KR20240108545A - Methods for protecting porcine fetuses from infection with virus - Google Patents

Abstract

How to protect pig fetuses from porcine reproductive and respiratory syndrome virus (PRRSV) infection. The method involves breeding a female porcine animal with a male porcine animal. The female swine animal comprises a modified chromosomal sequence in both alleles of its CD163 gene, wherein the modified chromosomal sequence does not include any modified chromosomal sequence in all alleles of its CD163 gene. Reduces the susceptibility of female swine animals to PRRSV infection compared to the susceptibility of female swine animals to PRRSV infection. The male porcine animal contains at least one wild type CD163 allele.

Description

{METHODS FOR PROTECTING PORCINE FETUSES FROM INFECTION WITH VIRUS}

Reference to electronically submitted sequence listing

An official copy of the Sequence Listing will be submitted electronically via EFS-Web as an ASCII-format sequence listing with a file titled "SEQ LISTING 18054WO", formed on March 27, 2018 and having a size of 175.5 kilobytes, together with the specification. It has been applied for. The sequence listing contained in this ASCII-format document is part of the specification and is hereby incorporated by reference in its entirety.

field of invention

The present invention relates to a method of protecting porcine fetuses from porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Background of the invention

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). The clinical disease syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection was first reported in the United States in 1987 (Keffaber, 1989) and later in Europe in 1990 (Wensvoort et al., 1991). Infection with PRRSV causes respiratory illness including cough and fever, reproductive problems during the third trimester, and reduced growth capacity. This virus is also involved in various polymicrobial disease syndrome interactions, maintaining lifelong asymptomatic infection (Rowland et al., 2012). Losses are the result of respiratory disease, poor growth performance, reproductive failure and uterine infection in young pigs (Keffaber, 1989).

The reproductive form of these diseases accounts for an estimated 45% of losses due to miscarriage, fetal death and respiratory disease in newborns. The most severe form of reproductive PRRS can cause up to 90% fetal/neonatal mortality with increased maternal mortality. The reproductive form of PRRS occurs after infection of a pregnant sow or sow at about day 90 of a 114-day gestation period (Christianson et al., 1993; Rowland, 2010). After an initial stage of replication in maternal macrophages, the virus crosses the placenta and begins to actively infect the fetus. The virus initially infects only a small number of fetuses and then horizontally spreads the virus from fetus to fetus (Wilkinson et al., 2016). The exact mechanism by which the virus crosses the placenta is not known, but may be similar to infecting “Trojan horse” macrophages previously described for lactate dehydrogenase-enhancing virus (LDV) (Cafruny, 1996). Unlike alveolar macrophages in adult animals, the primary site of PRRSV replication in the fetus is the thymus (Rowland, 2003). Because pig fetuses become immunocompetent at approximately day 70 of gestation, PRRSV infection occurs in a fetal immune environment containing functional B and T cells (Rowland, 2003; Rowland, 2010).

Pigs that survive uterine infection become a persistent source of virus for downstream production steps, creating endemic infected herds (Rowland, et al., 2003). The most severe form of reproductive disease is associated with a group of highly virulent isolates termed atypical PRRSV (Halbur et al., 1997; Mengeling et al., 1998). Interestingly, many atypical PRRSV isolates came from PRRS-vaccinated farms (Key et al., 2001). In 2006, an atypical virus called highly pathogenic PRRSV (HP-PRRSV) appeared in China and continues to decimate the country's pig population (Tian et al., 2007). Since a standard commercial breeding facility has approximately 5,000 sows, an outbreak of reproductive PRRS with high mortality rates could have a devastating impact. To ensure sustainability of pork production and food security, reproductive PRRS control solutions remain a priority. Vaccines have been unable to control the disease primarily due to genetic diversity within the structural proteins of the virus (Shi et al., 2010). In reality, intensive biosecurity measures provide only a means of protecting breeding flocks.

Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the Arterividae family along with murine lactate dehydrogenase-enhanced virus, simian hemorrhagic fever virus, and equine arteritis virus. Structurally, arteriviruses resemble togaviruses, but are similar to coronaviruses and possess an inherent 3'-co-terminal set of subgenomic mRNAs with a common leader and poly-A tail. replicated through Arteriviruses share important characteristics 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 place all PRRSV isolates into one of two genotypes, type 2 or type 1, respectively. Although the two genotypes possess only about 70% identity at the nucleotide level (Nelsen et al., 1999), both share a tropism for CD163-positive cells, establish long-term infection, and produce similar clinical signs.

CD163 is a type 1 membrane protein of 130 kDa consisting of nine scavenger receptor cysteine-rich (SRCR) domains and two spacer domains, along with a transmembrane domain and a short cytoplasmic tail (Fabriek et al., 2005). Porcine CD163 has 17 exons encoding a peptide signal sequence followed by nine SRCR domains, two linker domains (also known as proline serine threonine (PST) domains located behind SRCR 6 and SRCR 9), and a cytoplasmic domain followed by a short cytoplasmic tail. Contains Surface expression of CD163 is restricted to cells of the monocyte-macrophage lineage. In addition to functioning as a viral receptor, CD163 exhibits several important functions related to maintaining normal homeostasis. For example, after infection or tissue damage, CD163 functions as a scavenger molecule, removing haptoglobin-hemoglobin complexes from the blood (Kristiansen et al., 2001). As a result, heme breakdown products regulate the associated inflammatory response (Fabriek et al., 2005). HbHp clearance is the main function of CD163, which is located in SRCR 3 (Madsen et al., 2004). Metabolites released by macrophages after HbHp degradation include bilirubin, CO, and free iron. One of the important functions of CD163 is the prevention of oxidative toxicity caused by free hemoglobin (Kristiansen et al., 2001; Soares et al., 2009).

Other important functions of C163 include erythroblast adhesion (SRCR2), TWEAK (tumor necrosis factor-like weak inducer of apoptosis) receptor (SRCR1-4 and 6-9), bacterial receptor (SRCR5), and as African swine virus receptor. functions (Sanchez-Torres et al. 2003). CD163 may also act as an immune-modulator (discussed in Van Gorp et al. 2010).

CD163 was first described as a receptor for PRRSV by Calvert et al (2007). Transfection of non-permissive cell lines with CD163 cDNA from a variety of species, including apes, humans, canines, and mice, allows cells to become permissive to PRRSV infection (Calvert et al., 2007). In addition to CD163, a second receptor protein, CD169 (also known as sialoadhesin or SIGLEC1), is the major PRRSV receptor involved in forming the initial interaction with the GP5-matrix (M) heterodimer, a major protein on the virion surface. confirmed (Delputte et al., 2002). In this model, the subsequent interaction between CD163 and the GP2, 3, 4 heterotrimer in the endosomal compartment mediates unenvelopment of the viral genome and release into the cytoplasm (Van Breedam et al., 2010, Allende et al., 1999). Previous models describing PRRSV infection of alveolar macrophages identified SIGLEC1 (CD169) as the primary viral receptor on the surface of macrophages; A previous study using SIGLEC1 ^−/− pigs showed no differences in viral replication compared to wild-type pigs (Prather et al., 2013). These results supported previous in vitro studies showing that PRRSV-resistant cell lines lacking surface CD169 and CD163 supported viral replication following transfection with the CD163 plasmid (Welch et al., 2010).

Many characteristics of both PRRSV pathogenesis (especially at the molecular level) and epidemiology are insufficiently understood, making control efforts difficult. Currently, producers often vaccinate swine with modified-live-attenuated strains or killed virus vaccines against PRRSV, but current vaccines often do not provide satisfactory protection. This is due to both strain variation and inappropriate stimulation of the immune system. In addition to concerns regarding the efficacy of available PRRSV vaccines, it is likely that the modified live vaccines currently in use will persist and accumulate mutations in individual pigs and herds of swine, as demonstrated by virulent site isolates following experimental infection of pigs (Rowland et al., 1999). There is strong evidence that it can (Mengeling et al. 1999). Additionally, the vaccine virus was shown to be shed in the semen of vaccinated boars (Christopher-Hennings et al., 1997). As an alternative to vaccination, some experts are advocating a “check and remove” strategy from breeding flocks (Dee et al., 1998). Successful use of this strategy depends on removal of all pigs acutely or persistently infected with PRRSV and then stringent controls to prevent reintroduction of the virus. The difficulty and high cost associated with this strategy is that little is known about the pathogenesis of persistent PRRSV infection and therefore there are no reliable techniques to identify persistently infected pigs.

As can be seen, there is a need in the art to develop strategies to induce PRRSV resistance in animals. Additionally, there is a particular need for technologies to protect fetuses from PRRSV infection while in the womb and prevent PRRSV transmission from mother to fetus.

BRIEF SUMMARY OF THE INVENTION

A method of protecting porcine fetuses from porcine reproductive and respiratory syndrome virus (PRRSV) infection is provided. The method involves breeding a female porcine animal with a male porcine animal. The female swine animal comprises a modified chromosomal sequence in both alleles of its CD163 gene, wherein the modified chromosomal sequence does not include any modified chromosomal sequence in all alleles of its CD163 gene. Reduces the susceptibility of female swine animals to PRRSV infection compared to their susceptibility to infection by PRRSV. The male porcine animal contains at least one wild type CD163 allele.

Other purposes and features will be partly apparent and partly pointed out below.

Brief description of the drawing
Figure 1. Targeting vector and CRISPR used to modify CD163. Panel A depicts wild-type exons 7, 8, and 9 of the CD163 gene targeted for modification using CRISPR. Panel B shows a targeting vector designed to replace porcine exon 7 (SRCR5, the porcine domain of CD163) with DNA encoding human SRCR8 of CD163L. This targeting vector was used for transfection with drug selection by G418. PCR primers for long-range, left arm, and right arm assays are indicated by arrows for 1230, 3752, 8791, 7765, and 7775. Panel C shows the same targeting vector as shown in panel B but with the Neo cassette removed. This targeting vector has already been used to target CD163 in neomycin-resistant cells. Primers used in the small deletion assay are indicated by arrows and labeled GCD163F and GCD163R. Panel D highlights the exons targeted by CRISPR. The positions of CRISPR 10, 131, 256, and 282 are indicated by downward-pointing arrows in exon 7. The CRISPR number indicates the number of base pairs at the intron-exon junction of intron 6 and exon 7.
Figure 2. Targeting vector and CRISPR used to modify CD1D. Panel A shows CD1D targeted for modification by CRISPR. Wild-type exons 3, 4, 5, 6, and 7 of the gene are shown. Panel B shows a targeting vector designed to replace exon 3 with the selection marker Neo . This targeting vector was used in combination with CRISPR to modify CD1D. PCR primers for the long-range, left arm, and right arm assays are indicated by arrows for 3991, 4363, 7373, and 12806. Panel C shows the exons targeted by CRISPR. The positions of CRISPR 4800, 5350, 5620 and 5626 are indicated by downward-pointing arrows in exon 3. Primers used in the small deletion assay are indicated by arrows and labeled GCD1DF and GCD1DR.
Figure 3. Production of CD163 and CD1D knockout pigs by CRISPR/Cas9 and SCNT. A) Targeted deletion of CD163 in somatic cells following transfection with CRISPR/Cas9 and donor DNA. The wild type (WT) genotype results in a 6545 base pair (bp) band. Lanes 1-6 represent six different colonies from a single transfection using CRISPR 10 with donor DNA containing Cas9 and 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 the WT allele and a small deletion or biallelic variant of both alleles. The exact transformation of each colony was determined only by sequencing of the colonies used for SCNT. Faint WT bands in some lanes may indicate cross-contamination of fetal fibroblasts from adjacent WT colonies. NTC = no template control. B) Targeted deletion of CD1D in somatic cells following transfection with CRISPR/Cas9 and donor DNA. The WT genotype results in an 8729 bp band. Lanes 1-4 represent colonies with a 500-2000 bp deletion of CD1D. Lane 4 appears to be a WT colony. NTC = no template control. C) Images of CD163 knockout pigs produced by SCNT during the study. This male piglet contains a homozygous 1506 bp deletion of CD163. D) Images of CD1D pigs produced during the study. This piglet contains a 1653 bp deletion in CD1D. E) Genotype of two SCNT littermates containing a 1506 bp deletion of CD163. Lanes 1-3 (litter 63) and lanes 1-4 (litter 64) represent the genotype for each piglet from each litter. Sow represents females that received SCNT embryos and WT represents WT controls. NTC = no template control. F) Genotypes of two SCNT littermates containing a 1653 bp deletion of CD1D. Lanes 1-7 (Litter 158) and Lanes 1-4 (Litter 159) represent the genotype for each piglet.
Figure 4. Effect of CRISPR/Cas9 system in porcine embryos. A) Frequency of blastocyst formation after injection of different concentrations of 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 the expression of eGFP in blastocysts when introduced into the zygote. Initial magnification X4. C) Types of mutations in eGFP generated using the CRISPR/Cas9 system: WT genotype (SEQ ID NO:16), #1 (SEQ ID NO:17), #2 (SEQ ID NO:18), and #3 (SEQ ID NO:18) :19).
Figure 5. Effect of the CRISPR/Cas9 system targeting CD163 in porcine embryos. A) Examples of mutations generated in CD163 by the CRISPR/Cas9 system: WT 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 tested by DNA sequencing showed mutations in CD163 (18/18). CRISPR 131 is highlighted in bold. B) Sequencing reads of homozygous deletions by the CRISPR/Cas9 system. Images represent #1-4 of panel A with a 2bp deletion of CD163.
Figure 6. Effect of CRISPR/Cas9 system when two types of CRISPR are introduced. A) PCR amplification of CD163 from blastocysts injected with CRISPR/Cas9 as zygotes. Lanes 1, 3, 6, and 12 show the designed deletion between two different CRISPRs. B) PCR amplification of CD1D from blastocysts injected with CRISPR/Cas9 as zygotes. CD1D showed a lower deletion frequency as determined by gel electrophoresis compared to CD163 (3/23); Lanes 1, 8, and 15 show clear deletions in CD1D. C) The CRISPR/Cas9 system successfully targeted two genes when the system was provided with two CRISPR targeting CD163 and eGFP. Modifications of CD163 and eGFP are indicated as follows: CD163 WT (SEQ ID NO:24), CD163 #1 (SEQ ID NO:25), CD163 #2 (SEQ ID NO:26), CD163 #3 (SEQ ID NO:27) , eGFP WT (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 #3 (SEQ ID NO: Number:32).
Figure 7. CD163 knockout pig generated by the CRISPR/Cas9 system injected into the zygote. A) PCR amplification of CD163 from knockout pigs; Clear signs of deletion were detected in littermates 67-2 and 67-4. B) Image of a CD163 knockout pig with a surrogate mother. All animals are healthy and show no abnormalities. C) Genotype of CD163 knockout pigs. The wild type (WT) sequence is indicated as SEQ ID NO:33. Two animals (from litters 67-1 (SEQ ID NO:34) and 67-3 (SEQ ID NO:37)) carry homozygous deletions or insertions in CD163. The other two animals (from litters 67-2 and 67-4) have biallelic variants of CD163: #67-2 A1 (SEQ ID NO:35), #67-2 A2 (SEQ ID NO:36) , #67-4 A1 (SEQ ID NO: 38), and #67-4 a2 (SEQ ID NO: 39). The deletion was created by introducing two different CRISPRs into the Cas9 system. Animals obtained from zygote injections for CD163 did not show a mosaic genotype.
Figure 8. CD1D knockout pig generated by the CRISPR/Cas9 system injected into the zygote. A) PCR amplification of CD1D from knockout pigs; 166-1 shows a mosaic genotype for CD1D. 166-2, 166-3, and 166-4 do not show size changes to the amplicons, but sequencing of the amplicons show variations. WT FF = wild-type fetal fibroblasts. B) PCR amplification of the long-range assay showed a clear deletion of one allele in piglets 166-1 and 166-2. C) Image of a CD1D knockout pig with a surrogate mother. D) Sequence data from CD1D knockout pigs; WT (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.
Figure 9. Clinical signs during acute PRRSV infection. Results of daily assessment for fever and presence of respiratory signs for CD163 +/+ (n=6) and CD163 -/- (n=3).
Figure 10. Lung histopathology during acute PRRSV infection. Representative photomicrographs of H and E stained tissues from wild-type and knockout pigs. Left panel shows edema and infiltration of mononuclear cells. The right panel from a knockout pig shows the lung structure of a normal lung.
Figure 11. Viremia in various genotypes. Note that CD163-/-piglet data lies along the X-axis.
Figure 12. Antibody production in null, wild-type, and uncharacterized allele pigs.
Figure 13. Cell surface expression of CD163 in individual pigs. The lines toward the right in the Uncharacterized A, Uncharacterized B, and CD163 +/+ panels represent CD163 antibodies, while the lines toward the left of these panels represent the no antibody control (background). Note that in CD163-/- animals, CD163 staining overlaps with background controls, and that CD163 staining of the uncharacterized allele is approximately half-way between WT levels and background (also, this is on a logarithmic scale, so less than ~10%). (Note).
Figure 14. Levels of CD169 on alveolar macrophages from three representative pigs and no antibody control (FITC labeled anti-CD169).
Figure 15. Viremia in various genotypes. Note that the Δ43 amino acid piglet data lies along the X-axis.
Figure 16. Genomic sequence of wild-type CD163 exons 7-10 (SEQ ID NO: 47) used as reference sequence. The sequence includes 3000 bp from upstream of exon 7 to the last base of exon 10. The underlined regions show the positions of exons 7, 8, 9, and 10, respectively.
Figure 17. CD163 variant diagram showing several CD163 chromosomal variants, predicted protein products for each variant, and relative macrophage expression for each variant as measured by levels of surface CD163 in porcine alveolar macrophages (PAM). . Black areas represent introns and white areas represent exons. The hatched region represents the h CD163L1 exon 11 mimic, a homolog of porcine exon 7. The gray area represents the synthesized intron with the PGK Neo construct.
Figure 18. Diagram of porcine CD163 protein and gene sequence. A) CD163 protein SRCR (oval) and PST (square) domains and corresponding gene exons. B) Comparison of porcine CD163 SRCR 5 (SEQ ID NO: 120) and human CD163L1 SRCR 8 (SEQ ID NO: 121) homolog.
Figure 19. Representative results for surface expression of CD163 and CD169 on PAMs of wild-type and CD163-transformed pigs. Panels AE show the results for CD163 modifications as described in Figure 17. Pooled data for d7 (1467) and d7 (1280) are shown in panel D.
Figure 20. Serum haptoglobin levels in wild-type and CD163-transformed pigs.
Figure 21. Relative permissibility of wild-type and HL11m PAMs to infection by type 2 PRRSV isolates.
Figure 22. Infection of CD163 transformed pigs with type 1 and type 2 PRRSV isolates.
Figure 23. Viral load for WT and CD163-transformed pigs infected with type 2 virus.
Figure 24. Fetal outcome after maternal infection with PRRSV. The numbers on the left identify each dam (“Dam Number”; see Table 16 below). Below each female number in parentheses are PRRS PCR results in serum measured in log ₁₀ templates per reaction. “N” is negative for PRRSV nucleic acid (Ct>39). Fetuses are identified by their number and relative position within each uterine horn. Asterisks identify fetal PCR samples obtained from abdominal fluid. Numbers below each fetus are PRRS PCR results of fetal serum (log ₁₀ templates per reaction). Numbers within each circle indicate the presence of anatomical pathology: 1) normal fetus; 2) small fetus; 3) placental changes such as abruption and/or necrosis; 4) meconium-stained fetus; 5) Fetal death and necrosis. Lowercase letters identify the genotype of the individual fetus (see Table 16). Symbol interpretation: a, A/A; b, C/A; c, B/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 was necrotic; nd, genotype not determined.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of protecting porcine fetuses from porcine reproductive and respiratory syndrome virus (PRRSV) infection. Pigs with inactivating mutations in both alleles of the CD163 gene are resistant to PRRSV infection. CD163-positive fetuses (e.g., fetuses with one or two wild-type CD163 alleles) may be protected from PRRSV infection while in utero as long as the female carries an inactivating mutation in both alleles of the CD163 gene. was discovered unexpectedly. Thus, for example, a female carrying an inactivating mutation in both alleles of the CD163 gene could be crossed with a male carrying two wild-type CD163 alleles, and the resulting heterozygous embryos would be protected from PRRSV infection. will be.

Justice

When introducing elements of the invention or preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “the” are intended to mean that there is one or more elements.

The term “and/or” means any one of the items, any combination of items, or all items associated with the term.

As used herein, the term “breeding” refers to the joining of male and female gametes so that fertilization occurs. Such bonding may occur by mating (mating) or by artificial methods in vitro or in vivo . Such artificial methods include, but are not limited to, artificial semen injection, surgically assisted artificial semen injection, in vitro fertilization, intracytoplasmic sperm injection, zona pellucida drilling, in vitro culture of fertilized oocytes, ovarian transplantation, and ovarian division. Includes. As used herein, the term “breeding” also includes the transfer of fertilized oocytes into the reproductive tract of a female animal.

The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than those listed.

The term “CRISPR” stands for “clustered regularly interspaced short palindromic repeats.” CRISPR systems include type I, type II, and type III CRISPR systems.

The term “Cas” refers to “CRISPR associated protein.” Cas proteins include, but are not limited to, Cas9 family member proteins, Cas6 family member proteins (e.g., Csy4 and Cas6), and Cas5 family member proteins.

The term “Cas9” generally refers to at least about 5%, 10%, 20%, 30%, 40%, 50% relative to a wild-type Cas9 polypeptide (e.g., Cas9 from S. pyogenes). , may refer to a polypeptide having 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity. Exemplary Cas9 sequences are provided by SEQ ID NOs: 1-256 and 795-1346 in US Patent Publication No. 2016/0046963. SEQ ID NOS: 1-256 and 795-1346 of US Patent Publication No. 2016/0046963 are incorporated herein by reference. “Cas9” refers to up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% relative to the wild type Cas9 polypeptide (e.g. from S. pyogenes ). , may refer to a polypeptide having 90%, or 100% sequence identity and/or sequence similarity. “Cas9” may refer to a wild-type or modified form of the Cas9 protein containing amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

The term “Cas5” generally refers to at least about 5%, 10%, 20%, 30%, 40%, 50% of the wild-type exemplary Cas5 polypeptide (e.g., Cas5 from D. vulgaris). %, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity. An exemplary Cas5 sequence is provided in Figure 42 of US Patent Publication No. 2016/0046963. Figure 42 of US Patent Publication No. 2016/0046963 is incorporated herein by reference. “Cas5” generally refers to up to about 5%, 10%, 20%, 30%, 40%, 50 % , 60%, 70%, may refer to a polypeptide having 80%, 90%, or 100% sequence identity and/or sequence similarity. “Cas5” may refer to a wild-type or modified form of the Cas5 protein, which may include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

The term “Cas6” generally refers to at least about 5%, 10%, 20%, 30%, 40% relative to a wild type exemplary Cas6 polypeptide (e.g., Cas6 from T. thermophilus). , may refer to a polypeptide having 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity. An exemplary Cas6 sequence is provided in Figure 41 of U.S. Patent Publication No. 2016/0046963. Figure 41 of U.S. Patent Publication No. 2016/0046963 is incorporated herein by reference. “Cas6” refers to up to about 5%, 10%, 20%, 30%, 40%, 50%, 60% , may refer to a polypeptide having 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity. “Cas6” may refer to a wild-type or modified form of the Cas6 protein, which may include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

The terms “CRISPR/Cas9” or “CRISPR/Cas9 system” refer to the Cas9 protein, or derivatives thereof, and one or more non-coding RNAs capable of providing the functions of CRISPR RNA (crRNA) and a trans-activating RNA for Cas9 ( tracrRNA) refers to a programmable nuclease for genetic engineering. crRNA and tracrRNA can be used individually or combined to produce a “guide RNA” (gRNA). crRNA or gRNA provides a complementary sequence to the genomic target.

Reference herein to a deletion of the nucleotide sequence from nucleotide x to nucleotide y means that all nucleotides within that range, including x and y, have been deleted. Thus, for example, the phrase "11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 compared to SEQ ID NO: 47" means that each of nucleotides 3,317 to 3,147, including nucleotides 3,317 and 3,147, is deleted.

An animal's "resistance" to a disease is a characteristic of the animal, in which the animal avoids disease symptoms that are the result of animal-pathogen interactions, such as the interaction between swine animals and PRRSV. That is, the pathogen prevents the animal disease and associated disease symptoms from occurring, or alternatively reduces the incidence and/or severity of clinical signs or reduces clinical symptoms. Those skilled in the art will understand that the methods disclosed herein can be used in conjunction with other compositions and methods available in the art to protect animals from pathogen attack.

As used herein, “gene editing,” “genetically edited,” “genetically edited,” and “gene editing effector” are also referred to as “molecular scissors,” “homing endonuclease,” or “targeting endonuclease.” Also referred to as "clease", it refers to the use of homing techniques by naturally occurring or artificially engineered nucleases. Nucleases create specific double-stranded chromosome breaks (DSBs) at desired locations in the genome, and in some cases utilize the cell's endogenous mechanisms to initiate homologous recombination (HR) and/or non-homologous end-joining (NHEJ). Repairs amputations induced by natural processes. Gene editing effectors include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR) system (e.g., the CRISPR/Cas9 system). , and meganucleases (e.g., meganucleases re-engineered into homing endonucleases). The term also includes the use of transformation procedures and techniques, including, for example, when the alterations are deletions or relatively small insertions (typically less than 20 nt) and/or do not introduce DNA from a foreign species. The term also includes progeny animals, such as those produced by sexual or asexual breeding from an initial gene-edited animal.

The terms “genome manipulation”, “genetic engineering”, “genetically engineered”, “genetically altered”, “genetic alteration”, “genome modification”, “genome modification” and “genome modified” refer to specific nucleic acid sequences. It may refer to altering the genome by deletion, insertion, mutation, or substitution. Changes may vary depending on the gene or location. In genome engineering, nucleases can be used to cleave nucleic acids to create altered sites. Manipulation of non-genomic nucleic acids is also contemplated. A protein containing a nuclease domain can bind to and cleave a target nucleic acid by forming a complex with the nucleic acid-targeting nucleic acid. In one example, cleavage may introduce a double-strand break in the target nucleic acid. Nucleic acids can be repaired, for example, by the endogenous non-homologous end joining (NHEJ) machinery. In a further example, a nucleic acid fragment may be inserted. Nucleic Acid-Targeting Modification of nucleic acids and site-directed polypeptides can introduce new functions for use in genome manipulation.

As used herein, “homing DNA technology,” “homing technology,” and “homing endonuclease” refer to certain molecules being used in the zinc finger (ZF) protein, transcription activator-like effector (TALE) meganuclease, and CRISPR system. (e.g., CRISPR/Cas9 system).

The terms “increased resistance” and “reduced susceptibility” herein mean, but are not limited to, a statistically significant reduction in the incidence and/or severity of clinical signs or symptoms associated with infection by a pathogen. For example, “increased resistance” or “reduced susceptibility” refers to clinical signs or symptoms associated with infection by PRRSV in animals containing a chromosomal sequence altered in the CD163 gene protein compared to control animals with unmodified chromosomal sequence. It may refer to a statistically significant reduction in the incidence and/or severity of clinical symptoms. The term “statistically significant reduction in clinical symptoms” includes, but is not limited to, the frequency of occurrence of at least one clinical symptom in a group of transformed subjects of at least 10%, preferably at least 10%, over a non-transformed control group after challenge with an infectious agent. means at least 20% lower, more preferably at least 30% lower, even more preferably at least 50% lower, and even more preferably at least 70% lower.

“Knockout” means disruption of the structure or regulatory mechanism of a gene. Knockouts can be generated through homologous recombination in targeting vectors, replacement vectors, or hit-and-run vectors, or through random insertion in gene trap vectors, resulting in complete, partial, or conditional loss of gene function. there is.

As used herein, the term “mutation” includes an alteration in the nucleotide sequence of a polynucleotide, such as a gene or coding DNA sequence (CDS), compared to the wild-type sequence. This term includes, but is not limited to, substitutions, insertions, frame shifts, deletions, inversions, translocations, duplications, splice-donor site mutations, point-mutations, and the like.

