KR20180033548A - Pathogen-resistant animal having a modified CD163 gene - Google Patents

Abstract

A non-human animal and its progeny are provided that comprise at least one modified chromosomal sequence in the gene encoding the CD163 protein. An animal cell containing such a modified chromosomal sequence is also provided. Animals and cells have increased resistance to pathogens including porcine reproductive and respiratory syndrome virus (PRRSV). Animals and offspring have chromosomal aberrations of the CD163 gene. The present invention further relates to breeding methods for producing pathogen-resistant animals and populations of animals made using such methods.

Description

Pathogen-resistant animal having a modified CD163 gene

The present invention relates to non-human animals and their progeny comprising at least one modified chromosomal sequence in a gene encoding the CD163 protein. The invention also relates to animal cells containing such modified chromosomal sequences. Animals and cells have increased resistance to pathogens including porcine reproductive and respiratory syndrome virus (PRRSV). The animal and offspring have a chromosomal modification of the CD163 gene so that PRRSV entry and replication are suppressed and thus the animal is resistant to the virus-induced diseases and syndromes. The present invention also relates to breeding methods for producing pathogenic-resistant animals and populations of animals made using such methods. The present invention also relates to a method for genetic editing of CD163 accompanied by the development of pathogens resistant animals, founder animals and lines such as direct injection of embryos and pathogenic bacteria such as PRRSV.

The porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the group of mammalian arteriviruses, which also include murine lactic dehydrogenase-increasing viruses, monkey hemorrhagic fever viruses and hemorrhagic viruses. The Atari viruses share important characteristics related to viral outbreaks, including the ability to cause tropism for macrophages and severe disease and persistent infection. Clinical disease syndromes caused by infection with porcine reproductive and respiratory syndrome virus (PRRSV) were first reported in the United States in 1987 (Keffaber, 1989) and then reported in Europe in 1990 (Wensvoort et al., 1991). Infection with PRRSV results in respiratory illness including cough and fever, reproductive failure during the late pregnancy, and poor growth rate. The virus is also involved in various bacterial disease syndrome interactions while maintaining a lifetime asymptomatic infection (Rowland et al., 2012).

Since its advent, PRRS has been the most important disease of commercial pigs in North America, Europe and Asia, and is not the only disease in Australia and Antarctica. In North America, PRRSV-related losses are estimated to be $ 664 million per year for producers (Holtkamp et al., 2013). In 2006, a more serious form of disease, known as high-pathogenic PRRS (HP-PRRS), killed pig populations throughout China. Genetic diversity has limited the development of vaccines needed to effectively control and eliminate disease. Gene selection for natural tolerance may be an option, but results have so far been limited (Boddicker et al., 2014).

A previous model describing PRRSV infection of alveolar macrophages has identified SIGLEC1 (CD169) as the primary viral receptor on the surface of macrophages; However, previous studies using SIGLEC1 ^{- / -} pigs showed no difference in viral replication compared to wild-type pigs.

The lack of understanding of many of the features of both PRRSV (at the molecular level in particular) and animal epidemiology (epizootiology) makes control efforts difficult. Current producers often vaccinate pigs for PRRSV with modified-attenuated live host or viral vaccines, but current vaccines often fail to provide satisfactory protection. This is due to both the mutation of the strain and the improper stimulation of the immune system. In addition to concerns about the efficacy of the available PRRSV vaccine, the presently used variant, as evidenced by a virulent field isolate following experimental infection of pigs (Rowland et al., Virology, 259: 262-266 (1999) (Mengeling et al., Am. J. Vet. Res., 60 (3): 334-340 (1999)) can survive in the individual pigs and pig swarms and can increase mutations. In addition, vaccine viruses have emerged from semen in vaccinated birds (Christopher-Hennings et al., Am. J. Vet. Res, 58 (1): 40-45 (1997)). As an alternative to vaccination, some experts are advocating a "test and removal" strategy in the breeding herd (Dee and Molitor, Vet. Rec., 143: 474-476 (1998)). The successful use of this strategy depends on strict control to prevent the reintroduction of the virus following removal of all pigs acutely or continuously infected with PRRSV. The difficulties and high costs associated with this strategy are that little is known about the onset of persistent PRRSV infection and therefore there is no reliable technique 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.

