HL11m pigs could not sustain infection with Type 1 PRRSV but retained the ability to infect with Type 2 viruses. the difference was not reflected in pigs. With respect to the results shown in FIG. 23: Here, the viral load was determined by calculating the area under the curve (AUC) for each pig during the 35 day infection period. Calculation of AUC was performed using log ₁₀ PCR viremia obtained on days 0, 4, 7, 10, 14, 21, 28 and 35 after infection. The horizontal lines show the mean and standard deviation. Key words: wild type = wild type pigs, HL11 = HL11m genotype pigs; Null = CD163-null genotype.

DISCUSSION

[00304] CD163 is a macrophage surface protein important for removing excess hemoglobin from the blood and modulating inflammation in response to tissue injury. It also acts as a viral receptor. CD163 is involved in both pro- and anti-inflammatory responses (Van Gorp et al., 2010). CD163-positive macrophages are classified in the alternatively activated group of M2 macrophages, which are generally described as highly phagocytic and anti-inflammatory. M2 macrophages are involved in cleaning and recovery after tissue mechanical damage or infection (Stein et al., 1992). For anti-inflammatory properties, CD163 expression is upregulated by anti-inflammatory proteins such as IL-10 (Sulahian, et al., 2002). During inflammation, CD163 reduces inflammation by reducing the oxidative process by removing circulating heme from the blood. Heme degradation products such as biliverdin, bilirubin, and carbon monoxide are powerful anti-inflammatory molecules (Soares and Bach, 2009 and Jeney et al., 2002). With regard to pro-inflammatory effects, cross-linking of CD163 on the surface of macrophages by an anti-CD163 antibody or bacteria results in localized release of pro-inflammatory cytokines, including IL-6, GM-CSF, TNFα, and IL-1β (Van den Heuvel et al., 1999 and Fabriek et al. , 2009).

[00305] GE pigs lacking CD163 cannot support replication of the Type 2 PRRSV isolate (Whitworth et al., 2016). In this study, in vitro infection studies demonstrate resistance of CD163 phenotype-null macrophages to a broad panel of Type 1 and Type 2 PRRSV isolates, further increasing resistance and potentially including all PRRSV isolates (Table 15). Resistance of macrophages CD163-null phenotype to Type 1 and Type 2 viruses was confirmed in vivo (Fig. 22 and Fig. 23). Based on these results, the contribution of other PRRSV receptors previously described in the literature can be excluded (Zhang and Yoo, 2015). For example, Shanmukhappa et al. (2007) showed that non-permissive BHK cells transfected with the CD151 plasmid acquired the ability to maintain PRRSV replication, and incubation with a polyclonal anti-CD151 antibody was shown to significantly reduce infection of MARC-145 cells. In addition, a monkey cell line, SJPL, originally developed for use in propagating swine influenza viruses, has previously been shown to support PRRSV replication (Provost, et al., 2012). Important properties of the SJPL cell line included the presence of CD151 and the absence of sialoadhesin and CD163. Taken together, these data provided strong evidence that CD151 alone is sufficient to support PRRSV replication. The results of this study showing the absence of PRRSV infection in macrophages and CD163 phenotype null pigs indicate that CD151 as an alternative receptor for PRRSV is not biologically relevant.

[00306] Viral GP2a and GP4 proteins that form part of the GP2a, GP3, GP4 heterotrimer complex on the surface of PRRSV can be coprecipitated with CD 163 in affinity adsorption assays from cells transfected with GP2 and GP4 plasmids (Das, et al., 2009). Presumably, GP2 and GP4 form an interaction with one or more CD 163 SRCR domains. In vitro infectivity assays involving a porcine CD163 cDNA backbone containing a domain exchange between porcine SRCR 5 and a homologue from hCD163-Ll SRCR 8 further localized a region in SRCR 5 used by Type 1 viruses (Van Gorp, et al., 2010). It is interesting to suggest that a stable interaction between GP2/GP4 and CD163 occurs through SRCR 5. Additional viral glycoproteins such as GP3 and GP5 may further stabilize the virus-receptor complex or may act as co-receptor molecules. The requirement for SRCR 5 was explored in this study by infecting macrophages and pigs bearing the HL11m allele, which recreated the CD163L1 SRCR 8 domain exchange by replacing the 33 bp in exon 7 of the pig. The HL11m allele also included a neomycin cassette to select cells positive for the genetic change (FIG. 17). HL11m pigs expressed CD163 on AMC, although to a lesser extent than wild-type AMC (Figure 19, compare panels A and E). The decrease in expression was probably due to the presence of the neomycin cassette, which was located between the exon 11 mimetic and the next intron. HL11m pigs were not permissive for Type 1 virus infection, confirming the importance of SRCR 5. However, HL11m macrophages and HL11m pigs did support infection with Type 2 viruses. virus compared to wild-type macrophages (Table 15 and Fig. 17). Decreased permissivity may be due to reduced levels of CD163 on HL11m macrophages coupled with reduced affinity of the virus for the modified CD 163 protein. Suggesting that Type 2 viruses require SRCR 5, and that L1 SRCR 8 may function as a suitable substitute, lower affinity can be explained by the difference in peptide sequences between human SRCR 8 and porcine SRCR 5 (see Fig. 18, panel B). However, the reduced permissivity of AMS was not translated into pigs. The mean viremia for HL11t pigs was not significantly different compared to WT pigs (FIG. 23). In addition to AMS, PRRSV infection of intravascular, septal, and lymph node macrophages contributes to viremia (Lawson et al., 1997 and Morgan et al., 2014). The potential contribution of these and other CD163-positive cell populations to the maintenance of total viral load in HL11m pigs deserves further study.

[00307] Although CD163 plasmids with deletions of SRCR domains are stably expressed in HEK cells (Van Gorp et al. surface expression of CD 163 (Fig. 19, panel D) Because mAB 2A10 used for flow cytometry recognizes three N-terminal domains of SRCR (Van Gorp et al., 2010) and possibly the 7th and 8th domains (Sanchez, et al., 1999), the lack of detection was not due to deletion of the 2A10 epitope in mutated proteins.While a small amount of CD163 expression could be detected on AMS from some d7(129) pigs (see Fig. 19, panel C), the amount of protein expressed was not sufficient to support PRRSV infection in AMS or in pigs The absence of CD163 expression in exon 7 and 8 deletion mutants is not fully understood, but is probably the result of mRNA and/or protein degradation.

[00308] In 2003, CD163 was identified as the receptor for African swine fever virus (ASFV; Sanchez-Torres et al., 2003). This conclusion was based on the observation that infected macrophages have a mature CD163-positive phenotype and anti-CD163 antibodies such as 2A10 block macrophage infection with ASFV in vitro. Whether CD163 null pigs are resistant to ASFV infection remains to be determined.

[00309] Cell culture models incorporating PRRSV receptor modifications have provided valuable insight into the mechanisms of PRRSV entry, replication and pathogenesis. One of the unique aspects of this study was to conduct a parallel in vivo experiment in pigs with modified receptors. This research is essential to the possibility of developing preventive treatments for one of the most serious diseases the global swine industry has ever faced.

Example 4 Maternal CD163 knockout protects fetuses from porcine reproductive and respiratory syndrome virus (PRRSV) infection.

[00310] Examples 1 to 3 above demonstrate that CD163 complete knockout (KO) pigs lack CD163 expression on macrophages and are unable to sustain PRRSV infection (see also Whitworth et al., 2016; Wells et al., 2017). Because CD163 expression is a dominant trait and is inherited in the classical Mendelian fashion, offspring with normal CD163 expression and function can be obtained by crossing a CD163 ^-/- knockout female pig with a wild-type CD163 ^+/+ male. For this study, young CD163 knockout pigs were crossed with wild-type boars to produce heterozygous CD163 ^+/- fetuses to determine whether the presence of the CD163 knockout genotype in the mother would be sufficient to protect fetuses after maternal infection with PRRSV. In this study, in CD163 receptor complete knockout sows, CD163-positive fetuses produced between 109 days of gestation or 20 days post maternal infection were completely protected from PRRSV. The results demonstrate a practical means of eliminating the reproductive disease associated with PRRSV, which is a major source of economic hardship for agriculture.

Materials and methods

[00311] Editing the CD163 gene. The CRISPR/Cas9 methods used to generate all knockout alleles are detailed in Examples 1-3 above. The experiments described in this example used wild-type and knockout or heterozygous animals obtained as described in Examples 1-3 and having the alleles described in Table 16. Each of these alleles is described in Examples 1-3 and in PCT Publication No. WO 2017/023570, which is incorporated herein by reference in its entirety. Specific changes for alleles B, D, and E are also described in Whitworth et al., 2014, and specific changes in allele C (2 bp inset) are described in Whitworth et al., 2016. All alleles described in Table 16 are were identified based on DNA sequencing. The knockout genotype was confirmed by the absence of CD163 expression, which was measured by staining alveolar macrophages with anti-CD163 monoclonal antibodies, 2A10, as described above in Example 2.

[00312] PRRSV infection. The PRRSV strain used in this study, NVSL 97-7895 (NVSL), is a laboratory strain isolated in 1997 from a herd in Southeast Iowa, USA, that was experiencing a PRRSV abortion storm (Halbur et al., 1997) . The virus, maintained as a low passage number isolate, was propagated and titrated on MARC-145 cells. On days 89-91 of gestation, young pigs were inoculated with 10 ⁵ TCID ₅₀ virus diluted in 5 ml culture medium. Half of the inoculum was administered intramuscularly and the remainder was administered intranasally. All young pigs were kept in an environment that allowed continuous exposure to the virus shed by infected pens. Blood samples were taken from young pigs before infection, seven days after inoculation (dpi) and at the time of euthanasia. PRRSV nucleic acid was measured by isolation of total RNA from serum followed by PRRSV real-time reverse transcriptase PCR (Tetracore, Rockville, MD). A standard curve was generated using the quantitation standards supplied with the RT-PCR kit. The results are reported as log ₁₀ matrices per 25 µl of reaction, which is approximately the number of viral RNA matrices per ml of blood.

RESULTS

[00313] A detailed description of the knockout alleles used in this study is shown in Table 16 above. Each knockout allele had a mutation in exon 7 that was predicted to result in a codon frameshift followed by a premature stop codon in the mRNA. Matings between wild-type and CD163 knockout parents are summarized in Table 17. The first group of three sows that served as positive infection controls were CD163 ^+/+ females bearing CD163 ^+/+ fetuses (group++/++). The second group (- -/+-) consisted of CD163 ^-/- sows bearing CD163 ^+/- fetuses. In this group, CD163 ^-/- sows were unable to maintain PRRS replication, while CD163 ^+/- fetuses remain susceptible to PRRS infection. And finally, the third group (- -/- -) consisted of CD163 ^-/- sows bearing CD 163"" fetuses. In the last group, both sows and fetuses had to be resistant to infection.

[00314] Clinical signs in infected wild-type sows included lethargy and temporary anorexia. Knockout sows had no clinical signs. During the study period, one wild type sow, #139, aborted on day 106 (15 dpi). PRRSV nucleic acid measured 7 days post-inoculation showed a viremia level for Sow #139 of 5.5 log ₁₀ templates per reaction, indicating a productive PRRSV infection. Between 15 and 20 days post-inoculation, all remaining sows were euthanized and the uterine horns removed immediately. Starting at the tip of each horn, fetuses and placentas were removed and assessed for anatomical pathology. A blood sample was taken from each fetus. If blood could not be obtained, a fluid sample was taken from the abdomen. The number of fetuses recovered from each sow is shown in Table 17. For wild-type CD163 (Group++/++) (including the aborted sow), the number of fetuses was 16, 14 and 12 (mean = 14.0). CD163 knockout sows bearing CD163 ^+/- fetuses (group - -/+ -) produced 14, 17 and 11 fetuses (mean = 13.6). For CD163 knockout females bearing CD163 knockout fetuses (group - -/- -), the number of fetuses was 7 and 9. Results for fetal viremia and macropathology are presented in FIG. 24 and in Table 18.

[00315] FIG. 24 depicts each fetal outcome after maternal infection with PRRSV. The numbers on the left identify each sow. Below each sow in brackets is the result of serum PRRS PCR measured as log ₁₀ templates per reaction. "N" indicates a negative PRRSV nucleic acid result (Ct>39). Fruits are identified by number and relative position in each uterine horn. Asterisks denote fetal PCR samples obtained from abdominal fluid. The number below each fetus is the fetal serum PRRS PCR result (log ₁₀ matrices per reaction). The number in each circle indicates the presence of anatomical pathology: 1) normal fetus; 2) a small fetus; 3) placental changes such as placental abruption and/or necrosis; 4) a fetus stained with meconium; 5) the fetus is dead and necrotic. Lowercase letters indicate the genotype of individual fetuses (see Table 17). Designations: a, A/A; b, C/A; s, V/A; d, E/A; e, B/C; f, B/D; g, D/C; h, D/D; i, E/C; j, E/D; ND, not determined because the fetus is necrotic; nd, the genotype was not determined.

[00316] At the anatomical level, 50% and 72% of fetuses from two CD163 wild-type (++/++) sows, #138 and #140, had some degree of pathology, including smaller than normal fetuses ( 11% of all fetuses), fetuses with detached or necrotic placenta (14%), meconium stained (7%), and dead and necrotic fetuses (25%). Pathological observations are typical of reproductive PRRS. The same litters showed high levels of PRRSV infection, with 92% of fetuses testing positive for PRRSV nucleic acid. PCR results for fetuses from wild-type sows illustrate two important features of PRRSV fetal infection. First, there was a large difference in the concentration of virus found in the blood serum between fetuses, as a result of infection of the fetuses at different times. Secondly, the level of viremia did not always correlate with pathology. For example, fetus #5 from sow #138 had a high level of viremia (7.3 log ₁₀ matrices per reaction) but the fetus was still unaffected. The reason for the discrepancy between viremia and pathology is unclear. One possibility is that the fetal pathology is the result of maternal tissue damage and is not related to the level of fetal viremia. In the field, these normal but infected newborn piglets can act as "superspreaders", allowing PRRSV to spread rapidly throughout the production system. For the --/+- group (sows #84, 87 and 122) all fetuses looked normal, with the minor exception of two fetuses that were smaller than the other littermates. The smaller than normal size is likely due to crowding in the uterine horn, which reduces the surface area of the placenta, thereby limiting the growth of the developing fetus. All mothers and fetuses in the -/+- group were negative for the presence of PRRSV nucleic acid. In the last group - -/- - there was no visible pathology and all sows (#86 and 121) and fetuses were negative for PRRSV nucleic acid.

[00317] The results of this study clearly demonstrate that the absence of CD163 in the sow is sufficient to protect a fetus susceptible to PRRSV. Although CD163-positive offspring from CD163 knockout sows are susceptible to the virus immediately after birth, protection against PRRSV in utero provides a means to eliminate a major source of economic loss and animal suffering.

[00318] The examples disclosed herein are provided by way of illustration and are not intended to limit the scope of the invention.

[00319] In view of the foregoing, it will be seen that several objectives of the invention are achieved and other useful results are achieved.

[00320] Since various changes can be made to the above products and methods without deviating from the scope of the invention, it is intended that all materials contained in the above description and shown in the accompanying drawing(s) should be interpreted as illustrative and not in restrictive sense.

LINKS

Allende R, et al., 1999. North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions. J. Gen. Virol. 80: 307 315. Bauer BK, et al., Arginine supplementation in vitro increases porcine embryo development and affects mRN A transcript expression. Reprod Fertil Dev 2011; 23:107.

Beaton BP, et al., Compound transgenics: recombinase-mediated gene stacking. In: Pinkert CA (ed.). Transgenic Animal Technology: A Laboratory Handbook, 3rd ed. Waltham, MA: Elsevier; 2014:565-578.

Benfield DA, et al., 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus (Isolate ATCC VR-2332). J Vet Diag Invest 4:127-133.

Boddicker, et al., 2014. Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection. Genetics, selection, evolution: GSE46, 18.

Borg NA, et al., CD1d-1ipid- antigen recognition by the semi-invariant NKT T-cell receptor. Nature 2007; 448:44^19.

Brinster RL, et al., Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci U S A 1985; 82:4438-1442.

Cafruny, WA, et al, Trojan Horse macrophages: studies with the murine lactate dehydrogenase-elevating virus and implications for sexually transmitted virus infection. J. Gen. Virol. 77(12): 3005-3012(1996).

Calvert JG, et al., 2007. CD163 expression confers susceptibility to porcine reproductive and respiratory viruses syndrome. J Virol. 81:7371-7379.

Carter DB, et al., Phenotyping of transgenic cloned piglets. Cloning Stem Cells 2002; 4:131-145. Cong L, et al., Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339:819 823.

Christianson, W. T. et al. Experimental reproduction of swine infertility and respiratory syndrome in pregnant sows. American Journal of Veterinary Research 53, 485-488 (1993). Christopher-Hennings, et al., Effects of a modified-live virus vaccine against porcine reproductive and respiratory syndrome in boars, American J. Veterinary Research 58(1), 40 45 (1997). Dai Y, et al., Targeted disruption of the alphal,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002; 20:251-255.

Das PB, et al., 2009. The minor envelope glycoproteins GP2a and GP4 of porcine reproductive and respiratory syndrome virus interact with the receptor CD 163. J Virol. 84:1731-40. Dee et al., Elimination of porcine reproductive and respiratory syndrome virus using a test and removal process, The Veterinary Record 143, 474-476 (1998).

Delputte PL, et al., 2002. Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparin-like receptor on porcine alveolar macrophages. J Virol. 76:4312-4320.

Etzerodt, A., et al., 2013. Plasma clearance of hemoglobin and haptoglobin in mice and effect of CD163 gene targeting disruption. Antioxidants & redox signaling 18, 2254-2263. Etzerodt, A., et al., 2013. CD163 and inflammation: biological, diagnostic, and therapeutic aspects. Antioxidants & redox signaling 18, 2352-2363.

Fabriek BO, et al., 2005. The macrophage scavenger receptor CD163. Immunobiology. 210:153-160.

Fabriek BO, et al., 2009. The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria. Blood. 113:887-892.

Gaj T, et al., ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013; 31:397^105.

Gaudreault N, et al., 2009. Factors affecting the permissiveness of porcine alveolar macrophages for porcine reproductive and respiratory syndrome virus. Archive Virol. 154:133-136. Graversen, J.H., et al., 2012. Targeting the hemoglobin scavenger receptor CD163 in macrophages highly increases the anti-inflammatory potency of dexamethasone. Molecular therapy: the journal of the American Society of Gene Therapy 20, 1550-1558.

Groenen, M.A., et al., 2012. Analyzes of pig genomes provide insight into porcine demography and evolution. Nature 491, 393-398.

Hai T, et al., One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 2014; 24:372-375.

Halbur, P.G., et al., 1995. Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Veterinary Pathology 32, 648-660.

Halbur, P. & Bush, E. B. Update on abortion storms and sow mortality. Journal of Swine Health and Production 5, 73 (1997).