As used herein, “reduction in the incidence and/or severity of clinical signs” or “reduction in clinical signs” includes, but is not limited to, a decrease in the number of infected subjects in a group compared to a wild-type infection, the number of subjects showing clinical signs of infection. means reducing or eliminating the number, or reducing the severity of any clinical signs present in one or more subjects. For example, these terms include any clinical manifestation consisting of a reduction in symptoms of infection, pulmonary pathology, viremia, antibody production, pathogen load reduction, pathogen shedding, reduced pathogen transmission, or any clinical sign of PRRSV. Preferably these clinical signs are reduced by at least 10% in one or more animals of the invention compared to infected subjects without alterations in the CD163 gene. More preferably clinical signs are reduced in the subject of the invention by at least 20%, preferably by at least 30%, more preferably by at least 40%, and even more preferably by at least 50%.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domain is involved in the binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also called a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. Zinc fingers and TALE binding domains can be “engineered” to bind predetermined nucleotide sequences, for example, through manipulation (altering one or more amino acids) of the naturally occurring zinc fingers or the recognition helix region of the TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are non-naturally occurring proteins. A non-limiting example of a method for manipulating DNA-binding proteins is design and selection. Engineered DNA-binding proteins are proteins that do not occur in nature, whose design/construction is primarily based on rational criteria. Reasonable design criteria include the application of alternative rules and computer algorithms to process information in databases storing information and binding data from existing ZFP and/or TALE designs. See, for example, US Patent No. 6,140,081; 6,453,242; and 6,534,261; See also WO 98/53058; WO 98/53059; WO 98/53060; See WO 02/016536 and WO 03/016496 and US Publication No. 20110301073.

“Zinc finger DNA binding protein” (or binding domain) is a protein that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of the amino acid sequence within the binding domain whose structure is stabilized through the coordination of zinc ions or larger proteins. It is a domain within a protein. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

“Selected” zinc finger proteins, or TALEs, are proteins that are not found in nature, whose production primarily occurs in empirical processes such as phage display, interaction traps, or hybrid selection. See, for example, US Patent No. 5,789,538; U.S. Patent No. 5,925,523; U.S. Patent No. 6,007,988; US Patent No. 6,013,453; US Patent No. 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; See WO 01/60970 WO 01/88197, WO 02/099084 and US Publication No. 20110301073.

Various other terms are defined below.

How to Protect Pig Fetuses from PRRSV Infection

Methods for protecting porcine fetuses from porcine reproductive and respiratory syndrome virus (PRRSV) infection are provided. The method involves breeding a female porcine animal with a male porcine animal. The female swine animal comprises a modified chromosomal sequence in both alleles of its CD163 gene, wherein the modified chromosomal sequence does not include any modified chromosomal sequence in all alleles of its CD163 gene. Reduces the susceptibility of female swine animals to PRRSV infection compared to their susceptibility to infection by PRRSV. The male porcine animal contains at least one wild type CD163 allele.

In the methods described herein, the modified chromosomal sequence may be a sequence that is altered such that CD163 protein function associated with PRRSV infection is impaired, reduced, or eliminated. Accordingly, the female porcine animals used in the methods described herein may be referred to as “knock-out” animals.

Male swine animals may contain two wild-type CD163 alleles.

As used herein, the term “wild-type CD163 allele” means that the sequence of the CD163 allele is the sequence found in nature, or that the sequence of the CD163 allele has one or more mutations that do not substantially impair CD163 activity (e.g. , insertion, deletion, or substitution). Accordingly, the wild-type CD163 allele may contain polymorphisms and/or mutations as long as the polymorphisms or mutations do not substantially impair CD163 activity.

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.

Accordingly, in the methods described herein, the modified chromosomal sequence reduces the susceptibility of the female swine animal to type cl PRRSV virus, type 2 PRRSV, or both type 1 and type 2 PRRSV viruses.

The modified chromosome sequences are 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.

The female porcine animal may include a genetically edited female porcine animal.

The genetically edited female swine animal may be an animal edited using a homing endonuclease. The homing endonuclease may be a naturally occurring endonuclease, but is preferably a rationally designed, non-naturally occurring homing endonuclease having a DNA recognition sequence designed to target a chromosomal sequence in the gene encoding the CD163 protein. am.

Accordingly, the homing endonuclease may be a designed homing endonuclease. Homing endonucleases include, for example, the clustered regularly interspaced short palindromic repeat (CRISPR) system, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and recombinase fusion proteins. , meganuclease, or any combination thereof.

The homing nuclease preferably comprises a CRISPR system. Examples of CRISPR systems that can be used to generate female porcine animals for use in the methods described herein include, but are not limited to, CRISPR/Cas9, CRISPR/Cas5, and CRISPR/Cas6. The use of the CRISPR system to generate genetically edited animals is discussed further below.

In any of the methods described herein, the female porcine animal may comprise the same modified chromosomal sequence on both alleles of the CD163 gene.

Alternatively, the female swine animal may comprise a first modified chromosomal sequence in a first allele of the CD163 gene and a second modified chromosomal sequence in a second allele of the CD163 gene, wherein the first and second modifications The chromosomal sequences are different from each other.

In any of the methods described herein, each allele of the CD163 gene in a female porcine animal may comprise an insertion, deletion, or combination thereof.

For example, at least one allele of the CD163 gene in a female swine animal may contain a deletion.

Alleles of at least one CD163 gene may contain an in-frame deletion.

At least one allele of the CD163 gene in the female swine animal may contain the insertion.

In the methods described herein, the modified chromosomal sequence preferably results in reduced CD163 protein production or activity compared to CD163 protein production or activity in a female porcine animal without the modified chromosomal sequence.

Preferably, the modified chromosomal sequence substantially results in no production of functional CD163 protein by the female porcine animal. “Substantially no functional CD163 protein” means that the level of CD163 protein in the animal, progeny, or cell is undetectable, or, if detectable, is lower than the level observed in the animal, progeny, or cell that does not contain the modified chromosomal sequence. It means at least less than about 90%, preferably less than about 95%, more preferably less than about 98%, and even more preferably less than about 99%.

For example, in any of the methods described herein, the female porcine animal does not produce CD163 protein.

In any of the methods described herein, each allele of the CD163 gene of the female swine animal has a modification in exon 7, a modification in exon 8, a modification in the intron adjacent to exon 7 or exon 8, or any of these. May include combinations.

For example, one or both alleles of the CD163 gene in a female swine animal may include a modification in exon 7 of the CD163 gene.

Variations in exon 7 may include deletions.

In any of the methods described herein wherein the allele of the CD163 gene in the female porcine animal comprises a deletion, the deletion may comprise an in-frame deletion.

Modifications in exon 7 may include insertions.

In any of the methods described herein, altered chromosomal sequences in one or both alleles of the CD163 gene in a female porcine animal may result in miscoding.

If the altered chromosomal sequence results in a miscoding in an allele of the CD163 gene, the miscoding may result in a premature stop codon downstream of the miscoding in the allele of the CD163 gene.

In any of the methods described herein, at least one of the alleles of the CD163 gene in the female swine animal comprises a modification selected from the group consisting of: nucleotides 3,137 to 3,147 compared to the reference sequence SEQ ID NO: 47. 11 base pair deletion; a 377 base pair deletion from nucleotide 2,573 to nucleotide 2,949 compared to the reference sequence SEQ ID NO: 47 on the same allele and a 2 base pair insertion between nucleotides 3,149 and 3,150 compared to the reference sequence SEQ ID NO: 47; A 124 base pair deletion from nucleotide 3,024 to nucleotide 3,147 compared to the reference sequence SEQ ID NO: 47; A 123 base pair deletion from nucleotide 3,024 to nucleotide 3,146 compared to the reference sequence SEQ ID NO: 47; 1 base pair insertion between nucleotides 3,147 and 3,148 compared to reference sequence SEQ ID NO: 47; A 130 base pair deletion from nucleotide 3,030 to nucleotide 3,159 compared to the reference sequence SEQ ID NO: 47; A 132 base pair deletion from nucleotide 3,030 to nucleotide 3,161 compared to the reference sequence SEQ ID NO: 47; A 1506 base pair deletion from nucleotide 1,525 to nucleotide 3,030 compared to reference sequence SEQ ID NO: 47; 7 base pair insertion between nucleotides 3,148 and 3,149 compared to reference sequence SEQ ID NO: 47; A 1280 base pair deletion from nucleotide 2,818 to nucleotide 4,097 compared to reference sequence SEQ ID NO: 47; A 1373 base pair deletion from nucleotide 2,724 to nucleotide 4,096 compared to reference sequence SEQ ID NO: 47; A 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897 compared to reference sequence SEQ ID NO: 47; A 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488, and compared to the reference sequence SEQ ID NO: 47 with an additional 129 base pair deletion in exon 7 from nucleotide 3,044 to nucleotide 3,172; A 28 base pair deletion from nucleotide 3,145 to nucleotide 3,172 compared to reference sequence SEQ ID NO: 47; 1387 base pair deletion from nucleotide 3,145 to nucleotide 4,531 compared to reference sequence SEQ ID NO: 47; A 1382 base pair deletion from nucleotide 3,113 to nucleotide 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113; A 1720 base pair deletion from nucleotide 2,440 to nucleotide 4,160 compared to reference sequence SEQ ID NO: 47; A 452 base pair deletion from nucleotide 3,015 to nucleotide 3,466 compared to the reference sequence SEQ ID NO: 47; and any combination thereof.

At least one of the alleles of the CD163 gene in a female swine animal may contain an 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal has a 377 base pair deletion from nucleotide 2,573 to nucleotide 2,949 compared to the reference sequence SEQ ID NO: 47 on the same allele, It may contain a two base pair insertion between nucleotides 3,149 and 3,150.

The variant comprises a two base pair insertion between nucleotides 3,149 and 3,150 compared to the reference sequence SEQ ID NO: 47 along with a 377 base pair deletion from nucleotide 2,573 to nucleotide 2,949 compared to the reference sequence SEQ ID NO: 47 on the same allele. If so, the two base pair insertion may include dinucleotide AG.

At least one of the alleles of the CD163 gene in a female swine animal may contain a 124 base pair deletion from nucleotide 3,024 to nucleotide 3,147 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal may comprise a 123 base pair deletion from nucleotide 3,024 to nucleotide 3,146 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal may comprise a 1 base pair insertion between nucleotides 3,147 and 3,148 compared to the reference sequence SEQ ID NO:47.

If the modification includes a one base pair insertion between nucleotides 3,147 and 3,148 compared to the reference sequence SEQ ID NO:47, then the one base pair insertion may include a single adenine residue.

At least one of the alleles of the CD163 gene in a female swine animal may comprise a 130 base pair deletion from nucleotide 3,030 to nucleotide 3,159 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal may comprise a 132 base pair deletion from nucleotide 3,030 to nucleotide 3,161 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal may comprise a 1506 base pair deletion from nucleotide 1,525 to nucleotide 3,030 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal may contain a 7 base pair insertion between nucleotides 3,148 and 3,149 compared to the reference sequence SEQ ID NO:47.

If the modification includes a 7 base pair insertion between nucleotides 3,148 and 3,149 compared to the reference sequence SEQ ID NO: 47, then the 7 base pair insertion may include the sequence TACTACT (SEQ ID NO: 115).

At least one of the alleles of the CD163 gene in a female porcine animal may contain a 1280 base pair deletion from nucleotide 2,818 to nucleotide 4,097 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal may contain a 1373 base pair deletion from nucleotide 2,724 to nucleotide 4,096 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female porcine animal may contain a 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal comprises a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence comprises 12 base pairs starting at nucleotide 488. replaced by an insertion and an additional 129 base pair deletion in exon 7 from nucleotide 3,044 to nucleotide 3,172 compared to the reference sequence SEQ ID NO:47.

If the modification includes a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 compared to the reference sequence SEQ ID NO: 47, then the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488, and the reference sequence sequence There are an additional 129 base pair deletion in exon 7 from nucleotide 3,044 to nucleotide 3,172 compared to number: 47, where the 12 base pair insertion may include the sequence TGTGGAGAATTC (SEQ ID NO: 116).

At least one of the alleles of the CD163 gene in a female swine animal may contain a 28 base pair deletion from nucleotide 3,145 to nucleotide 3,172 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal may contain a 1387 base pair deletion from nucleotide 3,145 to nucleotide 4,531 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in female swine animals is a 1382 base pair deletion from nucleotide 3,113 to nucleotide 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence has an 11 base pair insertion starting at nucleotide 3,113. replaced, may include the base pair deletion.

If the variant includes a deletion of 1382 base pairs from nucleotide 3,113 to nucleotide 4,494 compared to the reference sequence SEQ ID NO: 47, then the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113, and the 11 base pair insertion is It may include the sequence AGCCAGCGTGC (SEQ ID NO: 117).

At least one of the alleles of the CD163 gene in a female porcine animal may contain a 1720 base pair deletion from nucleotide 2,440 to nucleotide 4,160 compared to the reference sequence SEQ ID NO:47.

At least one of the alleles of the CD163 gene in a female swine animal may comprise a 452 base pair deletion from nucleotide 3,015 to nucleotide 3,466 compared to the reference sequence SEQ ID NO:47.

For example, in a female swine animal, at least one of the alleles of the CD163 gene comprises a modification selected from the group consisting of: a 7 base pair insertion between nucleotides 3,148 and 3,149 compared to the reference sequence SEQ ID NO: 47; a 377 base pair deletion from nucleotide 2,573 to nucleotide 2,949 compared to the reference sequence SEQ ID NO: 47 on the same allele and a 2 base pair insertion between nucleotides 3,149 and 3,150 compared to the reference sequence SEQ ID NO: 47; An 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 compared to reference sequence SEQ ID NO: 47; A 1382 base pair deletion from nucleotide 3,113 to nucleotide 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113; and any combination thereof.

The CD163 gene in a female swine animal may contain any combination of the modified chromosomal sequences described herein.

For example, a female swine animal has in one allele of the CD163 gene a 7 base pair insertion between nucleotides 3,148 and 3,149 compared to the reference sequence SEQ ID NO:47; and in the other allele of the CD163 gene, a 377 base pair deletion from nucleotide 2,573 to nucleotide 2,949 compared to the reference sequence SEQ ID NO: 47, together with 2 base pairs between nucleotides 3,149 and 3,150 compared to the reference sequence SEQ ID NO: 47. May contain insertions.

A female swine animal has a 1382 base pair deletion from nucleotide 3,113 to nucleotide 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113 in one allele of the CD163 gene. wherein the base pair deletion; and in the other allele of the CD163 gene, a 377 base pair deletion from nucleotide 2,573 to nucleotide 2,949 compared to the reference sequence SEQ ID NO: 47, together with 2 base pairs between nucleotides 3,149 and 3,150 compared to the reference sequence SEQ ID NO: 47. May contain insertions.

Female swine animals have a 7 base pair insertion in one allele of the CD163 gene between nucleotides 3,148 and 3,149 compared to the reference sequence SEQ ID NO: 47; and an 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 compared to the reference sequence SEQ ID NO: 47 in the other allele of the CD163 gene.

A female swine animal has a 1382 base pair deletion from nucleotide 3,113 to nucleotide 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113 in one allele of the CD163 gene. wherein the base pair deletion; and an 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 compared to the reference sequence SEQ ID NO: 47 in the other allele of the CD163 gene.

In any of the methods described herein, the allele of the CD163 gene of the female swine animal may comprise a chromosomal sequence having at least 80% sequence identity to SEQ ID NO: 47 in said chromosomal sequence region outside the insertion or deletion. .

The allele of the CD163 gene in a female porcine animal may comprise a chromosomal sequence having at least 85% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence outside the insertion or deletion.

The allele of the CD163 gene in a female swine animal may comprise a chromosomal sequence with at least 90% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence outside the insertion or deletion.

The allele of the CD163 gene in a female porcine animal may comprise a chromosomal sequence having at least 95% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence outside the insertion or deletion.

The allele of the CD163 gene in a female swine animal may comprise a chromosomal sequence having at least 98% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence outside the insertion or deletion.

The allele of the CD163 gene in a female swine animal may comprise a chromosomal sequence having at least 99% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence outside the insertion or deletion.

The allele of the CD163 gene in a female swine animal may comprise a chromosomal sequence with at least 99.9% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence outside the insertion or deletion.

The allele of the CD163 gene in a female swine animal may comprise a chromosomal sequence having at least 100% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence outside the insertion or deletion.

In any of the methods described herein, the female swine animal has one or both alleles of the CD163 gene SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 , 110, 111, 112, 113, 114, or 119.

For example, a female porcine animal may comprise a chromosomal sequence comprising SEQ ID NO: 99, 102, 103, or 113 in one or both alleles of the CD163 gene.

Alleles of the CD163 gene in female pig animals are SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 119. It may include any combination of chromosomal sequences comprising. Therefore, female pig animals have SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 on one allele of the CD163 gene. , or chromosomal sequence comprising any one of 119 and other alleles of the CD163 gene SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, or 119.

For example, a female swine animal may comprise 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 the other allele of the CD163 gene.

The female swine animal may comprise 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 the other allele of the CD163 gene.

The female swine animal may comprise 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 the other allele of the CD163 gene.

The female swine animal may comprise 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 the other allele of the CD163 gene.

In any of the methods described herein, breeding will produce one or more embryos comprising a chromosomal sequence modified in a single allele of the CD163 gene. Because the female porcine animal used in the methods described herein contains altered chromosomal sequences in both alleles of its CD163 gene, the female porcine animal is bred with a male porcine animal containing at least one wild-type CD163 allele. This will produce fetuses that inherit a CD163 allele containing a modified chromosomal sequence from a female swine animal and a wild-type CD163 allele from a male swine animal. Therefore, breeding will produce embryos heterozygous for the modified CD163 chromosomal sequence. If a male animal contains two wild-type CD163 alleles, any embryos produced as a result of breeding will be heterozygous for the modified CD163 chromosomal sequence.

Fetuses produced by breeding will have reduced susceptibility to PRRSV infection while in utero compared to intrauterine fetuses in wild-type female swine animals.

In any of the methods described herein, breeding may include crossing (mating) a female porcine animal with a male porcine animal.

In any of the methods described herein, breeding may include artificially injecting the female animal with sperm obtained from a male animal.

In any of the methods described herein, breeding may include transferring fertilized oocytes into the reproductive tract of the female porcine animal.

In a method where breeding involves transferring fertilized oocytes into the reproductive tract of the female porcine animal, the fertilized oocytes can be produced by in vitro fertilization of the oocytes with sperm obtained from a male porcine animal.

In vitro fertilization may involve injecting sperm obtained from a male porcine animal into the cytoplasm of an oocyte.

When breeding involves transferring a fertilized oocyte into the reproductive tract of the female porcine animal, the oocyte may be an oocyte derived from a porcine female animal, such that the oocyte has both alleles of its CD163 gene. Contains altered chromosomal sequences. Alternatively, the oocytes may be oocytes derived from a different female porcine animal that contains a modified chromosomal sequence in both alleles of its CD163 gene, wherein the modified chromosomal sequence is a modified chromosomal sequence in both alleles of its CD163 gene. reduces the susceptibility of the animal to infection by PRRSV compared to the susceptibility to infection by PRRSV of female swine animals that do not contain any modified chromosomal sequence. Thus, for example, any oocyte with an altered chromosomal sequence in both alleles of the CD163 gene (e.g., a knockout of both alleles of the C163 gene) can be used.

However, if breeding involves transferring fertilized oocytes into the reproductive tract of the female porcine animal, the oocytes need not contain any modified chromosomal sequence in the alleles of their CD163 gene. Oocytes containing two wild-type CD163 alleles can be used. Oocytes containing two wild-type CD163 alleles can be fertilized with sperm obtained from a male porcine animal, wherein the sperm contains two wild-type CD163 alleles to produce a fertilized oocyte containing two wild-type CD163 alleles. Includes. Such fertilized oocytes (containing two wild-type CD163 alleles) are transferred to the reproductive tract of a female swine animal containing altered chromosomal sequences in both alleles of its CD163 gene, resulting in embryos. will be protected from PRRSV infection.

Alternatively, if breeding involves transferring fertilized oocytes into the reproductive tract of the female porcine animal, the fertilized oocytes may contain a modified chromosomal sequence in a single allele of its CD163 gene. Such fertilized oocytes may also be transferred to the reproductive tract of a female porcine animal containing the altered chromosomal sequence in both alleles of its CD163 gene. When producing a fetus that is protected from PRRSV infection.

Accordingly, if breeding involves transferring fertilized oocytes into the reproductive tract of the female porcine animal, the fertilized oocytes will have a chromosomal sequence modified in both alleles of its CD163 gene (e.g., its CD163 A knockout of both alleles of a gene) may include a chromosomal sequence altered in only one allele of its CD163 gene, or may include only the wild-type CD163 allele.

Affinity Tag

An “affinity tag” may be 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 bound by a molecule (e.g., a small molecule, protein, or covalently bound). The affinity tag may be a non-natural sequence. A peptide affinity tag may include a peptide. The peptide affinity tag can be one that can be part of a splitting system (e.g., two inactive peptide fragments can be joined together in trans to form an active affinity tag). Nucleic acid affinity tags can include nucleic acids. A nucleic acid affinity tag may be a sequence capable of selectively binding to a known nucleic acid sequence (e.g., through hybridization). A nucleic acid affinity tag may be a sequence that can selectively bind to a protein. Affinity tags can be fused to native proteins. The affinity tag may be fused to a nucleotide sequence.

Sometimes, one, two, or multiple affinity tags can be fused to a native protein or nucleotide sequence. Affinity tags can be introduced into nucleic acid-targeting nucleic acids using methods of in vitro or in vivo transcription. Nucleic acid affinity tags include, for example, chemical tags, RNA-binding protein binding sequences, DNA-binding protein binding sequences, sequences hybridizable to affinity-tagged polynucleotides, synthetic RNA aptamers, or synthetic DNA aptamers. It can be included. Examples of chemical nucleic acid affinity tags may include, but are not limited to, biotin, fluorescent dyes, and ribo-nucleotriphosphate containing digoxenin. Examples of protein-binding nucleic acid affinity tags may include, but are not limited to, an MS2 binding sequence, a U1A binding sequence, a stem-loop binding protein sequence, a boxB sequence, an eIF4A sequence, or any sequence recognized by an RNA binding protein. You can. Examples of nucleic acid affinity-tagged oligonucleotides include, but are not limited to, biotinylated oligonucleotides, 2, 4-dinitrophenyl oligonucleotides, fluorescein oligonucleotides, and primary amine-conjugated oligonucleotides. You can.

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

Nucleic acid affinity tags may include RNA sequences that can be linked by site-directed polypeptides. Site-directed polypeptides may be conditionally enzymatically inactive. The RNA sequence may include a sequence that can be bound by a member of a type I, type II, and/or type III CRISPR system. The RNA sequence is a LAMP family member protein. RNA sequences can be linked by Cas9 family member proteins, Cas6 family member proteins (eg, Csy4, Cas6). RNA sequences can be linked by Cas5 family member proteins (eg, Cas5). For example, Csy4 can bind to specific RNA hairpin sequences with high affinity (Kd ~50 pM) and cleave RNA at the 3' site of the hairpin.

Nucleic acid affinity tags may include DNA sequences that can be linked by site-directed polypeptides. Site-directed polypeptides are conditionally enzymatically inactive. The DNA sequence may include sequences that can be linked by members of type I, type II, and/or type III CRISPR systems. DNA sequences can be joined by the Argonaut protein. DNA sequences can be bound by proteins containing zinc finger domains, TALE domains, or any other DNA-binding domain.

Nucleic acid affinity tags may include ribozyme sequences. Suitable ribozymes include peptidyl transferase 23 SrRNA, RnaseP, group I intron, group II intron, GIR1 branching ribozyme, leadzyme, hairpin ribozyme, hammerhead ribozyme, HDV ribozyme, CPEB3 ribozyme, and VS ribozyme. , glmS ribozyme, CoTC ribozyme, and synthetic ribozyme.

Peptide affinity tags are tags that can be used for tracking or purification (e.g., fluorescent proteins such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato; His tag, (e.g., 6XHis tag) a hemagglutinin (HA) tag; a GST tag; a chitin binding tag; a streptavidin tag;

Both nucleic acid and peptide affinity tags include small molecule tags such as biotin, or digitoxin, and fluorescent label tags such as fluorescein, rhodamine, Alexa fluor dye, cyanine3 dye, and cyanine5 dye. can do.

A nucleic acid affinity tag may be located 5' of a nucleic acid (e.g., a nucleic acid-targeting nucleic acid). The nucleic acid affinity tag may be located 3' to the nucleic acid. Nucleic acid affinity tags can be located 5' and 3' of the nucleic acid. A nucleic acid affinity tag can be located within a nucleic acid. The peptide affinity tag may be located at the N-terminus of the polypeptide sequence. The peptide affinity tag may be located at the C-terminus of the 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 nucleic acid and/or polypeptide sequences.

capture agent

As used herein, “capture agent” generally refers to an agent capable of purifying polypeptides and/or nucleic acids. The capture agent can be a biologically active molecule or substance (e.g., any biological material found in nature or synthetically, and cells, viruses, subcellular particles, antibodies, immunoglobulins, antigens, lipoproteins, glycoproteins, peptides). , polypeptides, protein complexes, (strept)avidin-biotin complexes, ligands, receptors, or proteins, including more specifically small molecules, aptamers, nucleic acids, DNA, RNA, peptide nucleic acids, oligosaccharides, polysaccharides, lipopolysaccharides. (including, but not limited to, hydrodes, cellular metabolites, haptens, pharmacologically active substances, alkaloids, steroids, vitamins, amino acids, and saccharides). In some embodiments, the capture agent can include an affinity tag. In some embodiments, a capture agent can preferentially bind to a target polypeptide or nucleic acid of interest. The capture agent can float freely in the mixture. Capturing agents can be bound to particles (e.g. beads, microbeads, nanoparticles). The capture agent may be bound to a solid or semi-solid surface. In some cases, the capture agent is irreversibly bound to the target. In other cases, the capture agent is reversibly bound to the target (eg, if the target is eluted by use of a chemical such as imidazole).

DNA-binding polypeptide

Site-specific integration can be achieved, for example, using factors that can recognize and bind to specific nucleotide sequences in the genome of the host organism. For example, many proteins contain polypeptide domains that can recognize and bind DNA in a site-specific manner. The DNA sequence recognized by the DNA-binding polypeptide may be referred to as the “target” sequence. Polypeptide domains that are capable of recognizing and binding DNA in a site-specific manner are generally correctly folded and function independently, allowing the domain to function in a site-specific manner even when expressed in a polypeptide other than the native isolated protein. binds to DNA. Similarly, target sequences for recognition and binding by DNA-binding polypeptides are generally present on large DNA structures (e.g., chromosomes), especially if the site where the target sequence is located is accessible to soluble cellular proteins. Even known sites (e.g. genes) can be recognized and bound by such polypeptides.

DNA-binding polypeptides identified from naturally occurring proteins typically bind distinct nucleotide sequences or motifs (e.g., a consensus recognition sequence), but many such DNA-binding polypeptides are used to recognize different nucleotide sequences or motifs. Methods for modifying binding polypeptides exist and are known in the art. DNA-binding polypeptides include, for example and without limitation: a zinc finger DNA-binding domain; leucine zipper; UPA DNA-binding domain; GAL4; TAL; LexA; Tet inhibitor; LacI; and steroid hormone receptors.

For example, the DNA-binding polypeptide may have zinc fingers. Individual zinc finger motifs can be designed to target and specifically bind to any broad DNA site. Canonical Cys ₂ His ₂ (and non-canonical Cys ₃ His) zinc finger polypeptides bind DNA by inserting an α-helix into the major groove of the target DNA double helix. Recognition of DNA by zinc fingers is modular; Each finger primarily contacts three consecutive base pairs on the target, and several key residues within the polypeptide mediate recognition. By including multiple zinc finger DNA-binding domains in the targeting endonuclease, the DNA-binding specificity of the targeting endonuclease can be further increased (and thus also increases the specificity of any gene regulatory effect conferred thereby). can be). See, for example, Urnov et al. (2005) Nature 435:646-51. Accordingly, one or more zinc finger DNA-binding polypeptides can be engineered and used to allow a targeting endonuclease introduced into a host cell to interact with a unique DNA sequence within the genome of the host cell.

Preferably, the zinc finger protein is non-naturally occurring in the sense that it is engineered to bind to a selected target site. 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. Biotechnology. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Patent No. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication No. 2005/0064474; 2007/0218528; Please refer to 2005/0267061.

Engineered zinc finger binding domains can have novel binding specificities compared to naturally occurring zinc finger proteins. Methods of operation include, but are not limited to, rational design and selection of various types. Rational design includes, for example, using a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, wherein each triplet or quadruplet nucleotide has a specific triplet or quadruplet sequence. It involves one or more amino acid sequences of zinc fingers that bind to. For example, U.S. See Patent Nos. 6,453,242 and 6,534,261.