A non-human animal, its offspring, and animal cells comprising at least one modified chromosomal sequence in the gene encoding the CD163 protein are provided.

Reproduction methods are also provided for producing an animal or lineage with reduced susceptibility to infection by pathogenic bacteria. The method comprises genetically transforming the oocyte or sperm cell to introduce a modified chromosomal sequence into the gene encoding the CD163 protein into at least one of the oocyte and sperm cell and selecting a chromosomal sequence modified to the gene encoding the CD163 protein And modifying the oocyte with sperm cells to produce a fertilized egg containing the fertilized egg. Alternatively, the method includes genetically transforming the embryo to introduce a modified chromosomal sequence into the gene encoding the CD163 protein into the fertilized egg. The method comprises the steps of transplanting an embryo into a surrogate female animal (where the pregnancy and termination produce a progeny animal), screening offspring for susceptibility to pathogens, and transforming the chromosome of the gene encoding the CD163 protein Further comprising the step of selecting offspring having reduced susceptibility to pathogens compared to animals not comprising the sequence.

Populations of animals made by the breeding methods described above are also provided.

Methods of increasing the resistance of livestock animals to infection with pathogens are also provided. The method comprises encoding at least one chromosomal sequence from a gene encoding the CD163 protein such that CD163 protein production or activity is reduced relative to CD63 protein production or activity in a livestock animal that does not contain an edited chromosomal sequence in the gene encoding the CD163 protein Editing.

Modifications to the chromosomal sequence in the gene encoding the CD163 protein provided herein may be used to identify an animal, offspring, cell, or population (e.g., a porcine animal, offspring, or rodent) against a pathogen (e.g., a virus such as a porcine reproductive and respiratory syndrome virus Cells or populations).

In any of the porcine animals, offspring, cells, and methods provided herein, the modification of the chromosomal sequence in the gene encoding the CD163 protein results in the deletion of 11 base pairs of nucleotide 3,137 to 3,147 compared to reference sequence SEQ ID NO: 47; Two base pair insertions of nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and a 377 base pair deletion of nucleotides 2,573 to 2,949 compared to reference sequence SEQ ID NO: 47 on the same allele; Deletion of 124 base pairs of nucleotides 3,024 to 3,147 compared to reference sequence SEQ ID NO: 47; 123 base pair deletion of 3,024 nucleotides to 3,147 nucleotides relative to reference sequence SEQ ID NO: 47; One base pair insertion between nucleotides 3,147 and 3,148 compared to reference sequence SEQ ID NO: 47; 130 base pairs deletion of 3,030 nucleotides to 3,159 nucleotides relative to reference sequence SEQ ID NO: 47; 132 base pair deletions of 3,030 nucleotides to 3,161 nucleotides relative to reference sequence SEQ ID NO: 47; 1506 base pair deletions of nucleotides 1,525 to 3,030 compared to reference sequence SEQ ID NO: 47; Insertion of 7 base pairs between 3,148 nucleotides and 3,149 nucleotides relative to reference sequence SEQ ID NO: 47; A 1280 base pair deletion of the nucleotide 2,818 to 4,097 nucleotides relative to the reference sequence SEQ ID NO: 47; 1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47; 1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47; 1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides); 28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47; 1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47; 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113; Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47; Or a combination thereof.

Isolated nucleic acids are also provided. The isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 47; (b) a nucleotide sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47; And (c) the cDNA sequence of (a) or (b).

Additional isolated nucleic acids are also provided. The isolated nucleic acid comprises SEQ ID NOS: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,

Other objects and features will become apparent to a certain extent and to some extent hereinafter.