Hammer RE, et al., Production of transgenic rabbits, sheep and pigs by microinjection. Nature 1985;315:680-683.

Hauschild J, et al., Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci USA 2011; 108:12013-12017.

Holfkamp, D.J., et al., 2013. Assessment of the economic impact of porcine reproductive and respiratory syndrome virus on United States pork producers. Journal of Swine Health and Production 21, 72-84.

Hwang WY, et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 2013; 31:227-229.

Jeney V, et al., 2002. Pro-oxidant and cytotoxic effects of circulating heme. Blood. 100:879-887. Keffaber, K.K., 1989. Reproductive failure of unknown etiology. American Association of Swine Practitioners Newsletter 1, 1-10.

Key, K. F. et al. Genetic variation and phylogenetic analyzes of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates. Veterinary Microbiology 83, 249-263 (2001).

Kim, H.S., et al., 1993. Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch Virol 133, 477-483. Kristiansen M, et al., 2001. Identification of the haemoglobin scavenger receptor. Nature. 409:198-201.

Kwon DN, et al., Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs. Sci Rep 2013; 3:1981.

Ladinig, A., et al., 2015. Pathogenicity of three type 2 porcine reproductive and respiratory syndrome virus strains in experimentally inoculated pregnant gilts. Virus Res 203, 24-35. Lai L, et al., Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295:1089-1092.

Lai L, et al., Creating genetically modified pigs by using nuclear transfer. Reprod Biol Endocrinol 2003; 1:82.

Lai L, et al., Production of cloned pigs by using somatic cells as donors. Cloning Stem Cells 2003; 5:233-241.

Lawson S.R., et al., 1997. Porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with MAC-387 staining at 21 days post-infection. VirusRes. 51:105-113.

Lee K, et al., Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency. Proc Natl Acad Sci USA 2014; 111:7260-7265.

Lee K, et al., Piglets produced from cloned blastocysts cultured in vitro with GM-CSF. Mol Reprod Dev2013; 80:145-154.

Li D, et al., Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol 2013; 31:681 683.

Lillico SG, et al., Live pigs produced from genome edited zygotes. Sci Rep 2013; 3:2847. Machaty Z, et al., Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal. Biol Reprod 1997; 57:1123 1127.

Madsen M, et al., 2004. Molecular characterization of the haptoglobin-hemoglobin receptor CD 163. Ligand binding properties of the scavenger receptor cysteine-rich domain region. J Biol Chem. 279:51561-51567.

Mengeling, W. L. et al. Clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus isolated from field cases of "atypical" PRRS. American Journal of Veterinary Research. 59, 1540-1544 (1998).

Merck, The Merck Veterinary Manual.

http://www.merckmanuals.com/vet/appendixes/reference guides/normal rectal temperature ranges.html.

Miao, Y.L., et al., 2009. Centrosome abnormalities during porcine oocyte aging. Environmental and molecular mutagenesis 50, 666-671.

Morgan SB, et al., 2014. Pathology and Virus Distribution in the Lung and Lymphoid Tissues of Pigs Experimentally Inoculated with Three Distinct Type 1 PRRS Virus Isolates of Varying Pathogenicity. Transboundary Emerge Dis. Nov. 10. pp. 1-11.

Nelsen CJ, et al., 1999. Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents. J. Virol. 73, 270-280.

Neumann EJ, et al., Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J Am Vet Med Assoc 2005; 227:385-392. Niu Y, et al., Generation of gene-modified Cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156:836-843.

Patience, J.F., et al., 1989. Swine Nutrition Guide, in: Center, P.S. (Ed.), University of Saskatchewan, Saskatoon, CA, pp.149-171.

Patton JP, et al., 2008. Modulation of CD 163 receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages. VirusRes. 140:161-171. Plagemann, P. G. W. Lactate dehydrogenase-elevating virus and related viruses. In Fields Virology, 3rd Ed, Fields, B. ed. Lippincott-Raven, Philadelphia, pp 1105-1120 (1996). Prather RS, et al., An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/ internalization of the porcine reproductive and respiratory syndrome virus. J Virol 2013; 87:9538 9546.

Prather, R.S., et al., 2013. An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus. J Virol 87, 9538-9546.

Provost C, et al., 2012. Identification of a new cell line permissive to porcine reproductive and respiratory syndrome virus infection and replication which is phenotypically distinct from M ARC-145 cell line. Virol J. 9:267.

Reed JL, et al., 1938. A simple method of estimating fifty percent endpoints. The American Journal of Hygiene 27:493-197.

Ross JW, et al., Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts. Transgenic Res 2010; 19:611-620.

Rowland, R.R.R., et al., The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332, Virology 259, 262-266 (1999).

Rowland, R.R.R. et al. Lymphotropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero. Veterinary Microbiology 96, 219-235 (2003).

Rowland, R.R.R. The interaction between PRRSV and the late gestation pig fetus. Virus Research 154, 114-122 (2010).

Rowland, R.R., et al., 2012. Control of porcine reproductive and respiratory syndrome (PRRS) through genetic improvements in disease resistance and tolerance. Frontiers in genetics 3, 260. Sanchez C, et al., 1999. The porcine 2A10 antigen is homologous to human CD163 and related to macrophage differentiation. Journal of Immunology 162:5230-5237.

Sanchez-Torres C, et al., 2003. Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection. Arch Virol. 148:2307-2323. Schaer, C.A., et al., 2006a. Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin. Circulation research 99, 943-950.

Schaer, D.J., et al., 2006b. CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin. Blood 107, 373-380.

Schaer, D.J., et al., 2006c. Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis. European journal of haematology 77, 432-436.

Shanmukhappa, K, et al., 2007. Role of CD151, A tetraspanin, in porcine reproductive and respiratory syndrome virus infection. Virol J. 4:62.

Shimozawa N, et al., Abnormalities in cloned mice are not transmitted to the progeny. Genesis 2002; 34:203-207.

Shi, M. et al. Phylogeny-based evolutionary, demographical, and geographical dissection of North American type 2 porcine reproductive and respiratory syndrome viruses. Journal of Virology 84, 8700-8711 (2010).

Smit, AFA, et al., RepeatMasker Open-3.0. 1996-2010. Current Version: open-4.0.5 (RMLib: 20140131 and Dfam: 1.2). http://www.repeatmasker.org&gt. CD163: accessed July 25, 2014; CDID: accessed August 27, 2013.

Soares MP, et al., 2009. Heme oxygenase-1: from biology to therapeutic potential. Trends Mol Med. 15:50-58.

Stein M, et al., 1992. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunological macrophage activation. J Exp Med. 176:287-292. Stephenson R, et al., 2015. Multiplex serology for common viral infections in feral pigs in Hawaii between 2007 and 2010. Accepted for publication. J Wildlife Dis 51:239-2343. Sulahian TH1, et al., 2002. 2002. Human monocytes express CD163, which is upregulated by IL-10 and identical to p155. Cytokine. 2000 12:1312-1321.

Terns MP, et al., CRISPR-based adaptive immune systems. Curr Opin Microbiol 2011; 14:321 327.

Tian, K. et al. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in

China and molecular dissection of the unique hallmark. PLOS One. 2, e526 (2007).

Trible, B.R., et al., 2012. Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2. J Virol 86, 13508-13514.

U.S. Department of Agriculture and U.S. Department of Health and Human Services. Dietary Guidelines for Americans, 2010. 7th Edition,

Van Breedam W, et al., Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. J Gen Virol 2010; 91:1659 1667.

Van Breedam, W., et al., 2010. Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. J Gen Virol 91, 1659-1667.

Van den Heuvel MM, et al., 1999. Regulation of CD 163 on human macrophages: cross-linking of CD163 induces signaling and activation. J Leukoc Biol. 66:858-866.

van den Hoff MJ, et al., Electroporation in 'intracellular' buffer increases cell survival. Nucleic Acids Res 1992; 20:2902.

Van Gorp, H., et al., 2010. Scavenger receptor CD163, a Jack-of-all-trades and potential target for cell-directed therapy. Mol Immunol 47, 1650-1660.

Van Gorp H, et al., 2010. Identification of the CD 163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus. J Virol. 84:3101-3105.

Walters EM, et al., Advancing swine models for human health and diseases. Mo Med 2013; 110:212-215.

Wang H, et al., One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013; 153:910-918.

Welch SK, et al., 2010. A brief review of CD163 and its role in PRRSV infection. VirusRes. 154:98-103.

Wells, K.D., et al., 2014. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod 91, 78.

Wells, K.D. et al. Substitution of porcine CD163 SRCR domain 5 with a CD163-like homolog confers resistance of pigs to genotype1 but not genotype 2 porcine reproductive and respiratory syndrome (PRRS) viruses. Journal of Virology In press (2017).

Wensvoort, G., et al., 1991. Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Veterinary Quarterly 13, 121-130.

Whitworth KM, et al., 2016. CD163 facilitates both entry and replication of porcine reproductive and respiratory syndrome virus. nature biotech. 34:20-22.

Whitworth KM, et al., Method of oocyte activation affects cloning efficiency in pigs. Mol Reprod Dev 2009;76:490-500.

Whitworth et al. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos, Biology of Reproduction 91(3): 1 13 (2014). Whitworth KM, et al., Activation method does not alter abnormal placental gene expression and development in cloned pigs. Mol Reprod Dev 2010; 77:1016-1030.

Whitworth KM, et al., Scriptaid corrects gene expression of a few aberrantly reprogrammed transcripts in nuclear transfer pig blastocyst stage embryos. Cell Reprogram 2011; 13:191-204. Whitworth, K.M., et al., 2014. Use ofthe CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod 91, 78.

Whyte JJ, et al., Genetic modifications of pigs for medicine and agriculture. Mol Reprod Dev 2011;78:879-891.

Whyte JJ, et al., Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78:2.

Wiedenheft B, et al., RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012; 482:331-338.

Wilkinson et al. Genome-wide analysis of the transcriptional response to porcine reproductive and respiratory syndrome virus infection at the maternal/fetal interface and in the fetus. BMC Genomics 17, 383 (2016).

Whitworth, K.M. et al. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nature Biotechnology 34, 20-22 (2016).

Winckler, C, et al., 2001. The reliability and repeatability of a lameness scoring system for use as an indicator of welfare in dairy cattle. Acta Agricultura Scandinavica Section A. Animal Science, 103-107.

Yang D, et al., Generation of PPARgamma mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning. Cell Res 2011; 21:979-982.

Yoshioka K, et al., Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 2002; 66:112-119.

Zhang Q, et al., 2015. PRRS virus receptors and their role for pathogenesis. Vet Microbiol. 177:229-241.

Zhao J, et al., Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell Reprogram 2010; 12:75 83. Zhao J, et al., Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biol Reprod 2009; 81:525 530.

--->

SEQUENCE LIST

<110> Curators of the University of Missouri

Prather, Randall

Wells, Kevin

Whitworth, Christine

<120> WAYS TO PROTECT PIGS FRUIT FROM INFECTION WITH THE REPRODUCTIVE VIRUS

AND PIG RESPIRATORY SYNDROME (PRRSV)