Exemplary selection methods including phage display and two-hybrid systems are described in the U.S. Pat. Patent No. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; Disclosed in WO 01/88197 and GB 2,338,237. Additionally, improvements in binding specificity for zinc finger binding domains have been described, for example, in WO 02/077227.

Additionally, as disclosed in these and other references, zinc finger domains and/or multi-finger zinc finger proteins may be linked together using any suitable linker sequence, including, for example, a linker of five or more amino acids in length. You can. Additionally, U.S. For exemplary linker sequences six or more amino acids in length, see Patent Nos. 6,479,626; 6,903,185; and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

Selection of Target Site: ZFPs and methods for the design and construction of fusion proteins (and polynucleotides encoding them) are known to those skilled in the art and are available in the U.S. Patent No. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; It is described in detail in WO 02/016536 and WO 03/016496.

Additionally, as disclosed in these and other references, zinc finger domains and/or multi-finger zinc finger proteins may be linked together using any suitable linker sequence, including, for example, a linker of five or more amino acids in length. You can. Additionally, for exemplary linker sequences six or more amino acids in length, the U.S. Pat. Patent No. 6,479,626; 6,903,185; and 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

When a porcine female animal for use in the methods described herein has been genetically edited using a zinc-finger nuclease, the female animal has at least one RNA molecule encoding a targeted zinc finger nuclease and, optionally, , can be produced using a process that includes introducing at least one accessory polynucleotide into the embryo or cell. The method further comprises incubating the embryo or cell to allow expression of the zinc finger nuclease, wherein the double-strand break introduced into the targeted chromosomal sequence by the zinc finger nuclease is error-prone non-homologous. Repaired by end-joining DNA repair process or homology-directed DNA repair process. The method of editing chromosomal sequences encoding proteins involved in germline development using targeted zinc finger nuclease technology is rapid, accurate, and highly efficient.

Alternatively, the DNA-binding polypeptide is the DNA-binding domain from GAL4. GAL4 is a modular cross-activator in Saccharomyces cerevisiae , but also acts as a cross-activator 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 regulation of these metabolic enzymes is mediated by the interaction between the positive regulatory protein GAL4 and a 17 bp symmetrical DNA sequence (upstream activation sequence (UAS)) to which GAL4 specifically binds.

Native GAL4 consists of 881 amino acid residues with a molecular mass of 99 kDa. GAL4 contains functionally autonomous domains, whose combined activity accounts for 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 N-terminal 65 amino acids of GAL4 contain the GAL4 DNA-binding domain. 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 on which the DNA binding domain resides. The coordinated cation-containing domain interacts with and recognizes the conserved CCG triplet at each end of the 17 bp UAS for direct contact with the major groove of the DNA helix. Marmorstein et al. (1992) Nature 356:408-14. The DNA-binding function of the protein positions the C-terminal transcriptional activation domain in the vicinity of the promoter so that the activation domain can direct transcription.

Additional DNA-binding polypeptides that can be used include, for example and without limitation, binding sequences from AVRBS3-inducible genes; A consensus binding sequence from an AVRBS3-inducible gene or a synthetic binding sequence engineered therefrom (e.g., UPA DNA-binding domain); TAL; LexA (see, e.g., Brent & Ptashne (1985), supra); LacR (see, e.g., Labow et al. (1990) Mol. Cell. Biol. 10:3343-56; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88(12):5072-6); Steroid hormone receptor (Ellliston et al. (1990) J. Biol. Chem. 265:11517-121); Tet repressor (U.S. Pat. No. 6,271,341) and a mutated Tet repressor that binds a tet operator sequence in the presence but not in the absence of tetracycline (Tc); DNA-binding domain of NF-kappaB; and Wang et al. (1994) Proc., utilizing a fusion of GAL4, hormone receptor, and VP16. Natl. Acad. Sci. It includes components of the control system described in USA 91(17):8180-4.

One or more DNA-binding domains of the nucleases used in the methods and compositions described herein may comprise naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domains. See, for example, US Patent Publication No. 2011/0301073.

Alternatively, the nuclease may include the CRISPR system. For example, nucleases may include the CRISPR/Cas system.

The (CRISPR-related) system evolved in bacteria and archaea as an adaptive immune system to defend against viral attacks. Upon exposure to a virus, a short segment of viral DNA is integrated into the CRISPR locus. RNA is transcribed from the part of the CRISPR locus that contains the viral sequence. RNA containing sequences complementary to the viral genome mediates targeting of Cas proteins (e.g., Cas9 proteins) to sequences in the viral genome. The Cas protein cleaves and silences the viral target. Recently, the CRISPR/Cas system has been applied to genome editing in eukaryotic cells. Introducing a site-specific double strand break (DSB) can alter the target sequence through one of two endogenous DNA repair mechanisms: non-homologous end-joining (NHEJ) or 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).

The CRISPR/Cas system comprises a CRISPR (clustered regularly interspaced short palindromic repeat) locus encoding the RNA component of the system and a Cas (CRISPR-related) locus encoding the protein (Jansen et al., 2002. Mol 43: 1565-1575; Nucleic Acids Res. 2006. Haft et al., 2005. . The CRISPR locus of a microbial host contains a combination of Cas genes and non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage.

Type II CRISPR is one of the best characterized systems and performs targeted DNA double-strand breaks in nature in four sequential steps. First, two non-coding RNAs, a pre-crRNA array and a tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of pre-crRNA and mediates the processing of pre-crRNA into mature crRNA containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex binds Cas9 via Watson-Crick base pairing between the spacer of the crRNA and the protospacer of the target DNA flanked by a protospacer adjacent motif (PAM), an additional requirement for target recognition. is guided to the target DNA. Finally, Cas9 mediates cleavage of the target DNA, creating a double-strand break within the protospacer.

For use of the CRISPR/Cas system to create targeted insertions and deletions, two non-coding RNAs (crRNA and TracrRNA) can be replaced with a single RNA called a guide RNA (gRNA). The activity of the CRISPR/Cas system consists of three steps: (i) insertion of exogenous DNA sequences into the CRISPR array to prevent future attacks in a process called “adaptation”, (ii) expression of the associated proteins, as well as the array. expression and processing, followed by (iii) RNA-mediated interference with foreign nucleic acids. In bacterial cells, several Cas proteins are involved in the native functions of the CRISPR/Cas system and play a role in functions such as foreign DNA insertion.

Cas proteins may be “functional derivatives” of naturally occurring Cas proteins. A “functional derivative” of a native sequence polypeptide is a compound that has qualitative biological properties in common with the native sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of native sequence and derivatives of native sequence polypeptides and fragments thereof, provided that they have a biological activity in common with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of the functional derivative to hydrolyze the DNA substrate into fragments. The term “derivative” includes all amino acid sequence variants, covalent modifications, and fusions thereof of a polypeptide. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, and covalent modifications of Cas proteins or fragments thereof. Cas proteins, including Cas proteins or fragments thereof, as well as derivatives of Cas proteins or fragments thereof, can be obtained from cells, chemically synthesized, or obtained by a combination of these two procedures. A cell may be a cell that naturally produces a Cas protein, or may naturally produce a Cas protein and produce an endogenous Cas protein at a higher expression level, or may produce a Cas protein from an exogenously introduced nucleic acid in which the nucleic acid encodes a Cas that is the same or different from the endogenous Cas. It may be a cell that has been genetically engineered to produce In some cases, cells are genetically engineered to produce Cas proteins rather than naturally producing them.

If female porcine animals for use in the methods described herein have been genetically edited using the CRISPR system, the CRISPR/Cas9 system can be used to generate female porcine animals. Cas9 can be used to deliver proteins directly into cells to edit genome sequences. Alternatively, mRNA encoding Cas9 can be delivered to the cell, or a gene that provides for expression of mRNA encoding Cas9 can be delivered to the cell. Additionally, target-specific crRNA and tracrRNA can be delivered directly to the cell or target-specific gRNA(s) can be present in the cell (these RNAs can alternatively be produced by genes configured to express these RNAs). . Selection of target sites and design of crRNA/gRNA are 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.

DNA-binding polypeptides are capable of specifically recognizing and binding to target nucleotide sequences contained within the genomic nucleic acid of a host organism. Any number of individual instances of the target nucleotide sequence may, in some instances, be found in the host genome. The target nucleotide sequence may be rare within the genome of the organism (e.g., about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about Less than 1 copy(s) may be present in the genome). For example, the target nucleotide sequence may be located at a unique site within the organism's genome. Target nucleotide sequences may be randomly distributed throughout the genome, for example and without limitation, with respect to each other; Located in different linkage groups within the genome; located in the same linkage group; located on different chromosomes; located on the same chromosome; is located in the genome at a site where it is expressed under similar conditions in an organism (e.g., under the control of the same or substantially functionally identical regulatory elements); They may be located closely together in the genome (e.g., the target sequences may be comprised within nucleic acids that are concatemerically integrated at a genomic locus).

Targeting of endonucleases

A DNA-binding polypeptide that specifically recognizes and binds to a target nucleotide sequence may be included within the chimeric polypeptide to confer specific binding to the target sequence on the chimeric polypeptide. In an example, such a chimeric polypeptide may include, for example and without limitation, a nuclease, recombinase, and/or ligase polypeptide, as the polypeptide is described above. Chimeric polypeptides, including DNA-binding polypeptides and nuclease, recombinase, and/or ligase polypeptides, may also include other functional polypeptide motifs and/or domains, such as, but not limited to: A spacer sequence positioned between functional polypeptides in a chimeric protein; leader peptide; peptides that target the fusion protein to an organelle (e.g., the nucleus); polypeptides cleaved by cellular enzymes; Peptide tags (e.g., Myc, His, etc.); and other amino acid sequences that do not interfere with the function of the chimeric polypeptide.

Functional polypeptides (e.g., DNA-binding polypeptides and nuclease polypeptides) in a chimeric polypeptide can be operably linked. The functional polypeptides of the chimeric polypeptide may be operably linked by expression from a single polynucleotide encoding at least the functional polypeptide to be linked together in frame to generate a chimeric gene encoding the chimeric protein. Alternatively, the functional polypeptides of the chimeric polypeptide may be operably linked by other means, such as by cross-linking of independently expressed polypeptides.

A DNA-binding polypeptide, or guide RNA, that specifically recognizes and binds to a target nucleotide sequence may be included in a naturally occurring isolated protein (or a mutant thereof), wherein the naturally isolated protein or a mutant thereof also contains a nuclease. Includes polypeptides (and may also include recombinase and/or ligase polypeptides). Examples of such isolated proteins include TALENs, recombinases (e.g., Cre, Hin, Tre, and FLP recombinases), RNA-guided CRISPR/Cas9, and meganucleases.

As used herein, the term “targeting endonuclease” refers to isolated proteins and mutants thereof, natural or engineered, including DNA-binding polypeptides or guide RNAs and nuclease polypeptides, as well as DNA-binding polypeptides. Refers to a polypeptide or a chimeric polypeptide containing a guide RNA and a nuclease. specifically recognizes a target nucleotide sequence contained within the CD163 locus (e.g., because the target sequence is contained within the native sequence of the locus, or because the target sequence has been introduced into the locus, e.g., by recombination) and Any targeting endonuclease comprising a DNA-binding polypeptide or guide RNA that binds to can be used.

Some examples of suitable chimeric polypeptides include, but are not limited to, combinations of the following polypeptides: zinc finger DNA-binding polypeptides; FokI nuclease polypeptide; TALE domain; leucine zipper; transcription factor DNA-binding motif; and, for example and without limitation, recognition of isolated DNA from TALENs, recombinases (e.g., Cre, Hin, RecA, Tre, and FLP recombinases), RNA-guided CRISPR/Cas9, meganucleases, and /or cleavage domain; and others known to those skilled in the art. Specific examples include chimeric proteins comprising site-specific DNA binding polypeptides and nuclease polypeptides. Chimeric polypeptides can be manipulated by methods known to those skilled in the art to alter the recognition sequences of DNA-binding polypeptides contained within the chimeric polypeptide to target the chimeric polypeptide to a specific nucleotide sequence of interest.

The chimeric polypeptide may include a DNA-binding domain (eg, zinc fingers, TAL-effector domain, etc.) and a nuclease (cleavage) domain. The cleavage domain may be 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 from a different nuclease, or a meganuclease DNA-binding domain and a cleavage domain. It may be heterogeneous for the domain. Heterologous cleavage domains can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which cleavage domains can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. For example, 2002-2003 Catalog, New England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res. See 25:3379-3388. Additional enzymes that cleave DNA are known (e.g., 51 nuclease; mung bean nuclease; pancreatic DNAse I; micrococcal nuclease; yeast HO endonuclease; 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 hemi-domains.

Similarly, the cleavage half-domain may be derived from any of the nucleases described above or portions thereof that require dimerization for cleavage activity. Generally, if the fusion protein contains a cleavage half-domain, two fusion proteins are required for cleavage. Alternatively, a single protein containing two truncated half-domains can be used. The two cleavage half-domains may be derived from the same endonuclease (or functional fragment thereof), or each cleavage half-domain may be derived from a different endonuclease (or functional fragment thereof). Additionally, the target sites for the two fusion proteins are preferably such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation relative to each other such that the cleavage half-domains are, for example, , are positioned relative to each other so that they can form functional cleavage domains by dimerization. Therefore, the proximal edge of the target site may be separated by 5-8 nucleotides or 15-18 nucleotides. However, any integer number of nucleotides or nucleotide pairs (e.g., 2 to 50 nucleotide pairs or more) may intervene between the two target sites. Typically, the cleavage site is between target sites.

Restriction endonucleases (restriction enzymes) exist in many species and can bind sequence-specifically to DNA (recognition site), e.g. one or more exogenous sequences (donor/transgene) to bind (target). DNA can be cut at or near the binding site to allow integration at or near the site. Certain restriction enzymes (e.g., type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme Fok I catalyzes double-strand breaks in DNA at 9 nucleotides in the recognition site on one strand and 13 nucleotides in the recognition site on the other strand. See, for example, US Patent No. 5,356,802; 5,436,150 and 5,487,994; In addition, Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. See 269:31,978-31,982. Accordingly, the fusion protein may comprise a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains that may or may not be engineered.

An exemplary type IIS restriction enzyme, whose cleavage domain is separable from the binding domain, is Fok I. This particular enzyme acts as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Accordingly, for the purposes of this disclosure, the portion of the Fok I enzyme used in the disclosed fusion proteins is considered the cleavage half-domain. Therefore, for targeted double-strand cleavage and/or targeted replacement of cellular sequences using zinc finger-FokI fusions, two fusion proteins each containing a FokI cleavage half-domain are combined with a catalytically active cleavage domain. It can be used to reconstruct. Alternatively, a single polypeptide molecule containing a DNA binding domain and two Fok I cleavage half-domains can also be used.

A cleavage domain or cleavage half-domain can be any portion of a protein that possesses cleavage activity or the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in US Patent Publication No. 2007/0134796. Additional restriction enzymes also include separable binding and cleavage domains, which are contemplated by this disclosure. For example, Roberts et al. (2003) Nucleic Acids Res. See 31:418-420.

Cleavage domains include, for example, US Patent Publication No. 2005/0064474; It may comprise one or more engineered truncated half-domains (also called dimerization domain mutants) that minimize or prevent homodimerization, as described in 2006/0188987 and 2008/0131962.

Alternatively, nucleases can be assembled in vivo at nucleic acid target sites using so-called “split-enzyme” techniques (see, for example, US Patent Publication No. 20090068164). Components of such a cleavage enzyme can be expressed on separate expression constructs, or the individual components can be linked in one open reading frame separated by, for example, a self-cleaving 2A peptide or an IRES sequence. . The components may be individual zinc finger binding domains or domains of a meganuclease nucleic acid binding domain.

zinc finger nuclease

Chimeric polypeptides are custom-designed zinc finger nucleases (ZFNs) designed to deliver targeted site-specific double-stranded DNA breaks into which exogenous nucleic acids, or donor DNA, can be incorporated (see US Patent Publication 2010/0257638). . ZFNs are chimeric polypeptides containing a non-specific cleavage domain from a restriction endonuclease (eg, FokI) and a zinc finger DNA-binding domain polypeptide. For example, Huang et al. (1996) J. Protein Chem. 15:481-9; Kim et al. (1997a) Proc. Natl. Acad. Sci. USA 94:3616-20; Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-60; Kim et al. (1994) Proc Natl. Acad. Sci. USA 91:883-7; Kim et al. (1997b) Proc. Natl. Acad. Sci. USA 94:12875-9; Kim et al. (1997c) Gene 203:43-9; Kim 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. See 27:674-81. ZFNs may contain non-canonical zinc finger DNA binding domains (see US Patent Publication 2008/0182332). The FokI restriction endonuclease must dimerize through the nuclease domain to cleave DNA and introduce double-strand breaks. As a result, ZFNs containing a nuclease domain from such endonucleases also require dimerization of the nuclease domain 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. Dimerization of ZFNs can be promoted by two adjacent, oppositely oriented DNA-binding sites. Id.

A method for site-specific integration of an exogenous nucleic acid into at least one CD163 locus of a host includes introducing a ZFN into a cell of the host, wherein the ZFN recognizes and binds to a target nucleotide sequence, and The nucleotide sequence is contained within at least one CD163 locus of the host. In certain instances, the target nucleotide sequence is not contained within the host's genome at any location other than at least one CD163 locus. For example, a DNA-binding polypeptide of a ZFN can be engineered to recognize and bind a target nucleotide sequence identified within at least one CD163 locus (e.g., by sequencing the CD163 locus). A method for site-specific integration of an exogenous nucleic acid into at least one CD163 performance locus of a host comprising introducing a ZFN into a cell of the host may also include introducing the exogenous nucleic acid into the cell, the exogenous nucleic acid comprising: Recombination into a host's nucleic acid containing at least one CD163 locus is facilitated by site-specific recognition and binding of the ZFN to the target sequence (and subsequent cleavage of the nucleic acid containing the CD163 locus).

Optional exogenous nucleic acid for integration at the CD163 locus

Exogenous nucleic acids for integration at the CD163 locus include: exogenous nucleic acids for site-specific integration at at least one CD163 locus, including, but not limited to, ORFs; A nucleic acid comprising a nucleotide sequence encoding a targeting endonuclease; and vectors comprising one or both of the foregoing. Accordingly, a particular nucleic acid includes a nucleotide sequence encoding a polypeptide, a structural nucleotide sequence, and/or a DNA-binding polypeptide recognition and binding site.

Selective exogenous nucleic acid molecules for site-specific integration

As mentioned above, the insertion of an exogenous sequence (also called a “donor sequence” or “donor” or “transgene”) provides for, for example, the expression of a polypeptide, modification of a mutant gene or increased expression of a wild-type gene. . It will be readily apparent that the donor sequence is not identical to the genomic sequence from which it is typically placed. The donor sequence may contain non-homologous sequences flanked by two regions of homology to allow efficient homology-directed repair (HDR) at the position of interest. Additionally, the donor sequence may include a vector molecule containing a sequence that is not homologous to the region of interest in cellular chromatin. The donor molecule may contain several, discrete regions of homology to cellular chromatin. For example, for targeted insertion of a sequence that is not normally present in the region of interest, the sequence can be present in a donor nucleic acid molecule and flanked by regions of homology to the sequence in the region of interest.

The donor polynucleotide may be DNA or RNA, single-stranded 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. If introduced in linear form, the ends of the donor sequence may be protected (e.g., 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. For example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; See Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and modification of internucleotide linkages such as phosphorothioate, phosphoramidate, and O-methyl ribose or deoxy. Including the use of ribose residues.

The polynucleotide can be introduced into the cell as part of a vector molecule with additional sequences, for example, an origin of replication, a promoter, and genes encoding antibiotic resistance. Additionally, the donor polynucleotide can be introduced as a naked nucleic acid, such as a nucleic acid complexed with an agent such as a liposome or poloxamer, or as a nucleic acid complexed with a virus (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus, and integrase It can be transmitted by defective lentivirus (IDLV).

The donor is generally integrated such that its expression is driven by an endogenous promoter at the site of integration, i.e. a promoter that drives expression of the endogenous gene into which the donor is integrated (e.g., CD163). However, it will be clear that the donor may comprise a promoter and/or enhancer, for example a constitutive promoter or an inducible or tissue specific promoter.

Additionally, although not required for expression, exogenous sequences may also include transcriptional or translational control sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding the 2A peptide, and/or polyadenylation signals. there is.

Exogenous nucleic acids that can be integrated into at least one CD163 locus in a site-specific manner to modify the CD163 locus include, for example and without limitation, nucleic acids comprising a nucleotide sequence encoding a polypeptide of interest; Nucleic acids containing agronomic genes; A nucleic acid comprising a nucleotide sequence encoding an RNAi molecule; or a nucleic acid that destroys the CD163 gene.

An exogenous nucleic acid can be integrated at the CD163 locus, modifying the CD163 locus, wherein the nucleic acid comprises 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, a polypeptide of interest (e.g., a foreign protein) is expressed in commercial quantities from a nucleotide sequence encoding the polypeptide of interest. In such examples, the polypeptide of interest may be extracted from host cells, tissues, or biomass.

Nucleic acid molecule comprising a nucleotide sequence encoding a targeting endonuclease

The nucleotide sequence encoding the targeting endonuclease can be engineered by manipulation (e.g., ligation) of the native nucleotide sequence encoding the polypeptide comprised within the targeting 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 the gene corresponding to the DNA-binding polypeptide, and that nucleotide sequence may be a DNA-binding polypeptide. It can be used as an element of a nucleotide sequence encoding a targeting endonuclease, including a peptide. Alternatively, the amino acid sequence of the targeting endonuclease can be used to infer the nucleotide sequence encoding the targeting endonuclease, for example, according to the degeneracy of the genetic code.

In an exemplary nucleic acid molecule comprising a nucleotide sequence encoding a targeting endonuclease, the last codon of a first polynucleotide sequence encoding a nuclease polypeptide, and a second polynucleotide sequence encoding a DNA-binding polypeptide. The first codon of may be separated by any number of nucleotide triplets, for example, without coding for an intron or “STOP”. Likewise, the last codon of the nucleotide sequence encoding the first polynucleotide sequence encoding the DNA-binding polypeptide, and the first codon of the second polynucleotide sequence encoding the nuclease polypeptide may also be any number of nucleotide triplets. can be separated by The last codon (i.e., 3' last in the nucleic acid sequence) of the first polynucleotide sequence encoding a nuclease polypeptide and the second polynucleotide sequence encoding a DNA-binding polypeptide is either immediately adjacent thereto or is connected to a synthetic nucleotide linker. the first codon of an additional polynucleotide coding sequence separated therefrom by only a short peptide sequence, such as that encoded by (e.g., a nucleotide linker that can be used to effect fusion) and a phase-register ) can be fused in. Examples of such additional polynucleotide sequences include, by way of example and without limitation, tags, targeting peptides, and enzymatic cleavage sites. Likewise, the 5'most (in the nucleic acid sequence) first codon of the first and second polynucleotide sequences is the last codon of an additional polynucleotide coding sequence immediately adjacent thereto or separated therefrom by only a short peptide sequence. Can be fused in phase-register.

Sequences that separate polynucleotide sequences encoding functional polypeptides (e.g., DNA-binding polypeptides and nuclease polypeptides) from targeting endonucleases include, for example, the amino acid sequence encoded in the targeting endonuclease. It may consist of any sequence that does not significantly alter the translation of the clease. Due to the autonomous nature of known nuclease polypeptides and known DNA-binding polypeptides, intervening sequences will not interfere with the respective functions of these structures.

Different knockout methods

Various other techniques known in the art can be used to inactivate genes to create knockout animals and/or to introduce nucleic acid constructs into animals to produce progenitor animals and to create animal lines, wherein the knockouts or nucleic acid constructs are generated. is integrated into the genome. Such techniques include, but are not limited to, pronuclear microinjection (US Pat. No. 4,873,191), retroviral mediated gene transfer to the germ line (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82 , 6148-1652), gene targeting to embryonic stem cells (Thompson et al. (1989) Cell 56, 313-321), electroporation of embryos (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 somatic cells, such as cumulus cells. or in vitro transformation of mammary cells, or adult, fetal, or embryonic stem cells, followed by nuclear transfer (Wilmut et al. (1997) Nature 385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374). do. Pronuclear microinjection, sperm-mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques. A genome-modified animal is one in which all cells, including germ line cells, have been modified. When methods are used to produce modified mosaic animals, the animals can be inbred and genomically modified progeny selected. For example, cloning can be used to create mosaic animals in which cells are modified in the blastocyst state, and genomic modifications can occur when a single cell is modified. Animals modified to fail to reach sexual maturity may be homozygous or heterozygous for the modification, depending on the specific approach used. If a specific gene is inactivated by a knockout variant, homozygosity will generally be required. When a specific gene is inactivated by RNA interference or dominant negative strategies, heterozygosity is often appropriate.

Typically, in embryo/zygote microinjection, a nucleic acid construct or mRNA is introduced into a fertilized egg; A one or two cell fertilized egg serves as the nuclear structure that contains the genetic material of the sperm head and the fertilized egg is visible within the protoplasm. Pronuclear stage fertilized eggs can be obtained in vitro or in vivo (i.e., surgically retrieved from the oviduct of a donor animal). In vitro fertilized eggs can be produced as follows. For example, porcine ovaries may be collected from slaughterhouses and maintained at 22-28°C during transport. Ovaries can be cleaned and isolated for follicular aspiration, and follicles ranging from 4-8 mm can be aspirated into a 50 mL conical centrifuge tube using an 18 gauge needle under vacuum. Follicular fluid and aspirated oocytes can be rinsed through a pre-filter using commercial TL-HEPES (Minitube, Verona, Wis.). Select oocytes surrounded by a dense mass of cumulus cells and incubate at 38.7°C in humidified air. and 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid, 50 μM 2-mercaptoethanol, 0.5 mg/ml cAMP, pregnant mare serum for approximately 22 hours at 5% CO _{2 .} were placed in TCM-199 oocyte maturation medium (Minitube, Verona, Wis.) supplemented with 10 IU/mL each of ropinene (PMSG) and human chorionic gonadotropin (hCG). Oocytes can then be transferred to fresh TCM-199 maturation medium containing cAMP, PMSG or hCG and incubated for an additional 22 hours. Mature oocytes can be vortexed in 0.1% hyaluronidase for 1 min to dissociate cumulus cells.

For porcines, mature oocytes can be fertilized in 500 μl minitube PORCPRO IVF medium system (Minitube, Verona, Wis.) in minitube 5-well fertilization dishes. In preparation for in vitro fertilization (IVF), freshly-collected or frozen boar semen can be cleaned and resuspended up to 400,000 sperm in PORCPRO IVF medium. Sperm concentration can be analyzed by computer-assisted semen analysis (SPERMVISION, Minitube, Verona, Wis.). The final in vitro semen injection can be performed in a 10 μl volume at a final concentration of approximately 40 motile sperm/oocytes depending on the boar. All fertilized oocytes can be incubated for 6 hours at 38.7°C in a 5.0% CO ₂ atmosphere. Six hours after semen injection, the putative zygote can be washed twice in NCSU-23 and transferred to 0.5 mL of the same medium. This system can typically produce 20-30% blastocysts in most boars at a polysperm insemination semen injection rate of 10-30%.

Linearized nucleic acid constructs or mRNA can be injected into either the pronucleus or the cytoplasm. The injected fertilized eggs can then be transferred to the recipient female (e.g., in the oviduct of the recipient female) and develop in the recipient female to produce transgenic or genetically edited animals. In particular, the pronucleus can be visualized by centrifuging in vitro fertilized embryos at 15,000 xg for 5 minutes to precipitate lipids. Embryos can be injected using an Eppendorf FEMTOJET injector and cultured until blastocysts are formed. The rate and quality of embryo excision and blastocyst formation can be recorded.

Embryos can be surgically transferred to the uterus of an asynchronous recipient. Typically, 100-200 (e.g., 150-200) embryos can be deposited at the ampullary-isthmus junction of the oviduct using a 5.5-inch TOMCAT® catheter. After surgery, a real-time pregnancy ultrasound can be performed.