Figure 1. CRISPR and targeting vector used to transform CD163. Panel A shows the 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 (the porcine domain SRCR5 of CD163) with DNA encoding human SRCR8 of CD163L. This targeting vector is used for drug selection and transfection by G418. PCR primers for long range, left arm and right arm assays are labeled with arrows for 1230, 3752, 8791, 7765 and 7775. Panel C shows the same targeting vector as shown in panel B, but here the Neo cassette was removed. This targeting vector was used to target CD163 in cells already neomycin resistant. Primers used in small deletion assays are illustrated by the arrows and labeled GCD163F and GCD163R. Panel D highlights exons targeted by CRISPR. The positions of CRISPR 10, 131, 256 and 282 are indicated by downward arrows on the exon 7. The number of CRISPRs represents the number of base pairs from intron-exon junctions of intron 6 and exon 7.
Figure 2. CRISPR and targeting vector used to transform CD1D. Panel A shows wild-type exons 3, 4, 5, 6 and 7 of the CD1D gene targeted for transformation by CRISPR. Panel B shows the targeting vector designed to replace exon 3 with the selection marker Neo . This targeting vector was used to modify CD1D with CRISPR. PCR primers for remote, left arm and right arm probes are labeled with arrows for 3991, 4363, 7373 and 12806. Panel C shows the exon targeted by the CRISPR. The positions of the CRISPR 4800, 5350, 5620 and 5626 are indicated by downward arrows on the exon 3. Primers used in small deletion assays are illustrated by arrows and are labeled GCD1DF and GCD1DR. The number of CRISPRs represents the number of base pairs from intron-exon junctions of intron 6 and exon 7.
Figure 3. Reproduction of CD163 and CD1D knockout pigs by CRISPR / Cas9 and SCNT. A) Targeted deletion of CD163 in somatic cells after transfection with CRISPR / Cas9 and donor DNA. The wild type (WT) genotype causes a 6545 base pair (bp) band. Lanes 1-6 represent six different colonies from a single transfection with donor DNA containing CRISPR 10 and Neo with Cas9. Lanes 1, 4, and 5 show large homozygous deletions of 1500-2000 bp. Lane 2 shows less homozygous deletion. Lanes 3 and 6 represent the double allelic variation of both the WT allele and the small deletion or allele. The exact transformation of each colony was determined only by sequencing of the colonies used in the SCNT. Faint WT bands in some of the lanes may indicate cross-contamination of fetal fibroblasts from adjacent WT colonies. NTC = No template control. B) Targeted deletion of CD1D in somatic cells after transfection with CRISPR / Cas9 and donor DNA. The WT genotype causes a 8729 bp band. Lanes 1-4 represent colonies with 500-2000 bp deletion of CD1D. Lane 4 seems to be a WT colony. NTC ¼ No mold adjustment. C) Images of CD163 knockout pigs produced by SCNT during the study. These male piglets contain a homozygous 1506 bp deletion of CD163. D) Images of CD1D pigs produced during the study. These piglets contain a 1653 bp deletion of CD1D. E) Genotypes of two SCNT litters containing a 1506 bp deletion of CD163. Lanes 1 to 3 (litter size 63) and lanes 1-4 (litter size 64) show the genotype for each litter from each litter. Sow represents the recipient female of the SCNT embryo, and WT represents the WT control. NTC = no mold adjustment. F) Genotypes of two SCNT litters containing a 1653 bp deletion of CD1D. Lanes 1 to 7 (litter 158) and lanes 1-4 (litter 159) represent the genotype for each litter.
Figure 4. Effect of CRISPR / Cas9 system in porcine embryos. A) Frequency of blastocyst formation after injection of different concentrations of CRISPR / Cas9 system to zygote. 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. Original magnification X4. C) Type of mutation for eGFP generated using CRISPR / Cas9 system: WT genotype (SEQ ID NO: 16), # 1 (SEQ ID NO: 17), # 2 (SEQ ID NO: .
Figure 5. Effect of CRISPR / Cas9 system in targeting CD163 in porcine embryos. Examples of mutations generated on CD163 by the CRISPR / Cas9 system are: WT genotype (SEQ ID NO: 20), # 1-1 (SEQ ID NO: 21), # 1-4 (SEQ ID NO: 22) (SEQ ID NO: 23). All embryos tested by DNA sequencing showed mutations on CD163 (18/18). CRISPR 131 is highlighted in bold. B) Sequence analysis of homozygous deletions caused by the CRISPR / Cas9 system. Image shows # 1-4 from panel A with 2 bp deletion of CD163.