<130> UMO 18054.WO

<160> 121

<170> Patent Version 3.5

<210> 1

<211> 23

<212> DNA

<213> Sus scrofa

<400> 1

ggaaacccag gctggttgga ggg 23

<210> 2

<211> 23

<212> DNA

<213> Sus scrofa

<400> 2

ggaactacag tgcggcactg tgg 23

<210> 3

<211> 23

<212> DNA

<213> Sus scrofa

<400> 3

cagtagcacc ccgccctgac ggg 23

<210> 4

<211> 23

<212> DNA

<213> Sus scrofa

<400> 4

tgtagccaca gcagggacgt cgg 23

<210> 5

<211> 23

<212> DNA

<213> Sus scrofa

<400> 5

ccagcctcgc ccagcgacat ggg 23

<210> 6

<211> 23

<212> DNA

<213> Sus scrofa

<400> 6

ctttcattta tctgaactca ggg 23

<210> 7

<211> 23

<212> DNA

<213> Sus scrofa

<400> 7

ttatctgaac tcagggtccc cgg 23

<210> 8

<211> 23

<212> DNA

<213> Sus scrofa

<400> 8

cagctgcagc atatatttaa ggg 23

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 9

ctcctcgccc ttgctcacca tgg 23

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 10

gaccaggatg ggcaccaccc cgg 23

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 11

ctctccctca ctctaaccta ctt 23

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 12

tatttctctc acatggccag tc 22

<210> 13

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 13

ctctccctca ctctaaccta ctt 23

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 14

gactggccat gtgagagaaa ta 22

<210> 15

<211> 27767

<212> DNA

<213> Sus scrofa

<400> 15

atacaagtgc cttttacaga caatctgcac aagttatttg ttagacatat ttgattatag 60

aattaatatt aaaaggggtt ataacaatca agcattgata

tttactttag ttttttgaca taactgtgta actattgcga tttttttatt cctaatgtaa 180

ttagttcaaa acaaagtgca gaaatttaaa atattcaatt caacaacagt atataagtca 240

atattccccc cttaaattttt tacaaatctt tagggagtgt ttctcaattt ctcaatttct 300

ttggttgttt catgtcccat atggaagaaa acatgggtgt gaaagggaag cttactcttt 360

tgattacttc ccttttctgg ttgactccac ctccattatg aagcctttct gtatttttgt 420

ggaagtgaaa tgatttttag aattcttagt ggttctcttc ttcaggagaa catttctagg 480

taataataca agaagattta aatggcataa aaccttggaa tggacaaact cagaatggtg 540

ctacatgaaa actctggatc tgcaggtaaa atcttctcat ttattctata tttacctttt 600

aaatagagtg tagcaatatt ccgacagtca atcaatctga tttaatagtg attggcatct 660

ggagaagaag taacagggaa aaaggcaata agctttataa ggggaacttt tatcttccat 720

agactcaaaa ttgaagacgt gactagaaga ttgctagatt tggcatcagt tttgtaaaat 780

tgctgaggtg aaattaagta agggatgaaa attaactaaa ttgtgttgag tatgaaacta 840

gtagttgtta gaaaagatag aacatgaagg aatgaatatt gattgaaagt tgatgaccta 900

gaggacattt agactaacac ctctgagtgt caaagtctaa tttatgattt acatcgatgc 960

1020

tttcacatga aagcctggat tataaatgca cagttcagtg acctatctca gaggagtgac 1080

tgccatagca ttttttttgt ctttttgcct tcagagccac agcaacgcgg gatccgaagc 1140

cgcgtctgcg acccacacca cagctcacgg caatgccgga tctttaaccc actgagcgag 1200

gccggggatc gaacccgcag tctcatggtt cctagtagga ttcgttaacc actgcgccac 1260

gacgggaact cctaccatag catttttact tttaagttac tgttggttta gagtaagaag 1320

1380

ctcaaatata aattttgtcg ctttttcctc caatgtattg ttcaactact atttagcagg 1440

ccactgtgcc aggtactggt gaaactggtg aacatgatag atgtaattca ttccctcatg 1500

gaactttcca tctaacaatg tggatcaggt aggcttggag atgagaatgc cagtggttga 1560

ctatgactct gtggctgaag ggagagctac tcacttcgta gtttcatcaa tgtctttttg 1620

1680

tttctcaccc ctaggatgcc tataaacatg agtattttca aagctacttc actgaggtta 1740

1800

aatttccatt gtggcttggt ggttaaagac cctgcattgt ctctgtgagg atgtgggttc 1860

aatctctggc ctcattcagt gagtgggtta aggatctggt gtcgctgcaa gctgtggcta 1920

agatcccaca ttgccatgtc tgtggtgtag actggcacct ggagctctga tttgaccaca 1980

2040

cttgtttgga ccttcgttaa tctcaaacct ttggaaccat ctctcctcca aaacctcctt 2100

2160

ccatttaagt tccttcactt tggctgtagt cgctgttctc agtgcctgct tggtcactag 2220

ttctcttggt gagtactttg acaaatttac ttgtaaccga gcccaactgt gacaagaaac 2280

actgaaaagc aaataattgc tcctgaagtc tagatagcat ctaaaaacat gcttcatggt 2340

ttcaagggatc atatattgaa accccaggga tcctctagag tcgacctgca gcatgcaggg 2400

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 2460

gggggggggg gggggggggg gggggggggg gggggggggg gtgcataagg aaagactatc 2520

tcaacgtctt attcctcagc ttacattaga tttgaaactc tagtcaccta aaatgcaaat 2580

ctcatttact taccatcaga gatattaatg acctatagaa ttcagcataa ataaagtttc 2640

atgtatggat attagcttat ggttctagtc actgctaatt gaaacctgtg atattgctgt 2700

ttgttttgac tcctatgaaa taacattctc ccattgtacc atggatgggt ccagaaacat 2760

ttctcaaatc ctggcttgaa aaaataaata agtaatctaa agaataataa ttctctactt 2820

gctctttgaa tcttgaccaa ttgctgcatt tacctattgt tacaggagga aaagacaagg 2880

agctgaggct aacgggtggt gaaaacaagt gctctggaag agtggaggtg aaagtgcagg 2940

aggagtgggg aactgtgtgt aataatggct gggacatgga tgtggtctct gttgtttgta 3000

ggcagctggg atgtccaact gctatcaaag ccactggatg ggctaatttt agtgcaggtt 3060

ctggacgcat ttggatggat catgtttctt gtcgagggaa tgagtcagct ctctgggact 3120

gcaaacatga tggatgggga aagcataact gtactcacca acaggatgct ggagtaacct 3180

gctcaggtaa gacatacaca aataagtcaa gcctatacat gaaatgcttt gtgggaaaaa 3240

atgtatagat gagttaaaaa caaaaaggaa ccagttttct ataagtcatc tagtccatgt 3300

ataaaattac ccaatccatt actaaaagac cacttctggt attttacaca tgacaaagcc 3360

catattaaaa aaaaaaaatt cagaagagat tctgaatgct ataataaatg agcaagtgac 3420

tagcttcaat tttatattag gtcattctac cttctacttc tacatgaaaa tatcataatg 3480

tctaagttaa ttccttgtcc cctttcccaa taaagcactg ctttcatgca ctggcctatg 3540

3600

cttagcactg atcctttttt gttgttgttg gaggaagaat gttttgcaaa gtagaattgc 3660

3720

tttcataagt ttcacctcat atgcccttac aataactcag gaagttatat gttagacctt 3780

3840

tctctttgct catgtcattg acctttatgc agccctctta cctcccacct ttctattaca 3900

gaccatctcc tccataggac tggtgttaga aagtactaat ctctacccag gcattgtggt 3960

gcaatgtggg cagcacaggc tggtatctag aaaaatgctg aagtgaattc cagctcagct 4020

gctcgttaat actatcgttt taagtaagct gttcaatcct ttgaaattca ctttctgagc 4080

actcagtgat ataataaatg tagagctact ggtacactgt ctggtatgta ataggtgtta 4140

ccaattaacc ttagtttcct catgggtcac tggttctcat tacctagaca actcatttct 4200

ctttcttcct ctttctcttt ctccattctc ctcctccttc ttcctcttct tcttgtctgt 4260

tattgttata tcattttgct gagaaagtta agaaataaca actctaacct ctacatcgac 4320

cacctagagc aaagttaaaa ataataataa accttgccag actcttacta taattgttgc 4380

tgtctataga gttgactgtt taagttaaga catcagtata tatttttaat ttttgtgttt 4440

tttttttcat acttttacat gaggatcctt tatataagga tgagttaaac aaacttgatt 4500

tttgaagttt atacccctga ggctcaactg cataataata gaaagggatc catagcctct 4560

caaggactta actagtttca tgagttttca gaatctgaat ttctgagatt ctccacccca 4620

attaaagctc aagcctcaga acatatatcc ttctcttggt aaattctatt cttatcacat 4680

gcgtaataat aaaaaagaga gatgttggag acagattttt ttcctcacat tctgtctcta 4740

ctgttttcta ggtgtttgat tctgtgttat ttaacctcag tttgcttatc tgtgaagtag 4800

ggattatggt aataacatat aatgctttat gttgtaaaga ctaaagaaga tagcatatgt 4860

aacacatttg gaacagggaa tgcatattttt gattgtgagc tcttattatt attaccattc 4920

agccctaata aaaatcttgg taagtggaag gctttggatt tcagaacttt taaaatctaa 4980

ttactttttc aaaaaagaac ttcttagggt tttttttttt taaccacaaa gtgtttctat 5040

tttttaggtg tcccaaaatt tcgttccaaa tatctttttc tcagatattt tagtcctcat 5100

agaacaccta gggatagtgg atagagaaaa ttttctttat taaaaagctg ttctttgcta 5160

5220

5280

tgcaaactat tatgcatact tatatatttt tacttttttc ctgtctttta acttccaaat 5340

tcaacttcag acaattattc atgcactaaa ctgtttgtag taagaaagat taaaattaaa 5400

aaattaacca ttcaacaaat gactggtttg ccatttttac tactttgttg tatgaacaat 5460

ttttttttct acaaatgaat actttgagtc tgatttatcc attcctacat aaaagtttt 5520

actatatctt agtattggaa ggaaacaaaa caaaacacaa tgtaaatttt aatctataaa 5580

ttttgggggg gtaaatatac atagatgaaa gtcttaacca ttaattagag tcaaaagatt 5640

aaaattctcc aatatgtgaa cttaggctgc atccaaaatg aagcatcatt tttaaggaca 5700

gcatcaaaag tgaccagagg aattttactt tctttctttt tttttttttt gaattttagt 5760

ttctaaactc acttctgaat aaatacaact tctaaattct cgtcttttct ctactctaga 5820

tggatctgat ttagagatga ggctggtgaa tggaggaaac cggtgcttag gaagaataga 5880

agtcaaattt caaggaacgt ggggaacagt gtgtgatgat cacttcaaca taaatcatgc 5940

ttctgtggtt tgtaaacaac ttgaatgtgg aagtgctgtc agtttctctg gttcagctaa 6000

ttttggagaa ggttctggac caatctggtt tgatgatctt gtatgcaatg gaaatgagtc 6060

agctctctgg aactgcaaac atgaaggatg gggaaagcac aattgcgatc atgctgagga 6120

tgctggagtg atttgcttaa gtaaggactg acctgggttt gttctgttct ccatgagagg 6180

gcaaaaaaag gggagtaaaa gtcttaaaag ctcaaactgt taaaaacata atgatgattg 6240

6300

6360

aggtccaagt catgaggtta gaaggaaatg ttcagagaat agcccattcc ctcagccctc 6420

6480

6540

tcacattacc caccctttcc tactatagtc aggggggggg gggggggggg gggggggggg 6600

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 6660

gggggggggg gtgaaaaaag aaccaaacaa tttcaacaaa aaaccaaaca attccaacaa 6720

aattggtcca ataagcaaac ctctagataa atttcagtgc cctggatgtt ttgttaggaa 6780

ctcttcctac aatgcgtgct ttccattctg aaaagtccta tctacttgcc tgatccactt 6840

ctccttccat cctaaacgat tttcagtggt agtatattac tgttgtctct gtctctactt 6900

atatatcttc cccttttcac tcactcctct caggtacagc tcttcagttt gcccttattc 6960

ttgtttcctt gtcaatgact tgttttgtgt ccctcttaca gatggagcag acctgaaact 7020

gagagtggta gatggagtca ctgaatgttc aggaagattg gaagtgaaat tccaaggaga 7080

atggggaaca atctgtgatg atggctggga tagtgatgat gccgctgtgg catgtaagca 7140

actgggaggt ccaactgctg tcactgccat tgtcgagtta acgccagtga gggactggac 7200

acattggctc acacacatac agccatgaca cgatctgctc tatggtccga tgattaaagg 7260

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 7320

gggggggggg gggggggggg gggggggggg ggggggggag aagagctggt ggacatttct 7380

ggaaaggaac caaaacccgg aagggccttg ttcttcagga tttgggatgg attggggagg 7440

gagaaaattg tttctaatat ttcttggtgg gaattctttt acagttgtga caaatctttc 7500

acatattctt catttgagta gtttggaggg ttgtctgact gttttctata ataaatgtcc 7560

caagtgctat gaggtaccac atttcaaatt ctaattctac ctgaagctcc aaaaagacaa 7620

aatgttatag gtcttttctt tatatctaat ttgcttatgg tttttagcca ttgacaattt 7680

ttttttctta actcttgaaa ctataaccct atttctaacc aaattcatgt tctatactgg 7740

ctcttcaaaa acccaggaga tgggaaagcc agaatctcca gtgtttcagc ttctgggaag 7800

7860

gtatgtttat tttgcagatg gatcagatct ggaactgaga cttaaaggtg gaggcagcca 7920

ctgtgctggg acagtggagg tggaaattca gaaactggta ggaaaagtgt gtgatagaag 7980

ctggggactg aaagaagctg atgtggtttg caggcagctg ggatgtggat ctgcactcaa 8040

aacatcatat caagtttatt ccaaaaccaa ggcaacaaac acatggctgt ttgtaagcag 8100

ctgtaatgga aatgaaactt ctctttggga ctgcaagaat tggcagtggg gtggacttag 8160

ttgtgatcac tatgacgaag ccaaaattac ctgctcaggt aagaatttca atcaatgtgt 8220

taggaaattg cattctactt tcttttacat gtagctgtcc agttttccca gcaccacttg 8280

ttgaagagac tgtcttttct tcatcatata gtcctacatc ctttgtcata aattaattga 8340

ccataggtgt gtgggtttatctgggctc tctattctgt tcctttgatc tatatgtctg 8400

tttttatgcc agcaccatgc tgttttgatt actatagctt tgtagtatca tctgaagtca 8460

ggaaacatga ttcctccagc tttgttcttc tttctcaaga ttgttttgtc tattcagagt 8520

ttatgttccc atgcagattt aatttttaaa tttatttaat ttttattttt tatttttaat 8580

ttaaattaat ttaaattttt tatttcccaa cgtacagcca agggggccag ggtaaccttt 8640

acatgtatac attaaaaatt tcaggttttt cccccaccca ttttctttctg ttggcaagta 8700

aatttttgaa caaagtttcc caatgctttt taaggggaat tcccttgggg gggggggggg 8760

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 8820

gggggggggg gggggggggg gggggggggg gggggggggg agacgaaatt gactatattt 8880

tctttgttgg gaatctttta cagttgtgac aaatctttca catattcttc atttgagtag 8940

tttggagggt tgtctgactg ttttctataa taaatgtccc aagtgctatg aggtaccaca 9000

tttcaaattc taattctacc tgaagctcca aaaagacaaa atgttatagg tcttttcttt 9060

atatctaatt tgcttatggt ttttagccat tgacaatttt ttttttcttaa ctcttgaaac 9120

tataacccta tttctaacca aattcatgtt ctatactggc tcttcaaaaa cccaggagat 9180

gggaaagcca gaatctccag tgtttcagct tctgggaagg agcaagtttt taaaaatacc 9240

ctctggggagc taaattccac atgtatctat ggcctaagtg tatgtttatt ttgcagatgg 9300

atcagatctg gaactgagac ttaaaggtgg aggcagccac tgtgctggga cagtggaggt 9360

ggaaattcag aaactggtag gaaaagtgtg tgatagaagc tggggactga aagaagctga 9420

tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa acatcatatc aagtttattc 9480