In somatic cell nuclear transfer, transformed or gene-edited cells, such as embryonic blastomeres, fetal fibroblasts, adult otic fibroblasts, or granulosa cells, containing the nucleic acid constructs described above, are introduced into the enucleated oocyte to combine the cells. can be established. Oocytes can be enucleated by partial zona pellucida incision near the polar body followed by extrusion of the cytoplasm from the incision area. Typically, an injection pipette with a sharply beveled tip is used to inject transformed or genetically edited cells into enucleated oocytes arrested in meiosis 2. In some conventions, an oocyte arrested in meiosis-2 is called a zygote. After generating pig or bovine embryos (e.g., by fusing and activating oocytes), the embryos are transferred to the oviduct of a recipient female approximately 20 to 24 hours after activation. See, for example, Cibelli et al. (1998) Science 280, 1256-1258 and US Patent Nos. 6,548,741, 7,547,816, 7,989,657, or 6,211,429. In pigs, female recipients can be tested for pregnancy approximately 20-21 days after embryo transfer.

Standard breeding techniques can be used to generate animals homozygous for the inactivated gene from an initially heterozygous progenitor animal. However, homozygosity may not be necessary. Gene-edited pigs described herein can be housed with other pigs of interest.

Once gene-edited animals are generated, inactivation of endogenous nucleic acids can be assessed using standard techniques. Initial screening can be performed by Southern blot analysis to determine whether inactivation has occurred. For descriptions of Southern analysis, see Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; See sections 9.37-9.52 of N.Y. Polymerase chain reaction (PCR) technology can also be used for initial screening. PCR refers to the procedure or technique by which target nucleic acids are amplified. Generally, sequence information at or beyond the end of the region of interest is used to design oligonucleotide primers that are identical or similar in sequence to the opposite strand of the template to be amplified. PCR can be used to amplify specific sequences of RNA as well as DNA, including sequences of total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides in length, but can vary from 10 nucleotides to several hundred nucleotides in length. PCR is described, for example, in PCR Primer: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Nucleic acids may also be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or may be nucleic acid sequence-based. 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 processed individually for PCR, Southern hybridization and splinkerette PCR analysis (see, e.g., Dupuy et al. Proc Natl Acad Sci USA (2002) 99:4495).

interfering RNA

A variety of interfering RNA (RNAi) systems are known. Double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous gene transcripts. The RNA-induced silencing complex (RISC) metabolizes dsRNA into small 21-23-nucleotide small interfering RNAs (siRNAs). RISC includes double-stranded RNAse (dsRNAse, e.g. Dicer) and ssRNAse (e.g. Argonaut 2 or Ago2). RISC uses the antisense strand as a guide to find cleavable targets. Both siRNA and microRNA (miRNA) are known. Methods for inactivating genes in genetically edited animals include inducing RNA interference to target genes and/or nucleic acids such that expression of the target genes and/or nucleic acids is reduced.

For example, an exogenous nucleic acid sequence can induce RNA interference with the nucleic acid encoding the polypeptide. For example, double-stranded small interfering RNA (siRNA) or small hairpin RNA (shRNA) homologous to the target DNA can be used to reduce expression of that DNA. Constructs for siRNA can be produced, for example, as described 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. Constructs for shRNA can be produced as described in McIntyre and Fanning (2006) BMC Biotechnology 6:1. Typically, shRNA is transcribed as a single-stranded RNA molecule containing complementary regions that anneal to form short hairpins.

The probability of finding a single individual functional siRNA or miRNA for a specific gene is high. For example, although the predictability of a particular sequence in siRNA is about 50%, numerous interfering RNAs can be made with confidence that at least one of them will be effective.

In vitro cells expressing RNAi targeting the gene encoding CD163, in vivo cells, or genetically edited animals such as domestic animals can be used. RNAi may be selected from the group consisting of, for example, siRNA, shRNA, dsRNA, RISC and miRNA.

inductive system

The CD163 gene can be inactivated using an inducible system. A variety of inducible systems are known that allow spatial and temporal control of gene inactivation. Several have been demonstrated to function in vivo in porcine animals.

An example of an inducible system is the tetracycline (tet)-on promoter system, which can be used to regulate transcription of nucleic acids. In this system, a mutated Tet repressor (TetR) is fused to the activation domain of the herpes simplex virus VP 16 trans-activator protein to generate a tetracycline-controlled transcriptional activator (tTA) that is regulated by tet or doxycycline (dox). do. In the absence of antibiotics, transcription is minimal, but in the presence of tet or dox, transcription is induced. Alternative inducible systems include the ecdysone or rapamycin systems. Ecdysone is an insect molting hormone whose production is controlled by a heterodimer of the ecdysone receptor, and the ultraspiracle gene (USP) product. Expression is induced by treatment with ecdysone or an ecdysone analog such as muristerone A. Agents administered to animals to trigger the inducible system are called inducing agents.

Among the more commonly used inducible systems are the tetracycline-inducible system and the Cre/loxP recombinase system (constitutive or inducible). Tetracycline-inducible systems include tetracycline-controlled cross-activator (tTA)/reverse tTA (rtTA). Methods for using these systems in vivo involve generating two strains of genetically edited animals. An animal strain expresses an activator (tTA, rtTA, or Cre recombinase) under the control of a selected promoter. Another animal strain expresses the receptor, where the expression of the gene of interest (or the gene to be altered) is under the control of a target sequence for a tTA/rtTA crossactivator (or flanked by a loxP sequence). When two animals mate, gene expression is controlled.

The tetracycline-dependent regulatory system (tet system) consists of two components: a tTA/rtTA-dependent promoter that controls the expression of downstream cDNA in a tetracycline-dependent manner and a tetracycline-controlled cross-activator (tTA or rtTA). In the absence of tetracycline or its derivatives (such as doxycycline), tTA binds to the tetO sequence, allowing tTA-dependent transcriptional activation of the promoter. However, in the presence of doxycycline, tTA cannot interact with its target, and transcription does not occur. The tet system using tTA is called tet-OFF because tetracycline or doxycycline enables transcriptional down-regulation. Administration of tetracycline or its derivatives allows temporal control of transgene expression in vivo . rtTA is a variant of tTA that does not function in the absence of doxycycline but requires the presence of a ligand for co-activation. Therefore, this tet system is called tet-ON. The tet system has been used in vivo for the inducible expression of several transgenes encoding, for example, reporter genes, oncogenes, or proteins involved in signaling cascades.

The Cre/lox system uses Cre recombinase to catalyze site-specific recombination by crossover between two separate Cre recognition sequences, the loxP site. DNA sequences introduced between two loxP sequences (aka floxed DNA) are cleaved by Cre-mediated recombination. Controlling Cre expression in transgenic and/or genetically edited animals using spatial control (using tissue-specific or cell-specific promoters) or temporal control (using an inducible system) allows for DNA incision between two loxP sites. It is controlled. One application is for conditional gene inactivation (conditional knockout). Another approach is for protein overexpression, in which a floxed stop codon is inserted between the promoter sequence and the DNA of interest. Genetically edited animals do not express the transgene until Cre is expressed, thereby excising the floxed stop codon. This system has been applied to tissue-specific tumorigenesis and controlled antigen receptor expression in B lymphocytes. Inducible Cre recombinase was also developed. Inducible Cre recombinase is activated only by administration of exogenous ligand. Inducible Cre recombinase is a fusion protein comprising the original Cre recombinase and a specific ligand-binding domain. The functional activity of Cre recombinase depends on external ligands that can bind to this specific domain in the fusion protein.

In vitro cells containing the CD163 gene under the control of an inducible system, in vivo cells, or genetically edited animals such as domestic animals can be used. Chromosomal alterations in animals can be genomic or mosaic. The inducible system may be selected from the group consisting of, for example, Tet-On, Tet-Off, Cre-lox, and Hif1 alpha.

Vectors and Nucleic Acids

Various nucleic acids can be introduced into cells for knockout purposes, to inactivate genes, to achieve expression of genes, or for other purposes. As used herein, the term nucleic acid includes DNA, RNA, and nucleic acid analogs, and nucleic acids that are double-stranded or single-stranded (i.e., sense or antisense single stranded). Nucleic acid analogs can be modified in the base moiety, sugar moiety, or phosphate backbone to improve, for example, the stability, hybridization, or solubility of the nucleic acid. Modifications in the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-oxycytidine for deoxycytidine. do. Modification of the sugar moiety involves modification of the 2'hydroxyl of the ribose sugar to form a 2'-O-methyl or 2'-O-allyl saccharide. The deoxyribose phosphate backbone can be modified to produce a morpholino nucleic acid, wherein each base moiety is linked to a six-membered circle, morpholino ring, or peptide nucleic acid, and the deoxyphosphate backbone is replaced by a pseudopeptide backbone. and 4 bases are maintained. Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7(3):187; and Hyrup et al. (1996) Bioorgan. Med. Chem. See 4:5. Additionally, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

The target nucleic acid sequence can be operably linked to a regulatory region such as a promoter. The regulatory region may be a porcine regulatory region or may be from another species. As used herein, operably linked refers to the positioning of a regulatory region relative to a nucleic acid sequence in a manner that allows or promotes transcription of the target nucleic acid.

Any type of promoter can be operably linked to a target nucleic acid sequence. Examples of promoters include, but are not limited to, tissue-specific promoters, constitutive promoters, inducible promoters, and promoters that may or may not respond to specific stimuli. Suitable tissue-specific promoters can result in preferential expression of nucleic acid transcripts in beta cells and include, for example, the human insulin promoter. Other tissue-specific promoters may, for example, result in preferential expression in hepatocytes or cardiac tissue and include the albumin or alpha-myosin heavy chain promoters, respectively. Promoters that promote expression of nucleic acid molecules without significant tissue or temporal-specificity (i.e., constitutive promoters) can be used. For example, beta-actin promoters such as chicken beta-actin gene promoter, ubiquitin promoter, miniCAGs promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or 3-phosphoglycerate kinase (PGK) promoter may be used. Additionally, viral promoters such as the herpes simplex virus thymidine kinase (HSV-TK) promoter, SV40 promoter, or cytomegalovirus (CMV) promoter may be used. For example, a fusion of the chicken beta actin gene promoter with the CMV enhancer can be used as the promoter. For example, Xu et al. (2001) Hum. Gene Ther. 12:563; and Kiwaki et al. (1996) Hum. Gene Ther. See 7:821.

Additional regulatory regions that may be useful in nucleic acid constructs may include, but are not limited to, polyadenylation sequences, translational control sequences (e.g., internal ribosome entry segments, IRES), enhancers, inducible elements, or introns. there is. Such regulatory regions may increase expression by influencing transcription, mRNA stability, translation efficiency, etc., but may not be necessary. Such regulatory regions may be included in the nucleic acid construct as desired to achieve optimal expression of the nucleic acid in the cell(s). However, sufficient expression can sometimes be obtained without such additional elements.

Nucleic acid constructs encoding signal peptides or selectable markers may be used. Signal peptides can be used to direct the encoded polypeptide to a specific cellular location (e.g., the cell surface). Non-limiting examples of selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphatase (neo, G418, APH), dihydrofolate reductase (DHFR), hygro Includes mycin-B-phosphotransferase, thymidine kinase (TK), and xanthine-guanine phosphoribosyltransferase (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.

Sequences encoding selectable markers may be flanked by recognition sequences for recombinase enzymes such as Cre or Flp. For example, a selectable marker can be flanked by a loxP recognition site (a 34-bp recognition site recognized by Cre recombinase) or an FRT recognition site so that the selectable marker can be cleaved from the construct. Orban, et al., for a review of Cre/lox technology, Proc. Natl. Acad. Sci. (1992) 89:6861, and Brand and Dymecki, Dev. See Cell (2004) 6:7. Transposons containing a Cre- or Flp-activating transgene blocked by a selectable marker gene can also be used to obtain animals with conditional expression of the transgene. For example, the promoter that drives expression of the marker/transgene may be ubiquitous or tissue-specific, which may result in ubiquitous or tissue-specific expression of the marker in F0 animals (e.g., pigs). . Tissue-specific activation of a transgene can be achieved, for example, by crossing a pig ubiquitously expressing a marker-disrupted transgene with a pig expressing Cre or Flp in a tissue-specific manner, or by crossing a pig expressing Cre or Flp in a tissue-specific manner. This can be achieved by crossing a pig expressing the marker-disrupted transgene in this way with a pig expressing ubiquitously Cre or Flp recombinase. Controlled expression of the transgene or controlled excision of the marker allows expression of the transgene.

Exogenous nucleic acids can encode polypeptides. The nucleic acid sequence encoding the polypeptide includes a tag sequence encoding a “tag” designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate localization or detection). The tag sequence can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include glutathione S-transferase (GST) and FLAG ^™ tag (Kodak, New Haven, Conn.).

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

Nucleic acid constructs can be prepared using a variety of techniques, for example, in germ cells such as oocytes or egg yolks, progenitor cells, adult or embryonic stem cells, primordial germ cells, kidney cells such as PK-15 cells, islet cells, beta cells, liver. The cells can be introduced into any type of embryonic, fetal, or adult animal cell, including fibroblasts such as dermal fibroblasts. Non-limiting examples of techniques include the use of transposon systems, recombinant viruses that can infect cells, or liposomes or other non-viral methods such as electroporation, microinjection, or calcium phosphate precipitation that can deliver nucleic acids to cells. Includes.

In a transposon system, the transcription unit of the nucleic acid construct, i.e., the regulatory region operably linked to an exogenous nucleic acid sequence, is flanked by inverted repeat sequences of the transposon. For example, Sleeping Beauty (see, US Patent No. 6,613,752 and US Publication No. 2005/0003542); Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31:6873); Tol2 (Kawakami (2007) Genome Biology 8(Suppl.1):S7; Minos (Pavlopoulos et al. (2007) Genome Biology 8(Suppl.1):S2); Hsmar1 (Miskey et al. (2007)) Mol Cell Biol 27:4589); Several transposon systems have been developed to introduce nucleic acids into cells, including mouse, human, and porcine cells. Sleeping Beauty transposons are particularly useful. The transposase may be delivered as a protein, encoded on the same nucleic acid construct as the exogenous nucleic acid, introduced on a separate nucleic acid construct, or as mRNA (e.g., in vitro -transcribed and capped). mRNA).

Insulator elements can also be included in nucleic acid constructs to maintain expression of exogenous nucleic acids and to suppress unwanted transcription of host genes. For example, U.S. See publication number 2004/0203158. Typically, insulator elements flank each side of the transcription unit and are located within the inverted repeat sequence of the transposon. Non-limiting examples of insulator elements include matrix attachment region- (MAR) type insulator elements and boundary-type insulator elements. For example, U.S. Patent Nos. 6,395,549, 5,731,178, 6,100,448, and 5,610,053, and U.S. Pat. See publication number 2004/0203158.

Nucleic acids can be incorporated into vectors. Vector is a broad term that includes any specific DNA segment designed to transfer target DNA from a carrier. A vector may be referred to as an expression vector, or vector system, which is a set of components required to insert DNA into a genome or other targeted DNA sequence such as an episome, plasmid, or even a viral/phage DNA segment. Vector systems such as viral vectors (e.g., retroviruses, adeno-associated viruses, and integrated phage viruses), and non-viral vectors (e.g., transposons) used for gene transfer in animals have two basic components: It has: 1) a vector containing DNA (or RNA reverse transcribed into cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and the DNA target sequence and inserts the vector into the target DNA sequence. . Vectors most often contain one or more expression cassettes containing one or more expression control sequences, which may be another DNA sequence or mRNA, a DNA sequence that controls and regulates transcription and/or translation, respectively.

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, suitable promoters and optional enhancers, the necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non-transcribed sequences. Examples of vectors include: plasmids (which can also be carriers of another type of vector), adenoviruses, adeno-associated viruses (AAV), lentiviruses (e.g., modified HIV-1, SIV or FIV), retroviruses (e.g., ASV, ALV, or MoMLV), and transposons (e.g., Sleeping Beauty, P-element, Tol-2, Frog Prince, piggyBac).

As used herein, the term nucleic acid includes both RNA and DNA, including, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, as well as naturally occurring and chemically modified nucleic acids; For example, it refers to a synthetic base or alternative framework. Nucleic acid molecules can be double-stranded or single-stranded (i.e., sense or antisense single stranded).

Having described the invention in detail, it will be apparent that modifications and changes may be made without departing from the scope of the invention as defined in the appended claims.

Example

The following non-limiting examples are provided to further illustrate the invention.

Example 1: Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro derived oocytes and embryos

Homing endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related Recent reports describing components within the (Cas9) system suggest that genetic engineering (GE) of pigs may now be more efficient. Targeted homing endonucleases can induce double-strand breaks (DSBs) at specific locations in the genome, either through nonhomologous end joining (NHEJ) or homologously, when donor DNA is provided. Random mutations can occur through stimulation of homologous recombination (HR). Targeted modification of the genome through HR can be achieved using homing endonucleases if donor DNA is provided with a targeted nuclease. After introducing specific modifications into somatic cells, these cells were used to produce GE pigs for various purposes through SCNT. Therefore, homing endonucleases are useful tools in the generation of GE pigs. Among different homing endonucleases, the CRISPR/Cas9 system adapted from prokaryotes used as a defense mechanism appears to be an effective approach. In nature, the Cas9 system consists of three components: an RNA (∼20 bases) containing a region complementary to the target sequence (cis-repressed RNA [crRNA]), an RNA containing a region complementary to the crRNA (trans). -activating crRNA [tracrRNA]), and Cas9, the enzymatic protein component of this complex. A single guide RNA (guide RNA, gRNA) can be produced to perform the role of base-paired crRNA and tracrRNA. The gRNA/protein complex can scan the genome and catalyze DSBs in regions complementary to the crRNA/gRNA. Unlike other designed nucleases, only short oligomers need to be designed to fabricate the reagents needed to target the gene of interest, whereas a series of cloning steps are required to assemble ZFNs and TALENs.

In contrast to current standard methods for gene disruption, the use of designed nucleases provides the opportunity to use zygotes as starting material for GE. Standard methods for gene disruption in livestock include HR in cultured cells and subsequent reconstitution of the embryo by somatic cell nuclear transfer (SCNT). Because cloned animals produced through SCNT sometimes show signs of developmental defects, progeny of SCNT/GE founders are generally studied to avoid confounding SCNT abnormalities and phenotypes that may arise if founder animals are used in experiments. It is used in Considering the longer gestation period and higher husbandry costs of pigs compared to rodents, there are time and cost advantages in reducing the need for breeding. Recent reports have demonstrated that direct injection of ZFNs and TALENs into pig zygotes can disrupt endogenous genes and produce piglets with desired mutations. However, only about 10% of piglets exhibited biallelic variants of the target genes, and some displayed mosaic genotypes. A recent article demonstrated that the CRISPR/Cas9 system can induce mutations in developing embryos and produce GE pigs with higher efficiency than ZFNs or TALENs. However, GE pigs produced from the CRISPR/Cas9 system also had a mosaic genotype. Additionally, all of the above-mentioned studies used in vivo derived zygotes for experiments, which requires intensive labor and a large number of sows to obtain a sufficient number of zygotes.

This example describes an efficient approach using the CRISPR/Cas9 system in generating GE pigs via SCNT following injection of in vitro derived zygotes and modification of somatic cells. Two endogenous genes (CD163 and CD1D) and one transgene (eGFP) were targeted, and only in vitro derived oocytes or zygotes were used for SCNT or RNA injection, respectively. CD163 appears to be required for productive infection by porcine reproductive and respiratory syndrome virus, a virus known to cause significant economic losses to the swine industry. CD1D is considered a nonclassical major histocompatibility complex protein and is involved in presenting lipid antigens to invariant natural killer T cells. Pigs lacking these genes were designed as models for agriculture and biomedicine. The eGFP transgene was used as a target for preliminary proof-of-concept experiments and optimization of the method.

Materials and Methods

Chemistry and Reagents . Unless otherwise noted, all chemicals used in this study were purchased from Sigma.

Design of gRNA to construct specific CRISPR

To ensure that CRISPR results in a DSB in wild-type CD163 but not in the domain swap targeting vector, the guide RNA was placed in a region within exon 7 of CD163 that is unique to wild-type CD163 and not present in the domain swap targeting vector (described below). It was designed for. There were only four positions at which the targeting vector would introduce single nucleotide polymorphisms (SNPs) altering the S. pyogenes ( Spy ) protospacer adjacent motif (PAM). All four targets were selected, including:

(SEQ ID NO: 1) GGAAACCCAGGCTGGTTGGA gGG (CRISPR 10),

(SEQ ID NO: 2) GGAACTACAGTGCGGCACTG tGG (CRISPR 131),

(SEQ ID NO: 3) CAGTAGCACCCCGCCCTGAC gGG (CRISPR 256) and

(SEQ ID NO: 4) TGTAGCCACAGCAGGGACGT cGG (CRISPR 282).

PAMs can be identified by bold font in each gRNA.

For CD1D mutations, the search for CRISPR targets was arbitrarily restricted to the coding strand within the first 1000 bp of the primary transcript. However, RepeatMasker [26] (“pig” repeat library) identified a repeat element starting at base 943 of the primary transcript. Thereafter, the search for CRISPR targets was limited to the first 942 bp of the primary transcript. Since the last Spy PAM is located at base 873, the search was further restricted to the first 873 bp of the primary transcript. The first target (CRISPR 4800) was selected because it overlaps with the start codon located at base 42 in the primary transcript (CCAGCCTCGCCCAGCGACAT gGG (SEQ ID NO: 5)). Two additional targets (CRISPR 5620 and 5626) were selected because they were furthest from the first choice within the randomly selected region (CTTTCATTTATCTGAACTCA gGG (SEQ ID NO: 6)) and TTATCTGAACTCAGGGTCCC cGG (SEQ ID NO: 7). These targets overlap. With respect to the start codon, the closest Spy PAM was located in a simple sequence containing broadly homopolymeric sequences as determined by visual assessment. The fourth target (CRISPR 5350) was selected because it was the closest target that did not contain an extensive homopolymeric region with respect to the first target selection (CAGCTGCAGCATATATTTAA gGG (SEQ ID NO:8)). The specificity of the designed crRNA was confirmed by searching similar porcine sequences in GenBank. The oligonucleotides (Table 1) were annealed and cloned into the p330X vector containing two expression cassettes: a human codon-optimized S. pyogenes (h Spy ) Cas9 and a chimeric guide RNA. P330X was digested with BbsI (New England Biolabs) according to the Zhang laboratory protocol (http://www.addgene.org/crispr/zhang/).

To target eGFP , two specific gRNAs targeting the eGFP coding sequence were designed within the first 60 bp of the eGFP start codon. eGFP 1 and eGFP 2 The gRNA was on the antisense strand, and eGFP1 targeted the start codon directly. The eGFP 1 gRNA sequence was CTCCTCGCCCTTGCTCACCA tGG (SEQ ID NO: 9), and the eGFP 2 gRNA sequence was GACCAGGATGGGCACCACCC cGG (SEQ ID NO: 10).

Synthesis of donor DNA for CD163 and CD1D genes

To ensure an isogenic match between the targeting vector and the transfected cell line, both porcine CD163 and CD1D were amplified by PCR from DNA isolated from fetal fibroblasts to be used for subsequent transfections. Briefly, a 9538 bp fragment of CD163 was amplified using LA taq (Clontech) using the forward primer CTCTCCCTCACTCTAACCTACTT (SEQ ID NO: 11), and the reverse primer TATTTCTCTCACATGGCCAGTC (SEQ ID NO: 12). The fragment was DNA sequence verified and used to construct a domain-swap targeting vector (Figure 1). This vector contained 33 point mutations in exon 7 to encode the same amino acid sequence as human CD163L from exon 11. The replaced exon was 315 bp. Additionally, the subsequent intron was replaced with a modified myostatin intron B that contains a selectable marker gene that can be removed with Cre-recombinase (Cre) and has previously demonstrated normal splicing when it has a retained loxP site ( Wells, unpublished results). The long arm of the construct was 3469 bp and contained a domain swap DS exon. The short arm was 1578 bp and included exons 7 and 8 (Figure 1, panel B). This plasmid was used to replace the coding region of exon 7 in the first transfection experiment, allowing selection of targeting events through a selection marker (G418). If targeting has occurred, the marker can be deleted by Cre-recombinase. The CD163 DS-targeting vector was then modified for use with cell lines already containing the Neo-disrupted SIGLEC1 gene, which cannot be Cre deleted. In this targeting vector, the Neo cassette, loxP, and myostatin intron B were removed, leaving only the DS exons in the WT long and short arms (Figure 1, panel C).

The genomic sequence for porcine CD1D was amplified with LA taq using the forward primer CTCTCCCTCACTCTAACCTACTT (SEQ ID NO: 13) and the reverse primer GACTGGCCATGTGAGAGAAATA (SEQ ID NO: 14), generating an 8729 bp fragment. The fragment was DNA sequenced and used to construct the targeting vector shown in Figure 2. The Neo cassette is under the control of the phosphoglycerol kinase (PGK) promoter and is flanked by loxP sequences 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 HR occurs, exons 3, 4, and 5 will be removed and replaced with the Neo cassette. If NHEJ repair occurs incorrectly, exon 3 will be destroyed.

Fetal fibroblast collection

To generate cell lines, porcine fetal tissue was collected on day 35 of gestation. Two wild type (WT) male and female fetal fibroblast cell lines were established from a Large White domestic hybrid. Male and female fetal fibroblasts previously modified to contain the Neo cassette (SIGLEC1-/- genetics) were also used in these studies. Fetal fibroblasts were collected as described with slight modifications. Minced tissue from each fetus was incubated in 20 mL of digestion medium (L-glutamine and 1 g/L D-glucose supplemented with 200 units/ml collagenase and 25 Kunitz units/ml DNAseI) for 5 h at 38.5°C [Cellgro ] was digested in Dulbecco's modified Eagle's medium [DMEM] containing . After digestion, fetal fibroblasts were washed and cultured with DMEM, 15% fetal bovine serum (FBS), and 40 μg/ml gentamicin. After overnight incubation, cells were trypsinized, frozen at -80°C in aliquots in FBS with 10% dimethyl sulfoxide, and stored in liquid nitrogen.

Cell transfection and genotyping

Transfection conditions were essentially as previously reported. Donor DNA was always used at an aliquot of 1 μg along with various amounts of CRISPR/Cas9 plasmid (listed below). Donor DNA was linearized with MLUI (CD163) (NEB) or AFLII (CD1D) (NEB) before transfection. The gender of the established cell lines was determined by PCR as previously described before transfection. Male and female cell lines were transfected and genomic modification data were analyzed together between transfections. Fetal fibroblast cell lines of similar passage number (2-4) were cultured for 2 days and 1 g of L-glutamine supplemented with 15% FBS, 2.5 ng/ml basic fibroblast growth factor, and 10 mg/ml gentamicin. /L grown to 75%-85% confluency in DMEM containing D-glucose (Cellgro). Fibroblasts were washed with phosphate-buffered saline (PBS, Life Technologies) and trypsinized. As soon as the cells were detached, they were permeated with electroporation medium (75% cytosalt [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 (LifeTechnologies). rinsed with Cell concentration was quantified using a hemocytometer. Cells ^were pelleted at 600 Each electroporation used 200 μl of cells in a 2 mm gap cuvette with three (1 msec) square wave pulses administered via BTX ECM 2001 at 250 V. After electroporation, cells were resuspended in DMEM as described above. For selection, 600 μg/ml G418 (Life Technologies) was added 24 h after transfection, and the medium was changed on day 7. Colonies were picked 14 days after transfection. Fetal fibroblasts were plated at 10,000 cells/plate if G418 selection was used and 50 cells/plate if G418 selection was not used. Fetal fibroblast colonies were collected by applying a 10 mm autoclaved cloning cylinder sealed around each colony by autoclaved vacuum grease. Colonies were rinsed with PBS, harvested via trypsin; It was resuspended in DMEM culture medium. A portion (1/3) of the resuspended colonies was transferred to a 96-well PCR plate, and the remaining (2/3) cells were cultured in the wells of a 24-well plate. The cell pellet was resuspended in 6 μl of lysis buffer (40 mM Tris, pH 8.9, 0.9% Triton Proteinase K was inactivated by incubation for 10 minutes, followed by incubation at 85°C for 10 minutes.

PCR screening for DS and large and small deletions

Detection of HR-directed repair . Mutations on CD163 or CD1D were confirmed using long-range PCR. Three different PCR assays were used to confirm HR events: PCR amplification of a region spanning from the CD163 or CD1D sequence in the donor DNA to the endogenous CD163 or CD1D sequence on the left or right and a large region of CD163 or CD1D containing the designed donor DNA. Long-distance PCR to amplify. An increase in the size of the PCR product to 1.8 kb (CD1D) or 3.5 kb (CD163) resulting from the addition of an exogenous Neo sequence was considered evidence of HR-directed repair of the gene. All PCR conditions included an initial denaturation at 95°C for 2 minutes, followed by 33 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 68°C for 7-10 minutes. LA taq was used in all analyzes according to the manufacturer's recommendations. Primers are shown in Table 2.