Figure 6. The effect of the CRISPR / Cas9 system when introduced as two types of CRISPR. A) PCR amplification of CD163 in blastocysts injected with CRISPR / Cas9 as zygote. Lanes 1, 3, 6, and 12 show the designated deletion between two different CRISPRs. B) PCR amplification of CD1D in blastocysts injected with CRISPR / Cas9 as zygote. CD1D had a lower frequency of deletion as measured by gel electrophoresis when compared to CD163 (3/23); Lanes 1, 8, and 15 show apparent deletion of CD1D. C) The CRISPR / Cas9 system successfully targeted two genes when two CRISPRs targeting CD163 and eGFP were provided to the system. CD163 and eGFP are shown: CD163 WT (SEQ ID NO: 24), CD163 # 1 (SEQ ID NO: 25), CD163 # 2 (SEQ ID NO: 26), CD163 # 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: 32).
Figure 7. CD163 knockout pig produced by the CRISPR / Cas9 system injected into the zygote. A) PCR amplification of CD163 from knockout pigs; Clear signs of deletion were detected in cubs 67-2 and 67-4. B) Video of CD163 knockout pig with surrogate mother. All animals are healthy and show no signs of abnormality. C) Genotype of CD163 knockout pig. Gt; (WT) < / RTI > sequence SEQ ID NO: 33. Two animals, one to three times young (from SEQ ID NO: 34) and 67-3 (SEQ ID NO: 37), have homozygous deletions or insertions in CD163. The other two animals (from cubs 67-2 and 67-4) have a dual allelic variant 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 caused by the introduction of two different CRISPR systems with Cas9. No animal from zygote infusion for CD163 showed a mosaic genotype.
Figure 8. CD1D knockout pig produced by the CRISPR / Cas9 system injected into the zygote. A) PCR amplification of CD1D from knockout pigs; 166-1 shows the mosaic genotype for CD1D. 166-2, 166-3, and 166-4 did not show any change in size for the amplicon, but showed modifications from the sequence analysis of the amplicon. WT FF = wild type fetal fibroblast. B) PCR amplification of the remote assay showed a clear deletion of one allele in piglets 166-1 and 166-2. C) Images of CD1D knockout pigs with surrogate mothers. D) Sequence data of CD1D knock-out 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 3.2 (SEQ ID NO: 45), and # 166-4 (SEQ ID NO: 46). The atg start codon in exon 3 is shown in lower case in bold.
9. Clinical signs during acute PRRSV infection. Results for daily evaluation of respiratory signs and presence of heat for CD163 + / + (n = 6) and CD163 - / - (n = 3)
10. Lung histopathology during acute PRRSV infection. Representative photomicrographs of H and E stained tissue from wild-type and knockout pigs. The left panel shows infiltration of mononuclear cells and edema. The right panel from the knockout pig shows the lung structure of the normal lung.
Figure 11. Viral hemoglobin in various genotypes. Note that CD163 - / - piglet data lies along the X axis.
Figure 12. Antibody production in unreacted, wild type and non-characterized alleles.
Figure 13. Cell surface expression of CD163 in individual pigs. Lines that seem to be directed to the right in non-characterized A, uncharacterized B, and CD163 + / + panels represent CD163 antibodies, whereas lines that appear to be directed to the left side of these panels show no antibody control (background). It is noted that CD163 staining in CD163 - / - animals overlaps with background control, and CD163 staining in non-characterized alleles is roughly intermediate between WT levels and background (logarithmic scale, thus less than ~ 10% ).
Figure 14. Levels of CD169 on the alveolar macrophages from three representative pig and non-antibody controls (FITC-labeled anti-CD169).
Figure 15. Viral hemoglobin in various genotypes. Note that the Δ43 amino acid piglet data lies along the X-axis.
16. Genomic sequence of wild type CD163 exons 7-10 used as reference sequence (SEQ ID NO: 47). The sequence includes the last base of exon 7 at 3000 bp upstream or exon 10. The underlined regions show the positions of exons 7, 8, 9, and 10, respectively.