caaaaccaag gcaacaaaca catggctgtt tgtaagcagc tgtaatggaa atgaaacttc 9540

tctttgggac tgcaagaatt ggcagtgggg tggacttagt tgtgatcact atgacgaaac 9600

caaaattacc tgctcaggta agaatttcaa tcaatgtgtt aggaaaattg cattctactt 9660

tcttttacat gtagctgtcc agttttccca gcaccacttg ttgaaaaaac tgtctttttc 9720

ttcatcatat agtcctacat cccttggcca taaattaatt gaccataagg ggtgtgggtt 9780

taatatccgg ggctcctcaa ttcgggtccc ttggatccta aaagccggtt ttataacccg 9840

acacatggcc tgtttttgac taaataaaac ctttggaaaa caatcccgaa ggtcgaggaa 9900

catggaatcc ccccaacaaa ggaccttctt tccccaaaaa tgcggctcag ccaactcaaa 9960

aagattttat gaatcacaaa ccgcacatta tcttcctaaa attactattc ctatgtttta 10020

atttgcaaag tcattccgat atagttggcg cagagtaact catttagata tccaccccac 10080

cagttcctca ctcaagtaag gggggggggg gggggggggg gggggggggg gggggggggg 10140

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggc 10200

ccccatgtga gattttgtgt gtcctttaag agtggagtct ctatttccca ctgctctctg 10260

gttctcccca aagtaagccc tgctggcttt caaaacttct gggagcttgc cttcttggta 10320

taggactcct gggctaggga gtctaatgtt tggcttagac cccttactgc ttgggaagaa 10380

10440

tgttctgtgt tctacccctc ctatccatct tgttgtggtt ccttctttat atctttagtt 10500

gtagaaaagt ttttcttatc aacagttgct ctgtaaattg taacttgggt gtacacctag 10560

taggaggtga gctcagggtc ttcctactct gccatcttgg ccatgtcctc taaacatttt 10620

ggtgtatttc actgcaacct ttttaaaaat ctcaaaagtg agctgtgatt ggctagtctt 10680

gtggataatc tctagcattt gatgctaatc atatttatac aaatactttg ttgaaaagtg 10740

atgccttttt aactattatt aaaaaacgta ttgacataac tattgctatt atactgaaaa 10800

10860

tagggtggaa attttatgtg gtccatatcc cctaaccagt ggctttacac caggcacatc 10920

ctaactaaga tctgctccca agtgtcttcc ctgatgcttt aaattgtgtt acatggaaac 10980

11040

ttttgctttt caacattttt tctttttaaa agaagaaatt tatttgtttt tttaaattttt 11100

aatggccacg gcatatggaa gttctcaggc cagggataga attcaagcca caggtgcgac 11160

ccatgccaca actgctgcaa caccagatcc tttaacccac tgcaccaggc cagggattga 11220

agccttgcct tactgacaat ctgagccact tcagtcagat aaagaaattt cttcattaag 11280

cagagtattc acatggttta aacttcaaaa tattaaagtg taaactcttt ccccaccact 11340

gtccccagct caccaactct acttaccaca gacaactgat gtggttaggg tatttaaata 11400

gtaaatccaa gaaaatataa acaaatccgt atatataggt ttcaccccat tttattatcc 11460

taatgttgca tatcatataa actatactgt cccttgggta ttcacttagt aaaatatttt 11520

11580

ttttctcatc ggtaggggggg gggggggggg gggggggggg gggggggggg gggggggggg 11640

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggaatgggg 11700

11760

gggccacact tttctataga aaggggatta tttaaaaggg tctgaaaaag aatttttttt 11820

tcgaaagggg aaatatttgg cctaacttag tcacataagc catgttctct ggcaagttag 11880

11940

actccgcatt ggttagggaa ggggaagtat attggaattc ggaaaattcc ttccaaatta 12000

12060

cattaaaagg gaaaacacct ggagtggtaa gaaaaaagtt ttttttttctc tttttttttt 12120

ttttttttta atggccacat ctgtggcatg tgaagttccc aggctaggggg tcgaatagga 12180

gctacagctg ccagcttgca ccacagccac aacaatgcca gagccaagcc tcatctgcga 12240

cctataccac aactcatggc aatgctggtt ccttaacccc ctgagtgagg cctggggtca 12300

aacccacatc ctcatggata ctaaccggct ttgttaccgc tgagccatga gggaaactcc 12360

ctttttctca ttgaaaataa gtcaaataga taagcagctt aaggctgttt gggtgattct 12420

gtggtccagt aattatcaaa tcctactgga caagaataga gaatgtgcaa atgagggaac 12480

gtgttggtga gatcaggctc tgcccactga gctatcctct gtcatgggcc ctgtgctgtt 12540

ctcagagctg tacttcctag ggcattgttc tcatttcaat tctgagttca gtgtggagag 12600

12660

gatccttgga ttgctttaca gttgagtcat cattaactag tgtcacttgc cttcaaagtc 12720

agcaaaataa ttgtctccaa actagtaggc ttctagtgta tttgctttaa tccaatgcca 12780

tgtgaaagta acatggtcaa agaataagtt atataccttg acctaccctg tgaccaggct 12840

cttcctctta atttattgac cactgcctta aggtcatttg aaaccatggg tttgggagga 12900

tagagcatca 12960

ttttttttta aactggggta cagtttattt acagtgttgt gtcaatttct gctgtacagc 13020

acagtgaccc agtcatacac atacatacat tctttttctc atactatctt caattttatt 13080

13140

gttttttttt tccccccaat cctgcaattt ctaaattata agactctcaa ttagccgtat 13200

ataacagctg caggcacagg atgtctccct cacaaaattg gtatttttcc ttccatttct 13260

tcttgcagtt tggctatttc ttgtctgagt tcatctctct ttttaagtgt taaaaagggc 13320

13380

tacttttccc aaatgtcatc caacttttca gcatcagttt ggacatggtt ttcttttcaa 13440

ggtggtattt ctctaatgtc acttgaataa caagactcgt tagttctcca ggctacaata 13500

13560

tattcctgga acacctcact tttttttttt ttttggtct tttttatagcc ataaccatgg 13620

catatggagg ttcccaggct aggggtctaa tctgagcttt agccactggc ccatgccaca 13680

gccacagcca tgccacatct gagccacatc tgtgaccttt tccacagctc acagaaacac 13740

cagatcccta acccactgag tgaggccagg ggtcaaacct gtaacccttc catggttcct 13800

agtcagattc gttcctctgt accacgatgg gaattcctaa tacctcactt atgataacac 13860

attctgaatt atttaggatt ctattatact gcatgtaata gaaatcccaa atagcaaaat 13920

13980

ctttgctttc ctctgtgaat tcccttttttt gttatttc tatagctttt ggaaacactt 14040

ttacttatt gggggggcct agatttttaa ccctctcctt gttttttctag aaatagagtt 14100

tataatttta tttcttcatt tacttgatac tttcaagaga ttcccaggaa aaaaattatg 14160

gaaatactgt ctctgtgcct gccaagttca aactaagaat tgtataatct gttttaattc 14220

ttaagcattt atagatgaca aggctttgtg tctgataggg gccagcgaac tcagtaaaga 14280

gggaagatga gaaagataat ggcaagaatt tatccctgaa gtgtagtttt gacaaaccag 14340

tcacaaagag gtctaagaaa ttttggtcac aaagttgttt tgaatcccag gcattttatt 14400

tgcaatgatt gcatatgttc tggaaaggac atctgaacct aagaaatagt tcatttgcat 14460

14520

tcagagtctg gaaggaataa attatgaaaa tgtattaatg cttctttaaa ccatattgta 14580

tatttatcta ttactaaaca aaaagaagta gctctattta tttatttatt tatttattta 14640

tttatgtctt ttgtctcttt agggccacac ctgtggcata tggaggttcc caggctagag 14700

gtccaattgg agatgtagca gccagcctat gccagagcca ccgcaacacg ggatctgagc 14760

cacgtctgtg acttacacca cagctcacag caacgcctga tcctcaaccc actgagcgag 14820

gccagggatc gaacccatgt cctcatggat gctagttggg ttcgttaact gctgagccat 14880

gatgggaact ccaaattaat tatttcttat atttgttctt catatattca tttctataga 14940

aagaaataaa tacagattca gttaatgatg gcaggtaaaa gcttaactta ttaatcaaag 15000

gagttaatcc aggcacaaaa attcaattca tggctctctg ttaaaattta ggtataggtt 15060

tagcaggaag aaaaggttag tagatgcaga ctattacatt tagaatggat ggacaatgaa 15120

gtcctactat acagcacagg gaactatatc caatctcttg ggatagaata tgatggaaga 15180

15240

gtgtgtgtgt gtgtgactgg gtcaccctgc ggcacagcag aaattggcag aacattgtaa 15300

atcaactata ctttaatagg aaaaatactt ttaagggcta aatttccaat attctaacca 15360

tgtacacaga gtaaatgtca taaggatgcc agtctgtgta gagattgatg tgttactagc 15420

agattcatga aataaaggct gaggatgtag tccccaagtc acttctgagt ggaagaattt 15480

15540

ggacttgttt tcaagtactt gacaaaattt caccattaaa gagaaatttg tgggagttcc 15600

catcgtggct cagtggaaac aaatccaact aggaaccatg aggttgtggg tttgatccct 15660

ggcctcactc agtgggttaa ggatccggtg ttgccgtgag ctgtggtgta ggttgcagac 15720

acggttctga tcctgcgttg ctgtggctgt ggctgtggtg taggccagca gcaaacagct 15780

ctgattagac ccctagcctg gaaacctcca tatgccacag gtgcagccct aaaaagacaa 15840

15900

15960

tttgagactg atctagaaga ctagagatta caagaaatat ggatttggct cactctaaga 16020

aactgctttc attccaaggt ttgggtctat ccaaaagtgg aatagaatca tatgaatact 16080

agtttatgag tatttagtga gaggaatttc aagctcaaat aatgattcag caagattaaa 16140

ttaaggaggg aattttcctt gtggctgagt gggttaagga cccaatgttg tctctgtgag 16200

gatgtaggtt ccatcctggg ctttgctcat taggttaagg atctggcatt gctgcagctc 16260

agacccagtg ctgccctggt tgtggcttag gccaaagctg cagctccaat tcaatctctg 16320

gcctgggaac ctccatgtgc tacaaggtgc ggccttaaaa ggaaaaaaaa aaaattaaat 16380

16440

agttgtgttt agaaagcaag atgttcttca actcaaattt gggagggaac ttgtttcata 16500

catttttaat ggataagtgg caaaattttc atgctgaggt gatctatagt gttgtaatgc 16560

agaatatagt cagatcttga acattttagg aagttggtga gggccaattg tgtatctgtg 16620

16680

aatgcatttc ctttctttaa aagataaatt 16740

16800

actcttacct tgatgattgc gctcttaacc tggcaaagat tgtctttaaa atctgagctc 16860

catgtcttct gctttatttc tggtgtgcct ttgactccag attacagtaa atggaggact 16920

gagtataggg ctaaaaagta gagagaatgg atgcatatta tctgtggtct ccaatgtgat 16980

gaatgaagta ggcaaatact caaaggaaag agaaagcatg ctccaagaat tatgggttcc 17040

agaaggcaaa gtcccagaat tgtctccagg gaaggacagg gaggtctaga atcggctaag 17100

cccactgtag gcagaaaaac caagaggcat gaatggcttc cctttctcac ttttcactct 17160

ctggcttact cctatcatga aggaaaatat tggaatcata ttctccctca ccgaaatgct 17220

atttttcagc ccacaggaaa cccaggctgg ttggagggga cattccctgc tctggtcgtg 17280

ttgaagtaca acatggagac acgtggggca ccgtctgtga ttctgacttc tctctggagg 17340

cggccagcgt gctgtgcagg gaactacagt gcggcactgt ggtttccctc ctggggggag 17400

ctcactttgg agaaggaagt ggacagatct gggctgaaga attccagtgt gaggggcacg 17460

agtcccacct ttcactctgc ccagtagcac cccgccctga cgggacatgt agccacagca 17520

gggacgtcgg cgtagtctgc tcaagtgaga cccagggaat gtgttcactt tgttcccatg 17580

ccatgaagag ggtagggtta ggtagtcaca gacatctttt taaagccctg tctccttcca 17640

ggatacacac aaatccgctt ggtgaatggc aagaccccat gtgaaggaag agtggagctc 17700

aacattcttg ggtcctgggg gtccctctgc aactctcact gggacatgga agatgcccat 17760

gttttatgcc agcagcttaa atgtggagtt gccctttcta tcccgggagg agcacctttt 17820

gggaaaggaa gtgagcaggt ctggaggcac atgtttcact gcactgggac tgagaagcac 17880

atgggagatt gttccgtcac tgctctgggc gcatcactct gttcttcagg gcaagtggcc 17940

tctgtaatct gctcaggtaa gagaataagg gcagccagtg atgagccact catgacggtg 18000

ccttaagagt gggtgtacct aggagttccc attgtggctc agtggtaaca aactcgactg 18060

gtatccatga gggtatgggt ttgatccctg gccttgctca atgggttaag gatccagcat 18120

tgctgtgagc tgtggtatag gttgcagact ctgctcaggt cccatgttgc tgtgattgtg 18180

gtgtaggctg actgctgcag cttcaatttg acccctagcc cgggaatttc cataggccac 18240

18300

gtgaagaaca gatgtaaaag ggaagtgaaa gggattcccc cattctgagg gattgtgaga 18360

agtgtgccag aatattaact tcatttgact tgttacaggg aaagtaaact tgactttcac 18420

ggacctccta gttacctggt gcttactata tgtcttctca gagtacctga ttcattccca 18480

18540

cactccagct gatcttcctg acacagctgt ggcaaccctg gatcctttaa ccaactgtgc 18600

caggctggag atcaaaccta agcctctgca gcaacccaag ctgctgcagt cagattttta 18660

accccctgtg ccactgtggg tatctccgat attttgtatc ttctgtgact gagtggtttg 18720

ctgtttgcag ggaaccagag tcagacacta tccccgtgca attcatcatc ctcggaccca 18780

tcaagctcta ttatttcaga agaaaatggt gttgcctgca taggtgagaa tcagtgacca 18840

acctatgaaa atgatctcaa tcctctgaaa tgcattttat tcatgtttta tttcctcttt 18900

gcagggagtg gtcaacttcg cctggtcgat ggaggtggtc gttgtgctgg gagagtagag 18960

gtctatcatg agggctcctg gggcaccatc tgtgatgaca gctgggacct gaatgatgcc 19020

catgtggtgt gcaaacagct gagctgtgga tgggccatta atgccactgg ttctgctcat 19080

tttggggaag gaacagggcc catttggctg gatgagataa actgtaatgg aaaagaatct 19140

catatttggc aatgccactc acatggttgg gggcggcaca attgcaggca taaggaggat 19200

gcaggagtca tctgctcggg taagttctgc acatcacttc gggttacagt gatttaagaa 19260

acaactaagg tggggcaaag ggtagtgagg catatccatc agagcaaatt ccttgaaata 19320

cggactcaga gggaaccatt gtgagattga ggttcccaga ggtgtggatt taatgaatta 19380

gtgttacctc atgtacaagg tagtatacta ccagaaagat aaaaattcag aagcgagttt 19440

gcagcaaaac tcatagggag aacttctttt ataaataata tgaagctgga tatttagtgc 19500

accacctgat gaccacttta ttaataaata aagagttcct gttgtggcgc agcggaaatg 19560

aatccgacaa ataatcatga gtttgcgggt ttgatccctg acctcgctca gtgggttggg 19620

gatctggtgt tgccatgagc tgtggtgtag gtcgcagatg ctgcttggat cctgctttgc 19680

tgtggctgtg gtataggctt gtggctacag ctccgatttg accgctagcc tgggaacctc 19740

catatgctgc gggggtggcc ctcaaaagaa aaataaataa ataagtaaat aaataagtag 19800

tttaaaaagg acaagaagaa atatatttgg tgttatattc tacagagaca aagataatca 19860

ccatgcccga ttgatttttc aaggcatata aatgagacgt catgggagca aaaatggtca 19920

taatacaatg cccttgtttt gtgtacatgg taagatttta gaaagcattg tgaggtaaaa 19980

20040

atgccggaag ttattttttt aatgtctctt ttagagtcgc acatgcggca tatggaggtt 20100

cccaggctag gggtcgaatc agagctatag ccactggctt atggcacagc cacaacaaca 20160

ctagatctga gccacatcag cgacctatac tatagctcat ggcaatgcca gatccttaac 20220

ctactgagcc aagccatggg tcaaatccag gtcctcacgg atcctaggca aattcatttc 20280

tgctgagcca cgaagggaac tcctcagaag tgattttgat gttactttct tttcatgaca 20340

20400

aaggtaatgt atacaaaaca gtggttttac catctgagta tctcgctaaa ttttaactat 20460

caaggacaat tgccaaaaaa aaaaaaaaaa gagagagaga gagaacagaa tagggttatg 20520

aagctaaaat cacagggtta tgaagctaaa atcacagtaa tttagggaga aaaaaatcca 20580

20640

ataccggtgg tattttaaag cagtccataa gtataaaatc caaggctaaa aaaccagaag 20700

gtatttgtag aacaaatata ttttaataag ctctaccaag tcatccagaa gctattaaag 20760

aattactggt cactgacata gtgtacctgt tttcaaggcc attcttacat cagaataaag 20820

ggagagcacc ctctgaatct tcagaaaaga tgtgaaagtg ctaattctct atttcatccc 20880

agagttcatg tctctgagac tgatcagtga aaacagcaga gagacctgtg cagggcgcct 20940

ggaagtttt tacaacggag cttggggcag cgttggcaag aatagcatgt ctccagccac 21000

agtgggggtg gtatgcaggc agctaggctg tgcagacaga ggggacatca gccctgcatc 21060

ttcagacaag acagtgtcca ggcacatgtg ggtggacaat gttcagtgtc ctaaaggacc 21120

tgacacacta tggcagtgcc catcatctcc atggaagaag agactggcca gcccctcaga 21180

ggagacatgg atcacatgtg ccagtgagta tccattcttt agcgccactg ttatcttctg 21240

atctacctaa gcagaagttt tataatctgt agttaatccc tattctacct ggatgatggg 21300

attcattctg tttaatttgg tgtgcaggta ttcagcatca gtgatcattt tcccaaagac 21360

catcatgctc tgatggtctt ctcaaaagtt ctaatcagtt gcttcctccg tgaacagttg 21420

aggagcagag aatatgtaat tcagaatttg actattgaat catcccattt ttctttcaca 21480

tagtcttttg ttgcactgaa tataaggaga gaagcagtca gaaagatcaa tcctgaatta 21540

tttctccatt ctacatctgt tttaaatttc aaaaaaaaaa attgttatag gtgatttaca 21600

atgtctgtca atttctgctc tacagcaaag tgacccagtt atttacatat acattctgtt 21660

21720

atgcatttat gttaacttta ttcacctgat gttttctaag tcatactgag attcttatgc 21780

ccaggatgga atacacctgg tttgctggaa agacatgtgc tttcataaag acgaattttg 21840

gaaaaaatat aaaatttaaa aggcccatta aataagcaaa gttttaagag atttcaaaaa 21900

aaatttcatc tctctctttt cctctttgac ctcttgggca cgttcatctt ctcaaatatg 21960

atcttggtgt ttctgacttt tcagacaaaa taagacttca agaaggaaac actaattgtt 22020

ctggacgtgt ggagatctgg tacggaggtt cctggggcac tgtgtgtgac gactcctggg 22080

accttgaaga tgctcaggtg gtgtgccgac agctgggctg tggctcagct ttggaggcag 22140

gaaaagaggc cgcatttggc caggggactg ggcccatatg gctcaatgaa gtgaagtgca 22200

aggggaatga aacctccttg tgggattgtc ctgccagatc ctggggccac agtgactgtg 22260

gacacaagga ggatgctgct gtgacgtgtt caggtgaggg cagagagtct ggattgagct 22320

tggaagctct ggcagcaaag agagggtggg cggtgacctg cattgggtaa agattggaag 22380

gtccagccta aggatctggt ggtggggggga gacatgatgt ttcagtctga agaatgatga 22440

aaacctgtgt ggttacgcat gggccttcgc cgaggaaagg gacataacta ccatgtatcc 22500

tcctgcagag ggaggaagaa ctaggggatt ctagttttgt gtgggaagga gcagtttact 22560

tggctcagga ggcactaaag gctcagatag gaaacagaga tctgttccat tcttactccc 22620

agaactgatt ctcttctctt ttctcctaca gaaattgcaa agagccgaga atccctacat 22680

gccacaggta tatcaaaaag tttaagaaca tgggacccat tgtctgcatt ttgtggaatc 22740

cctcttatta agacattctg ggtcagaagt tctgaggatt tgacatttac ttcagctatc 22800

tgttatctta cccaagagag ggatggtaac taggaaccca ggtcttttag ctaagacatt 22860

atcacctctt gtgatgttta cttgttctca ggtcgctcat cttttgttgc acttgcaatc 22920

tttggggtca ttctgttggc ctgtctcatc gcattcctca tttggactca gaagcgaaga 22980

cagaggcagc ggctctcagg tctgaacaaa attacggtct ctctaatgtt tctatgggag 23040

aagaagcctc tctggataat aaaacaaaaa aattacattc aagtatcagt tggccagaaa 23100

23160

gaaaaggcac attggctgta ctgatgatac ctgcactcgc tatgtatgtt taatggggga 23220

23280

ccccccaaac atttattttc tcttactata tacttattac taatttaact cttctgtcaa 23340

gccatgtgct aggttctgaa gatggttcag acttggataa ccaagtgctt ttgttttcat 23400