Small deletion analysis (NHEJ) . Small deletions were determined by PCR amplifying CD163 or CD1D flanking the protruding cleavage site introduced by the CRISPR/Cas9 system. The sizes of the amplicons were 435 bp and 1244 bp for CD163 and CD1D, respectively. Lysates from both embryonic and fetal fibroblasts were PCR amplified with LA taq. PCR conditions for the assay were an initial denaturation at 95°C for 2 minutes, followed by 33 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute. For genotyping of transfected cells, insertions and deletions (INDELs) were confirmed by separating PCR amplicons by agarose gel electrophoresis. For embryo genotyping, the resulting PCR products were subsequently DNA sequenced to confirm small deletions using the forward primer used in PCR. Primer information is shown in Table 3.

Somatic cell nuclear transfer (SCNT)

To produce SCNT embryos, sow-derived oocytes (ART, Inc.) or gilt-derived oocytes from a local slaughterhouse were used. Sow-derived oocytes were cultured in maturation medium (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 μg/ml TCM-199 with mM cysteine, 10% porcine follicular fluid, and 25 ng/ml gentamicin) overnight and transferred to fresh medium after 24 hours. After 40-42 hours of maturation, cumulus cells were removed from the oocytes by vortexing in the presence of 0.1% hyaluronidase. Oocytes from young sows were matured for in vitro fertilization (IVF) as described below. During manipulation, oocytes were cultured in manipulation medium supplemented with 7.0 μg/ml cytochalasin B (TCM- with 0.6 mM NaHCO ₃ , 2.9 mM Hepes, 30 mM NaCl, 10 ng/ml gentamicin, and 3 mg/ml BSA). 199 [Life Technologies], with an osmolality of 305 mOsm). The polar body was removed along with a portion of the adjacent cytoplasm, presumed to contain the metaphase II plate, and the donor cell was placed into the perivitelline space using a thin glass capillary. Afterwards, the reconstituted embryos were incubated with fusion medium (0.3 M mannitol, 0.1 mM CaCl ₂ , 0.1 mM MgCl ) with two DC pulses (1 s apart) for 30 s at 1.2 kV/cm using a BTX electric cell manipulator (Harvard Apparatus). ₂ , and 0.5 mM Hepes). After fusion, fused embryos were fully activated using 200 μM thimerosal for 10 min and 8 mM dithiothreitol for 30 min in the dark. Embryos were then incubated for 14–16 h in modified porcine zygote medium PZM3-MU1 with 0.5 μM Scriptaid (S7817; Sigma-Aldrich), a histone deacetylase inhibitor, as previously described.

In vitro fertilization (IVF)

For IVF, ovaries from prepubescent young sows were obtained from an abattoir (Farmland Foods Inc.). Immature oocytes were aspirated from medium-sized (3-6 mm) follicles using an 18-gauge hypodermic needle attached to a 10 ml syringe. Then, oocytes with evenly dark cytoplasm and intact surrounding cumulus cells were selected for maturation. Approximately 50 cumulus oocyte complexes were incubated in ₅₀₀ μl of maturation medium containing 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng/ml EGF, were placed in wells containing 0.5 μg/ml luteinizing hormone (LH), 0.5 μg/ml FSH, 10 ng/ml gentamicin (APP Pharm), and TCM-199 (Invitrogen) with 0.1% polyvinyl alcohol. At the end of maturation, surrounding cumulus cells were removed from the oocyte by vortexing for 3 min in the presence of 0.1% hyaluronidase. Afterwards, in vitro matured oocytes were cultured in groups of 25-30 oocytes in IVF medium (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 modified Tris-buffered medium containing 2 mg/ml bovine serum albumin [BSA]). One 100 μl frozen semen pellet was thawed in 3 mL of Dulbecco's PBS supplemented with 0.1% BSA. Frozen WT or fresh eGFP semen was washed by centrifugation at 650 3 g for 20 min in 60% Percoll and in modified Tris-buffered medium for 10 min. In some cases, freshly collected semen heterozygous for the previously described eGFP transgene was washed three times in PBS. Afterwards, the semen pellet was resuspended at ^0.5 Fifty microliters of semen suspension was introduced into the droplet containing the oocytes. Gametes were co-cultured at 38.5°C for 5 hours in an atmosphere of 5% CO ₂ in air. After fertilization, embryos were incubated in PZM3-MU1 at 38.5°C and 5% CO ₂ in air.

embryo transfer

To produce GE CD163 or CD1D pigs, the resulting embryos were transferred into surrogates 1 day (SCNT) or 6 days (injected zygotes) after the first estrus. For day 6 transplantation, zygotes were cultured for an additional 5 days in PZM3-MU1 in the presence of 10 ng/ml ps48 (Stemgent, Inc.). Embryos were surgically implanted into the ampullary-isthmic junction of the surrogate mother's oviduct.

In vitro synthesis of RNA for the CRISPR/Cas9 system

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

Microinjection of designed CRISPR/Cas9 system in zygotes

Messenger RNA encoding Cas9 and gRNA was injected into the cytoplasm of fertilized oocytes (putative zygotes) at 14 h postfertilization using a FemtoJet microinjector (Eppendorf). Microinjections were performed in engineered medium on a heated stage of a Nikon inverted microscope (Nikon Corporation; Tokyo, Japan). The injected zygotes were then transferred into PZM3-MU1 with 10 ng/ml ps48 until further use.

statistical analysis

The number of colonies with a modified genome was classified as 1, and the number of colonies with an unmodified genome was classified as 0. Differences were determined using PROC GLM (SAS), and a P-value of 0.05 was considered significant. Means were calculated as least square means. Data are expressed as numerical mean ± SEM.

result

CRISPR/Cas9-mediated knockout of CD163 and CD1D in somatic cells.

The efficiency of four different CRISPR plasmids (guides 10, 131, 256, and 282) targeting CD163 was tested with an amount of 2 μg/μl of donor DNA (Table 5). CRISPR 282 resulted in significantly more average colony formation than CRISPR 10 and 256 treatments (P < 0.05). From the long-range PCR analysis described above, a large deletion ranging from 503 bp to 1506 bp was found instead of the originally intended DS through HR (Figure 3, panel A). This was not expected, because previous reports on other DNA-editing systems showed much smaller deletions of 6-333 bp using ZFNs in pigs. CRISPR 10 and a mixture of all four CRISPRs resulted in a greater number of colonies with modified genomes than CRISPR 256 and 282 (Table 5, P < 0.002). Transfection with plasmids containing Neo but no homology to CD163 and CRISPR 10 did not result in colonies showing large deletions. Interestingly, one monoallelic deletion was also detected when donor DNA was introduced without any CRISPR. This analysis likely represents an underestimate of the mutation rate because transfected somatic cells were not screened for any potential small deletions by sequencing that could not be detected on agarose gels.

The initial goal was to obtain a domain swap (DS)-targeting event by HR against CD163, but CRISPR did not increase the efficiency of targeting CD163. It should be noted that various combinations of these targeting vectors were used to modify CD163 by HR by traditional transfection and resulted in 0 targeting events after screening 3399 colonies (Whitworth and Prather, unpublished results). Transfection with CRISPR 10 and DS-targeting vector as donor DNA yielded two pigs with full DS resulting from HR containing all 33 mutations to be introduced.

Next, we tested the efficiency of CRISPR/Cas9-induced mutagenesis without drug selection. The fetal fibroblast cell line used in this study already had integration of the Neo resistance cassette and knockout of SIGLEC1. We also tested whether the ratio of CRISPR/Cas9 and donor DNA increases genomic modifications or results in toxic effects at high concentrations. CRISPR 131 was selected for this test because in previous experiments it resulted in a high number of total colonies and an increased percentage of colonies with modified genomes. Increasing the amount of CRISPR 131 DNA from 3:1 to 20:1 had no significant effect on fetal fibroblast viability. The percentage of colonies with genomes modified by NHEJ was not significantly different between the various CRISPR concentrations, but the 10:1 ratio had the highest number of NHEJs (Table 6, P = 0.33). Even at the highest ratio of CRISPR DNA to donor DNA (20:1), no HR was observed.

Based on this experience, targeted destruction of CD1D in somatic cells was attempted. Four different CRISPRs were designed and tested in both male and female cells. Although modifications of CD1D could be detected from three of the applied CRISPRs, use of CRISPR 5350 did not result in modifications of CD1D with deletions large enough to be detected by agarose gel electrophoresis (Table 7). Interestingly, no genetic changes were obtained through HR, even though donor DNA was provided. However, large deletions were observed similar to CD163 knockout experiments (Figure 3, panel B). Targeted modification of CD1D with large deletions was not detected when CRISPR/Cas9 was not used with donor DNA. Modification of CD1D from CRISPR/Cas9 guided targeting was 4/121 and 3/28 in male and female colonies of cells, respectively. Only INDELs detectable by agarose gel electrophoresis were included in the transfection data.

Production of CD163 and CD1D pigs via SCNT using GE cells

Cells expressing alterations in CD163 or CD1D were used for SCNT to produce CD163 and CD1D knockout pigs (Figure 3). Male and female fetal fibers transfected using the CRISPR/Cas9 system for 7 embryo transfers (CD163 Table 8), 6 embryo transfers (no CD163-Neo), and 5 embryo transfers (CD1D) into recipient gilts. This was performed using SCNT embryos from mother cells. Of the recipient gilts, 6 (CD163), 2 (CD163-Neo None), and 4 (CD1D) (Table 9) remained pregnant until parturition date, 85.7% each. The pregnancy rate was maintained at 33.3% and 80%. Among the CD163 recipients, 5 gave birth to healthy piglets by cesarean section. One animal (O044) gave birth naturally. The litter size ranged from 1 to 8. Four pigs were euthanized due to growth failure after birth. One piglet was euthanized due to severe cleft palate. All remaining piglets appear healthy (Figure 3, Panel C). Two litters of male piglets generated from fetal fibroblasts transfected with CRISPR 10 and donor DNA described in Figure 3, panel B, had a 30 bp deletion in exon 7 adjacent to CRISPR 10 and an additional 1476 bp deletion in the previous intron. Therefore, the intron 6/exon 7 junction of CD163 was removed (Figure 3, panel E). Genotypes and predicted translations are summarized in Table 10. One male piglet and one female litter (4 piglets) were obtained from the CD163-Neo no transfection of previously modified SIGLEC1 cells. All five piglets were double knockouts for SIGLEC1 and CD163. Male piglets are biallelic in CD163 with a 28 bp deletion in exon 7 on one allele and a 1387 bp deletion on the other allele containing a partial deletion of exon 7 and a complete deletion of exon 8 and the proceeding intron. It had a genetic mutation, so the intron-exon junction was removed. The female piglet had a biallelic mutation in CD163, comprising a 1382 bp deletion with an 11 bp insertion on one allele and a 1720 bp deletion of CD163 on the other allele. A summary of CD163 modifications and predicted translations can be found in Table 10. Translations predicted by CD1D modifications and CRISPR modifications can be found in Table 11. Briefly, one female and two male litters were born, producing 13 piglets. One piglet died shortly after birth. Twelve of the 13 piglets contained a biallelic or homozygous deletion of CD1D (Figure 3, panel F). One piglet was WT.

Efficiency of the CRISPR/Cas9 system in pig zygotes

Based on targeted destruction of CD163 and CD1D in somatic cells using the CRISPR/Cas9 system, this approach was applied to porcine embryogenesis. First, the effectiveness of the CRISPR/Cas9 system was tested in developing embryos. The CRISPR/Cas9 system targeting eGFP was introduced into zygotes fertilized with semen from boars heterozygous for the eGFP transgene. After injection, subsequent embryos expressing eGFP were monitored. Various concentrations of the CRISPR/Cas9 system were tested, and cytotoxicity of the delivered CRISPR/Cas9 system was observed (Figure 4, panel A). Embryonic development after CRISPR/Cas9 injection was lower compared to the control group. However, all concentrations of CRISPR/Cas9 investigated were effective in generating variants of eGFP, as no embryos with eGFP expression were found in the CRISPR/Cas9 injected group (Figure 4, panel B). Among uninjected control embryos, 67.7% were green, indicating expression of eGFP. When individual blastocysts were genotyped, a small mutation was identified near the CRISPR binding site (Figure 4, panel C). Based on toxicity and effectiveness, 10 ng/μl of gRNA and Cas9 mRNA were used in the following experiments.

When a CRISPR/Cas9 component designed to target CD163 was introduced into a putative zygote, targeted editing of the gene was observed in subsequent blastocysts. When individual blastocysts were genotyped for mutations in CD163, specific mutations were found in all embryos (100% GE efficiency). More importantly, although embryos with homozygous or biallelic variants (8/18 and 3/18, respectively) could be found (Figure 5), mosaic (monoallelic variant) genotypes were also detected (4/ 18 embryos). A subset of embryos (8/10) from the pool were injected with 2 ng/μl Cas9 and 10 ng/μl CRISPR and no differences in mutagenesis efficiency were found. Next, based on the in vitro results, two CRISPRs representing different gRNAs were introduced to disrupt CD163 or CD1D during embryogenesis to induce specific deletion of target genes. As a result, the designed deletion of CD163 and CD1D could be successfully induced by introducing two guides. An engineered deletion is defined as a deletion that removes the genomic sequence between the two introduced guides. Of the two embryos that received CRISPR targeting CD163, all but one resulted in targeted modification of CD163. Additionally, 5/13 embryos were found to have engineered deletions in CD163 (Figure 6, panel A), and 10/13 embryos were found to have alterations in CD163 in a homozygous or biallelic manner. Targeting CD1D with two CRISPRs was also effective, as all embryos (23/23) showed alterations in CD1D. However, the engineered deletion of CD1D could only be detected in two embryos (2/23) (Figure 6, panel B). Five of the 23 embryos with a mosaic genotype were also found, but the remaining embryos had homozygous or biallelic variants of CD1D. Finally, we tested whether multiple genes could be targeted by the CRISPR/Cas9 system within the same embryo. For this purpose, targeting of both CD163 and eGFP was performed in zygotes fertilized with heterozygous eGFP semen. When blastocysts from the injected embryos were genotyped for CD163 and eGFP, CD163 and eGFP were found to be successfully targeted during embryogenesis. Sequencing results demonstrated that multiple genes could be targeted by introducing multiple CRISPRs with Cas9 (Figure 6, panel C).

Production of CD163 and CD1D mutants from CRISPR/Cas9 injected zygotes

Based on success from previous in vitro studies, we produced some CRISPR/Cas9 injected zygotes and implanted 46-55 blastocysts per recipient (this number has been shown to be effective for producing pigs from in vitro derived embryos). Because it appeared). Four embryo transfers were performed, two each for CD163 and CD1D, and pregnancies were obtained for each variant. Four healthy piglets with variants in CD163 were produced (Table 8). All piglets, litter 67 from recipient sow ID O083, exhibited homozygous or biallelic variation of CD163 (Figure 7). Two piglets displayed a designed deletion of CD163 by two CRISPR transfers. All piglets were healthy. For CD1D, one pregnancy also produced 1 female and 3 males with 4 piglets (litter 166 from recipient sow identifier O165) (Table 9). One piglet (166-1) had a mosaic mutation in CD1D containing a 362 bp deletion that completely removed exon 3 containing the start codon (Figure 8). One piglet had a 6 bp insertion with a 2 bp mismatch on one allele and a large deletion on the other allele. Two additional piglets had biallelic single bp insertions. No mosaic mutations were detected for CD163.

Argument

Increasing the efficiency of GE pig production could have far-reaching implications by providing more GE pigs for agricultural and biomedical applications. The aforementioned data show that GE pigs with specific mutations can be produced with high efficiency by using the CRISPR/Cas9 system. The CRISPR/Cas9 system has been successfully applied to edit genes in both somatic cells and preimplantation embryos.

When the CRISPR/Cas9 system was introduced into somatic cells, it successfully induced targeted destruction of target genes by NHEJ, but did not increase the ability to target by HR. The targeting efficiency of individual CRISPR/Cas9 in somatic cells was variable, indicating that the design of the guide may affect targeting efficiency. In particular, no targeted modification of CD1D was found when CRISPR 5350 and Cas9 were introduced into somatic cells. This suggests that it may be beneficial to design multiple gRNAs and verify their efficiency before producing pigs. The reason for the lack of HR-directed repair in the presence of donor DNA is not yet clear. After screening 886 colonies (both CD163 and CD1D) transfected with CRISPR and donor DNA, only one colony had evidence for a partial HR event. The results demonstrate that the CRISPR/Cas9 system works with introduced donor DNA to produce unexpectedly large deletions on target genes but does not increase HR efficiency for these two specific targeting vectors. However, the specific mechanism for the observation of large deletions is unknown. Our previous report suggested that donor DNA could be effectively used with ZFNs to induce HR-directed repair. Similarly, an increase in targeting efficiency was observed when donor DNA was used with the CRISPR/Cas9 system, but full HR-directed repair was not observed. In previous studies using ZFNs, it was observed that targeted modifications can occur through a combination of HR and NHEJ, since partial recombination of the introduced donor DNA was found after inducing DSBs by ZFNs. One explanation could be that the HR and NHEJ pathways are not independent but act together to complete the repair process after DSBs are induced by homing endonucleases. Although no statistical differences were found in the results of these experiments, higher concentrations of CRISPR may improve targeting efficiency in somatic cells. This may suggest that CRISPR is a limiting factor in the CRISPR/Cas9 system, but further validation is needed. Targeted cells were successfully used to produce GE pigs via SCNT, indicating that application of CRISPR/Cas9 does not affect the ability of the cells to be cloned. Some piglets were euthanized due to health issues, but this is common in SCNT-induced piglets.

When the CRISPR/Cas9 system was introduced into developing embryos by zygote injection, nearly 100% of embryos and pigs contained INDELs within the targeted gene, demonstrating that the technology is highly effective during embryogenesis. The efficiencies observed during this study exceed the frequencies reported in other studies utilizing homing endonucleases during embryogenesis. The decrease in the number of embryos reaching the blastocyst stage suggested that the concentration of CRISPR/Cas9 introduced in this study may be toxic to embryos. Further optimization of the delivery system could improve the overall efficiency of the process by increasing embryo viability. The nearly 100% mutagenicity rate observed here differed from previous reports in CRISPR/Cas9-mediated knockouts in pigs; Differences in efficiency between studies may be due to the combination of guides and targets selected. In this study, lower concentrations of CRISPR/Cas9 (10 ng/μl each) were effective in generating mutations in developing embryos and producing GE pigs. The concentrations are lower than those previously reported in porcine zygotes (125 ng/μl Cas9 and 12.5 ng/μl CRISPR). Lower concentrations of CRISPR/Cas9 components may be beneficial to the developing embryo, as introducing excess nucleic acid into the developing embryo can be toxic. Some mosaic genotypes were observed in CRISPR/Cas9-injected embryos from in vitro assays; Only one piglet produced through this approach had a mosaic genotype. Potentially, injecting CRISPR/Cas9 components may be more effective than introducing other homing endonucleases, as mosaic genotypes have been considered a major obstacle to using the CRISPR/Cas9 system in zygotes. Another advantage of using the CRISPR/Cas9 system demonstrated by our results is that CD163 knockout pigs produced from IVF-derived zygotes injected with the CRISPR/Cas9 system were not lost, whereas several piglets originating from SCNT were lost after a few days. It is said that he was euthanized. This suggests that the technology can not only bypass the need for SCNT when generating knockout pigs, but also overcome common health problems associated with SCNT. Now that injection of CRISPR/Cas9 mRNA into zygotes has been optimized, future experiments will also include co-injection of donor DNA.

This study demonstrates that introducing two CRISPRs together with Cas9 in the zygote can induce chromosomal deletions in the developing embryo and produce pigs with the intended deletion, i.e., a specific deletion between the two CRISPR guides. . This engineered deletion can be beneficial because it allows the size of the deletion to be specified without relying on random events caused by NHEJ. In particular, if there are insertions/deletions of multiples of 3 nucleotides caused by homing endonucleases, the mutation may actually result in a hypomorphic mutation since no frameshift will occur. However, by introducing two CRISPRs, it is possible to cause larger deletions that are more likely to produce non-functional proteins. Interestingly, CD1D CRISPR was designed to span a larger region within the genome than CD163; There was a 124 bp distance between CD163 CRISPR 10 and 131, while for CD1D there was a 550 bp distance between CRISPR 4800 and 5350. Longer distances between CRISPRs were not as effective in generating deletions as shown in the study. However, because this study included only a limited number of observations and there is a need to consider the efficacy of individual CRISPRs, which are not covered herein, further studies are needed to determine the relationship between the distance between CRISPRs and their likelihood of causing the intended deletion. need.

The CRISPR/Cas9 system was also effective in simultaneously targeting two genes within the same embryo, with the only additional step being the introduction of one additional CRISPR along with the crRNA. This illustrates that it is easy to destroy multiple genes compared to other homing endonucleases. These results suggest that this technique can be used to target gene clusters or gene families that may have compensatory effects and thus demonstrate that it is difficult to determine the role of individual genes unless all genes are disrupted. The results demonstrate that CRISPR/Cas9 technology can be applied to generate GE pigs by increasing gene targeting efficiency in somatic cells and by direct zygote injection.

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

Porcine reproductive and respiratory syndrome virus (PRRSV) has ravaged the swine industry over the past 25 years. Speculation regarding the mode of viral entry included both SIGLEC1 and CD163. Knockout of SIGLEC1 did not affect the response to viral challenge, but in this example CD163 null animals appear to show no clinical signs of infection, lung pathology, viremia or antibody production, all hallmarks of PRRSV infection. Not only has the vector of PRRSV introduction been identified; Strategies have been described to prevent significant economic loss and animal suffering if similarly produced animals were to enter the food supply.

Materials and Methods

Genotyping

Genotyping was based on both DNA sequencing and mRNA sequencing. The boar (sire) genotype had an 11 bp deletion in one allele, which upon translation predicted 45 amino acids to domain 5, resulting in a premature stop codon at amino acid 64. The other allele contained a 2 bp addition in exon 7 and a 377 bp deletion in the intron preceding exon 7, which upon translation predicted the first 49 amino acids of domain 5, resulting in a premature stop code at amino acid 85. One sow had a 7 bp addition on one allele, which upon translation predicted 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 by PCR or long-range 6.3 kb PCR. The other three sows were clones and had a 129 bp deletion in exon 7, which was predicted to result in a deletion of 43 amino acids from domain 5. The other allele has not been characterized (B).

The growth of PRRSV in the culture and production of viral inoculum for swine infection is covered under approved IBC support 973.

Isolate NVSL 97-7895, a type A strain of PRRSV (GenBank # AF325691 2001-02-11), was grown as described in the approved IBC protocol 973. This laboratory isolate has been used in experimental research for approximately 20 years (Ladinig et al., 2015). A second isolate was used in the second test KS06-72109 as previously described (Prather et al., 2013).

Infection of pigs with PRRSV

A standardized infection protocol for PRRSV was used to infect pigs. PRRS of approximately 10 ⁴ TCID50 administered by intramuscular (IM) and intranasal (IN) routes to 3-week-old piglets. The virus was inoculated. Pigs are monitored daily, and pigs showing disease symptoms are treated according to the recommendations of CMG veterinarians. Pigs showing severe distress and at risk of succumbing to infection are humanely euthanized and samples collected. To eliminate bias in evaluation or treatment, staff and veterinarians were blinded to the pigs' genetic status. PRRSV is present in body fluids during infection; Therefore, blood samples were collected and stored at -80°C until measured to determine the amount or degree of viremia in each pig. At the end of the experiment, pigs were weighed, humanely euthanized, and tissues were collected, fixed in 10% buffered formalin, embedded in paraffin, and processed for histopathology by a licensed pathologist.

Phenotypic scores of challenge-infected pigs

The pigs' phenotype was blindly scored daily as follows: What is the pig's attitude? Attitude Score: 0: BAR, 1: QAR, 2: slightly depressed, 3: depressed, 4: moribund. What is the pig's body condition? Body condition score: 1: emaciated, 2: thin, 3: ideal, 4: lean, 5: overweight/obese. What is the rectal temperature of a pig? Normal body temperature 101.6-103.6°F (fever is considered ≥104°F). Are you lame (level)? Which leg? Assess for joint swelling and hoof lesions (check the bottom and sides of the hoof). Limping Score: 1: No limping, 2: Walking is slightly uneven, some joints appear stiff, but no limping. 3: Mild limping, slight limping when walking, 4: Moderate limping, apparent limping including toe touching lame, 5: Severe limping, inability to bear weight on leg, standing and walking. Needs stimulation. Do you have difficulty breathing (grade)? Do you breathe with your mouth open? Is there any nasal discharge (color of discharge, amount of discharge: mild/moderate/severe)? Have you noticed an animal coughing? Is there eye discharge? Breathing Score: 0: normal, 1: mild dyspnea and/or tachypnea when stressed (handled), 2: mild dyspnea and/or tachypnea at rest, 3: moderate dyspnea when stressed (handled) and/or tachypnea, 4: moderate dyspnea and/or tachypnea at rest, 5: severe dyspnea and/or tachypnea when stressed (handled), 6: severe dyspnea and/or tachypnea at rest. Is there evidence of diarrhea (grade) or vomiting? Is there blood or mucus? Diarrhea score: 0: no visible feces, 1: normal stool, 2: soft but formable stool (consistency of soft yogurt, producing cow patties), 3: brown/tan liquid diarrhea with particulate fecal material, 4 : Brown/tan liquid diarrhea without particulate fecal material, 5: Liquid diarrhea that appears similar to water.

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 temperature were analyzed using separate ANOVA based on genotype as treatment.

Measurement of PRRSV viremia

Viremia was determined using two approaches. Virus titrations were performed by adding serial 1:10 dilutions of serum to confluent MARC-145 cells in 96 well-plates. Serum was diluted in Eagle's minimal essential medium supplemented with 8% fetal bovine serum, penicillin, streptomycin, and amphotericin B, as previously described (Prather et al., 2013). Cells were examined for the presence of cytopathic effects using a microscope after 4 days of incubation. The highest dilution showing a cytopathic effect was scored as the titration endpoint. To measure viral nucleic acid, total RNA was isolated from serum using the Life Technologies MagMAX-96 viral RNA isolation kit. 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 5.8 μl of RNA from serum. A standard curve was created by preparing serial dilutions of the RNA control supplied in the kit (Tetracore). Record the number of templates per PCR.

SIGLEC1 and CD163 staining of PAM cells

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

Measurement of PRRSV-specific Ig

To measure PRRSV-specific Ig, recombinant PRRSV N protein was expressed in bacteria (Trible et al., 2012) and conjugated to magnetic Luminex beads using a kit (Luminex Corporation). N protein-bound beads were diluted to 2,500 beads/50 μl in phosphate-buffered saline containing 10% goat serum and placed in wells of a 96-well round-bottom polystyrene plate. Serum was diluted 1:400 in phosphate-buffered saline containing 10% goat serum, and 50 μl was added to duplicate wells and incubated for 30 min with gentle shaking at room temperature. Next, the plate was washed (3 Intracellular purified goat anti-porcine secondary antibody (IgG, Jackson ImmunoResearch) or biotin-labeled affinity purified goat anti-porcine IgM (KPL) was added. Plates were washed (3X) after 30 min of incubation, followed by addition of 50 μl of streptavidin-conjugated phycoerythrin (2 μg/ml in phosphate-buffered saline containing 10% goat serum (Moss, Inc.)). did. After 30 min, the plates were washed, and the microspheres were resuspended in 100 μl of phosphate-buffered saline containing 10% goat serum and analyzed using MAGPIX and Luminex xPONENT 4.2 software. Record the mean fluorescence intensity (MFI).

result

Mutations of CD163 were generated using CRISPR/Cas9 technology as described above in Example 1. Several founder animals were produced from zygote injection and somatic cell nuclear transfer. Some of these founders were crossed to produce offspring to be studied. Single founder males were crossed with females of two genotypes. The founder male (67-1) had an 11 bp deletion in exon 7 on one allele and a 2 bp addition in exon 7 on the other allele (and a 377 bp deletion in the previous intron), and null animals ( CD163 ^−/− ) was predicted to be. One founder female (65-1) had a 7 bp addition in exon 7 in one allele and an uncharacterized corresponding allele and was therefore heterozygous for the knockout ( CD163 ^-/? ). It was predicted. The second founder female genotype (three clonal animals) contained an as yet uncharacterized allele and an allele with a 129 bp deletion within exon 7. This deletion is predicted to result in the deletion of 43 amino acids in domain 5. Crosses between these animals resulted in piglets that inherited both a null allele from the boar and either a 43 amino acid deletion or an uncharacterized allele from the sow. In addition to wild-type piglets, which served as positive controls for virus challenge, this resulted in four additional genotypes (Table 12).