Methods are provided herein for producing animal and genetically modified animals that have a modification of the CD163 gene and are resistant to PRRSV and other related respiratory virus infections. The animal has a chromosome modification (insertion or deletion) that inactivates or otherwise modulates CD163 gene activity. CD163 is required for PRRSV entry into cells and for viral replication. Therefore, unreacted CD163 animals are resistant to PRRSV infection when challenged. Such animals can be generated using any of a number of protocols using genetic editing.

Also provided herein are methods of using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas system, a transcription activator-like effector nuclease (TALEN), a ZFN (zinc finger nuclease), a recombinant enzyme fusion protein, Or a meganuclease may be used to specifically bind to a chromosome surface region of a cell in a porcine animal cell or a porcine embryo to induce double-stranded DNA degradation or otherwise alter the activity of the CD163 gene or protein therein A method for producing a porcine animal is provided, including introducing an agent that activates or reduces.

Also described herein are the uses of one or more specific CD163 loci in conjunction with polypeptides capable of cleaving and / or integrating specific nucleic acid sequences within the CD163 locus. Examples of uses of the CD163 gene in combination with a polypeptide or RNA capable of performing cleavage and / or integration of the CD163 locus include a zinc finger protein, a meganuclease, a TAL domain, TALENs, an RNA-guided CRISPR / Cas recombinase, Zippers, and others known in the art. Specific examples include chimeric ("fusion") proteins including site-specific DNA binding domain polypeptides and truncation domain polypeptides (eg, nuclease), such as ZFN comprising a zinc-finger polypeptide and a FokI nuclease polypeptide Proteins. Disclosed herein are polypeptides comprising a DNA-binding domain that specifically binds to the CD163 gene. Such polypeptides may also be capable of inducing double-strand breaks in which the polypeptides are targeted and / or contain nucleases (truncation) domains or half-domains (e. G., Modified DNA-binding A homing endonuclease that includes a homing endonuclease domain), and / or a ligation domain. The DNA-binding domain targeting the CD163 locus may be a DNA-cleaving functional domain. The polypeptide can be used to introduce an exogenous nucleic acid into the genome of a host organism (e. G., Animal species) at one or more CD163 loci. The DNA-binding domain may comprise a zinc finger protein with one or more zinc finger (e.g., 2,3, 4,5, 6,7, 8, 9 or more zinc fingers) (Non-naturally occurring) to bind to the sequence of SEQ ID NO. Any of the zinc finger proteins described herein can bind to a target site (e.g., an enhancer or other expression element) within the coding sequence of the target gene or within an adjacent sequence. The zinc finger protein can bind to the target site in the CD163 gene.

Justice

Units, prefixes, and symbols may be displayed in SI-approved form. Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction; Amino acid sequences are written from left to right in the amino to carboxy direction. The numerical range recited in the specification includes a number defining the range and includes each integer within the defined range. Amino acids may be represented herein by commonly known three letter symbols or by the one letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Nucleotides, likewise, can be represented by commonly accepted single-letter codes. Unless otherwise provided, the terms software, electrical, and electronics used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined with reference to the specification as a whole.