23460

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23520

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23580

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23640

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23700

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23760

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23820

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23880

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23940

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 24000

gggggggggg gggggggggg gggggggggg gggggggggg ggggggggtt ggcggggcccc 24060

cctcgaggtc gacggtatcg ataagcttga tatcgaattc gtgagccaga ggacgagact 24120

agagatggat gatgactacg ttatgcttgc actgctgggg aaaagcacac atagggaggg 24180

aacgttttat tatgacccag tccctaacct atgacctctg ttatcagttt tctcaggagg 24240

agagaattct gtccatcaaa ttcaataccg ggagatgaat tcttgcctga aagcagatga 24300

aacggatatg ctaaatccct caggtccgtg ggttctttga ggggctgtag ccctggggtt 24360

cagatcagca gctgcagttg aggttgaggc atgctacttt gcatagcagt agaaagaaat 24420

ctcaactgta ataggaagct tgggatgcat atgaggaaga aaggcaagaa tgaactacaa 24480

attattctta gggaagataa aaattgcagt catggggaga cctctggctg agaggggccgt 24540

gattatttct gacagaggga ttatggagta gaatatgatg gcttggacct tttttcacta 24600

24660

24720

gtttaaattt tctatatctt tatcccttca gaagccatcc ctagtcacag gactagtctc 24780

atttcccatt atgtaatgct tctttctctg tctgttgact tctatttaga accagtgcac 24840

taaatctgcc tttaggaaca tacctctgct aggttgcaag aaatatccca ttccccactc 24900

actctgtgaa gactcaatgc ttctcaatat tccttacctc ctgagaggga cttgcctcac 24960

ttctttaatc caagggactc gatttttgcc aaaactaagt caggaaaacc tacataagac 25020

ataggaaaga cttgctgtgc ttcttaaacc ccactgtttg ttttcctaat tgtgaacagt 25080

atttttaaag ttaacaagag agcttctaag gcacttgagg ggagatctga tttatttccc 25140

agtaattatt ttcttccttt cagaaaattc cactgaataa gatggtttta acggatgtgg 25200

gactaatttt tgtgtctaaa tctcttccta tttctggatg aaaaaaagga gaccactctg 25260

aagtacaatg aaaaggaaaa tgggaattat aacctggtga ggtgagtagg aagaatttat 25320

25380

25440

atattcagaa tatgcaacaa ctttttataa ctatgacttc agtccatctt ttagttacat 25500

25560

25620

gtgaagatga gggctcgagc cctggcctca ctcagtgagt taaggatcta gcattgctgt 25680

aagctgcagc gtaggttgca gatagggctc agatccagtg ttgctgtggc tgtggcctca 25740

gttgcagctc tgattcaacc cttaggcgag gaacttccat atgcagcaaa tgtggccatt 25800

aaaaaaaaaa aaaacattat

atatagtgca tactctggta aagatagtat aggatactat aggaatatag aaagcttgcc 25920

tatgaaaatt tgggaagatt gtggaaaaga catctcaaaa tatggcatag aaaagaatca 25980

tttttcattc aaaaccgtgt attgaacata cttgtggtga 26040

aaaaattcag tgataaaaga tgctcttgac aaagacatgg 26100

ctgttgaata gaaggtctca ctgtcaatgt gtgggaatta tggacagcct atgtggacac 26160

agggaataga tgagactcta ggctggaagg ctgcattgag cccaataatg aatggtcctg 26220

26280

gaagattaag ctgaggcaag acacaatcag attgcctttt ataatttact ttcaggagga 26340

26400

cccgcctgcc cttttggggg gggggggggg gggggggggg gggggggggg gggggggggg 26460

gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggcagca 26520

ccaattttat tattggcggg aataaagaga aaaatgtaat ttcaaagatt gctgttggaa 26580

atgaggggtg tggtagcttt tggagaaagc attctggaga cttctattaa tttttttttt 26640

ttaagtgctt caaagatcct ttgatccaac aattctactc ctaaaaattt cttccataca 26700

26760

gtagtgcccc caaactggaa acaaagtgaa tatcagtcag tggggtagcg gctggaaaaa 26820

ttttagtgca cccaaccaac aaagaaaaac catgcacaaa aattcaataa atatcatctc 26880

acttttgtgt tcatgttatt gaatataatt aaacataatg tttacatcta taaaattatc 26940

27000

tgaatgactt ttgattgata atctcaaaca tatggatact attctgatgt aataaataat 27060

gattaaattt tttccctaaa gagtaatcac tactgaatcg ttgcctcaga atcatatgga 27120

ggtgctttta aaaaaggcat ttctgcactg ttgttctctg gaatagaagt aattcttatg 27180

27240

27300

cacatgtgca tatgaagaga aaagtaacat ttttttctat ttatgtcttt agttcagcct 27360

27420

aagattacaa tacagtcctc tgtctcctgg tattccaaag actgctgttg aatttctaaa 27480

aaatagattg gtgaatgtga ctactcaaag ttgtatgtaa gactttcaag ggcattaaat 27540

aaaaaagaat attgctgatt cttgttcttg attttctgaa tttctgaatc tcttattggg 27600

cttctaattt aaaaaaaaat atctgggcgc ccgcagatat cgaactcttg ggcagtgtga 27660

ccaaacgaag acatatccaa tcaagcatgc aaatggacca gcccactgta ctagcacgct 27720

gtggcagcca atctgaccga gaaagcagac aaccgcaggg agcaacg 27767

<210> 16

<211> 55

<212> DNA

<213> Sus scrofa

<400> 16

ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatc 55

<210> 17

<211> 54

<212> DNA

<213> Sus scrofa

<400> 17

ggtcgccacc atggccatga gcaagggcga ggagctgttc accggggtgg tgcc 54

<210> 18

<211> 48

<212> DNA

<213> Sus scrofa

<400> 18

ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggt 48

<210> 19

<211> 55

<212> DNA

<213> Sus scrofa

<400> 19

ggtcgccacc atggttgagc aagggcgagg agctgttcac cggggtggtg cccat 55

<210> 20

<211> 43

<212> DNA

<213> Sus scrofa

<400> 20

tgcagggaac tacagtgcgg cactgtggtt tccctcctgg ggg 43

<210> 21

<211> 60

<212> DNA

<213> Sus scrofa

<400> 21

tgcagggaac tacagtgcgg cactgtaaac cactactact gtggtttccc tcctgggggg 60

<210> 22

<211> 41

<212> DNA

<213> Sus scrofa

<400> 22

tgcagggaac tacagtgcgg ctgtggtttc cctcctgggg g 41

<210> 23

<211> 46

<212> DNA

<213> Sus scrofa

<400> 23

tgcagggaac tacagtgcgg aactactgtg gtttccctcc tggggg 46

<210> 24

<211> 31

<212> DNA

<213> Sus scrofa

<400> 24

gaaacccagg ctggttggag gggacattcc c 31

<210> 25

<211> 24

<212> DNA

<213> Sus scrofa

<400> 25

gaaacccagg ctggggacat tccc 24

<210> 26

<211> 13

<212> DNA

<213> Sus scrofa

<400> 26

aggggacatt ccc 13

<210> 27

<211> 13

<212> DNA

<213> Sus scrofa

<400> 27

gaaacccatt ccc 13

<210> 28

<211> 31

<212> DNA

<213> Sus scrofa

<400> 28

ggtcgccacc atggtgagca agggcgagga g 31

<210> 29

<211> 32

<212> DNA

<213> Sus scrofa

<400> 29

ggtcgccacc atggctgagc aagggcgagg ag 32

<210> 30

<211> 29

<212> DNA

<213> Sus scrofa

<400> 30

ggtcgccacc atggtgagg ggcgaggag 29

<210> 31

<211> 32

<212> DNA

<213> Sus scrofa

<400> 31

ggtcgccacc atggttgagc aagggcgagg ag 32

<210> 32

<211> 48

<212> DNA

<213> Sus scrofa

<400> 32

ggtcgccacc atggtgagca agggcgagga gaacccaggc tggttgga 48

<210> 33

<211> 49

<212> DNA

<213> Sus scrofa

<400> 33

tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggg 49

<210> 34

<211> 38

<212> DNA

<213> Sus scrofa

<400> 34

tgctgtgcag ggaactctgt ggtttccctc ctgggggg 38

<210> 35

<211> 22

<212> DNA

<213> Sus scrofa

<400> 35

ctgtggtttc cctcctgggg gg 22

<210> 36

<211> 23

<212> DNA

<213> Sus scrofa

<400> 36

actgtggttt ccctcctgggg ggg 23

<210> 37

<211> 50

<212> DNA

<213> Sus scrofa

<400> 37

tgctgtgcag ggaactacag tgcggcaact gtggtttccc tcctgggggg 50

<210> 38

<211> 10

<212> DNA

<213> Sus scrofa

<400> 38

tcctgggggg 10

<210> 39

<211> 8

<212> DNA

<213> Sus scrofa

<400> 39

ctgggggg 8

<210> 40

<211> 52

<212> DNA

<213> Sus scrofa

<400> 40

agagagcaga gccagcgact cgcccagcga catggggtac ctgccgtttg tg 52

<210> 41

<211> 33

<212> DNA

<213> Sus scrofa

<400> 41

agagagcaga gccagcgact cgcccagcga gat 33

<210> 42

<211> 30

<212> DNA

<213> Sus scrofa

<400> 42

agagagcaga gccagcgact cgcccagcga 30

<210> 43

<211> 50

<212> DNA

<213> Sus scrofa

<400> 43

agagccagcc tcgcccagca ggggtaccat ggggtacctg ccgtttgtgt 50

<210> 44

<211> 53

<212> DNA

<213> Sus scrofa

<400> 44

agagagcaga gccagcgact cgcccagcga gcagtgggta cctgccgttt gtg 53

<210> 45

<211> 53

<212> DNA

<213> Sus scrofa

<400> 45

agagagcaga gccagcgact cgcccagcga tcagtgggta cctgccgttt gtg 53

<210> 46

<211> 53

<212> DNA

<213> Sus scrofa

<400> 46

agagagcaga gccagcgact cgcccagcga acatggggta cctgccgttt gtg 53

<210> 47

<211> 4990

<212> DNA

<213> Sus scrofa

<400> 47

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060

aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120

tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggga gctcactttg 3180

gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc 3240

tttcactctg cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg 3300

gcgtagtctg ctcaagtgag acccagggaa tgtgttcact ttgttcccat gccatgaaga 3360

gggtagggtt aggtagtcac agacatcttt ttaaagccct gtctccttcc aggatacaca 3420

caaatccgct tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt 3480

gggtcctggg ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc 3540

cagcagctta aatgtggagt tgccctttct atcccgggag gagcacctttt tgggaaagga 3600

3660

tgttccgtca ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc 3720

tgctcaggta agagaataag ggcagccagt gatgagccac tcatgacggt gccttaagag 3780

tgggtgtacc taggagttcc cattgtggct cagtggtaac aaactcgact ggtatccatg 3840

agggtatggg tttgatccct ggccttgctc aatgggttaa ggatccagca ttgctgtgag 3900

ctgtggtata ggttgcagac tctgctcagg tcccatgttg ctgtgattgt ggtgtaggct 3960

gactgctgca gcttcaattt gacccctagc ccgggaattt ccataggcca cacgtgcagc 4020

actaaggaag gtgtgcctat agtgaagaac 4080

agatgtaaaa gggaagtgaa agggattccc ccattctgag ggattgtgag aagtgtgcca 4140

gaatattaac ttcatttgac ttgttacagg gaaagtaaac ttgactttca cggacctcct 4200

agttacctgg tgcttactat atgtcttctc agagtacctg attcattccc agcctggttg 4260

acccatcccc ctatctctat ggctatgttt atccagagca catctatcta acactccagc 4320

tgatcttcct gacacagctg tggcaaccct ggatccttta accaactgtg ccaggctgga 4380

gatcaaacct aagcctctgc agcaacccaa gctgctgcag tcagattttt aaccccctgt 4440

gccactgtgg gtatctccga tattttgtat cttctgtgac tgagtggttt gctgtttgca 4500

gggaaccaga gtcagacact atccccgtgc aattcatcat cctcggaccc atcaagctct 4560

attatttcag aagaaaatgg tgttgcctgc ataggtgaga atcagtgacc aacctatgaa 4620

aatgatctca atcctctgaa atgcatttta ttcatgtttt atttcctctt tgcagggagt 4680

ggtcaacttc gcctggtcga tggaggtggt cgttgtgctg ggagagtaga ggtctatcat 4740

gagggctcct ggggcaccat ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg 4800

tgcaaacagc tgagctgtgg atgggccatt aatgccactg gttctgctca ttttggggaa 4860

ggaacagggc ccatttggct ggatgagata aactgtaatg gaaaagaatc tcatatttgg 4920

caatgccact cacatggttg ggggcggcac aattgcaggc ataagggagga tgcaggagtc 4980

atctgctcgg 4990

<210> 48

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 48

caccggaaac ccaggctggt tgga 24

<210> 49

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 49

aaactccaac cagcctgggt ttcc 24

<210> 50

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 50

caccggaact acagtgcggc actg 24

<210> 51

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 51

aaaccagtgc cgcactgtag ttcc 24

<210> 52

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 52

caccgcagta gcaccccgcc ctgac 25

<210> 53

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 53

aaacgtcagg gcggggtgct actgc 25

<210> 54

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 54

caccgtgtag ccacagcagg gacgt 25

<210> 55

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 55

aaacacgtcc ctgctgtggc tacac 25

<210> 56

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 56

caccgccagc ctcgcccagc gacat 25

<210> 57

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 57

aaacatgtcg ctgggcgagg ctggc 25

<210> 58

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 58

caccgcagct gcagcatata tttaa 25

<210> 59

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 59

aaacttaaat atatgctgca gctgc 25

<210> 60

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 60

caccgctttc atttatctga actca 25

<210> 61

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 61

aaactgagtt cagataaatg aaagc 25

<210> 62

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 62

caccgttatc tgaactcagg gtccc 25

<210> 63

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 63

aaacgggacc ctgagttcag ataac 25

<210> 64

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 64

caccgctcct cgcccttgct cacca 25

<210> 65

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 65

aaactggtga gcaagggcga ggagc 25

<210> 66

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 66

caccggacca ggatggggcac caccc 25

<210> 67

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 67

aaacggggtgg tgcccatcct ggtcc 25

<210> 68

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 68

ttgttggaag gctcactgtc cttg 24

<210> 69

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 69

acaactaagg tggggcaaag 20

<210> 70

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 70

ttgttggaag gctcactgtc cttg 24

<210> 71

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 71

ggagctcaac attcttggggt cct 23

<210> 72

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 72

ggcaaaattt tcatgctgag gtg 23

<210> 73

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 73

gcacatcact tcgggttaca gtg 23

<210> 74

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 74

cccaagtatc ttcagttctg cag 23

<210> 75

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 75

tacaggtagg agagcctgtt ttg 23

<210> 76

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 76

cccaagtatc ttcagttctg cag 23

<210> 77

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 77

ctcaaaagga tgtaaaccct gga 23

<210> 78

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 78

tgttgatgtg gtttgtttgc cc 22

<210> 79

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 79

tacaggtagg agagcctgtt ttg 23

<210> 80

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 80

ggaggtctag aatcggctaa gcc 23

<210> 81

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 81

ggctacatgt ccgtcaggg 20

<210> 82

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 82

gcaggccact aggcagatga a 21

<210> 83

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 83

gagctgacac ccaagaagtt cct 23

<210> 84

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 84

ggctctagag cctctgctaa cc 22

<210> 85

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 85

ggacttgaag aagtcgtgct gc 22

<210> 86

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 86

taatacgact cactataggg agaatggact ataaggacca cgac 44

<210> 87

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 87

gcgagctcta ggaattctta c 21

<210> 88

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 88

ttaatacgac tcactatagg ctcctcgccc ttgctcacca 40

<210> 89

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 89

aaaagcaccg actcggtgcc 20

<210> 90

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 90

ttaatacgac tcactatagg aaacccaggc tggttgga 38

<210> 91

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 91

aaaagcaccg actcggtgcc 20

<210> 92

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 92

ttaatacgac tcactatagg aactacagtg cggcactg 38

<210> 93

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 93

aaaagcaccg actcggtgcc 20

<210> 94

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 94

ttaatacgac tcactatagg ccagcctcgc ccagcgacat 40

<210> 95

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 95

aaaagcaccg actcggtgcc 20

<210> 96

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 96

ttaatacgac tcactatagg cagctgcagc atatatttaa 40

<210> 97

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 97

aaaagcaccg actcggtgcc 20

<210> 98

<211> 3484

<212> DNA

<213> Sus scrofa

<400> 98

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggcacattc cctgctctgg tcgtgttgaa gtacaacatg 1560