At weaning, gene-edited piglets and wild-type age-matched piglets were transported to Kansas State University for PRRSV challenge. PRRSV challenge was performed as previously described (Prather et al., 2013). Piglets at 3 weeks of age were transferred to the challenge facility and maintained as a single group. All experiments began after approval from the Animal Use and Biological Safety Committee. After acclimation, pigs were challenged with NVSL 97-7895 (Ladinig et al., 2015), a PRRSV isolate grown on MARC-145 cells (Kim et al., 1993). Pigs were challenged with approximately 10 ⁵ TCID ₅₀ viruses. Half of the inoculum was delivered intramuscularly and the remainder intranasally. All infected pigs were kept in a single group, allowing continued exposure to the virus from infected pen mates. Blood samples were collected at various days up to 35 days post-infection and at termination at 35 days. Pigs were necropsied, and tissues were fixed in 10% buffered formalin, embedded in paraffin, and processed for histopathology. PRRSV-related clinical signs recorded during the course of infection included shortness of breath, anorexia, lethargy, and fever. Clinical sign results during the study period are summarized in Figure 9. As expected, Wild Type ( CD163 +/+ ) pigs showed early signs of PRRSV infection, which peaked between days 5 and 14 and persisted in the group for the remainder of the study. The percentage of febrile pigs peaked at approximately day 10. In contrast, null ( CD163 −/− ) piglets showed no evidence of clinical signs during the entire study period. Respiratory signs during acute PRRSV infection are reflected in significant histopathological changes in the lungs (Table 9). Infection of wild-type pigs showed histopathology consistent with PRRS, including interstitial edema with infiltration of mononuclear cells ( Fig. 10 ). In contrast, there was no evidence of lung changes in null ( CD163 −/− ) pigs. Sample sizes for various genotypes are small; Nonetheless, the 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 null (CD163). -/-) was 0 (n=3).

Maximum clinical signs correlated with blood PRRSV levels. Measurement of viral nucleic acid was performed by isolation of total RNA from serum followed by amplification of PRRSV RNA using a commercial reverse transcriptase real-time PRRSV PCR test (Tetracore, Rockville, MD). A standard curve was created by preparing serial dilutions of the PRRSV RNA control supplied in the RT-PCR kit, and the results were normalized to the number of templates per 50 μl PCR reaction. PRRSV isolates followed the course of PRRSV viremia in wild-type CD163 +/+ pigs (Figure 11). Viremia was evident on day 4, peaked on day 11, and declined until the end of the study. In contrast, viral RNA was not detected in CD163 ^−/− pigs at any time during the study period. Consistent with viremia, antibody production by null and uncharacterized allele pigs was detectable by day 14 and increased until day 28. There was no antibody production in null animals (Figure 12). Together, these data show that wild-type pigs support PRRSV replication with the development of clinical signs consistent with PRRS. In contrast, despite the pigs being inoculated and continuously exposed to infected cage mates, the knockout pigs did not develop viremia and clinical signs.

At the end of the study, porcine alveolar macrophages were removed 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 detected in CD163 +/+ wild-type animals (Figure 13). In contrast, CD163 −/− pigs showed only background levels of anti-CD163 staining, thus confirming the knockout phenotype. Expression levels for another macrophage marker CD169 were similar for both wild-type and knockout pigs (Figure 14). Other macrophage surface markers, including MHC II and CD172, were identical for both genotypes (data not shown).

Although the sample size was small, wild-type pigs significantly outperformed 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 ± There was a tendency to gain less body weight over the course of the experiment (average daily gain 0.81 kg ± 0.33, n=7) compared to the 0.16, n=3).

In the second test, six wild-type, six Δ43 amino acid, and six pigs with an uncharacterized allele (B) were inoculated as above except that piglets were inoculated using KS06-72109. The administration test was conducted as described. Similar to the NVSL data, wild-type and uncharacterized B piglets developed viremia. However, KS06 did not cause viremia in Δ43 amino acid pigs (Figure 15; Table 7).

Results and Conclusion

The most clinically relevant disease in the swine industry is PRRS. Although vaccination programs have been successful in preventing or ameliorating most porcine pathogens, PRRSV has proven to be more challenging. Here, CD163 is identified as an entry vector for this virus strain. Founder sows were created by injecting CRISPR/Cas9 into the zygote (Whitworth et al., 2014) and therefore are transgene-free. Additionally, one of the alleles from the sow (also generated using CRISPR/Cas9) does not contain the transgene. Therefore, piglet #40 has a 7 bp addition on one allele and an 11 bp deletion on the other allele, but does not carry the transgene. This virus-resistant allele of CD163 represents a small genome edit, considering that the porcine genome is approximately 2.8 billion bp (Groenen et al., 2012). If similarly created animals are introduced into the food supply, significant economic losses could be prevented.

Example 3: Genotype 1 porcine reproduction and increased resistance to PRRS virus in porcines with CD163 SRCR domain 5 replaced by human CD163-like homologous SRCR domain 8

CD163 is considered the major receptor for porcine reproductive and respiratory syndrome virus (PRRSV). In this study, pigs were genetically edited (GE) to have one of the following genotypes: complete knockout (KO) of CD163, deletion within CD163 scavenger receptor cysteine-rich (SRCR) domain 5, or deletion of SRCR domain 5. Replacement (domain swap) with a synthesized exon encoding a homolog of the human CD163-like (hCD163L1) SRCR 8 domain. Immunophenotyping of porcine alveolar macrophages (PAM) showed that pigs with KO or SRCR domain 5 deletion did not express CD163 and PAM did not support PRRSV infection. hCD163L1 PAM from pigs with a domain 8 homolog expressed CD163 and supported replication of genotype 2 viruses, but not genotype 1 viruses. Infection of CD163 -transformed pigs with representative type 1 and type 2 viruses produced similar results. Although type 1 and 2 viruses are considered genetically and phenotypically similar at several levels, including the requirement for CD163 as a receptor, the results demonstrate marked differences between PRRSV genotypes in recognition of the CD163 molecule.

Materials and Methods

Genomic variants in the pig CD163 gene

Experiments involving animals and viruses must be conducted in accordance with the Federation of Animal Science Societies Guide for the Care and Use of Agricultural Animals in Research and Teaching, the USDA Animal Welfare Act, and It was performed in accordance with animal welfare regulations and approved by the Kansas State University and University of Missouri Animal Care and Use Committees and Institutional Biosafety Committees. Mutants of CD163 used in this study were generated using CRISPR/Cas9 technology as described in previous examples. The mutations are schematized in Figure 17. The schematic genomic region shown in Figure 17 includes the sequence from intron 6 to intron 8 of the porcine CD163 gene. The introns and exons schematized in Figure 17 are not drawn to scale. Predicted protein products are illustrated to the right of each genomic structure. Relative macrophage expression as measured by the level of surface CD163 on PAM is shown on the far right of Figure 17. The black region represents the intron, the white region represents the exon, the hatched region represents the hCD163L1 exon 11 mimetic, a homolog of porcine exon 7, and the gray region represents the synthesis with the PGK Neo construct as shown in Figure 17. represents the intron.

CD163 gene construct KO-d7(11) , shown in Figure 17, has an 11 base pair deletion in exon 7 from nucleotide 3,137 to nucleotide 3,147. The CD163 gene construct KO-i7 ( 2 ) has a 377 base pair deletion in the intron upstream of exon 7 from nucleotide 2,573 to nucleotide 2,949 as well as a 2 base pair insertion between nucleotides 3,149 and 3,150. These edits are predicted to result in frameshift mutations and premature stop codons, resulting in only partial translation of the SRCR 5 and KO phenotypes. Three other mutations produced deletions in exon 7. The first, d7(129 ), has a 129 base pair deletion in exon 7 from nucleotide 3,044 to nucleotide 3,172. The d7(129 ) construct also has a deletion from nucleotide 488 to nucleotide 2,417 in exon 6, where the deleted sequence is replaced by a 12 bp insertion. The other two deletion constructs, d7(1467) and d7(1280) , have complete deletion of exons 7 and 8, as illustrated in Figure 17. d7(1467) has a 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897, and d7(1280) has a 1280 base pair deletion from nucleotide 2,818 to nucleotide 4,097. For these deletion constructs, other CD163 exons remained intact.

The final construct, HL11m , shown in Figure 17, was produced using a targeting event in which exon 7 was deleted and replaced with a synthetic exon encoding a homolog of SRCR 8 of the human CD163-like 1 protein (hCD163L1 domain 8 is the hCD163L1 exon coded as 11). The SRCR 8 peptide sequence was generated by changing 33 nucleotides in the porcine exon 7 sequence. A neomycin cassette was included in the synthesized exon to enable screening for variants. SEQ ID NO: 118 provides the nucleotide sequence for the HL11m construct in a region corresponding to the same region in the reference sequence SEQ ID NO: 47.

A diagram of the porcine CD163 protein and gene is provided in Figure 18. The CD163 protein SCRC (oval) and PST (square) domains along with the corresponding gene exons are shown in panel A of Figure 18. Peptide sequence comparison for porcine CD163 SRCR 5 (SEQ ID NO: 120) and human CD163 SRCR 8 homolog (SEQ ID NO: 121) is shown in panel B of Figure 18. The figure is based on GenBank accession numbers AJ311716 (porcine CD163) and GQ397482 (hCD163-L1).

virus

The panel of viruses used in this example is listed in Table 14. Isolates were propagated and titrated on MARC-145 cells (Kim et al., 1993). For titration, each virus was grown in MEM supplemented with 7% FBS, Pen-Strep (80 Units/ml and 80 μg/ml, respectively), 3 μg/ml FUNGIZONE (amphotericin B), and 25 mM HEPES. It was serially diluted with :10. Diluted samples were added in quadruplicate to confluent MARC-145 cells in 96 well plates to a final volume of 200 μl/well and incubated for 4 days at 37°C in 5% CO ₂ . The titration endpoint was identified as the last well with cytopathic effect (CPE). The 50% tissue culture infectious dose (TCID ₅₀ /ml) was calculated using the method previously described (Reed and Muench 1938).

Infection of alveolar macrophages

Preparation and infection of macrophages were performed as previously described ( Gaudreault et al., 2009 and Patton et al., 2008 ). The lungs were removed from the euthanized pig and washed by pouring 100 mL of cold phosphate-buffered saline (PBS) into the trachea. The trachea was clamped and the lungs were gently massaged. Alveolar contents were poured into a 50 ml centrifuge tube and stored on ice. Porcine alveolar macrophages (PAM) were sedimented by centrifugation at 1200 x g for 10 min at 4°C. The pellet was resuspended and washed once in cold sterile PBS. The cell pellet was resuspended in freezing medium containing 45% RPMI 1640, 45% fetal bovine serum (FBS), and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until use. Frozen cells were thawed on ice, counted, and adjusted to 5x10 ⁵ cells/ml in medium (RPMI 1640; RPMI-FBS supplemented with 10% FBS, PenStrep, and FUNGIZONE). Approximately 10 ³ PAMs per well were added to a 96 well plate and incubated overnight at 37° C. in 5% CO ₂ . Cells were washed gently to remove non-adherent cells. Serial 1:10 dilutions of virus were added to triplicate wells. After overnight incubation, cells were washed with PBS and fixed with 80% acetone for 10 min. After drying, wells were stained with PRRSV N-protein specific SDOW-17 mAb (Rural Technologies Inc.) diluted 1:1000 in PBS with 1% fish gelatin (PBS-FG; Sigma Aldrich). After 30 min incubation at 37°C, cells were washed with PBS and stained with ALEXAFLUOR 488 labeled anti-mouse IgG (Thermofisher Scientific) diluted 1:200 in PBS-FG. Plates were incubated for 30 minutes at 37°C in the dark, washed with PBS, and observed under a fluorescence microscope. The 50% tissue culture infectious dose (TCID ₅₀ )/ml was calculated according to the method previously described (Reed and Muench 1938).

Measurement of CD169 and CD163 surface expression on PAM

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

Measurement of PRRS viremia

RNA was isolated from 50 μl of serum using Ambion's MagMAX 96 virus isolation kit (Applied Biosystems) according to the manufacturer's instructions. PRRSV RNA was quantified using EZ-PRRSV MPX 4.0 real-time RT-PCR target specific reagent (Tetracore) performed according to the manufacturer's instructions. Each plate contained a set of Tetracore quantification standards and controls designed for use with RT-PCR reagents. PCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) in 96-well format using recommended cycling parameters. Results of the PCR analysis were reported as log ₁₀ PRRSV RNA copies per 50 μl 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 PRRSV antibodies

For detection of antibodies against PRRSV nucleocapsid (N) protein, microsphere fluorescence immunoassay (FMIA) was performed as previously described ( Stephenson et al., 2015 ). Recombinant PRRSV N protein was bound to carboxylated Luminex MAGPLEX polystyrene microsphere beads according to the manufacturer's instructions. For FMIA, approximately 2500 antigen-coated beads suspended in 50 μL PBS with 10% goat serum (PBS-GS) were placed in each well of a 96-well polystyrene round bottom plate. Serum was diluted 1:400 in PBS-GS, and 50 μl was added to each well. The plate 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 of PBS-GS. For detection of IgG, 50 μl of biotin-SP conjugated affinity purified goat anti-porcine secondary antibody (IgG, Jackson ImmunoReserch) was diluted to 2 μg/ml in PBS-GS and 100 μl was added to each well. Added. Plates were incubated for 30 minutes at room temperature, washed three times, and then 50 μl of streptavidin-conjugated phycoerythrin (2 μg/ml in PBS-GS; SAPE) was added. After 30 minutes, microspheres were washed, resuspended in 100 μl of PBS-GS, and analyzed using MAGPIX instruments (LUMINEX) and LUMINEX xPONENT 4.2 software. Mean fluorescence intensity (MFI) was calculated on a minimum of 100 microsphere beads.

Measurement of Haptoglobin (HP)

The amount of Hp in serum was measured using a porcine-specific 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 dilution solution, pipetted in duplicate into pre-coated anti-porcine Hp 96 well ELISA plates, incubated for 15 minutes at room temperature, and then washed three times. Anti-Hp-horseradish peroxidase (HRP) conjugate was added to each well and incubated for 15 minutes at room temperature in the dark. The plate was washed, and 100 μl 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. Plates were read at 450 nm on a Fluostar Omega filter based microplate reader (BMG Labtech).

result

Phenotypic characteristics of PAM in CD163-transformed pigs.

The forward and side scatter properties of cells in lung lavage material were used to gate mononuclear subpopulations of cells. Representative CD169 and CD163 staining results for the different chromosomal alterations shown in Figure 17 are shown in Figure 19. In the representative example shown in panel A of Figure 19, more than 91% of PAMs 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% of double-positive cells. As shown in panel B of Figure 19, PAM from CD163 KO pigs showed no evidence of CD163, but maintained normal surface levels of CD169. CD163 polypeptides derived from the d7(1467) and d7(1280) deletion genotypes were predicted to produce modified CD163 polypeptides immobilized on the PAM surface, but immunostaining results did not show surface expression of CD163 (Figure 19, panel (see D). Since MAb 2A10 recognizes epitopes located in the first three SRCR domains, the absence of detection was not a result of deletion of immunoreactive epitopes. The d7(129) genotype was predicted to have a 43 amino acid deletion in SRCR 5 (see Figure 17). In the example shown in panel C of Figure 19, only 2.4% of cells fell into the double-positive quadrant. Analysis of PAM from the nine d7 (129) pigs used in this study showed a percentage of double-positive cells ranging from 0% to 3.6% (mean = 0.9%). Surface expression of CD169 remained similar to WT PAM. For the purpose of this study, KO, d7(1467) , d7(1280) , and pigs with the d7(129) genotype were all classified as having the CD163-null phenotype.

The CD163 variant containing the hCD163L1 domain 8 peptide sequence HL11m showed dual expression of CD163 ⁺ and CD169 ⁺ on PAM (panel E of Figure 19). However, in all HL11m pigs analyzed in this study, surface expression of CD163 was significantly reduced compared to WT PAM. Levels of CD163 fell on a continuum of expression, from no detectable CD163 to pigs with intermediate levels of CD163. In the example shown in panel E of Figure 19, approximately 60% of the cells were in the double-positive quadrant, while 40% of the cells stained only for CD169. Analysis of PAM from a total of 24 HL11m pigs showed that 38 +/- 12% of PAM cells were positive for CD169 only and 54 +/- 14% were double-positive (CD169 ⁺ CD163 ⁺ ).

Circulating haptoglobin levels in WT and CD163 transformed pigs.

As a scavenging molecule, CD163 plays a role in removing HbHp complexes from the blood (Fabriek, et al., 2005; Kristiansen et al., 2001; and Madsen et al., 2004). The level of Hp in serum provides a convenient way to determine the overall functional properties of CD163-expressing macrophages. Hp levels in serum from WT, HL11m and CD163-null pigs were measured at 3 to 4 weeks of age immediately prior to PRRSV infection. Results presented in Figure 20 showed that serum from WT pigs had the lowest amount of Hb (mean A450=23+/-0.18, n=10). Means and standard deviations for each group are: WT, 0.23+/-0.18, n=10; HL11m , 1.63+/-0.8, n=11; and 2.06 +/-0.57 for the null group, n=9. The null group consisted of genotypes that did not express CD163 (CD163 null phenotype pigs). Hp measurements were performed on a single ELISA plate. Groups with the same letter were not significantly different (p>0.05, Kruskal-Wallis one-way ANOVA with Dunnett's post-test). The mean A450 values for WT pigs were significantly different from HL11m and CD163-null pigs (p < 0.05). Mean A450 values were lower in the HL11m group compared to the CD163-null group (A450 = 1.6+/-0.8 vs. 2.1+/-0.6), but the difference was not statistically significant. Because the interaction between HbHp and CD163 occurs through SRCR 3 (Madsen et al., 2004), the increase in circulating Hp in HL11m pigs compared to WT pigs is not a result of reduced affinity of CD163 for Hb/Hp. This was likely the result of a decrease in the number of CD163 ⁺ macrophages along with a decrease in CD163 expression on the remaining macrophages (see Panel E of Figure 19).

Infection of PAM with type 1 and type 2 viruses

The permissiveness of CD163 modified pigs to PRRSV was initially assessed by infecting PAM cells in vitro using a panel of six type 1 and nine type 2 PRRSV isolates (see Table 14 for list of viruses). Viruses within the panel represent different genotypes, as well as differences in nucleotide and peptide sequences, onset, and year of isolation. The data presented in Table 15 shows the results of an experiment using PAM from three pigs for each CD163 genotype group. The viruses listed correspond to the PRRSV isolates listed in Table 14. Results are expressed as mean +/- standard deviation of the percentage of infected PAM. CD163-null PAM was derived from pigs expressing the d7(129) allele (see Figures 17 and 19 for CD163 gene constructs and CD163 expression on PAM, respectively).

As expected, WT PAM was infected by all viruses. In contrast, pigs with the CD163-null phenotype were negative for infection by all viruses. Significant differences were observed in the response of PAM from HL11m pigs. While none of the type 1 viruses can infect HL11m PAM; All viruses within the type 2 panel infected HL11m PAM, albeit at much lower percentages compared to WT PAM.

Tolerability was also assessed by comparing viral titer endpoints between WT and HL11m PAM for the same type 2 virus. Results are shown for 2 WT and 2 HL11m pigs (Figure 21). Log ₁₀ TCID ₅₀ values were calculated based on infection of macrophage cultures with the same virus sample. Infection results represent two different pigs from each genotype. The viruses used for infection are listed in Table 14. The log ₁₀ TCID ₅₀ values for PAM from HL11m pigs were 1-3 log lower compared to WT PAM infected with the same virus. The only exception was infection with modified live virus vaccine strains. Taken together, the results suggest that PAMs from HL11m pigs have reduced susceptibility or tolerance to type 2 virus infection.

Infection of CD163 modified pigs with type 1 and type 2 viruses

Representative type 1 (SD13-15) (Figure 22, panel A, left graph) and type 2 (NVSL 97-7895) (Figure 22, panel A, left graph) WT (circles), HL11m (squares), and CD163-null (triangles) pigs. Panel A, right graph) were infected with the virus. Null phenotype pigs were derived from the KO and d(1567) alleles (see Figure 17). Pigs from three genotypes inoculated with the same virus were mixed together in one pen, allowing sequential exposure of CD163 modified pigs to viruses shed from WT cage littermates. The numbers of pigs infected with representative type 1 viruses were: WT (n=4), HL11m (n=5), and Null (n=3); and type 2 viruses: WT (n=4), HL11m (n=4), and Null (n=3). As shown in Figure 22, CD163-null pigs infected with type 1 or type 2 virus were negative for viremia at all time points and did not seroconvert. As expected, WT pigs were productively infected, with average viremia levels approaching 10 ⁶ strains per 50 μl PCR reaction at 7 days post-infection for both viruses. By day 14, all WT pigs had seroconverted (see Figure 22, Panel B). Consistent with the PAM infection results (Table 15), the five HL11m pigs infected with type 1 virus showed no evidence of viremia or PRRSV antibodies. All HL11m pigs infected with the type 2 isolate, NVSL, sustained infection and seroconverted (Figure 22, Panel B). The existence of reduced tolerance in HL11m pigs was uncertain. Mean viremia for 3 of 4 HL11m pigs was similar to WT pigs. However, in one HL11m pig #101 (open square in Figure 22, panel A right graph), viremia was significantly reduced compared to other pigs in the HL11m genotype group. The explanation for the 3 to 4 log reduction in viremia for pig #101 was unclear, but suggested that some HL11m pigs may be less permissive to PRRSV, an observation that supports the in vitro PAM infection results (Table 15). Because all pigs were inoculated with equal amounts of virus and remained mixed with the WT pigs, the lower viremia in pig #101 was not the result of a lower amount of virus or less exposure. Flow cytometry of macrophages showed that CD163 expression for pig #101 was comparable to other HL11m pigs (data not shown). There was no difference in the exon 11 mimic sequence.

Additional viral infection tests were performed using two viruses, NVSL 97-7895 and KS06-72109. The results are shown in Figure 23. Pigs were followed for 35 days post-infection, and data were reported as area under the curve (AUC) for viremia measurements taken at days 3, 7, 11, 14, 21, 28, and 35 post-infection. As shown in Figure 23, for NVSL, the average AUC value for 7 WT pigs infected with NVSL was 168 +/- 8 versus 165 +/- 15 for 7 HL11m pigs. For KS06, the average AUC values for 6 WT and 6 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). Taken together, the results showed that HL11m pigs failed to support type 1 PRRSV infection but maintained tolerance to type 2 virus infection. There was a reduction in PRRSV permissibility of PAM from HL11m pigs infected in vitro with type 2 isolates, but this difference was not transmitted to pigs. For the results shown in Figure 23, viral load was determined by calculating the area under the curve (AUC) for each pig over the 35 day infection period. AUC calculations were performed using log ₁₀ PCR viremia measurements taken at days 0, 4, 7, 10, 14, 21, 28, and 35 post infection. Horizontal lines show the mean and standard deviation. Symbols: WT = wild-type pig, HL11 = HL11m genotype pig; Null = CD163-null genotype.

Argument

CD163 is a macrophage surface protein that is important in removing excess Hb from the blood and controlling inflammation in response to tissue damage. It also functions as a viral receptor. CD163 participates in both pro-inflammatory and anti-inflammatory responses (Van Gorp et al., 2010). CD163-positive macrophages are located within the alternatively activated M2 group of macrophages, which are generally described as highly phagocytic and anti-inflammatory. M2 macrophages participate in clearance and repair after mechanical tissue damage or infection (Stein et al., 1992). In anti-inflammatory capacity, CD163 expression is upregulated by anti-inflammatory proteins such as IL-10 (Sulahian, et al., 2002). During inflammation, CD163 reduces inflammation by reducing oxidation through removal of circulating heme from the blood. Heme breakdown products such as bilverdin, bilirubin, and carbon monoxide are potent anti-inflammatory molecules (Soares and Bach, 2009 and Jeney et al., 2002). In the pro-inflammatory capacity, cross-linking of CD163 on the surface of macrophages by anti-CD163 antibodies 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).

GE pigs lacking CD163 are unable to support replication of type 2 PRRSV isolates (Whitworth et al., 2016). In this study, in vitro infection assays demonstrate resistance of CD163 null phenotype macrophages to a broad panel of type 1 and type 2 PRRSV isolates, potentially further expanding resistance to include all PRRSV isolates (Table 15) . Resistance of CD163-null phenotype macrophages to type 1 and type 2 viruses was confirmed in vivo (Figures 22 and 23). Based on these results, the contribution of other PRRSV receptors previously described in the literature (Zhang and Yoo, 2015) can be excluded. For example, Shanmukhappa et al. (2007) showed that non-permissive BHK cells transfected with the CD151 plasmid acquired the ability to support PRRSV replication, and incubation with a polyclonal anti-CD151 antibody resulted in the loss of MARC-145 cells. It was found that infections were significantly reduced. Additionally, SJPL, a simian cell line originally developed for use in propagating porcine influenza viruses, was previously shown to support PRRSV replication (Provost, et al., 2012). Key characteristics of the SJPL cell line included the presence of CD151 and the absence of sialoadhesin and CD163. Taken together, these data provided compelling evidence that the presence of CD151 alone is sufficient to support PRRSV replication. The absence of PRRSV infection in macrophages and pigs with a CD163 null phenotype indicates that CD151 is biologically irrelevant as an alternative receptor for PRRSV.

The viral proteins GP2a and GP4, which form part of the GP2a, GP3, and GP4 heterotrimeric complex on the PRRSV surface, can co-precipitate with CD163 in a pull-down assay from cells transfected with GP2 and GP4 plasmids. (Das, et al., 2009). Presumably, GP2 and GP4 form interactions with one or more CD163 SRCR domains. In vitro infectivity assays containing a porcine CD163 cDNA scaffold containing domain swaps between porcine SRCR 5 and the homologue of hCD163-L1 SRCR 8 further localized the region utilized by type 1 viruses to SRCR 5 (Van Gorp, et al., 2010). It is interesting to speculate that the stable interaction between GP2/GP4 and CD163 occurs through SRCR 5. Additional viral glycoproteins, such as GP3 and GP5, can further stabilize the virus-receptor complex or function as co-receptor molecules. The requirement for SRCR 5 was investigated in this study by infecting macrophages and pigs with the HL11m allele, which carried out a 33 bp substitution in pig exon 7 to recreate the CD163L1 SRCR 8 domain. The HL11m allele also contained a neomycin cassette for selection of cells positive for the genetic mutation (Figure 17). HL11m pigs expressed CD163 on PAM, albeit at reduced levels compared to WT PAM (Figure 19, compare panels A and E). The decreased expression was likely due to the presence of a neomycin cassette located between the exon 11 mimic and the following intron. HL11m pigs were not permissive to type 1 virus infection, confirming the importance of SRCR 5. However, HL11m macrophages and HL11m pigs did not support type 2 virus infection. Based on virus titration and percent infection results, PAM from HL11m pigs showed an overall reduction in tolerance to virus compared to WT macrophages (Table 15 and Figure 17). Reduced tolerance may be due to reduced levels of CD163 on HL11m macrophages, combined with reduced tropism of the virus for the modified CD163 protein. Assuming that type 2 viruses have a requirement for SRCR 5 and that L1 SRCR 8 can serve as a suitable replacement, the lower affinity may be explained by peptide sequence differences between human SRCR 8 and porcine SRCR 5 (Figure 18, see panel B). However, the reduced tolerability of PAM was not transferred to pigs. Mean viremia for HL11m pigs was not significantly different compared to WT pigs (Figure 23). In addition to PAM, PRRSV infection of intravascular, septal and lymphoid tissue macrophages contributes to viremia (Lawson et al., 1997 and Morgan et al., 2014). The potential contribution of these and other CD163-positive cell populations in maintaining overall viral load in HL11m pigs deserves further study.

CD163 plasmids with deletions of the SRCR domain are stably expressed in HEK cells (Van Gorp et al., 2010), but deletion of exons 7 and 8 in d7(1467) and d7(1280) results in a lack of detectable surface expression of CD163. (Figure 19, Panel D). The 2A10 mAB used for flow cytometry recognizes the three N-terminal SRCR domains (Van Gorp et al., 2010), and possibly the seventh and eighth domains (Sanchez, et al., 1999), so the absence of detection is indicative of the mutated protein. It was not due to removal of the 2A10 epitope. Although small amounts of CD163 expression could be detected on PAMs from some d7(129) pigs (see Figure 19, Panel C), the amount of protein expressed was not sufficient to support PRRSV infection in PAMs or pigs. The absence of CD163 expression in exon 7 and 8 deletion mutants is not fully understood, but is likely the result of mRNA and/or protein degradation.