As will be understood by those skilled in the art, for any and all purposes, especially in terms of providing a written description, all ranges cited herein are also to be construed to include any and all possible sub-ranges and sub-ranges thereof , As well as individual values constituting the range, in particular integer values. Quoted ranges include each particular value, integer, decimal, or identity in the range. Any recited range may be readily discerned as fully describing the same range and subdividing it into at least 1/2, 1/3, 1/4, 1/5, or 1/10 of the equivalent. As a non-limiting example, each of the ranges discussed herein may be readily subdivided into one third below, one third middle and one third above, and so on.

When introducing elements of the invention or the preferred embodiment (s) thereof, it is intended that the articles "a "," an ", "the" The terms " comprising ", " including ", and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

The term "and / or" means any of the items, any combination of the items, or both items associated with the term. The phrase "one or more" is readily understood by those skilled in the art, especially when read in the context of its use.

"Binding protein" is a protein that can bind to other molecules. Binding proteins can bind to, for example, DNA molecules (DNA-binding proteins), RNA molecules (RNA-binding proteins) and / or protein molecules (protein-binding proteins). In the case of protein-binding proteins, this can bind to itself (bind to form homodimers, homotrimers, etc.) and / or bind to one or more molecules of different proteins or proteins. Binding proteins can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activities.

The term " conservatively modified variants "applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variant" refers to a nucleic acid encoding an identical or conservatively modified variant of an amino acid sequence. Because of the degradation of the genetic code, a number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode amino acid alanine. Thus, at all positions where alanine is specified by the codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. This nucleic acid modification is a "silent variation" and represents a conservatively modified variant. All nucleic acid sequences herein encoding polypeptides also refer to all possible silent changes of the nucleic acid, with reference to the genetic code.

One of ordinary skill in the art will appreciate that each codon of the nucleic acid (except AUG, which is often the only codon for methionine; and often the UGG, which is the only codon for tryptophan), can be modified to yield functionally identical molecules. Thus, each asymptomatic variation of the nucleic acid encoding the polypeptide of the present invention is included in each described polypeptide sequence and is within the scope of the present invention.

With respect to the amino acid sequence, the skilled artisan will appreciate that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add or delete a single amino acid or a small percentage of amino acids in the coded sequence are & "Wherein the alteration will replace amino acids with chemically similar amino acids. Thus, the number of amino acid residues selected from the integer group of 1 to 15 can be varied in this way. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 changes may be made.

Conservatively modified variants typically provide biological activities similar to unmodified polypeptide sequences that derive them. For example, substrate specificity, enzyme activity, or ligand / receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein relative to its native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Each of the following six groups contains amino acids that are conservative substitutions for each other: [1] alanine (A), serine (S), threonine (T); [2] Aspartic acid (D), glutamic acid (E); [3] Asparagine (N), Glutamine (Q); [4] arginine (R), lysine (K); [5] isoleucine (I), leucine (L), methionine (M), valine (V); And [6] phenylalanine (F), trypsin (Y), tryptophan (W). See also Creighton (1984) Proteins W. H. Freeman and Company.

The term "CRISPR" refers to "clustered regularly interspaced short palindromic repeats" The term "Cas9" refers to CRISPR associated protein 9. The term "CRISPR / Cas9" or "CRISPR / Cas9 system" refers to a Cas9 protein, or derivative thereof, and CRISPR RNA (crRNA) And one or more non-coding RNAs capable of providing the function of a transcription-promoting RNA (tracrRNA). The crRNA and the tracrRNA produce a "guide RNA" (gRNA) The CRISPR / Cas9 system is further described below. The CRISPR / Cas9 system can be used in combination with the CRISPR / Cas9 system.