gagacacgtg gggcaccgtc tgtgattctg acttctctct ggaggcggcc agcgtgctgt 1620

gcagggaact acagtgcggc actgtggttt ccctcctggg gggagctcac tttggagaag 1680

gaagtggaca gatctgggct gaagaattcc agtgtgaggg gcacgagtcc cacctttcac 1740

tctgcccagt agcaccccgc cctgacggga catgtagcca cagcagggac gtcggcgtag 1800

tctgctcaag tgagacccag ggaatgtgtt cactttgttc ccatgccatg aagagggtag 1860

ggttaggtag tcacagacat ctttttaaag ccctgtctcc ttccaggata cacacaaatc 1920

cgcttggtga atggcaagac cccatgtgaa ggaagagtgg agctcaacat tcttgggtcc 1980

tgggggtccc tctgcaactc tcactgggac atggaagatg cccatgtttt atgccagcag 2040

cttaaatgtg gagttgccct ttctatcccg ggaggagcac cttttgggaa aggaagtgag 2100

caggtctgga ggcacatgtt tcactgcact gggactgaga agcacatggg agattgttcc 2160

gtcactgctc tgggcgcatc actctgttct tcagggcaag tggcctctgt aatctgctca 2220

ggtaagagaa taagggcagc cagtgatgag ccactcatga cggtgcctta agagtggggtg 2280

tacctaggag ttcccattgt ggctcagtgg taacaaactc gactggtatc catgagggta 2340

tgggtttgat ccctggcctt gctcaatggg ttaaggatcc agcattgctg tgagctgtgg 2400

tataggttgc agactctgct caggtcccat gttgctgtga ttgtggtgta ggctgactgc 2460

tgcagcttca atttgacccc tagcccggga atttccatag gccacacgtg cagcactaag 2520

2580

aaaagggaag tgaaagggat tcccccattc tgagggattg tgagaagtgt gccagaatat 2640

taacttcatt tgacttgtta cagggaaagt aaacttgact ttcacggacc tcctagttac 2700

ctggtgctta ctatatgtct tctcagagta cctgattcat tcccagcctg gttgacccat 2760

ccccctatct ctatggctat gtttatccag agcacatcta tctaacactc cagctgatct 2820

tcctgacaca gctgtggcaa ccctggatcc tttaaccaac tgtgccaggc tggagatcaa 2880

acctaagcct ctgcagcaac ccaagctgct gcagtcagat ttttaacccc ctgtgccact 2940

gtgggtatct ccgatatttt gtatcttctg tgactgagtg gtttgctgtt tgcagggaac 3000

cagagtcaga cactatcccc gtgcaattca tcatcctcgg acccatcaag ctctattatt 3060

tcagaagaaa atggtgttgc ctgcataggt gagaatcagt gaccaaccta tgaaaatgat 3120

ctcaatcctc tgaaatgcat tttattcatg ttttatttcc tctttgcagg gagtggtcaa 3180

cttcgcctgg tcgatggagg tggtcgttgt gctgggagag tagaggtcta tcatgagggc 3240

tcctggggca ccatctgtga tgacagctgg gacctgaatg atgcccatgt ggtgtgcaaa 3300

cagctgagct gtggatgggc cattaatgcc actggttctg ctcattttgg ggaaggaaca 3360

gggcccattt ggctggatga gataaactgt aatggaaaag aatctcatat ttggcaatgc 3420

cactcacatg gttgggggcg gcacaattgc aggcataagg aggatgcagg agtcatctgc 3480

tcgg 3484

<210> 99

<211> 4997

<212> DNA

<213> Sus scrofa

<400> 99

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060

aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120

tgctgtgcag ggaactacag tgcggctact actactgtgg tttccctcct ggggggagct 3180

cactttggag aaggaagtgg acagatctgg gctgaagaat tccagtgtga ggggcacgag 3240

tcccaccttt cactctgccc agtagcaccc cgccctgacg ggacatgtag ccacagcagg 3300

gacgtcggcg tagtctgctc aagtgagacc cagggaatgt gttcactttg ttcccatgcc 3360

atgaagaggg tagggttagg tagtcacaga catcttttta aagccctgtc tccttccagg 3420

atacacacaa atccgcttgg tgaatggcaa gaccccatgt gaaggaagag tggagctcaa 3480

cattcttggg tcctgggggt ccctctgcaa ctctcactgg gacatggaag atgcccatgt 3540

tttatgccag cagcttaaat gtggagttgc cctttctatc ccgggaggag caccttttgg 3600

gaaaggaagt gagcaggtct ggaggcacat gtttcactgc actgggactg agaagcacat 3660

gggagattgt tccgtcactg ctctgggcgc atcactctgt tcttcagggc aagtggcctc 3720

tgtaatctgc tcaggtaaga gaataagggc agccagtgat gagccactca tgacggtgcc 3780

ttaagagtgg gtgtacctag gagttcccat tgtggctcag tggtaacaaa ctcgactggt 3840

atccatgagg gtatgggttt gatccctggc cttgctcaat gggttaagga tccagcattg 3900

ctgtgagctg tggtataggt tgcagactct gctcaggtcc catgttgctg tgattgtggt 3960

gtaggctgac tgctgcagct tcaatttgac ccctagcccg ggaatttcca taggccacac 4020

4080

gaagaacaga tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag 4140

tgtgccagaa tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg 4200

acctcctagt tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc 4260

ctggttgacc catccccta tctctatggc tatgtttatc cagagcacat ctatctaaca 4320

ctccagctga tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca 4380

ggctggagat caaacctaag ccctgcagc aacccaagct gctgcagtca gatttttaac 4440

cccctgtgcc actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct 4500

gtttgcaggg aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc 4560

aagctctatt atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac 4620

ctatgaaaat gatctcaatc ctctgaaatg cattttattc atgttttatt tcctctttgc 4680

agggagtggt caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt 4740

ctatcatgag ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca 4800

tgtggtgtgc aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt 4860

tggggaagga acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca 4920

tatttggcaa tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc 4980

aggagtcatc tgctcgg 4997

<210> 100

<211> 3710

<212> DNA

<213> Sus scrofa

<400> 100

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaagggaa 2820

agggattccc ccattctgag ggattgtgag aagtgtgcca gaatattaac ttcatttgac 2880

ttgttacagg gaaagtaaac ttgactttca cggacctcct agttacctgg tgcttactat 2940

atgtcttctc agagtacctg attcattccc agcctggttg acccatcccc ctatctctat 3000

ggctatgttt atccagagca catctatcta acactccagc tgatcttcct gacacagctg 3060

tggcaaccct ggatccttta accaactgtg ccaggctgga gatcaaacct aagcctctgc 3120

agcaacccaa gctgctgcag tcagattttt aaccccctgt gccactgtgg gtatctccga 3180

tattttgtat cttctgtgac tgagtggttt gctgtttgca gggaaccaga gtcagacact 3240

atccccgtgc aattcatcat cctcggaccc atcaagctct attatttcag aagaaaatgg 3300

tgttgcctgc ataggtgaga atcagtgacc aacctatgaa aatgatctca atcctctgaa 3360

atgcatttta ttcatgtttt atttcctctt tgcagggagt ggtcaacttc gcctggtcga 3420

tggaggtggt cgttgtgctg ggagagtaga ggtctatcat gagggctcct ggggcaccat 3480

ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg 3540

atgggccatt aatgccactg gttctgctca ttttggggaa ggaacagggc ccatttggct 3600

ggatgagata aactgtaatg gaaaagaatc tcatatttgg caatgccact cacatggttg 3660

ggggcggcac aattgcaggc ataaggagga tgcaggagtc atctgctcgg 3710

<210> 101

<211> 3617

<212> DNA

<213> Sus scrofa

<400> 101

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

2760

tgtgccagaa tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg 2820

acctcctagt tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc 2880

ctggttgacc catccccta tctctatggc tatgtttatc cagagcacat ctatctaaca 2940

ctccagctga tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca 3000

ggctggagat caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac 3060

cccctgtgcc actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct 3120

gtttgcaggg aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc 3180

aagctctatt atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac 3240

ctatgaaaat gatctcaatc ctctgaaatg cattttattc atgttttatt tcctctttgc 3300

agggagtggt caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt 3360

ctatcatgag ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca 3420

tgtggtgtgc aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt 3480

tggggaagga acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca 3540

tatttggcaa tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc 3600

aggagtcatc tgctcgg 3617

<210> 102

<211> 4979

<212> DNA

<213> Sus scrofa

<400> 102

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060

aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120

tgctgtgcag ggaactctgt ggtttccctc ctggggggag ctcactttgg agaaggaagt 3180

ggacagatct gggctgaaga attccagtgt gaggggcacg agtcccacct ttcactctgc 3240

ccagtagcac cccgccctga cgggacatgt agccacagca gggacgtcgg cgtagtctgc 3300

tcaagtgaga cccagggaat gtgttcactt tgttcccatg ccatgaagag ggtagggtta 3360

ggtagtcaca gacatctttt taaagccctg tctccttcca ggatacacac aaatccgctt 3420

ggtgaatggc aagaccccat gtgaaggaag agtggagctc aacattcttg ggtcctgggg 3480

gtccctctgc aactctcact gggacatgga agatgcccat gttttatgcc agcagcttaa 3540

atgtggagtt gccctttcta tcccgggagg agcacctttt gggaaaggaa gtgagcaggt 3600

3660

tgctctgggc gcatcactct gttcttcagg gcaagtggcc tctgtaatct gctcaggtaa 3720

gagaataagg gcagccagtg atgagccact catgacggtg ccttaagagt gggtgtacct 3780

aggagttccc attgtggctc agtggtaaca aactcgactg gtatccatga gggtatgggt 3840

ttgatccctg gccttgctca atgggttaag gatccagcat tgctgtgagc tgtggtatag 3900

gttgcagact ctgctcaggt cccatgttgc tgtgattgtg gtgtaggctg actgctgcag 3960

cttcaatttg acccctagcc cgggaatttc cataggccac acgtgcagca ctaaggaagg 4020

4080

ggaagtgaaa gggattcccc cattctgagg gattgtgaga agtgtgccag aatattaact 4140

tcatttgact tgttacaggg aaagtaaact tgactttcac ggacctccta gttacctggt 4200

gcttactata tgtcttctca gagtacctga ttcattccca gcctggttga cccatccccc 4260

tatctctatg gctatgttta tccagagcac atctatctaa cactccagct gatcttcctg 4320

acacagctgt ggcaaccctg gatcctttaa ccaactgtgc caggctggag atcaaaccta 4380

agcctctgca gcaacccaag ctgctgcagt cagattttta accccctgtg ccactgtggg 4440

tatctccgat attttgtatc ttctgtgact gagtggtttg ctgtttgcag ggaaccagag 4500

tcagacacta tccccgtgca attcatcatc ctcggaccca tcaagctcta ttatttcaga 4560

agaaaatggt gttgcctgca taggtgagaa tcagtgacca acctatgaaa atgatctcaa 4620

4680

cctggtcgat ggaggtggtc gttgtgctgg gagagtagag gtctatcatg agggctcctg 4740

gggcaccatc tgtgatgaca gctgggacct gaatgatgcc catgtggtgt gcaaacagct 4800

gagctgtgga tgggccatta atgccactgg ttctgctcat tttggggaag gaacagggcc 4860

catttggctg gatgagataa actgtaatgg aaaagaatct catatttggc aatgccactc 4920

acatggttgg gggcggcaca attgcaggca taaggaggat gcaggagtca tctgctcgg 4979

<210> 103

<211> 4615

<212> DNA

<213> Sus scrofa

<400> 103

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aagaaggaaa 2580

atattggaat catattctcc ctcaccgaaa tgctattttt cagcccacag gaaacccagg 2640

ctggttggag gggacattcc ctgctctggt cgtgttgaag tacaacatgg agacacgtgg 2700

ggcaccgtct gtgattctga cttctctctg gaggcggcca gcgtgctgtg cagggaacta 2760

cagtgcggca cagtgtggtt tccctcctgg ggggagctca ctttggagaa ggaagtggac 2820

agatctgggc tgaagaattc cagtgtgagg ggcacgagtc ccacctttca ctctgcccag 2880

tagcaccccg ccctgacggg acatgtagcc acagcaggga cgtcggcgta gtctgctcaa 2940

gtgagaccca gggaatgtgt tcactttgtt cccatgccat gaagagggta gggttaggta 3000

gtcacagaca tctttttaaa gccctgtctc cttccaggat acacacaaat ccgcttggtg 3060

aatggcaaga ccccatgtga aggaagagtg gagctcaaca ttcttggggtc ctgggggtcc 3120

3180

ggagttgccc tttctatccc gggaggagca ccttttggga aaggaagtga gcaggtctgg 3240

aggcacatgt ttcactgcac tgggactgag aagcacatgg gagattgttc cgtcactgct 3300

ctgggcgcat cactctgttc ttcagggcaa gtggcctctg taatctgctc aggtaagaga 3360

ataagggcag ccagtgatga gccactcatg acggtgcctt aagagtggggt gtacctagga 3420

gttcccattg tggctcagtg gtaacaaact cgactggtat ccatgagggt atgggtttga 3480

tccctggcct tgctcaatgg gttaaggatc cagcattgct gtgagctgtg gtataggttg 3540

cagactctgc tcaggtccca tgttgctgtg attgtggtgt aggctgactg ctgcagcttc 3600

aatttgaccc ctagcccggg aatttccata ggccacacgt gcagcactaa ggaaggaaaa 3660

3720

gtgaaaggga ttcccccatt ctgagggatt gtgagaagtg tgccagaata ttaacttcat 3780

ttgacttgtt acagggaaag taaacttgac tttcacggac ctcctagtta cctggtgctt 3840

actatatgtc ttctcagagt acctgattca ttcccagcct ggttgaccca tccccctatc 3900

3960

agctgtggca accctggatc ctttaaccaa ctgtgccagg ctggagatca aacctaagcc 4020

tctgcagcaa cccaagctgc tgcagtcaga tttttaaccc cctgtgccac tgtgggtatc 4080

tccgatattt tgtatcttct gtgactgagt ggtttgctgt ttgcagggaa ccagagtcag 4140

acactatccc cgtgcaattc atcatcctcg gacccatcaa gctctattat ttcagaagaa 4200

aatggtgttg cctgcatagg tgagaatcag tgaccaacct atgaaaatga tctcaatcct 4260

ctgaaatgca tttttattcat gttttatttc ctctttgcag ggagtggtca acttcgcctg 4320

gtcgatggag gtggtcgttg tgctgggaga gtagaggtct atcatgaggg ctcctggggc 4380

accatctgtg atgacagctg ggacctgaat gatgcccatg tggtgtgcaa acagctgagc 4440

tgtggatggg ccattaatgc cactggttct gctcattttg gggaaggaac agggcccatt 4500

tggctggatg agataaactg taatggaaaa gaatctcata tttggcaatg ccactcacat 4560

ggttgggggc ggcacaattg caggcataag gaggatgcag gagtcatctg ctcgg 4615

<210> 104

<211> 4866

<212> DNA

<213> Sus scrofa

<400> 104

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttctgtggt ttccctcctg gggggagctc actttggaga 3060

aggaagtgga cagatctggg ctgaagaatt ccagtgtgag gggcacgagt cccacctttc 3120

actctgccca gtagcacccc gccctgacgg gacatgtagc cacagcaggg acgtcggcgt 3180

3240

agggttaggt agtcacagac atctttttaa agccctgtct ccttccagga tacacacaaa 3300

tccgcttggt gaatggcaag accccatgtg aaggaagagt ggagctcaac attcttgggt 3360

cctgggggtc cctctgcaac tctcactggg acatggaaga tgcccatgtt ttatgccagc 3420

agcttaaatg tggagttgcc ctttctatcc cgggaggagc accttttgggg aaaggaagtg 3480

agcaggtctg gaggcacatg tttcactgca ctgggactga gaagcacatg ggagattgtt 3540

ccgtcactgc tctgggcgca tcactctgtt cttcagggca agtggcctct gtaatctgct 3600

caggtaagag aataagggca gccagtgatg agccactcat gacggtgcct taagagtggg 3660

tgtacctagg agttcccatt gtggctcagt ggtaacaaac tcgactggta tccatgaggg 3720

tatgggtttg atccctggcc ttgctcaatg ggttaaggat ccagcattgc tgtgagctgt 3780

ggtataggtt gcagactctg ctcaggtccc atgttgctgt gattgtggtg taggctgact 3840

gctgcagctt caatttgacc cctagcccgg gaatttccat aggccacacg tgcagcacta 3900

aggaaggaaa aaaagaaaaa aaaaaaaaaa gagtgggtgt gcctatagtg aagaacagat 3960

gtaaaaggga agtgaaaggg attcccccat tctgagggat tgtgagaagt gtgccagaat 4020

4080

acctggtgct tactatatgt cttctcagag tacctgattc attcccagcc tggttgaccc 4140

atccccctat ctctatggct atgtttatcc agagcacatc tatctaacac tccagctgat 4200

cttcctgaca cagctgtggc aaccctggat cctttaacca actgtgccag gctggagatc 4260

aaacctaagc ctctgcagca acccaagctg ctgcagtcag atttttaacc ccctgtgcca 4320

4380

accagagtca gacactatcc ccgtgcaatt catcatcctc ggacccatca agctctatta 4440

tttcagaaga aaatggtgtt gcctgcatag gtgagaatca gtgaccaacc tatgaaaatg 4500

atctcaatcc tctgaaatgc attttattca tgttttattt cctctttgca gggagtggtc 4560

aacttcgcct ggtcgatgga ggtggtcgtt gtgctgggag agtagaggtc tatcatgagg 4620

gctcctgggg caccatctgt gatgacagct gggacctgaa tgatgcccat gtggtgtgca 4680

aacagctgag ctgtggatgg gccattaatg ccactggttc tgctcatttt ggggaaggaa 4740

cagggcccat ttggctggat gagataaact gtaatggaaa agaatctcat atttggcaat 4800

gccactcaca tggttggggg cggcacaatt gcaggcataa ggaggatgca ggagtcatct 4860

gctcgg 4866

<210> 105

<211> 4867

<212> DNA

<213> Sus scrofa

<400> 105

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttactgtgg tttccctcct ggggggagct cactttggag 3060

aaggaagtgg acagatctgg gctgaagaat tccagtgtga ggggcacgag tcccaccttt 3120

cactctgccc agtagcaccc cgccctgacg ggacatgtag ccacagcagg gacgtcggcg 3180

tagtctgctc aagtgagacc cagggaatgt gttcactttg ttcccatgcc atgaagaggg 3240

tagggttagg tagtcacaga catcttttta aagccctgtc tccttccagg atacacacaa 3300

atccgcttgg tgaatggcaa gaccccatgt gaaggaagag tggagctcaa cattcttggg 3360

tcctgggggt ccctctgcaa ctctcactgg gacatggaag atgcccatgt tttatgccag 3420

cagcttaaat gtggagttgc cctttctatc ccgggaggag caccttttgg gaaaggaagt 3480

gagcaggtct ggaggcacat gtttcactgc actgggactg agaagcacat gggagattgt 3540

tccgtcactg ctctgggcgc atcactctgt tcttcagggc aagtggcctc tgtaatctgc 3600

tcaggtaaga gaataagggc agccagtgat gagccactca tgacggtgcc ttaagagtgg 3660

gtgtacctag gagttcccat tgtggctcag tggtaacaaa ctcgactggt atccatgagg 3720

gtatgggttt gatccctggc cttgctcaat gggttaagga tccagcattg ctgtgagctg 3780

tggtataggt tgcagactct gctcaggtcc catgttgctg tgattgtggt gtaggctgac 3840

tgctgcagct tcaatttgac ccctagcccg ggaatttcca taggccacac gtgcagcact 3900

aaggaaggaa aaaaagaaaa aaaaaaaaaa agagtggggtg tgcctatagt gaagaacaga 3960

tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag tgtgccagaa 4020

tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg acctcctagt 4080

tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc ctggttgacc 4140

catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca ctccagctga 4200

tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca ggctggagat 4260

caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac cccctgtgcc 4320

actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct gtttgcaggg 4380

aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc aagctctatt 4440

atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac ctatgaaaat 4500

gatctcaatc ctctgaaatg cattttattc atgttttatt tcctctttgc agggagtggt 4560

caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt ctatcatgag 4620

ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca tgtggtgtgc 4680

aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt tggggaagga 4740

acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca tatttggcaa 4800

tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc aggagtcatc 4860

tgctcgg 4867

<210> 106

<211> 4991

<212> DNA

<213> Sus scrofa

<400> 106

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060

aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120

tgctgtgcag ggaactacag tgcggcaact gtggtttccc tcctgggggg agctcacttt 3180

ggagaaggaa gtggacagat ctgggctgaa gaattccagt gtgaggggca cgagtcccac 3240

ctttcactct gcccagtagc accccgccct gacgggacat gtagccacag cagggacgtc 3300

ggcgtagtct gctcaagtga gacccaggga atgtgttcac tttgttccca tgccatgaag 3360

agggtagggt taggtagtca cagacatctt tttaaagccc tgtctccttc caggatacac 3420

acaaatccgc ttggtgaatg gcaagacccc atgtgaagga agagtggagc tcaacattct 3480

tgggtcctgg gggtccctct gcaactctca ctgggacatg gaagatgccc atgttttatg 3540

ccagcagctt aaatgtggag ttgccctttc tatcccggga ggagcacctt ttgggaaagg 3600

3660

ttgttccgtc actgctctgg gcgcatcact ctgttcttca gggcaagtgg cctctgtaat 3720

ctgctcaggt aagagaataa gggcagccag tgatgagcca ctcatgacgg tgccttaaga 3780

3840

gagggtatgg gtttgatccc tggccttgct caatgggtta aggatccagc attgctgtga 3900

gctgtggtat aggttgcaga ctctgctcag gtcccatgtt gctgtgattg tggtgtaggc 3960

tgactgctgc agcttcaatt tgacccctag cccgggaatt tccataggcc acacgtgcag 4020

4080

cagatgtaaa agggaagtga aagggattcc cccattctga gggattgtga gaagtgtgcc 4140

agaatattaa cttcatttga cttgttacag ggaaagtaaa cttgactttc acggacctcc 4200

tagttacctg gtgcttacta tatgtcttct cagagtacct gattcattcc cagcctggtt 4260

gacccatccc cctatctcta tggctatgtt tatccagagc acatctatct aacactccag 4320

ctgatcttcc tgacacagct gtggcaaccc tggatccttt aaccaactgt gccaggctgg 4380

agatcaaacc taagcctctg cagcaaccca agctgctgca gtcagatttt taaccccctg 4440

tgccactgtg ggtatctccg atattttgta tcttctgtga ctgagtggtt tgctgtttgc 4500

agggaaccag agtcagacac tatccccgtg caattcatca tcctcggacc catcaagctc 4560

tattatttca gaagaaaatg gtgttgcctg cataggtgag aatcagtgac caacctatga 4620

aaatgatctc aatcctctga aatgcatttt attcatgttt tatttcctct ttgcagggag 4680

tggtcaactt cgcctggtcg atggaggtgg tcgttgtgct gggagagtag aggtctatca 4740

tgagggctcc tggggcacca tctgtgatga cagctgggac ctgaatgatg cccatgtggt 4800

gtgcaaacag ctgagctgtg gatggggccat taatgccact ggttctgctc attttgggga 4860

aggaacaggg cccatttggc tggatgagat aaactgtaat ggaaaagaat ctcatatttg 4920

gcaatgccac tcacatggtt gggggcggca caattgcagg cataaggagg atgcaggagt 4980

catctgctcg g 4991

<210> 107

<211> 4860

<212> DNA

<213> Sus scrofa

<400> 107

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggt cctgggggga gctcactttg gagaaggaag 3060

tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc tttcactctg 3120

3180

ctcaagtgag acccagggaa tgtgttcact ttgttcccat gccatgaaga gggtagggtt 3240

aggtagtcac agacatcttt ttaaagccct gtctccttcc aggatacaca caaatccgct 3300

tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt gggtcctggg 3360

ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc cagcagctta 3420

3480

3540

ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc tgctcaggta 3600

agagaataag ggcagccagt gatgagccac tcatgacggt gccttaagag tgggtgtacc 3660

taggagttcc cattgtggct cagtggtaac aaactcgact ggtatccatg agggtatggg 3720

3780

ggttgcagac tctgctcagg tcccatgttg ctgtgattgt ggtgtaggct gactgctgca 3840

gcttcaattt gacccctagc ccgggaattt ccataggcca cacgtgcagc actaaggaag 3900

3960

gggaagtgaa agggattccc ccattctgag ggattgtgag aagtgtgcca gaatattaac 4020

ttcatttgac ttgttacagg gaaagtaaac ttgactttca cggacctcct agttacctgg 4080

tgcttactat atgtcttctc agagtacctg attcattccc agcctggttg acccatcccc 4140

ctatctctat ggctatgttt atccagagca catctatcta acactccagc tgatcttcct 4200

gacacagctg tggcaaccct ggatccttta accaactgtg ccaggctgga gatcaaacct 4260

aagcctctgc agcaacccaa gctgctgcag tcagattttt aaccccctgt gccactgtgg 4320

gtatctccga tattttgtat cttctgtgac tgagtggttt gctgtttgca gggaaccaga 4380

gtcagacact atccccgtgc aattcatcat cctcggaccc atcaagctct atttttcag 4440

aagaaaatgg tgttgcctgc ataggtgaga atcagtgacc aacctatgaa aatgatctca 4500

atcctctgaa atgcatttta ttcatgtttt atttcctctt tgcagggagt ggtcaacttc 4560

gcctggtcga tggaggtggt cgttgtgctg ggagagtaga ggtctatcat gagggctcct 4620

ggggcaccat ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc 4680

tgagctgtgg atgggccatt aatgccactg gttctgctca ttttggggaa ggaacagggc 4740

ccatttggct ggatgagata aactgtaatg gaaaagaatc tcatatttgg caatgccact 4800

cacatggttg ggggcggcac aattgcaggc ataaggagga tgcaggagtc atctgctcgg 4860

<210> 108

<211> 4858

<212> DNA

<213> Sus scrofa

<400> 108

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggc tggggggagc tcactttgga gaaggaagtg 3060

gacagatctg ggctgaagaa ttccagtgtg aggggcacga gtcccacctt tcactctgcc 3120

cagtagcacc ccgccctgac gggacatgta gccacagcag ggacgtcggc gtagtctgct 3180

caagtgagac ccagggaatg tgttcacttt gttcccatgc catgaagagg gtagggttag 3240

gtagtcacag acatcttttt aaagccctgt ctccttccag gatacacaca aatccgcttg 3300

gtgaatggca agaccccatg tgaaggaaga gtggagctca acattcttgg gtcctggggg 3360

tccctctgca actctcactg ggacatggaa gatgcccatg ttttatgcca gcagcttaaa 3420

tgtggagttg ccctttctat cccgggagga gcaccttttg ggaaaggaag tgagcaggtc 3480

tggaggcaca tgtttcactg cactgggact gagaagcaca tgggagattg ttccgtcact 3540

gctctgggcg catcactctg ttcttcaggg caagtggcct ctgtaatctg ctcaggtaag 3600

agaataaggg cagccagtga tgagccactc atgacggtgc cttaagagtg ggtgtaccta 3660

ggagttccca ttgtggctca gtggtaacaa actcgactgg tatccatgag ggtatgggtt 3720

tgatccctgg ccttgctcaa tgggttaagg atccagcatt gctgtgagct gtggtatagg 3780

ttgcagactc tgctcaggtc ccatgttgct gtgattgtgg tgtaggctga ctgctgcagc 3840

ttcaatttga cccctagccc gggaatttcc ataggccaca cgtgcagcac taaggaagga 3900

3960

4020

catttgactt gttacaggga aagtaaactt gactttcacg gacctcctag ttacctggtg 4080

cttactatat gtcttctcag agtacctgat tcattcccag cctggttgac ccatccccct 4140

atctctatgg ctatgtttat ccagagcaca tctatctaac actccagctg atcttcctga 4200

cacagctgtg gcaaccctgg atcctttaac caactgtgcc aggctggaga tcaaacctaa 4260

gcctctgcag caacccaagc tgctgcagtc agatttttaa ccccctgtgc cactgtgggt 4320

atctccgata ttttgtatct tctgtgactg agtggtttgc tgtttgcagg gaaccagagt 4380

cagacactat ccccgtgcaa ttcatcatcc tcggacccat caagctctat tatttcagaa 4440

gaaaatggtg ttgcctgcat aggtgagaat cagtgaccaa cctatgaaaa tgatctcaat 4500

cctctgaaat gcattttatt catgttttat ttcctctttg cagggagtgg tcaacttcgc 4560

ctggtcgatg gaggtggtcg ttgtgctgggg agagtagagg tctatcatga gggctcctgg 4620

ggcaccatct gtgatgacag ctgggacctg aatgatgccc atgtggtgtg caaacagctg 4680

agctgtggat gggccattaa tgccactggt tctgctcatt ttggggaagg aacagggccc 4740

atttggctgg atgagataaa ctgtaatgga aaagaatctc atatttggca atgccactca 4800

catggttggg ggcggcacaa ttgcaggcat aaggaggatg caggagtcat ctgctcgg 4858

<210> 109

<211> 3523

<212> DNA

<213> Sus scrofa

<400> 109

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gagctgtggt ataggttgca gactctgctc 2460