In 2003, CD163 was identified as a 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 that anti-CD163 antibodies, such as 2A10, block ASFV infection of macrophages in vitro. Whether CD163-null pigs are resistant to ASFV infection has not yet been determined.

Cell culture models containing modifications to the PRRSV receptor have provided valuable insight into the mechanisms of PRRSV entry, replication, and pathogenesis. One unique aspect of this study was performing parallel experiments in vivo using receptor-modified pigs. This research has important implications for the feasibility of developing preventative treatments for one of the most serious diseases facing the global swine industry.

Example 4: Maternal CD163 Knockout of protects the fetus from porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Examples 1 to 3 above demonstrate that pigs with complete knockout (KO) of the CD163 gene lack CD163 expression on macrophages and fail to support PRRSV infection (see also Whitworth et al., 2016; Wells et al., 2017) . Because CD163 expression is a dominant trait and is inherited in classical Mendelian fashion, offspring with normal CD163 expression and function are KO CD163 ^−/− It can be derived by crossing female pigs with wild type (WT) CD163 ^+/+ males. For this study, to determine whether the presence of the CD163 KO genotype in the dam is sufficient to protect the fetus after maternal infection with PRRSV, CD163 KO young sows were crossed with WT boars to produce heterozygous CD163 ^+/- A fetus was produced. In this study, CD163 -positive fetuses recovered between day 109 of gestation or day 20 after maternal infection were fully protected against PRRSV in females with complete knockout of the CD163 receptor. The results demonstrate a practical means of eliminating PRRSV-related reproductive diseases, a major cause of economic hardship in agriculture.

Materials and Methods

CD163 gene editing. The CRISPR/Cas9 method used to generate all KO alleles is described in detail in Examples 1-3 above. Wild-type animals and knockouts, or heterozygous animals generated as described in Examples 1-3 and carrying the alleles listed in Table 16, were used in the experiments described in this Example. Each of these alleles is described in Examples 1-3 and PCT Publication No. WO 2017/023570, which are incorporated herein by reference in their entirety. Specific edits for alleles B, D and E are also described in Whitworth et al., 2014, and the specific edit in allele C (2 bp insertion) is described in Whitworth et al., 2016. All alleles listed in Table 16 were confirmed based on DNA sequencing. Knockout genotypes were confirmed by the absence of CD163 expression, which was determined by staining alveolar macrophages with the anti-CD163 mAb, 2A10, as described in Example 2 above.

PRRSV infection. The PRRSV strain used in this study, NVSL 97-7895 (NVSL), is a laboratory strain isolated in 1997 from a herd in southeastern Iowa, USA, that was experiencing a PRRS abortion storm (Halbur et al., 1997). Viruses maintained as low passage isolates were propagated and titrated in MARC-145 cells. On days 89 to 91 of pregnancy, young sows were inoculated with 10 ⁵ TCID ₅₀ virus diluted in 5 mL of culture medium. Half of the inoculum was administered by intramuscular injection and the remainder intranasally. All young sows were kept in an environment where they were continuously exposed to viruses shed by infected pen mates. Blood samples were collected from young sows before infection, 7 days post-inoculation (dpi), and at the time of euthanasia. Total RNA was isolated from serum and PRRSV nucleic acid was measured by reverse transcriptase real-time PRRSV PCR (Tetracore, Rockville, MD). A standard curve was created using the quantification standards provided in the RT-PCR kit. Results in 25 μl Reported as log ₁₀ templates per reaction, which approximates the number of viral RNA templates per ml of blood.

result

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 premature stop codon after a codon frameshift in the mRNA. Crosses between WT and CD163 KO parents are summarized in Table 17. The first group of three females, serving as positive infection controls, was CD163 ^+/+ CD163 ^+/+ females carrying fetuses (++/++ group). The second group (- -/+-) is CD163 ^+/- CD163 ^-/- carrying fetuses It was female. In this group, CD163 ^−/− The female PRRS cannot support replication, whereas CD163 ^+/- The fetus remains susceptible to PRRS infection. And, finally, the third group (- -/- -) is CD163 ^-/-- CD163 ^-/- carrying fetuses as a female It was composed. For the last group, both females and fetuses must be resistant to infection.

Clinical signs in infected wild type (WT) females included lethargy and transient anorexia. KO females showed no clinical signs. During the study period, one WT female, number 139, miscarried on day 106 of pregnancy (15 dpi). PRRSV nucleic acid measured at 7 dpi showed a viremia level of 5.5 log ₁₀ templates per reaction for 139 females, demonstrating the presence of a productive PRRSV infection. Between 15 and 20 dpi, all remaining females were euthanized and the uterine horns were immediately removed. Starting at the tip of each horn, the fetus and placenta were removed and evaluated for the presence of anatomical pathology. Blood samples were collected from each fetus. If blood could not be obtained, fluid samples were collected from the abdominal cavity. The number of fetuses recovered from each female is listed in Table 17. For the CD163 WT group (++/++) (including aborted females), the number of fetuses was 16, 14, and 12 (mean = 14.0). CD163 ^+/- CD163 KO females carrying fetuses (- -/+- groups) produced 14, 17 and 11 fetuses (mean = 13.6). For CD163 KO females with CD163 KO fetuses (- -/- - group), the number of fetuses was 7 and 9. Fetal viremia and gross pathology results are summarized in Figure 24 and Table 18.

Figure 24 depicts individual fetal outcomes following PRRSV maternal infection. The numbers on the left identify each female. Below each female in parentheses are the results of PRRS PCR in serum measured as log ₁₀ template per reaction. “N” is negative for PRRSV nucleic acid (Ct>39). Fetuses are identified by their number and relative position within each uterine horn. Asterisks identify fetal PCR samples obtained from abdominal fluid. Numbers below each fetus are PRRS PCR results in fetal serum (log ₁₀ templates per reaction). Numbers within each circle indicate the presence of anatomical pathology: 1) normal fetus; 2) small fetus; 3) Placental changes, such as detached placenta and/or necrosis; 4) fetus stained with meconium; 5) The fetus dies and becomes necrotic. Lowercase letters identify the genotype of the individual fetus (see Table 17). Symbol: a, A/A; b, C/A; c, B/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 was necrotic; nd, genotype not determined.

At the anatomical level, 50% and 72% of fetuses derived from two CD163 WT(++/++) females 138 and 140 were smaller than normal fetuses (11% of all fetuses), detached or Fetuses with necrotic placentas (14%), meconium stains (7%), and dead and necrotic fetuses (25%) exhibited some degree of pathology. Pathological observations are typical of reproductive PRRS. Identical littermates exhibited high rates of PRRSV infection, with 92% of fetuses testing positive for the presence of PRRSV nucleic acid. PCR results for fetuses from WT females illustrate two important characteristics of fetal PRRSV infection. First, there was wide variation between fetuses in the concentration of virus detected in serum, resulting in fetuses being infected at different times. Second, the level of viremia did not always correlate with pathology. For example, fetus 5 from female 138 had high levels of viremia (7.3 log ₁₀ templates per reaction), but the fetus appeared to be unaffected. The reason for the difference between viremia and pathology is unclear. One possibility is that fetal pathology is the result of tissue damage that occurs maternally and is unrelated to the level of fetal viremia. In the field, these normal but infected newborn piglets can function as “supershedders” promoting rapid spread of PRRSV throughout the production system. - In the -/+ -group (females 84, 87, and 122), all fetuses appeared normal except for two fetuses that were smaller than their littermates. The smaller than normal size is likely the result of crowding within the uterine horn, which reduces the surface area of the placenta, thus limiting the growth of the developing fetus. - -/+ - All females and fetuses in the group were negative for the presence of PRRSV nucleic acid. For the last group - -/- -, there was no visible pathology and all females (nos. 86 and 121) and fetuses were negative for PRRSV nucleic acid.

The results of this study demonstrate that the absence of CD163 in females is sufficient to protect PRRSV-susceptible fetuses. CD163-positive offspring derived from CD163 KO females are susceptible to the virus immediately after birth, but protection against PRRSV in utero provides a means of eliminating a major cause of economic loss and animal suffering.

The embodiments disclosed herein are provided by way of example and are not intended to limit the scope of the invention.

In light of the above, it will be seen that several objectives of the present invention have been achieved and other advantageous results have been obtained.

Since various changes may be made to the products and methods without departing from the scope of the present invention, all matters included in the above description and shown in the accompanying drawings should be construed as illustrative and not limiting.