References herein to deletion of the nucleotide sequence of nucleotides x to n means that all of the nucleotides in the range, including x and y, have been deleted. Thus, for example, the phrase "11 base pair deletion of 3,137 to 3,147 nucleotides relative to SEQ ID NO: 47" means that each of nucleotides 3,317 to 3,147 has been deleted, including nucleotides 3,317 and 3,147.

"Disease tolerance" is a characteristic of an animal in which the animal avoids symptoms of the disease that are the result of animal-pathogen interactions, such as the interaction between porcine animals and PRRSV. That is, prevent pathogens from causing animal disease and related disease symptoms, or alternatively, decrease the incidence and / or severity of clinical signs or decrease clinical symptoms. Those skilled in the art will appreciate that the compositions and methods disclosed herein can be used with other compositions and methods available in the art to protect animals from pathogenic attack.

With respect to a particular nucleic acid, "encoding" or "encoded" means including information about translation into a particular protein. The nucleic acid encoding the protein may comprise intervening sequences (e.g., introns) in the translated region of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information with which the protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by a nucleic acid using a "universal" genetic code. If the nucleic acid is produced or modified by synthesis, the known codon preference of the intended host in which the nucleic acid is to be expressed can be utilized.

As used herein, the terms "gene editing," "gene edited," "genetically edited," and "gene editiing effectors" Refers to the use of a homing technique using a naturally occurring or artificially engineered nuclease, also referred to as "homing endonuclease, " or" targeting endonuclease. &Quot; Nucleases generate specific double-stranded chromosome truncations (DSBs) at the desired location in the genome, which in some cases are homologous recombination (HR) and / or heterologous terminal-junction (NHEJ) Restoring the cutting induced by the natural process of. Genetic editing effectors include zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CAS9) system, and meganuclease And a re-engineered meganuclease as endonuclease). The term also encompasses the use of transgenic processes and techniques, including, for example, where the change is deletion or relatively small insertion (typically less than 20 nt) and / or where no DNA is introduced from foreign species . The term also includes offspring animals such as those produced by sexual crossing or asexual breeding from early genetically modified animals.

As used herein, "heterologous ", in relation to nucleic acids, refers to a nucleic acid that originates from, or is from the same species as, Is a modified nucleic acid. For example, one or both of the promoters operably linked to the heterologous structural gene are substantially modified from their original form if they are from a species that differs from that which derives the structural gene, or from the same species. A heterologous protein can originate from an exogenous species or, if it is from the same species, is substantially modified from its original form by intending human intervention.

As used herein, the terms " homing DNA technology ", "homing technology ", and" homing endonucleases "refer to a zinc finger (ZF) protein, a transcriptional activator-like effector (TALE) meganuclease, and a CRISPR / Cas9 system Including, but not limited to, the targeting of a particular molecule to a particular DNA sequence.

As used herein, the terms "increased tolerance" and "reduced susceptibility " refer to a statistically significant decrease in the incidence and / or severity of clinical signs or symptoms associated with infection by pathogens, . For example, "increased tolerance" or "reduced sensitivity" refers to an infection by PRRSV in an animal comprising at least one modified chromosomal sequence in a gene encoding the CD163 protein as compared to a control animal with a non- May indicate a statistically significant decrease in the incidence and / or severity of the relevant clinical symptoms or clinical symptoms. The term "statistically significant reduction in clinical symptoms" means that the frequency of occurrence of at least one clinical symptom in an edited group of subjects is at least 10%, preferably at least 20% %, More preferably at least 30%, even more preferably at least 50%, even more preferably at least 70% lower.

As used herein, the term "knock-in" means replacing the endogenous gene with the same endogenous gene as the transplantation gene or with some structural modification while maintaining the transcriptional regulation of the endogenous gene.

"Rust-out" refers to the disruption of the structure or regulation mechanism of a gene. The knock-out is generated by homologous recombination of the target vector, alternate vector or hit-and-run vector, or random insertion of the gene trap vector causing complete, partial or conditional loss of gene function .