aggtcccatg ttgctgtgat tgtggtgtag gctgactgct gcagcttcaa tttgacccct 2520

agcccgggaa tttccatagg ccacacgtgc agcactaagg aaggaaaaaa agaaaaaaaa 2580

2640

cccccattct gagggattgt gagaagtgtg ccagaatatt aacttcattt gacttgttac 2700

agggaaagta aacttgactt tcacggacct cctagttacc tggtgcttac tatatgtctt 2760

ctcagagtac ctgattcatt cccagcctgg ttgacccatc cccctatctc tatggctatg 2820

tttatccaga gcacatctat ctaacactcc agctgatctt cctgacacag ctgtggcaac 2880

cctggatcct ttaaccaact gtgccaggct ggagatcaaa cctaagcctc tgcagcaacc 2940

caagctgctg cagtcagatt tttaaccccc tgtgccactg tgggtatctc cgatattttg 3000

3060

tgcaattcat catcctcgga cccatcaagc tctattattt cagaagaaaa tggtgttgcc 3120

tgcataggtg agaatcagtg accaacctat gaaaatgatc tcaatcctct gaaatgcatt 3180

ttattcatgt tttatttcct ctttgcaggg agtggtcaac ttcgcctggt cgatggaggt 3240

ggtcgttgtg ctgggagagt agaggtctat catgagggct cctggggcac catctgtgat 3300

gacagctggg acctgaatga tgcccatgtg gtgtgcaaac agctgagctg tggatgggcc 3360

attaatgcca ctggttctgc tcattttggg gaaggaacag ggcccatttg gctggatgag 3420

ataaactgta atggaaaaga atctcatatt tggcaatgcc actcacatgg ttgggggcgg 3480

cacaattgca ggcataagga ggatgcagga gtcatctgct cgg 3523

<210> 110

<211> 3603

<212> DNA

<213> Sus scrofa

<400> 110

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgttgt ggagaattcc acaagaattc gtgttatttg acagcagtca tctttaaaag 540

gcatttgaga aagtccaatt tcaaatgcat ttccttttctt taaaagataa attgaagaaa 600

ataagtcttt atttcccaag taaattgaat tgcctctcag tctgttaaaa gaaactctta 660

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 720

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 780

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 840

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 900

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 960

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 1020

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 1080

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 1140

gagatgttgt ggagaattcc acaagaattc gtgttatttg acagcagtca tctttaaaag 1200

gcatttgaga aagtccaatt tcaaatgcat ttccttttctt taaaagataa attgaagaaa 1260

ataagtcttt atttcccaag taaattgaat tgcctctcag tctgttaaaa gaaactctta 1320

ccttgatgat tgcgctctta acctggcaaa gattgtcttt aaaatctgag ctccatgtct 1380

tctgctttat ttctggtgtg cctttgactc cagattacag taaatggagg actgagtata 1440

gggctaaaaa gtagagagaa tggatgcata ttatctgtgg tctccaatgt gatgaatgaa 1500

gtaggcaaat actcaaagga aagagaaagc atgctccaag aattatgggt tccagaaggc 1560

aaagtcccag aattgtctcc agggaaggac agggaggtct agaatcggct aagcccactg 1620

taggcagaaa aaccaagagg catgaatggc ttccctttct cacttttcac tctctggctt 1680

actcctatca tgaaggaaaa tattggaatc atattctccc tcaccgaaat gctatttttc 1740

agcccacagg aaacccaggc tggttggagg ggacattccc tgctctcact ttggagaagg 1800

aagtggacag atctgggctg aagaattcca gtgtgagggg cacgagtccc acctttcact 1860

ctgcccagta gcaccccgcc ctgacgggac atgtagccac agcagggacg tcggcgtagt 1920

ctgctcaagt gagacccagg gaatgtgttc actttgttcc catgccatga agagggtagg 1980

gttaggtagt cacagacatc tttttaaagc cctgtctcct tccaggatac acacaaatcc 2040

gcttggtgaa tggcaagacc ccatgtgaag gaagagtgga gctcaacatt cttgggtcct 2100

gggggtccct ctgcaactct cactgggaca tggaagatgc ccatgtttta tgccagcagc 2160

ttaaatgtgg agttgccctt tctatcccgg gaggagcacc ttttgggaaa ggaagtgagc 2220

aggtctggag gcacatgttt cactgcactg ggactgagaa gcacatggga gattgttccg 2280

tcactgctct gggcgcatca ctctgttctt cagggcaagt ggcctctgta atctgctcag 2340

gtaagagaat aagggcagcc agtgatgagc cactcatgac ggtgccttaa gagtgggtgt 2400

acctaggagt tcccattgtg gctcagtggt aacaaactcg actggtatcc atgagggtat 2460

gggtttgatc cctggccttg ctcaatgggt taaggatcca gcattgctgt gagctgtggt 2520

ataggttgca gactctgctc aggtcccatg ttgctgtgat tgtggtgtag gctgactgct 2580

2640

2700

aaagggaagt gaaagggatt cccccattct gagggattgt gagaagtgtg ccagaatatt 2760

aacttcattt gacttgttac agggaaagta aacttgactt tcacggacct cctagttacc 2820

tggtgcttac tatatgtctt ctcagagtac ctgattcatt cccagcctgg ttgacccatc 2880

cccctatctc tatggctatg tttatccaga gcacatctat ctaacactcc agctgatctt 2940

cctgacacag ctgtggcaac cctggatcct ttaaccaact gtgccaggct ggagatcaaa 3000

3060 cctaagcctc tgcagcaacc caagctgctg cagtcagatt

tgggtatctc cgatattttg tatcttctgt gactgagtgg tttgctgttt gcagggaacc 3120

agagtcagac actatccccg tgcaattcat catcctcgga cccatcaagc tctattattt 3180

cagaagaaaa tggtgttgcc tgcataggtg agaatcagtg accaacctat gaaaatgatc 3240

tcaatcctct gaaatgcatt ttattcatgt tttatttcct ctttgcaggg agtggtcaac 3300

ttcgcctggt cgatgggaggt ggtcgttgtg ctgggagagt agaggtctat catgagggct 3360

cctggggcac catctgtgat gacagctggg acctgaatga tgcccatgtg gtgtgcaaac 3420

agctgagctg tggatgggcc attaatgcca ctggttctgc tcattttggg gaaggaacag 3480

ggcccatttg gctggatgag ataaactgta atggaaaaga atctcatatt tggcaatgcc 3540

actcacatgg ttgggggcgg cacaattgca ggcataagga ggatgcagga gtcatctgct 3600

cgg 3603

<210> 111

<211> 4962

<212> DNA

<213> Sus scrofa

<400> 111

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060

aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120

tgctgtgcag ggaactacag tgcgtcactt tggagaagga agtggacaga tctgggctga 3180

agaattccag tgtgaggggc acgagtccca cctttcactc tgcccagtag caccccgccc 3240

tgacgggaca tgtagccaca gcagggacgt cggcgtagtc tgctcaagtg agacccaggg 3300

aatgtgttca ctttgttccc atgccatgaa gagggtaggg ttaggtagtc acagacatct 3360

ttttaaagcc ctgtctcctt ccaggataca cacaaatccg cttggtgaat ggcaagaccc 3420

catgtgaagg aagagtggag ctcaacattc ttgggtcctg ggggtccctc tgcaactctc 3480

actgggacat ggaagatgcc catgttttat gccagcagct taaatgtgga gttgcccttt 3540

ctatcccgggg aggagcacct tttgggaaag gaagtgagca ggtctggagg cacatgtttc 3600

actgcactgg gactgagaag cacatgggag attgttccgt cactgctctg ggcgcatcac 3660

tctgttcttc agggcaagtg gcctctgtaa tctgctcagg taagagaata agggcagcca 3720

gtgatgagcc actcatgacg gtgccttaag agtgggtgta cctaggagtt cccattgtgg 3780

ctcagtggta acaaactcga ctggtatcca tgagggtatg ggtttgatcc ctggccttgc 3840

tcaatgggtt aaggatccag cattgctgtg agctgtggta taggttgcag actctgctca 3900

ggtcccatgt tgctgtgatt gtggtgtagg ctgactgctg cagcttcaat ttgaccccta 3960

gcccgggaat ttccataggc cacacgtgca gcactaagga aggaaaaaaa gaaaaaaaaa 4020

aaaaaagagt gggtgtgcct atagtgaaga acagatgtaa aagggaagtg aaagggattc 4080

ccccattctg agggattgtg agaagtgtgc cagaatatta acttcatttg acttgttaca 4140

gggaaagtaa acttgacttt cacggacctc ctagttacct ggtgcttact atatgtcttc 4200

tcagagtacc tgattcattc ccagcctggt tgacccatcc ccctatctct atggctatgt 4260

ttatccagag cacatctatc taacactcca gctgatcttc ctgacacagc tgtggcaacc 4320

ctggatcctt taaccaactg tgccaggctg gagatcaaac ctaagcctct gcagcaaccc 4380

aagctgctgc agtcagattt ttaaccccct gtgccactgt gggtatctcc gatattttgt 4440

atcttctgtg actgagtggt ttgctgtttg cagggaacca gagtcagaca ctatccccgt 4500

gcaattcatc atcctcggac ccatcaagct ctattatttc agaagaaaat ggtgttgcct 4560

gcataggtga gaatcagtga ccaacctatg aaaatgatct caatcctctg aaatgcattt 4620

tattcatgtt ttatttcctc tttgcaggga gtggtcaact tcgcctggtc gatggaggtg 4680

gtcgttgtgc tgggagagta gaggtctatc atgagggctc ctggggcacc atctgtgatg 4740

acagctggga cctgaatgat gcccatgtgg tgtgcaaaca gctgagctgt ggatgggcca 4800

ttaatgccac tggttctgct cattttgggg aaggaacagg gcccatttgg ctggatgaga 4860

taaactgtaa tggaaaagaa tctcatattt ggcaatgcca ctcacatggt tgggggcggc 4920

acaattgcag gcataaggag gatgcaggag tcatctgctc gg 4962

<210> 112

<211> 3603

<212> DNA

<213> Sus scrofa

<400> 112

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060

aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120

tgctgtgcag ggaactacag tgcgattcat catcctcgga cccatcaagc tctattattt 3180

cagaagaaaa tggtgttgcc tgcataggtg agaatcagtg accaacctat gaaaatgatc 3240

tcaatcctct gaaatgcatt ttattcatgt tttatttcct ctttgcaggg agtggtcaac 3300

ttcgcctggt cgatgggaggt ggtcgttgtg ctgggagagt agaggtctat catgagggct 3360

cctggggcac catctgtgat gacagctggg acctgaatga tgcccatgtg gtgtgcaaac 3420

agctgagctg tggatgggcc attaatgcca ctggttctgc tcattttggg gaaggaacag 3480

ggcccatttg gctggatgag ataaactgta atggaaaaga atctcatatt tggcaatgcc 3540

actcacatgg ttgggggcgg cacaattgca ggcataagga ggatgcagga gtcatctgct 3600

cgg 3603

<210> 113

<211> 3619

<212> DNA

<213> Sus scrofa

<400> 113

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060

aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcagccagcg 3120

tgctttgcag ggaaccagag tcagacacta tccccgtgca attcatcatc ctcggaccca 3180

tcaagctcta ttatttcaga agaaaatggt gttgcctgca taggtgagaa tcagtgacca 3240

3300

gcagggagtg gtcaacttcg cctggtcgat ggaggtggtc gttgtgctgg gagagtagag 3360

gtctatcatg agggctcctg gggcaccatc tgtgatgaca gctgggacct gaatgatgcc 3420

catgtggtgt gcaaacagct gagctgtgga tgggccatta atgccactgg ttctgctcat 3480

tttggggaag gaacagggcc catttggctg gatgagataa actgtaatgg aaaagaatct 3540

catatttggc aatgccactc acatggttgg gggcggcaca attgcaggca taaggaggat 3600

gcaggagtca tctgctcgg 3619

<210> 114

<211> 3270

<212> DNA

<213> Sus scrofa

<400> 114

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac ttgttacagg gaaagtaaac 2460

ttgactttca cggacctcct agttacctgg tgcttactat atgtcttctc agagtacctg 2520

attcattccc agcctggttg acccatcccc ctatctctat ggctatgttt atccagagca 2580

catctatcta acactccagc tgatcttcct gacacagctg tggcaaccct ggatccttta 2640

accaactgtg ccaggctgga gatcaaacct aagcctctgc agcaacccaa gctgctgcag 2700

tcagattttt aaccccctgt gccactgtgg gtatctccga tattttgtat cttctgtgac 2760

tgagtggttt gctgtttgca gggaaccaga gtcagacact atccccgtgc aattcatcat 2820

cctcggaccc atcaagctct attatttcag aagaaaatgg tgttgcctgc ataggtgaga 2880

atcagtgacc aacctatgaa aatgatctca atcctctgaa atgcatttta ttcatgtttt 2940

atttcctctt tgcagggagt ggtcaacttc gcctggtcga tggaggtggt cgttgtgctg 3000

ggagagtaga ggtctatcat gagggctcct ggggcaccat ctgtgatgac agctgggacc 3060

tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg atgggccatt aatgccactg 3120

gttctgctca ttttggggaa ggaacagggc ccatttggct ggatgagata aactgtaatg 3180

gaaaagaatc tcatatttgg caatgccact cacatggttg ggggcggcac aattgcaggc 3240

ataaggagga tgcaggagtc atctgctcgg 3270

<210> 115

<211> 7

<212> DNA

<213> Sus scrofa

<400> 115

tactact 7

<210> 116

<211> 12

<212> DNA

<213> Sus scrofa

<400> 116

tgtggagaat tc 12

<210> 117

<211> 11

<212> DNA

<213> Sus scrofa

<400> 117

agccagcgtg c 11

<210> 118

<211> 8532

<212> DNA

<213> Sus scrofa

<400> 118

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actactacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccaggctg gttggagctg acatgccctg ctctggtcgt gttgaagtaa 3060