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SEQUENCE LISTING <110> Curators of the University of Missouri Prather, Randall Wells, Kevin Whitworth, Kristin <120> METHODS FOR PROTECTING PORCINE FETUSES FROM INFECTION WITH PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV) <130> UMO 18054.WO <160> 121 <170> PatentIn 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 atttaattat gtttgcctat 120 tttactttag ttttttgaca taactgtgta actattgcga tttttttat cctaatgta a 180 ttagttcaaa acaaagtgca gaaatttaaa atattcaatt caacaacagt atataagtca 240 atattccccc cttaaatttt tacaaatctt tagggagtgt ttctcaattt ctcaatttct 300 ttggttgttt catgtcccat atggaagaaa acatgggtgt gaaagggaag cttactcttt 36 0 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 ttta cctttt 600 aaatagagtg tagcaatatt ccgacagtca atcaatctga tttaatagtg attggcatct 660 ggagaagaag taacagggaa aaaggcaata agctttataa ggggaacttt tatcttccat 720 agactcaaaa ttgaagacgt gactagaaga ttgctagatt tggcatcagt tttgtaaaat 780 t gctgaggtg aaattaagta agggatgaaa attaactaaa ttgtgttgag tatgaaacta 840 gtagttgtta gaaaagatag aacatgaagg aatgaatatt gattgaaagt tgatgaccta 900 gaggacattt agactaacac ctctgagtgt caaagtctaa tttatgattt acatcgatgc 960 gttaaactca tttaacattc ttactttttt cccctcaagc atttaagctg aca 1020 tttcacatga aagcctggat tataaatgca cagttcagtg acctatctca gaggagtgac 1080 tgccatagca ttttttttgt ctttttgcct tcagagccac agcaacgcgg gatccgaagc 1140 cgcgtctgcg acccacacca cagctcacgg caatgccgg a tctttaaccc actgagcgag 1200 gccggggatc gaacccgcag tctcatggtt cctagtagga ttcgttaacc actgcgccac 1260 gacgggaact cctaccatag catttttact tttaagttac tgttggttta gagtaagaag 1320 gagaaatgag agtgatggag cgtttgctat atttggagac aaggtcctat attggaggtt 1380 ctcaaatata aattttgtcg ctttttcctc caatgtattg ttcaactact atttagca gg 1440 ccactgtgcc aggtactggt gaaactggtg aacatgatag atgtaattca ttccctcatg 1500 gaactttcca tctaacaatg tggatcaggt aggcttggag atgagaatgc cagtggttga 1560 ctatgactct gtggctgaag ggagagctac tcacttcgta gtttcatcaa tg tctttttg 1620 gttttccagg ttttaagccc tgctcttgca attcttttcc cttctccaac tttcttctaa 1680 tttctcaccc ctaggatgcc tataaacatg agtattttca aagctacttc actgaggtta 1740 tatgatcctg gtgtgaattt ttcctgcctg acttgccatt tagaaggaag tgtttcctgg 1800 aatttccatt gtggcttggt ggttaaagac cctgcattgt ctctgtgagg atgtgggt tc 1860 aatctctggc ctcattcagt gagtgggtta aggatctggt gtcgctgcaa gctgtggcta 1920 agatcccaca ttgccatgtc tgtggtgtag actggcacct ggagctctga tttgaccaca 1980 atcttaggaa cttcagatgt ggccataaaa aggaaaaaaa agttagga ag ggttttctgt 2040 cttgtttgga ccttcgttaa tctcaaacct ttggaaccat ctctcctcca aaacctcctt 2100 tgggtaagac tgtatgtttg ccctctctct tcttttcgca gactttagaa gatgttctgc 2160 ccatttaagt tccttcactt tggctgtagt cgctgttctc agtgcctgct tggtcactag 2220 ttctcttggt gagtactttg acaaatttac ttgtaaccga gcccaactgt ac 2280 actgaaaagc aaataattgc tcctgaagtc tagatagcat ctaaaaacat gcttcatggt 2340 ttcaaggatc atatattgaa accccaggga tcctctagag tcgacctgca gcatgcaggg 2400 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 2 460 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 tcctat gaaa taacattctc ccattgtacc atggatgggt ccagaaacat 2760 ttctcaaatc ctggcttgaa aaaataaata agtaatctaa agaataataa ttctctactt 2820 gctctttgaa tcttgaccaa ttgctgcatt tacctattgt tacaggagga aaagacaagg 2880 agctgaggtgg t 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 atgggga aagcataact gtactcacca acaggatgct ggagtaacct 3180 gctcaggtaa gacatacaca aataagtcaa gcctatacat gaaatgcttt gtgggaaaaa 3240 atgtatagat gagttaaaaa caaaaaggaa ccagttttct ataagtcatc tagtccatgt 3300 ataaaattac ccaatccatt c 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 aatcatgaac tttttgccct ttaactgatg atca acttac caaatcaaga aataaatatt 3600 cttagcactg atcctttttt gttgttgttg gaggaagaat gttttgcaaa gtagaattgc 3660 ttttttctgt ttaacagtgc tattcatttc atttacatgg tcgttttaat ttataaaaca 3720 tttcataagt ttcacctcat atgcc cttac aataactcag gaagttatat gttagacctt 3780 tctgctgaca aatcccagag tcatgtttct gacccagttc agattccttg gcttcccatt 3840 tctctttgct catgtcattg acctttatgc agccctctta cctcccacct ttctattaca 3900 gaccatctcc tccataggac tggtgttaga aagtactaat ctctacccag gcattgtggt 3960 gcaatgtggg cagcacaggc tggtatctag aaaaatgctg attc cagctcagct 4020 gctcgttaat actatcgttt taagtaagct gttcaatcct ttgaaattca ctttctgagc 4080 actcagtgat ataataaatg tagagctact ggtacactgt ctggtatgta ataggtgtta 4140 ccaattaacc ttagtttcct catgggtcac tggttctcat tacc tagaca 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 4 440 tttttttcat acttttacat gaggatcctt tatataagga tgagttaaac aaacttgatt 4500 tttgaagttt atacccctga ggctcaactg cataataata gaaagggatc catagcctct 4560 caaggactta actagtttca tgagttttca gaatctgaat ttctgagatt ctccacccca 462 0 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 ctaaaga tag catatgt 4860 aacacatttg gaacagggaa tgcatatttt gattgtgagc tcttattatt attaccattc 4920 agccctaata aaaatcttgg taagtggaag gctttggatt tcagaacttt taaaatctaa 4980 ttactttttc aaaaaagaac ttcttagggt tttttttttt taaccacaaa g tgtttctat 5040 tttttaggtg tcccaaaatt tcgttccaaa tatctttttc tcagatattt tagtcctcat 5100 agaacaccta gggatagtgg atagagaaaa ttttctttat taaaaagctg ttctttgcta 5160 aaaattgtag caggtacttt tgggaggggg gaaaactttg attcagaaac tgctaagaca 5220 tggagtgttt tgactaattt ttcctcaatt tttaatgttt tttataccat agggtacttt 5280 t gcaaactat tatgcatact tatatatttt tacttttttc ctgtctttta acttccaaat 5340 tcaacttcag acaattattc atgcactaaa ctgtttgtag taagaaagat taaaattaaa 5400 aaattaacca ttcaacaaat gactggtttg ccatttttac tactttgttg tatgaacaat 5460 tt ttttttct acaaatgaat actttgagtc tgatttatcc attcctacat aaaagttttt 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 tctt tctttt tttttttttt gaattttagt 5760 ttctaaactc acttctgaat aaatacaact tctaaattct cgtcttttct ctactctaga 5820 tggatctgat ttagagatga ggctggtgaa tggaggaaac cggtgcttag gaagaataga 5880 agtcaaattt caaggaacagt 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 tgctggag tg atttgcttaa gtaaggactg acctgggttt gttctgttct ccatgagagg 6180 gcaaaaaaag gggagtaaaa gtcttaaaag ctcaaactgt taaaaacata atgatgattg 6240 cttcttttat catcttatta ttatctaatt tcaggtcgaa attctagtac ctgtgcagtt 630 0 ttttacctta actgaaatta agataaatag gatagggagg aaggatgagc agtgacattt 6360 aggtccaagt catgaggtta gaaggaaatg ttcagagaat agcccattcc ctcagccctc 6420 aaagaaagaa agaaagaaaa agaaaaaaaaa aaagaaagct taactagaaa attttgttct 6480 ctggatgttt tagaggcaaa ccatcccttt atcattccta cctacaaagc cttctcttaa 6540 tcacattacc caccctttcc tactatagtc ggg gggggggggg gggggggggg 6600 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 6660 gggggggggg gtgaaaaaag aaccaaacaa tttcaacaaa aaaccaaaca attccaacaa 6720 aattggtcca ataagcaaac ctctagataa atttcagt gc 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 tgtgt ccctcttaca gatggagcag acctgaaact 7020 gagagtggta gatggagtca ctgaatgttc aggaagattg gaagtgaaat tccaaggaga 7080 atggggaaca atctgtgatg atggctggga tagtgatgat gccgctgtgg catgtaagca 7140 actgggaggt ccaactgctg tcact gccat 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 attgggg agg 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 gagcaagttt ttaaaaatac cctctgggag ctaaattcca catgtatcta tggcctaagt 7 860 gtatgtttat tttgcagatg gatcagatct ggaactgaga cttaaaggtg gaggcagcca 7920 ctgtgctggg acagtggagg tggaaattca gaaactggta ggaaaagtgt gtgatagaag 7980 ctggggactg aaagaagctg atgtggtttg caggcagctg ggatgtggat ctgcactcaa 8040 aacatcatat caagtttat 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 gac tgtcttttct tcatcatata gtcctacatc ctttgtcata aattaattga 8340 ccataggtgt gtgggtttat atctgggctc tctattctgt tcctttgatc tatatgtctg 8400 tttttatgcc agcaccatgc tgttttgatt actatagctt tgtagtatca tctgaagtca 846 0 ggaaacatga ttcctccagc tttgttcttc tttctcaaga ttgttttgtc tattcagagt 8520 ttatgttccc atgcagattt aatttttaaa tttatttaat ttttatttt tatttttaat 8580 ttaaattaat ttaaattttt tatttcccaa cgtacagcca agggggccag ggtaaccttt 8640 acatgtatac attaaaaatt tcaggttttt cccccaccca tttctttctg ttggcaagta 8700 aatttttgaa ca aagtttcc 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 tttttcttaa ctcttgaaac 9120 tataacccta tttctaacca aattcat gtt ctatactggc tcttcaaaaa cccaggagat 9180 gggaaagcca gaatctccag tgtttcagct tctgggaagg agcaagtttt taaaaatacc 9240 ctctgggagc taaattccac atgtatctat ggcctaagtg tatgtttatt ttgcagatgg 9300 atcagatctg gaact gagac 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 t tgtgatcact atgacgaaac 9600 caaaattacc tgctcaggta agaatttcaa tcaatgtgtt aggaaaaattg cattctactt 9660 tcttttacat gtagctgtcc agttttccca gcaccacttg ttgaaaaaac tgtctttttc 9720 ttcatcatat agtcctacat cccttggcca taa attaatt gaccataagg ggtgtgggtt 9780 taatatccgg ggctcctcaa ttcgggtccc ttggatccta aaagccggtt ttataacccg 9840 acacatggcc tgtttttgac taaataaaac ctttggaaaa caatcccgaa ggtcgaggaa 9900 catggaatcc ccccaacaaa ggaccttctt tcccccaaaaa tgcggctcag ccaactcaaa 9960 aagattttat gaatcacaaa ccgcacatta tcttcctaaa attactatt c ctatgtttta 10020 atttgcaaag tcattccgat atagttggcg cagagtaact catttagata tccaccccac 10080 cagttcctca ctcaagtaag gggggggggg gggggggggg gggggggggg 10140 gggggggggg gggggggggg gg gggggggg gg gggggggg gg gggggggg ggggggggc 10200 ccccatgtga gattttgtgt gtcctttaag agtggagtct ctatttccca ctgctctctg 10260 gttctcccca aagtaagccc tgctggcttt caaaacttct gggagcttgc cttcttggta 10320 taggactcct gggctaggga gtctaatgtt tggcttagac cccttactgc ttgggaagaa 10380 tctctgcaac tgtaatgaat tatcttccta tttgtgggtt gct gaggata tggtcttaac 10440 tgttctgtgt tctacccctc ctatccatct tgttgtggtt ccttctttat atctttagtt 10500 gtagaaaagt ttttcttatc aacagttgct ctgtaaattg taacttgggt gtacacctag 10560 taggaggtga gctcagggtc actct 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 gaaagacctt agagaaaata gcataagagc aaaaccatta aacatggaga catctagtca 10860 tagggt ggaa attttatgtg gtccatatcc cctaaccagt ggctttacac caggcacatc 10920 ctaactaaga tctgctccca agtgtcttcc ctgatgcttt aaattgtgtt acatggaaac 10980 tatcctttga tgaagaaatg caacctttta aaatacaaca ttgaaacttt tgtgctttaa 1 1040 ttttgctttt caacattttt tctttttaaa agaagaaatt tatttgtttt tttaaatttt 11100 aatggccacg gcatatggaa gttctcaggc cagggataga attcaagcca caggtgcgac 11160 ccatgccaca actgctgcaa caccagatcc tttaacccac tgcaccaggc cagggattga 11220 agccttgcct tactgacaat ctgagccact tcagtcagat aaagaaattt cttcattaag 11280 cagagt attc acatggttta aacttcaaaa tattaaagtg taaactcttt ccccaccact 11340 gtccccagct caccaactct acttaccaca gacaactgat gtggttaggg tatttaaata 11400 gtaaatccaa gaaaatataa acaaatccgt atatataggt ttcaccccat tttattatcc 11460 taatgttgca taa actatactgt cccttgggta ttcacttagt aaaatatttt 11520 gatcataatt tcctatcagt atttaaagag ctttctgaaa ttatttctgt ataacatttc 11580 ttttctcatc ggtagggggg gggggggggg gggggggggg gggggggggg gggggggggg 11640 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggaatggg 11700 aagaaaaaac cacca tggtt aatttttttt atccctctac acccgggaaa attacccttg 11760 gggccacact tttctataga aaggggatta tttaaaaggg tctgaaaaag aatttttttt 11820 tcgaaagggg aaatatttgg cctaacttag tcacataagc catgttctct ggcaagttag 11880 gtaacataca ttgtcat tgggggcaac aaaaacaatt ttccttttgg accttttggg 11940 actccgcatt ggttagggaa ggggaagtat attggaattc ggaaaattcc ttccaaatta 12000 aaaaaggttt gttattttca tattaaccta tttcatatta attagcatga attccagcgc 12060 cattaaaagg gaaaacacct ggagtggtaa gaaaaaagtt tttttttctc tttttttttt 12120 ttttttttta atgg ccacat ctgtggcatg tgaagttccc aggctagggg tcgaatagga 12180 gctacagctg ccagcttgca ccacagccac aacaatgcca gagccaagcc tcatctgcga 12240 cctataccac aactcatggc aatgctggtt ccttaacccc ctgagtgagg cctggggtca 12300 aaccca catc 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 ggcat tgttc tcatttcaat tctgagttca gtgtggagag 12600 tatacgtgtg tgggggctgc acgcttttca caacccactt tctgctgata ctgatttagg 12660 gatccttgga ttgctttaca gttgagtcat cattaactag tgtcacttgc cttcaaagtc 12720 a gcaaaataa ttgtctccaa actagtaggc ttctagtgta tttgctttaa tccaatgcca 12780 tgtgaaagta acatggtcaa agaataagtt atataccttg acctaccctg tgaccaggct 12840 cttcctctta atttattgac cactgcctta aggtcatttg aaaccatggg tttgggagga 12900 aggcaaggcc taaatcccgt ctttgttgga aggctcactg tccttgtctt tagagcatca 12960 ttttttttta aactggggta cagtttattt aca gtgttgt gtcaatttct gctgtacagc 13020 acagtgaccc agtcatacac atacatacat tctttttctc atactatctt caattttatt 13080 ttctgctaag tctgccattt tatcatcacc tcagtttgaa ggacaggata tttagagttt 13140 gttttttttt tccc cccaat cctgcaattt ctaaattata agactctcaa ttagccgtat 13200 ataacagctg caggcacagg atgtctccct cacaaaattg gtatttttcc ttccatttct 13260 tcttgcagtt tggctatttc ttgtctgagt tcatctctct ttttaagtgt taaaaagggc 13320 aaggaggatt catgctatgt caacattatg attttttctt ttctatactt gataagagta 13380 tacttttccc aaatgtcatc caacttttca gcatcagttt ggtt ttcttttcaa 13440 ggtggtattt ctctaatgtc acttgaataa caagactcgt tagttctcca ggctacaata 13500 tcctagtctg agtatattct gcatgttaat tctattcagc cacatccata atttaggttt 13560 tattcctgga acacctcact tttttttttt tttggtct ttttatagcc 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 atgata acac 13860 attctgaatt atttaggatt ctattatact gcatgtaata gaaatcccaa atagcaaaat 13920 ttgcaactta aggcaggttc ctgtctttac aaaatcatgt tttcctttgc tatatgtgca 13980 ctttgctttc ctctgtgaat tccctttttt gttatatttt ta tagctttt ggaaacactt 14040 ttacttattt gggggggcct agatttttaa ccctctcctt gtttttctag aaatagagtt 14100 tataatttta tttcttcatt tacttgatac tttcaagaga ttcccaggaa aaaaattatg 14160 gaaatactgt ctctgtgcct gccaagttca aactaagaat tgtataatct gttttaattc 14220 ttaagcattt atagatgaca aggctttgtg tctgataggg gccagcgaac tcagtaaaga 1428 0 gggaagatga gaaagataat ggcaagaatt tatccctgaa gtgtagtttt gacaaaccag 14340 tcacaaagag gtctaagaaa ttttggtcac aaagttgttt tgaatcccag gcattttatt 14400 tgcaatgatt gcatatgttc tggaaaggac atctgaacct aagaaatagtt gcat 14460 tgtgttatat tttactaagg tctgagaaat aatcttgaga tgagaatgaa ctctacttct 14520 tcagagtctg gaaggaataa attatgaaaa tgtattaatg cttctttaaa ccatattgta 14580 tatttatcta ttactaaaca aaaagaagta gctctattta tttatttat tatttattta 14640 tttatgtctt ttgtctcttt agggccacac ctgtggcata tggaggttcc caggctagag 14700 gtccaattgg tagca 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 caatctctt g ggatagaata tgatggaaga 15180 caaaatcaga acaagagagt atatatatat gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 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 ctcctttgtc ctggactcaa atattttagg ataaaggaaa aaagaagata tttatagaag 15540 ggacttgttt tcaagtactt gacaaaattt caccattaaa tg tgggagttcc 15600 catcgtggct cagtggaaac aaatccaact aggaaccatg aggttgtggg tttgatccct 15660 ggcctcactc agtgggttaa ggatccggtg ttgccgtgag ctgtggtgta ggttgcagac 15720 acggttctga tcctgcg ttg ctgtggctgt ggctgtggtg taggccagca gcaaacagct 15780 ctgattagac ccctagcctg gaaacctcca tatgccacag gtgcagccct aaaaagacaa 15840 aaaaagagaa aagacaaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaag 15900 aacccccaga ggtatttatt tgtttttgcc ttttttcact gactgttctt tgtttgtttg 15960 tttgagactg atctagaaga ctagagatta caagaaata t ggatttggct cactctaaga 16020 aactgctttc attccaaggt ttgggtctat ccaaaagtgg aatagaatca tatgaatact 16080 agtttatgag tatttagtga gaggaatttc aagctcaaat aatgattcag caagattaaa 16140 ttaaggaggg aattttcctt gtggctgagt cccaatgttg tctctgtgag 16200 gatgtaggtt ccatcctggg ctttgctcat taggttaagg atctggcatt gctgcagctc 16260 agacccagtg ctgccctggt tgtggcttag gccaaagctg cagctccaat tcaatctctg 16320 gcctgggaac ctccatgtgc tacaaggtgc ggccttaaaa ggaaaaaaaa aaaattaaat 16380 caaggactca agagtctttc attatttgtg ttgtggaagc ta tatttgtt ttaaagtctt 16440 agttgtgttt agaaagcaag atgttcttca actcaaattt gggagggaac ttgtttcata 16500 catttttaat ggataagtgg caaaattttc atgctgaggt gatctatagt gttgtaatgc 16560 agaatatagt cagatcttga acatttta gg aagttggtga gggccaattg tgtatctgtg 16620 ccatgctgat aagaatgtca agggatcaca agaattcgtg ttattgaca gcagtcatct 16680 ttaaaaggca tttgagaaag tccaatttca aatgcatttc ctttctttaa aagataaatt 16740 gaagaaaata agtctttatt tcccaagtaa attgaattgc ctctcagtct gttaaaagaa 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 1728 0 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 aacattct tg ggtcctgggg gtccctctgc aactctcact gggacatgga agatgcccat 17760 gttttatgcc agcagcttaa atgtggagtt gccctttcta tcccgggagg agcacctttt 17820 gggaaaggaa gtgagcaggt ctggaggcac atgtttcact gcactgggac tgagaag cac 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 tg tggtatag gttgcagact ctgctcaggt cccatgttgc tgtgattgtg 18180 gtgtaggctg actgctgcag cttcaatttg acccctagcc cgggaatttc cataggccac 18240 acgtgcagca ctaaggaagg aaaaaaagaa aaaaaaaaaa aaagagtggg tgtgcctata 1 8300 gtgaagaaca gatgtaaaag ggaagtgaaa gggattcccc cattctgagg gattgtgaga 18360 agtgtgccag aatattaact tcatttgact tgttacaggg aaagtaaact tgactttcac 18420 ggacctccta gttacctggt gcttactata tgtcttctca gagtacctga ttcattccca 18480 gcctggttga cccatccccc tatctctatg gctatgttta tccagagcac atctatctaa 18540 cactccagct gatcttcctg acacagctgt gg caaccctg gatcctttaa ccaactgtgc 18600 caggctggag atcaaaccta agcctctgca gcaacccaag ctgctgcagt cagattttta 18660 accccctgtg ccactgtggg tatctccgat attttgtatc ttctgtgact gagtggtttg 18720 ctgtttgcag g gaaccagag 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 tg tgatgaca gctgggacct gaatgatgcc 19020 catgtggtgt gcaaacagct gagctgtgga tgggccatta atgccactgg ttctgctcat 19080 tttggggaag gaacagggcc catttggctg gatgagataa actgtaatgg aaaagaatct 19140 catatttggc aatgccactc acatggttgg g ggcggcaca 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 aagcgagt tt 19440 gcagcaaaac tcatagggag aacttctttt ataaataata tgaagctgga tatttagtgc 19500 accacctgat gaccacttta ttaataaata aagagttcct gttgtggcgc agcggaaatg 19560 aatccgacaa ataatcatga gtttgcgggt ttgatccctg ctca 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 aagtgtactc agttataata tattggggaa aacagtacta tgagaagtaa aaaaatctac 20 040 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 tgctgagc ca cgaagggaac tcctcagaag tgattttgat gttactttct tttcatgaca 20340 aatctggtaa agtacataca catagaaact gaagtgtcag aaagggaaat atttcatttt 20400 aaggtaatgt atacaaaaca gtggttttac catctgagta tctcgctaaa ttttaactat 20460 caag gacaat tgccaaaaaa aaaaaaaaaaa gagagagaga gagaacagaa tagggttatg 20520 aagctaaaat cacagggtta tgaagctaaa atcacagtaa tttagggaga aaaaaatcca 20580 aagcatgtaa ttgataaaag gttctgagcc tttgtttgag atttagaatt caacttagaa 20640 ataccggtgg tattttaaag cagtccataa gtataaaatc caaggctaaa aaaccagaag 20700 gtatttgtag aacaaatata t tttaataag ctctaccaag tcatccagaa gctattaaag 20760 aattactggt cactgacata gtgtacctgt tttcaaggcc attcttacat cagaataaag 20820 ggagagcacc ctctgaatct tcagaaaaga tgtgaaagtg ctaattctct atttcatccc 20880 agagttcatg tctctgagac tgatca gtga aaacagcaga gagacctgtg cagggcgcct 20940 ggaagttttt tacaacggag cttggggcag cgttggcaag aatagcatgt ctccagccac 21000 agtgggggtg gtatgcaggc agctaggctg tgcagacaga ggggacatca gccctgcatc 21060 ttcagacaag acagtgtcca ggcacatgtg ggtggacaat gttcagtgtc ctaaaggacc 21120 acta tggcagtgcc catcatctcc atggaagaag agactggcca gcccctcaga 21180 ggagacatgg atcacatgtg ccagtgagta tccattcttt agcgccactg ttatcttctg 21240 atctacctaa gcagaagttt tataatctgt agttaatccc tattctacct ggatgatggg 21300 att cattctg 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 a aaaaaaaaa attgttatag gtgatttaca 21600 atgtctgtca atttctgctc tacagcaaag tgacccagtt atttacatat acattctgtt 21660 tctcatattt ttaaaccagg agatttctat ctgcctggcg gtttgaggga atttaacatt 21720 atgcatttat gttaacttta g ttttctaag 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 taagactt ca agaaggaaac actaattgtt 22020 ctggacgtgt ggagatctgg tacggaggtt cctggggcac tgtgtgtgac gactcctggg 22080 accttgaaga tgctcaggtg gtgtgccgac agctgggctg tggctcagct ttggaggcag 22140 gaaaagaggc tggc 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 ggtgggggga gacatgatgt ttcagtctga agaatgatga 22440 aaacctgtgt ggttacgcat gggccttcgc cgaggaaag gacataacta ccatgtatcc 22500 tcctgcagag ggaggaagaa ctaggggatt ctagttttgt gtgggaagga gcagtttact 22560 tggctcagga ggcactaaag gctcaga tag 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 228 60 atcacctctt gtgatgttta cttgttctca ggtcgctcat cttttgttgc acttgcaatc 22920 tttggggtca ttctgttggc ctgtctcatc gcattcctca tttggactca gaagcgaaga 22980 cagaggcagc ggctctcagg tctgaacggtct ct ctaatgtt tctatgggag 23040 aagaagcctc tctggataat aaaacaaaaa aattacattc aagtatcagt tggccagaaa 23100 gagggaacct agaagaggtt taagcagttt ctccgaaaca gggaacaaga attcagagaa 23160 gaaaaggcac attggctgta ctgatgatac ctgcactcgc tatgtatgtt taatggggga 23220 cagtagagaa ttgatagttt agaaggagta tgcttatatg gttctggatg aatcctgtat 23280 ccccccaaac atttattttc tcttactata tacttattac taatttaact cttctgtcaa 23340 gccatgtgct aggttctgaa gatggttcag acttggataa ccaagtgctt ttgttttcat 23400 ggaatttcca gtttagtgga agagataaat atgtaaaatgggg gggggggggg 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 gggg gggggg 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 ggcgggcccc 24060 cctcgaggtc gacggtatcg ataagcttga tatcgaattc gtgagccaga ggacgagact 24120 agagatggat gatgactac g 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 agagggccgt 24540 gattatttct gacagaggga ttatggagta gaatatgatg gct tggacct tttttcacta 24600 aaacaagtca gtcttctcaa aggtagttta gcttttcata tatctttcac agtttcttcc 24660 attcccattt cctgccattt tcctttctct aacttttatt tattatattt tttcctaaaa 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 c cactgaataa gatggtttta acggatgtgg 25200 gactaatttt tgtgtctaaa tctcttccta tttctggatg aaaaaaagga gaccactctg 25260 aagtacaatg aaaaggaaaa tgggaattat aacctggtga ggtgagtagg aagaatttat 25320 tcatcattgc tgaaaacagg tacattcctt ttgaaagttg agaactcctc tggtattaga 25380 aaaaaaaaaaa gaacgtatat acacatatat ttccatgtct atgtttatgt t tgtaaatcc 25440 atattcagaa tatgcaacaa ctttttataa ctatgacttc agtccatctt ttagttacat 25500 atatattcta aacaacaact attgctaaga gaagctgggt aagtaaatgt gaataaatct 25560 tctaaagata ttacaggaag ttcctgctgc ggctcagtgg aagact tgatgtcttt 25620 gtgaagatga gggctcgagc cctggcctca ctcagtgagt taaggatcta gcattgctgt 25680 aagctgcagc gtaggttgca gatagggctc agatccagtg ttgctgtggc tgtggcctca 25740 gttgcagctc tgattcaacc cttaggcgag gaacttccat atgcagcaaa tgtggccatt 25800 aaaaaaaaaa aaaacattat aggagtcatt tcataaaaga gataagacgt tagtt 25860 atatagtgca tactctggta aagatagtat aggatactat aggaatatag aaagcttgcc 25920 tatgaaaatt tgggaagatt gtggaaaaga catctcaaaa tatggcatag aaaagaatca 25980 tatctttgag gaacagtaag tttttcattc aaaaccgtgt attgaacata cttgt ggtga 26040 caagtggtgt cctgagtact aaaaattcag tgataaaaga tgctcttgac aaagacatgg 26100 ctgttgaata gaaggtctca ctgtcaatgt gtgggaatta tggacagcct atgtggacac 26160 agggaataga tgagactcta ggctggaagg ctgcattgag cccaataatg aatggtcctg 26220 tctgatatat ttcatgctca tattttattt tagggactat tggggaggtg gtgggttttg 262 80 gaagattaag ctgaggcaag acacaatcag attgcctttt ataatttact ttcaggagga 26340 aagtctaact aaaaaaagaa ttcgatatca agcttatcga taccgtcgac ctcgaggagg 26400 cccgcctgcc cttttggggg 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 gataaagc ca tttgtctgta tataacaaat agaagagaat tcctttttgc agccttgtta 26760 gtagtgcccc caaactggaa acaaagtgaa tatcagtcag tggggtagcg gctggaaaaaa 26820 ttttagtgca cccaaccaac aaagaaaaac catgcacaaa aattcaataa atatcatctc 26880 act tttgtgt tcatgttatt gaatataatt aaacataatg tttacatcta taaaattatc 26940 atatgtatac atgtaaagaa acattaaaac atttttaaca gactgtaaac ttgaggactg 27000 tgaatgactt ttgattgata atctcaaaca tatggatact attctgatgt aataaataat 27060 gattaaattt tttccctaaa gagtaatcac tactgaatcg ttgcctcaga atcatatgga 27120 ggtgctttta aaaaaggcact g ttgttctctg gaatagaagt aattcttatg 27180 tacactgaag tttgaaaatc attgcattta agtgttctgt tcaggaaagt agtgtgcttt 27240 ttaatatttg tgagtgaatg agtaacacaa tacattatat cacattttaa tgtaattcta 27300 cacatgtgca tatgaagaga aaagtaacat ttttttctat ttatgtcttt agttcagcct 27360 ttaagatacc ttgatgaaga cctggactat tgaatgagca agaatctgcc tcttacactg 27420 aagattacaa tacagtcctc tgtctcctgg tattccaaag actgctgttg aatttctaaa 27480 aaatagattg gtgaatgtga ctactcaaag ttgtatgtaa gactttcaag ggcattaaat 27540 aaaaaagaat attgctgatt cttgttcttg attttctgaa atc tcttattggg 27600 cttctaattt aaaaaaaaaat atctgggcgc ccgcagatat cgaactcttg ggcagtgtga 27660 ccaaacgaag acatatccaa tcaagcatgc aaatggacca gcccactgta ctagcacgct 27720gtggcagcca 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 ctgggggacat 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 gaaacccat 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 atggtgagag 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 ccctcctggg 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 gagcaccttt tgggaaagga 3600 agtgagcagg tctggaggca catgtttcac tgcactggga ctgagaagca catggggagat 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 gaaaaaaaaga aaaaaaaaaa aaaagagtgg 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 ataaggagga 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 ggatgggcac caccc 25 <210> 67 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotide <400> 67 aaacgggtgg 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 attcttgggt 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 cccgtcaggg 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 tggggggtccc 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 agagtgggtg 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 gaaggaaaaa aagaaaaaaa aaaaaaaaga gtgggtgtgc ctatagtgaa gaacagatgt 2580 aaaaggggaag tgaaaagggat 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 ttttattcc 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 aatggaaaaag 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 gggagatgt 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 gtgcagcact aaggaaggaa aaaaagaaaa aaaaaaaaaaa agagtgggtg tgcctatagt 4080 gaagaacaga tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag 4140 tgtgccagaa tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg 4200 acctcctagt tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc 4260 ctggttgacc catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca 4320 ctccagctga tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca 4380 ggctggagat caaacctaag cctctgcagc 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 cattttatattc atgttttatt tcctctttgc 4680 agggagtggt caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt 4740 ctatcatgag ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca 4800 tgtggtgtgc aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt 4860 tggggaaagga 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 ccattggct 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gattgaaagg gattccccca ttctgaggga ttgtgagaag 2760 tgtgccagaa tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg 2820 acctcctagt tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc 2880 ctggttgacc catcccccta 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 tggggaaagga 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 ctggaggcac atgtttcact gcactgggac tgagaagcac atgggagatt gttccgtcac 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 aaaaaaagaa aaaaaaaaaa aaagagtggg tgtgcctata gtgaagaaca gatgtaaaag 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 tcctctgaaa tgcattttat tcatgtttta tttcctcttt gcagggagtg gtcaacttcg 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac 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 agaacacgtgg 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 ttcttgggtc ctgggggtcc 3120 ctctgcaact ctcactggga catggaagat gcccatgttt tatgccagca gcttaaatgt 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 aagagtgggt 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 aaagaaaaaa aaaaaaaaag agtgggtgtg cctatagtga agaacagatg taaaagggaa 3720 gtgaaaggga ttcccccatt ctgagggatt gtgagaagtg tgccagaata ttaacttcat 3780 ttgacttgtt acagggaaag taaacttgac tttcacggac ctcctagtta cctggtgctt 3840 actatatgtc ttctcagagt acctgattca ttcccagcct ggttgaccca tccccctatc 3900 tctatggcta tgtttatcca gagcacatct atctaacact ccagctgatc ttcctgacac 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 ttttatcat gttttatttc ctctttgcag ggagtggtca acttcgcctg 4320 gtcgatggag gtggtcgttg tgctgggaga gtagaggtct atcatgaggg ctcctggggc 4380 acatctgtg 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 agtctgctca agtgagaccc agggaatgtg ttcactttgt tcccatgcca tgaagagggt 3240 agggttaggt agtcacagac atctttttaa agccctgtct ccttccagga tacacacaaa 3300 tccgcttggt gaatggcaag accccatgtg aaggaagagt ggagctcaac attcttgggt 3360 cctggggggtc cctctgcaac tctcactggg acatggaaga tgcccatgtt ttatgccagc 3420 agcttaaatg tggagttgcc ctttctatcc cgggaggagc accttttggg 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 aaaaaaaaaaa gagtgggtgt gcctatagtg aagaacagat 3960 gtaaaaggga agtgaaaggg attcccccat tctgagggat tgtgagaagt gtgccagaat 4020 attaacttca tttgacttgt tacagggaaa gtaaacttga ctttcacgga cctcctagtt 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 ctgtgggtat ctccgatatt ttgtatcttc tgtgactgag tggtttgctg tttgcaggga 4380 accagagtca gacactatcc ccgtgcaatt catcatcctc ggacccatca agctctatta 4440 tttcagaaga aaatggtgtt gcctgcatag gtgagaatca gtgaccaacc tatgaaaatg 4500 atctcaatcc tctgaaatgc attttatca tgttttatt 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 aaaaaaaaaaa agagtgggtg 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 cattttatattc atgttttat 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 aagtgagcag gtctgggaggc acatgtttca ctgcactggg actgagaagc acatggggaga 3660 ttgttccgtc actgctctgg gcgcatcact ctgttcttca gggcaagtgg cctctgtaat 3720 ctgctcaggt aagagaataa gggcagccag tgatgagcca ctcatgacgg tgccttaaga 3780 gtgggtgtac ctaggagttc ccattgtggc tcagtggtaa caaactcgac tggtatccat 3840 gagggtatgg gtttgatccc tggccttgct caatgggtta aggatccagc attgctgtga 3900 gctgtggtat aggttgcaga ctctgctcag gtcccatgtt gctgtgattg tggtgtaggc 3960 tgactgctgc agcttcaatt tgacccctag cccgggaatt tccataggcc acacgtgcag 4020 cactaaggaa ggaaaaaaag aaaaaaaaaaa aaaaagagtg ggtgtgccta tagtgaagaa 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 gatgggccat 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg gcgtagtctg 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 aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga agtgagcagg 3480 tctggaggca catgtttcac tgcactggga ctgagaagca catggggagat tgttccgtca 3540 ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc tgctcaggta 3600 agagaataag ggcagccagt gatgagccac tcatgacggt gccttaagag tgggtgtacc 3660 taggagttcc cattgtggct cagtggtaac aaactcgact ggtatccatg agggtatggg 3720 tttgatccct ggccttgctc aatgggttaa ggatccagca ttgctgtgag ctgtggtata 3780 ggttgcagac tctgctcagg tcccatgttg ctgtgattgt ggtgtaggct gactgctgca 3840 gcttcaattt gacccctagc ccgggaattt ccataggcca cacgtgcagc actaaggaag 3900 gaaaaaaaga aaaaaaaaaaa aaaagagtgg gtgtgcctat agtgaagaac agatgtaaaa 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 attatttcag 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 agacccccatg 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 aaaaaagaaa aaaaaaaaaaa aagagtgggt gtgcctatag tgaagaacag atgtaaaagg 3960 gaagtgaaag ggattccccc attctgaggg attgtgagaa gtgtgccaga atattaactt 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 caacccaaagc 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 gcattttat catgttttat ttcctctttg cagggagtgg tcaacttcgc 4560 ctggtcgatg gaggtggtcg ttgtgctggg 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 agaaaaaaaaa 2580 aaaaaaagag tgggtgtgcc tatagtgaag aacagatgta aaagggaagt gaaagggatt 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 tatcttctgt gactgagtgg tttgctgttt gcagggaacc agagtcagac actatccccg 3060 tgcaattcat catcctcgga cccatcaagc tctattattt cagaagaaaa tggtgttgcc 3120 tgcataggtg agaatcagtg accaacctat gaaaatgatc tcaatcctct gaaatgcatt 3180 ttattcatgt tttattcct 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 atggaaaaaga 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 actctacttc 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 gtgttattg acagcagtca tctttaaaag 540 gcatttgaga aagtccaatt tcaaatgcat ttcctttctt 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 actctacttc 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 gtgttattg acagcagtca tctttaaaag 1200 gcatttgaga aagtccaatt tcaaatgcat ttcctttctt 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 gcagcttcaa tttgacccct agcccgggaa tttccatagg ccacacgtgc agcactaagg 2640 aaggaaaaaa agaaaaaaaaa aaaaaaagag tgggtgtgcc tatagtgaag aacagatgta 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 cctaagcctc tgcagcaacc caagctgctg cagtcagatt tttaaccccc tgtgccactg 3060 tgggtatctc cgatattttg tatcttctgt gactgagtgg tttgctgttt gcagggaacc 3120 agagtcagac actatccccg tgcaattcat catcctcgga cccatcaagc tctattattt 3180 cagaagaaaa tggtgttgcc tgcataggtg agaatcagtg accaacctat gaaaatgatc 3240 tcaatcctct gaaatgcatt ttattcatgt tttattcct ctttgcaggg agtggtcaac 3300 ttcgcctggt cgatggaggt 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 ctatcccggg 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 aggaaaaaaaa 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 tggaaaaagaa 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 tttattcct ctttgcaggg agtggtcaac 3300 ttcgcctggt cgatggaggt 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 acctatgaaa atgatctcaa tcctctgaaa tgcattttat tcatgtttta tttcctcttt 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac 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 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtatttat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 gcgtagtctg ctcaagtaag agtttactga aaataacact cttaaaatct tgttatgttt 3360 ttattcataa tgtgaatgag tagtagtgga aaataactac cagtttccta agcttataac 3420 ttcgtatagc atacattata cgaagttata agcctgcagg aattctaccg ggtaggggag 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 caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg 3840 ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cgggaggcccg 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 tgagagggcc agaaccagga tggaggctgg 4440 cctggtggac caggatgacc tggatgaaga acaccagggc ctggcccctg ctgaactgtt 4500 tgccaggctc aaggcatcca tgccagatgg tgaggacctg gtggtgactc atggggatgc 4560 ctgcctgccc aacatcatgg tggaaaaatgg aaggttctct ggcttcattg actgtggcag 4620 gctgggagtg gctgacaggt accaggacat tgccctggca accagggaca ttgcagaaga 4680 gctgggggga gaatgggcag 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 aaaaaaaataa 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 tcactattct tttgcaagaa tttacttagg taatgccaac taatttattc caaggccaaa 5700 aagatgacaa tgtcttatat attataaaaa ctaataaaaa ccattttaaa acctagtata 5760 aatttaaagg tacttgctct tctggttcat ctcttctttt gtttacttct gctttcaaaa 5820 acttattat tgtgaccata ttctttactt ccatttatg ttataattta taagatacta 5880 tacttgcaag caataaatgt tatcttttta gcttttaaat ggtctcattt gaaaagaata 5940 tataattagt aagtcatagc tactttaaat aaaaacttat tctttaagag attaaacact 6000 tctccaagtg atctgttttt ctttaattaa aacgttatta actcccaaaa tgatgttatt 6060 gtttttttat aatcttaaat accaataatt accaggtcta ttttgatttt gatacaggat 6120 aaaaactact attaattact taagaatgtg ttctttttta tatgtaccat tttcatgatc 6180 aaagttggtg atatgactga ggttttgatt attattaaac agatagttaa tatgatatat 6240 tcctcatttt tccaaatgaa aggaaaaatg tcttatatgg aggaaaaagat tggggcaggg 6300 ggattagtaa attattactt aaatatctga ataggaggat ttttcaatga aaggataaag 6360 gaagaatgat tgtatcatct gaatctttcc ctccctttcc tggagtttgt cctttcaacc 6420 cagtatacct accactccct tcatcaccta ctttcccatt acagtcccta tgtgttgggt 6480 ggtaactatt ttgttttggt 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 caagaaaaaaa aaaaaaaaaaa ggcgaagacc tcagggaaat 6780 ttatattgct 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 gccagcagct taaatgtgga gttgcccttt ctatcccggg aggagcacct tttgggaaag 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 aggaaaaaaaa 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 tggaaaaagaa 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 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc 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 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 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 aggaaaaaaaa 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 gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg 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 cccgggagga gcaccttttg ggaaaggaag tgagcaggtc tggaggcaca tgtttcactg 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 aaaaaaaaaaa 3600 aagagtgggt gtgcctatag tgaagaacag atgtaaaagg gaagtgaaag ggattccccc 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> PRT <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> PRT <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 (22)

A method for providing protection against viral introduction to a porcine fetus containing a modified CD163 gene, comprising breeding a female porcine animal with a male porcine animal to produce a porcine fetus containing a modified CD163 gene, comprising:
The female swine animal comprises a modified chromosomal sequence in both alleles of its CD163 gene, wherein the modified chromosomal sequence is a female swine animal compared to a female swine animal that does not include the modified chromosomal sequence in both alleles of its CD163 gene. A method comprising causing a reduction in viral entry into the swine animal, thereby providing protection against viral entry into the porcine fetus. The method of claim 1 , wherein the male porcine animal comprises two wild type CD163 alleles. 2. The method of claim 1, wherein the female swine animal comprises a first modified chromosomal sequence in a first allele of the CD163 gene and a second modified chromosomal sequence in a second allele of the CD163 gene, The method, wherein the modified chromosomal sequences are different from each other. The method of claim 1, wherein each allele of the CD163 gene of the female swine animal comprises an insertion, deletion, or combination thereof. 5. The method of claim 4, wherein at least one allele of the CD163 gene of the female swine animal comprises a deletion. 5. The method of claim 4, wherein at least one allele of the CD163 gene of the female swine animal comprises an insertion. The method of claim 1 , wherein the modified chromosomal sequence results in reduced CD163 protein production or activity compared to CD163 protein production or activity in a female swine animal without the modified chromosomal sequence. The method of claim 1 , wherein breeding produces one or more embryos comprising a chromosomal sequence modified in a single allele of the CD163 gene. The method of claim 1 , wherein breeding comprises crossing a female swine animal with a male swine animal. The method of claim 1, wherein breeding includes artificially injecting sperm into a female animal with sperm obtained from a male animal. The method of claim 1 , wherein breeding comprises transferring the fertilized oocytes into the reproductive tract of a female swine animal. 12. The method of claim 11, wherein the fertilized oocyte is produced by in vitro fertilization of the oocyte with sperm obtained from a male porcine animal. The method of claim 1, wherein each allele of the CD163 gene of the female pig animal has a modification in exon 7, a modification in exon 8, a modification in an intron adjacent to exon 7 or exon 8, or any combination thereof. Including, method. 14. The method of claim 13, wherein one or both alleles of the CD163 gene in the female porcine animal comprise a modification in exon 7 of the CD163 gene. 14. The method of claim 13, wherein the modification in exon 7 comprises a deletion. 16. The method of claim 15, wherein the deletion comprises an in-frame deletion. 14. The method of claim 13, wherein the modification in exon 7 comprises an insertion. The method of claim 1 , wherein the altered chromosomal sequence in both alleles of the CD163 gene in the female swine animal results in miscoding. 19. The method of claim 18, wherein the miscoding results in a premature stop codon downstream of the miscoding in an allele of the CD163 gene. The method of claim 1, wherein at least one of the alleles of the CD163 gene in the female swine animal comprises a modification selected from the group consisting of:
An 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 compared to reference sequence SEQ ID NO: 47;
a 377 base pair deletion from nucleotide 2,573 to nucleotide 2,949 compared to the reference sequence SEQ ID NO: 47 on the same allele and a 2 base pair insertion between nucleotides 3,149 and 3,150 compared to the reference sequence SEQ ID NO: 47;
A 124 base pair deletion from nucleotide 3,024 to nucleotide 3,147 compared to the reference sequence SEQ ID NO: 47;
A 123 base pair deletion from nucleotide 3,024 to nucleotide 3,146 compared to the reference sequence SEQ ID NO: 47;
1 base pair insertion between nucleotides 3,147 and 3,148 compared to reference sequence SEQ ID NO: 47;
A 130 base pair deletion from nucleotide 3,030 to nucleotide 3,159 compared to the reference sequence SEQ ID NO: 47;
A 132 base pair deletion from nucleotide 3,030 to nucleotide 3,161 compared to the reference sequence SEQ ID NO: 47;
A 1506 base pair deletion from nucleotide 1,525 to nucleotide 3,030 compared to reference sequence SEQ ID NO: 47;
7 base pair insertion between nucleotides 3,148 and 3,149 compared to reference sequence SEQ ID NO: 47;
A 1280 base pair deletion from nucleotide 2,818 to nucleotide 4,097 compared to the reference sequence SEQ ID NO: 47;
A 1373 base pair deletion from nucleotide 2,724 to nucleotide 4,096 compared to the reference sequence SEQ ID NO: 47;
A 1467 base pair deletion from nucleotide 2,431 to nucleotide 3,897 compared to reference sequence SEQ ID NO: 47;
A 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488, nucleotides compared to the reference sequence SEQ ID NO: 47 the base pair deletion, with an additional 129 base pair deletion in exon 7 from 3,044 to nucleotide 3,172;
A 28 base pair deletion from nucleotide 3,145 to nucleotide 3,172 compared to reference sequence SEQ ID NO: 47;
1387 base pair deletion from nucleotide 3,145 to nucleotide 4,531 compared to reference sequence SEQ ID NO: 47;
A base pair deletion of 1382 base pairs from nucleotide 3,113 to nucleotide 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113;
A 1720 base pair deletion from nucleotide 2,440 to nucleotide 4,160 compared to reference sequence SEQ ID NO: 47;
A 452 base pair deletion from nucleotide 3,015 to nucleotide 3,466 compared to the reference sequence SEQ ID NO: 47;
and any combination thereof. The method of claim 1 , wherein the modified chromosomal sequence reduces viral entry into the female swine animal for type 1 PRRSV virus, type 2 PRRSV, or both type 1 and type 2 PRRSV viruses. The method of claim 21, wherein the modified chromosome sequence is 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.

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