aacatgcaga cacgtggggc tccgtctgtg attctgactt ctctctgcat gcggccaacg 3120

tgctgtgcag ggaactaaat tgcggcgatg cgatttccct ctcggtggga gatcactttg 3180

gaaaaggaaa tggactgacc tgggctgaaa aattccagtg tgaggggagc gagacccacc 3240

ttgcactctg cccaatagta caacaccctg aagacacatg tatccacagc agggaagtcg 3300

3360

ttattcataa tgtgaatgag tagtagtgga aaataactac cagtttccta agcttataac 3420

ttcgtatagc atacattata cgaagttata agcctgcagg aattctaccg ggtagggggag 3480

gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc 3540

tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct 3600

ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc 3660

cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt 3720

ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc 3780

3840

ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg 3900

gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg 3960

ggcctttcga cctgcagcca ccatggccat gattgaacag gatggcctgc atgcaggttc 4020

tccagctgcc tgggtggaga gactgtttgg ctatgactgg gcacagcaga ccattggttg 4080

ctctgatgca gcagtgttca gactgtcagc ccagggcagg ccagtcctgt ttgtgaagac 4140

agacctcagt ggggctctca atgagctgca ggatgaggct gccagactct cctggctggc 4200

aacaactggg gtcccctgtg cagctgtcct ggatgtggtc acagaagctg gaagggactg 4260

gctcctgctg ggtgaggtgc ctgggcagga cctcctgtcc tctcacctgg ctccagctga 4320

gaaagtgtca atcatggctg atgccatgag aagactccac accctggacc cagccacctg 4380

cccctttgac caccaggcca agcacaggat tgagaggggcc agaaccagga tggaggctgg 4440

cctggtggac caggatgacc tggatgaaga acaccagggc ctggcccctg ctgaactgtt 4500

tgccaggctc aaggcatcca tgccagatgg tgaggacctg gtggtgactc atggggatgc 4560

ctgcctgccc aacatcatgg tggaaaatgg aaggttctct ggcttcattg actgtggcag 4620

gctgggagtg gctgacaggt accaggacat tgccctggca accagggaca ttgcagaaga 4680

gctgggggga gaatggggcag acaggttcct ggtgctctat ggcattgcag cccctgactc 4740

ccagagaatt gccttctaca gactgctgga tgagttcttc taaatcgatt ataatcagcc 4800

ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc 4860

tgaaacataa aatgaatgca atgttgttgt taaacttgtt tattgcagct tataatggtt 4920

acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 4980

gttgtggttt gtccaaactc atcaatgtgg aaaataacta ccagtttcct aagcttataa 5040

cttcgtatag catacattat acgaagttat aagctagaca aaagtatctt accccaatgg 5100

tagccctgta cccaataaaa gtaggtgttc agtttcatat cctatgaaat accctcttga 5160

tacttttact ttgcatgagg atttagaaga aaaaagtttt actataatcc ttaacttagg 5220

aaattctttt gaattggaaa tgaaacacaa attgcttttc attgatatgc catatgatta 5280

tatgaataaa acatgaaatc ttcatattgg attctagtat atacccaagt aaatattttt 5340

tccctagaag agtgccaagt gtgttaaaac cttttggttt aataaagcag aaaaaaataa 5400

actctaaaaa tcataattaa aaatgaaatg cttttattta tagcaattaa ctacaacatg 5460

tttagactta catactatta aatataatat atttaagatc ccctcatgat aaatatgttc 5520

attattttgt aggctgttga tgcactaata tgtatgtaga ttactttgtg aattgccctt 5580

aataaaattt aaaactttag gctagtaaac ctgtaacact caacttagtt ctgaactatc 5640

5700

aagatgacaa tgtcttatat attataaaaa ctaataaaaa ccattttaaa acctagtata 5760

aatttaaagg tacttgctct tctggttcat ctcttctttt gtttacttct gctttcaaaa 5820

5880

tacttgcaag caataaatgt tatcttttta gcttttaaat ggtctcattt gaaaagaata 5940

tataattagt aagtcatagc tactttaaat aaaaacttat tctttaagag attaaacact 6000

6060

gtttttttat aatcttaaat accaataatt accaggtcta ttttgatttt gatacaggat 6120

6180

aaagttggtg atatgactga ggttttgatt attattaaac agatagttaa tatgatatat 6240

tcctcatttt tccaaatgaa aggaaaaatg tcttatatgg aggaaaagat tggggcaggg 6300

ggattagtaa attattactt aaatatctga ataggaggat ttttcaatga aaggataaag 6360

gaagaatgat tgtatcatct gaatctttcc ctccctttcc tggagtttgt ccttcaacc 6420

6480

ggtaactatt ttgttttgggt gttaatatcc aagtttccct taataacacc tagtgaatgg 6540

aggaaggatg agcataccta cccatcagac atatttagcc accatattta atcaacaagc 6600

atgaagaaag gaagctagcc tctccccttc ctttcctcct gcctctctct ctcttctctg 6660

tcctcgctcc ctttcttccc atcaatattt tcagagcacc tcttatgcgc caggcattgg 6720

gatactcaaa ctggaggaaa caagaaaaaa aaaaaaaaaa ggcgaagacc tcagggaaat 6780

tttattgct gctatatttt tttgagccta gtgtaaatta aaattcctta atgctgtgcc 6840

ttttaaaaac acaaataagc aaaatagttt atttcttcaa cagttaaatc cttagggtag 6900

gaaagtgatt caggatctat tgctactatt aactcttctt tcattttcac acaggataca 6960

cacaaatccg cttggtgaat ggcaagaccc catgtgaagg aagagtggag ctcaacattc 7020

ttgggtcctg ggggtccctc tgcaactctc actgggacat ggaagatgcc catgttttat 7080

7140

gaagtgagca ggtctggagg cacatgtttc actgcactgg gactgagaag cacatgggag 7200

attgttccgt cactgctctg ggcgcatcac tctgttcttc agggcaagtg gcctctgtaa 7260

tctgctcagg taagagaata agggcagcca gtgatgagcc actcatgacg gtgccttaag 7320

agtgggtgta cctaggagtt cccattgtgg ctcagtggta acaaactcga ctggtatcca 7380

tgagggtatg ggtttgatcc ctggccttgc tcaatgggtt aaggatccag cattgctgtg 7440

agctgtggta taggttgcag actctgctca ggtcccatgt tgctgtgatt gtggtgtagg 7500

ctgactgctg cagcttcaat ttgaccccta gcccgggaat ttccataggc cacacgtgca 7560

gcactaagga aggaaaaaaa gaaaaaaaaa aaaaaagagt gggtgtgcct atagtgaaga 7620

acagatgtaa aagggaagtg aaagggattc ccccattctg agggattgtg agaagtgtgc 7680

cagaatatta acttcatttg acttgttaca gggaaagtaa acttgacttt cacggacctc 7740

ctagttacct ggtgcttact atatgtcttc tcagagtacc tgattcattc ccagcctggt 7800

tgacccatcc ccctatctct atggctatgt ttatccagag cacatctatc taacactcca 7860

gctgatcttc ctgacacagc tgtggcaacc ctggatcctt taaccaactg tgccaggctg 7920

gagatcaaac ctaagcctct gcagcaaccc aagctgctgc agtcagattt ttaaccccct 7980

gtgccactgt gggtatctcc gatattttgt atcttctgtg actgagtggt ttgctgtttg 8040

cagggaacca gagtcagaca ctatccccgt gcaattcatc atcctcggac ccatcaagct 8100

ctattatttc agaagaaaat ggtgttgcct gcataggtga gaatcagtga ccaacctatg 8160

aaaatgatct caatcctctg aaatgcattt tattcatgtt ttatttcctc tttgcaggga 8220

gtggtcaact tcgcctggtc gatggaggtg gtcgttgtgc tgggagagta gaggtctatc 8280

atgagggctc ctggggcacc atctgtgatg acagctggga cctgaatgat gcccatgtgg 8340

tgtgcaaaca gctgagctgt ggatgggcca ttaatgccac tggttctgct cattttgggg 8400

aaggaacagg gcccatttgg ctggatgaga taaactgtaa tggaaaagaa tctcatattt 8460

ggcaatgcca ctcacatggt tgggggcggc acaattgcag gcataaggag gatgcaggag 8520

tcatctgctc gg 8532

<210> 119

<211> 4538

<212> DNA

<213> Sus scrofa

<400> 119

tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60

agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120

ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180

tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240

ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300

ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360

attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420

tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480

gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540

gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600

cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660

tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720

atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780

caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840

gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900

tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960

1020

tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080

actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140

agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200

1260

cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320

ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtgggagttc ccatcgtggc 1380

tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440

cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500

atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560

cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620

1680

1740

gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800

cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860

gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920

gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980

tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040

gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100

cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaa aaaaattaaa tcaaggactc 2160

aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220

tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280

tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340

tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400

taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460

atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520

aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580

ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640

tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700

gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760

aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820

agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880

ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940

tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000

cccacaggaa acccagctca acattcttgg gtcctggggg tccctctgca actctcactg 3060

ggacatggaa gatgcccatg ttttatgcca gcagcttaaa tgtggagttg ccctttctat 3120

3180

cactgggact gagaagcaca tgggagattg ttccgtcact gctctgggcg catcactctg 3240

ttcttcaggg caagtggcct ctgtaatctg ctcaggtaag agaataaggg cagccagtga 3300

tgagccactc atgacggtgc cttaagagtg ggtgtaccta ggagttccca ttgtggctca 3360

gtggtaacaa actcgactgg tatccatgag ggtatgggtt tgatccctgg ccttgctcaa 3420

tgggttaagg atccagcatt gctgtgagct gtggtatagg ttgcagactc tgctcaggtc 3480

ccatgttgct gtgattgtgg tgtaggctga ctgctgcagc ttcaatttga cccctagccc 3540

gggaatttcc ataggccaca cgtgcagcac taaggaagga aaaaaagaaa aaaaaaaaaa 3600

3660

attctgaggg attgtgagaa gtgtgccaga atattaactt catttgactt gttacaggga 3720

aagtaaactt gactttcacg gacctcctag ttacctggtg cttactatat gtcttctcag 3780

agtacctgat tcattcccag cctggttgac ccatccccct atctctatgg ctatgtttat 3840

ccagagcaca tctatctaac actccagctg atcttcctga cacagctgtg gcaaccctgg 3900

atcctttaac caactgtgcc aggctggaga tcaaacctaa gcctctgcag caacccaagc 3960

tgctgcagtc agatttttaa ccccctgtgc cactgtgggt atctccgata ttttgtatct 4020

tctgtgactg agtggtttgc tgtttgcagg gaaccagagt cagacactat ccccgtgcaa 4080

ttcatcatcc tcggacccat caagctctat tatttcagaa gaaaatggtg ttgcctgcat 4140

aggtgagaat cagtgaccaa cctatgaaaa tgatctcaat cctctgaaat gcattttatt 4200

catgttttat ttcctctttg cagggagtgg tcaacttcgc ctggtcgatg gaggtggtcg 4260

ttgtgctggg agagtagagg tctatcatga gggctcctgg ggcaccatct gtgatgacag 4320

ctgggacctg aatgatgccc atgtggtgtg caaacagctg agctgtggat gggccattaa 4380

tgccactggt tctgctcatt ttggggaagg aacagggccc atttggctgg atgagataaa 4440

ctgtaatgga aaagaatctc atatttggca atgccactca catggttggg ggcggcacaa 4500

ttgcaggcat aaggaggatg caggagtcat ctgctcgg 4538

<210> 120

<211> 101

<212> Protein

<213> Sus scrofa

<400> 120

Pro Arg Leu Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val

1 5 10 15

Gln His Gly Asp Thr Trp Gly Thr Val Cys Asp Ser Asp Phe Ser Leu

20 25 30

Glu Ala Ala Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val

35 40 45

Ser Leu Leu Gly Gly Ala His Phe Gly Glu Gly Ser Gly Gln Ile Trp

50 55 60

Ala Glu Glu Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys

65 70 75 80

Pro Val Ala Pro Arg Pro Asp Gly Thr Cys Ser His Ser Arg Asp Val

85 90 95

Gly Val Val Cys Ser

100

<210> 121

<211> 100

<212> Protein

<213> Homo sapiens

<400> 121

Leu Arg Leu Val Asp Gly Asp Ser Arg Cys Ala Gly Arg Val Glu Ile

1 5 10 15

Tyr His Asp Gly Phe Trp Gly Thr Ile Cys Asp Asp Gly Trp Asp Leu

20 25 30

Ser Asp Ala His Val Val Cys Gln Lys Leu Gly Cys Gly Val Ala Phe

35 40 45

Asn Ala Thr Val Ser Ala His Phe Gly Glu Gly Ser Gly Pro Ile Trp

50 55 60

Leu Asp Asp Leu Asn Cys Thr Gly Met Glu Ser His Leu Trp Gln Cys

65 70 75 80

Pro Ser Arg Gly Trp Gly Gln His Asp Cys Arg His Lys Glu Asp Ala

85 90 95

Gly Val Ile Cys

100

<---

Claims (64)

1. A method for protecting a pig fetus from infection with the porcine reproductive and respiratory syndrome virus (PRRSV), which includes breeding a female pig with a male pig, while: the female pig contains modified chromosomal sequences in both alleles of the CD163 gene, and the modified chromosomal sequence is an insertion in the gene encoding the CD163 protein, a deletion in the gene encoding the CD163 protein, or a combination thereof, wherein the insert is selected from a 7 base pair insert between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47, and a 2 base pair insert between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47; and inserting 1 base pair between nucleotides 3147 and 3148 compared to the reference sequence of SEQ ID NO: 47, and wherein the deletion is selected from an 11 base pair deletion from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47, a 1280 base pair deletion from nucleotide 2818 to nucleotide 4097 compared to the reference sequence of SEQ ID NO: 47, a deletion of 1930 base pairs nucleotide 488 to nucleotide 2417 compared to the reference sequence of SEQ ID NO: 47, which is replaced by a 12 base pair insert starting at nucleotide 488, a 129 base pair deletion in exon 7 from nucleotide 3044 to nucleotide 3172 compared to the reference sequence of SEQ ID NO: 47, deletions of 1467 base pairs from nucleotide 2431 to nucleotide 3897 compared to the reference sequence of SEQ ID NO: 47, deletions in frame in exon 7 of the gene encoding the CD163 protein, deletions of exons 7 and 8 of the gene encoding the CD163 protein, deletions in exon 7 of the gene encoding the CD163 protein, which leads to the introduction of a stop codon in exon 7, a 123 bp deletion from the nucleo 3024 to nucleotide 3146 compared to the reference sequence of SEQ ID NO: 47, deletions of 1373 base pairs from nucleotide 2724 to nucleotide 4096 compared to the reference sequence of SEQ ID NO: 47, deletions of 1720 base pairs from nucleotide 2440 to nucleotide 4160 compared to reference sequence SEQ ID NO: 47, 124 base pair deletions from nucleotide 3024 to nucleotide 3147 compared to the reference sequence SEQ ID NO: 47, 130 base pair deletions from nucleotide 3030 to nucleotide 3159 compared to the reference sequence SEQ ID NO: 47, deletions of 132 base pairs from nucleotide 3030 to nucleotide 3161 compared to the reference sequence of SEQ ID NO: 47, deletions of 28 base pairs from nucleotide 3145 to nucleotide 3172 compared to the reference sequence of SEQ ID NO: 47, wherein the modified chromosomal sequences reduce the susceptibility of the female pig to PRRSV infection compared to the susceptibility to PRRSV infection of the female pig that does not contain any of the modified chromosomal sequences in the alleles of the CD163 gene; and the male pig contains at least one wild-type CD163 allele. 2. The method of claim 1, wherein the male pig contains two wild-type CD163 alleles. 3. The method according to claim 1 or 2, characterized in that the modified chromosomal sequences reduce the sensitivity of the pig fetus to PRRSV Type 1, PRRSV Type 2, or both PRRSV Type 1 and Type 2. 4. The method according to claim 3, characterized in that the modified chromosomal sequences reduce the sensitivity of the female pig to a PRRSV isolate selected from the group consisting of NVSL 97-7895, KS06-72109, P129, VR2332, CO90, AZ25, MLV-ResPRRS, KS62-06274, KS483 (SD23983), CO84, SD13-15, Lelystad, 03-1059, 03-1060, SD01-08, 4353PZ and combinations thereof. 5. The method according to any one of paragraphs. 1-4, characterized in that the female pig includes a genetically modified female pig. 6. The method according to claim 5, characterized in that the female pig is genetically modified using homing endonuclease. 7. The method according to claim 6, characterized in that the homing endonuclease includes a constructed homing endonuclease. 8. The method according to claim 6 or 7, characterized in that the homing endonuclease includes a system of clustered short palindromic repeats at regular intervals (CRISPR), a transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN), a recombinase fusion protein , a meganuclease, or a combination of any of them. 9. The method according to any one of paragraphs. 1-8, characterized in that the female pig is genetically modified using the CRISPR system. 10. The method according to any one of paragraphs. 1–9, characterized in that the female pig contains the first modified chromosomal sequence in the first allele of the CD163 gene and the second modified chromosomal sequence in the second allele of the CD163 gene, and the first and second modified chromosomal sequences differ from each other. 11. The method according to any one of paragraphs. 1-10, characterized in that the modified chromosomal sequences cause a decrease in the production or activity of the CD163 protein compared to the production or activity of the CD163 protein in a female pig that lacks the modified chromosomal sequences. 12. The method according to any one of paragraphs. 1-11, characterized in that the modified chromosomal sequences lead to a virtual absence of the production of functional CD163 protein in the female pig. 13. The method according to any one of paragraphs. 1-12, characterized in that the female pig does not produce the CD163 protein. 14. The method according to any one of paragraphs. 1–13, characterized in that modified chromosomal sequences in one or both alleles of the female pig CD163 gene lead to incorrect coding. 15. The method of claim 14, wherein the miscoding results in a premature stop codon below the miscoding in the CD163 gene allele. 16. The method according to any one of paragraphs. 1-15, characterized in that: the modification includes a 2 bp insertion between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a 377 base pair deletion from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47 on the same allele, and insert of 2 base pairs includes dinucleotide AG; the modification includes a 1 base pair insert between nucleotides 3147 and 3148 compared to the reference sequence of SEQ ID NO: 47, and the 1 base pair insert contains a single adenine residue; the modification includes a 7 base pair insert between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47, and the 7 base pair insert contains the sequence TACTACT (SEQ ID NO: 115); the modification includes a 1930 base pair deletion from nucleotide 488 to nucleotide 2417 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by a 12 base pair insert starting at nucleotide 488, and there is an additional 129 base pair deletion in exon 7 from nucleotide 3044 to nucleotide 3172 compared to the reference sequence of SEQ ID NO: 47, and where the insert of 12 base pairs contains the sequence TGTGGAGAATTC (SEQ ID NO: 116); or the modification includes a deletion of 1382 base pairs from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113, and the insertion of 11 base pairs includes the sequence AGCCAGCGTGC (SEQ ID NO: 117). 17. The method according to claim 16, characterized in that at least one of the alleles of the female pig CD163 gene contains a modification selected from the group consisting of: insertion of 7 base pairs between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47; insertion of 2 base pairs between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a deletion of 377 base pairs from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47 on the same allele; deletions of 11 base pairs from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47; deletions of 1382 base pairs from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113; and combinations of any of them. 18. The method according to p. 17, characterized in that the female pig contains: (a) a 7 bp insertion between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47 in one allele of the CD163 gene; and a 2 bp insertion between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a 377 base pair deletion from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47, in another allele of the CD163 gene; (b) deletion of 1382 base pairs from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113 in one allele of the CD163 gene; and a 2 bp insertion between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a 377 base pair deletion from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47, in another allele of the CD163 gene; (c) a 7 bp insertion between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47 in one allele of the CD163 gene; and deletion of 11 base pairs from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47 in another allele of the CD163 gene; or (d) deletion of 1382 base pairs from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113 in one allele of the CD163 gene; and deletion of 11 base pairs from nucleotide 3137 to nucleotide 3147 compared to the reference sequence of SEQ ID NO: 47 in another allele of the CD163 gene. 19. The method according to p. 18, characterized in that the female pig contains: (a) a 7 bp insertion between nucleotide 3148 and nucleotide 3149 compared to the reference sequence of SEQ ID NO: 47 in one allele of the CD163 gene; and a 2 bp insertion between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a 377 base pair deletion from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47, in another allele of the CD163 gene; or (b) deletion of 1382 base pairs from nucleotide 3113 to nucleotide 4494 compared to the reference sequence of SEQ ID NO: 47, where the deleted sequence is replaced by an insertion of 11 base pairs starting at nucleotide 3113 in one allele of the CD163 gene; and insertion of 2 bp between nucleotides 3149 and 3150 compared to the reference sequence of SEQ ID NO: 47, with a deletion of 377 base pairs from nucleotide 2573 to nucleotide 2949 compared to the reference sequence of SEQ ID NO: 47, in another allele of the CD163 gene. 20. The method according to any one of claims 1 to 19, characterized in that the alleles of the CD163 gene of the female pig contain a chromosomal sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 47 in regions of said chromosomal sequence outside of the insertion or deletion. 21. The method according to any one of paragraphs. 1-20, characterized in that the female pig contains a chromosomal sequence including SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113 , 114 or 119 in one or both alleles of the CD163 gene. 22. The method according to claim 21, characterized in that the female pig contains a chromosomal sequence comprising SEQ ID NO: 99, 102, 103 or 113 in one or both alleles of the CD163 gene. 23. The method according to p. 22, characterized in that the female pig contains: (a) a chromosomal sequence comprising SEQ ID NO: 99 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 103 in another allele of the CD163 gene; (b) a chromosomal sequence comprising SEQ ID NO: 113 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 99 in another allele of the CD163 gene; (c) a chromosomal sequence comprising SEQ ID NO: 99 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 102 in another allele of the CD163 gene; or (d) a chromosomal sequence comprising SEQ ID NO: 113 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 102 in another allele of the CD163 gene. 24. The method according to p. 23, characterized in that the female pig contains: (a) a chromosomal sequence comprising SEQ ID NO: 99 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 103 in another allele of the CD163 gene; or (b) a chromosomal sequence comprising SEQ ID NO: 113 in one allele of the CD163 gene and a chromosomal sequence comprising SEQ ID NO: 99 in another allele of the CD163 gene. 25. The method according to any one of paragraphs. 1–24, characterized in that, as a result of breeding, one or more fetuses are obtained that contain a modified chromosomal sequence in a single allele of the CD163 gene. 26. The method of claim 25, wherein the fetuses have a reduced susceptibility to PRRSV infection in utero compared to fetuses in the womb of a wild-type female pig. 27. The method according to any one of paragraphs. 1-26, characterized in that breeding involves crossing a female pig with a male pig. 28. The method according to any one of paragraphs. 1-26, characterized in that the breeding includes the artificial insemination of the female with sperm obtained from the male. 29. The method according to any one of paragraphs. 1-26, characterized in that the breeding involves the transfer of a fertilized oocyte into the reproductive tract of a female pig. 30. The method according to claim 29, characterized in that the fertilized oocyte was obtained by in vitro fertilization of the oocyte with sperm obtained from a male pig. 31. The method according to claim 30, characterized in that in vitro fertilization includes intracytoplasmic injection of an oocyte with sperm obtained from a male pig.

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