The term "animal" includes any non-human animal, for example, a domesticated animal (e.g., a domestic animal). The term "livestock animal" refers to any animal that traditionally grows in a herd, such as a pig animal, a small animal (eg beef or cow), a sheep, a goat, a horse and an animal (eg horse or donkey) Camels, or birds (eg, chickens, turkeys, ducks, geese, guinea fowl, or freshly hatched birds). The term "animal animal" does not include rats, mice, or other rodents.

As used herein, the term "mutation " includes variations in the nucleotide sequence of a polynucleotide, such as, for example, a gene or a coding DNA sequence (CDS), relative to a wild-type sequence. The term includes, without limitation, substitutions, insertions, frame shifts, deletions, inversions, translocations, redundancies, splice-donor site mutations, point-mutations, and the like.

As used herein, "operably linked" includes reference to a functional linkage between two nucleic acid sequences, e. G., Promoter sequences and secondary sequences, wherein the promoter sequence is capable of initiating and mediating the transcription of a DNA sequence corresponding to a secondary sequence do. Generally, operatively linked means that the linked nucleic acid sequences are contiguous and, if necessary, link two protein coding regions adjacent and in the same decoding frame.

As used herein, "polynucleotide" includes references to deoxyribonucleotides, ribo polynucleotides, or conservatively modified variants; The term also encompasses nucleic acid molecules that have the essential property of a natural ribonucleotide in that they are hybridized under stringent hybridization conditions to substantially the same nucleotide sequence as the naturally occurring nucleotide and / or translated into the same amino acid (s) as the naturally occurring nucleotide (s) Can refer to analogs. The polynucleotide may be a native or heterologous structural gene or a full-length or subsequence of a regulatory gene. Unless otherwise indicated, the term includes reference to a particular sequence as well as its complementary sequence. Thus, DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide " as that term is intended in the present application. Moreover, DNA or RNA comprising a modified base such as an unusual base, such as inosine, or a tritylated base, as just two, are polynucleotides as the term is used herein. It will be appreciated that a wide variety of modifications have been made to DNA and RNA to provide various useful purposes known to those skilled in the art.

The term polynucleotide as used herein refers to such chemical, enzymatically or metabolically modified forms of polynucleotides as well as the chemical forms of DNA and RNA characteristic of cells and viruses, including simple and complex cells, among others .

The terms "polypeptide "," peptide "and" protein "are used interchangeably herein to denote polymers of amino acid residues. The terms can also be applied to conservatively modified variants and amino acid polymers wherein one or more amino acid residues are the artificial chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The essential property of these analogs of naturally occurring amino acids is that when incorporated into proteins, the protein is specifically reactive to antibodies directed to the same protein consisting entirely of naturally occurring amino acids.

The terms "polypeptide," "peptide," and "protein" also include variations that include, but are not limited to, glycation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP- . As is well known and well-known, it will be appreciated that the polypeptide is not always entirely linear. For example, the polypeptides may be branched as a result of ubiquitination, and they may have branching as a result of post-translational events, including events generally caused by natural processing events and naturally occurring human manipulations It may be circular. Circular, branched, and branched circular polypeptides can be synthesized by non-translation natural processes and entirely in a synthetic manner. The present invention also contemplates the use of both the methionine-containing and methionine-free amino terminal variants of the protein of the present invention.

As used herein, the term " reduction in the incidence and / or severity of clinical symptoms "or" reduction in clinical symptoms "refers to a decrease in the number of infected subjects in a group, , Or a reduction in the severity of any clinical symptoms present in one or more subjects. For example, these terms may include any clinical indication of infection, pulmonary pathology, viremia antibody production, reduction of pathogen load, pathogen shedding, reduction of pathogen transmission, or any clinical indication of symptoms of PRRSV . Preferably these clinical indications are reduced to at least 10% in one or more animals of the invention compared to an infected subject without a modification to the CD163 gene. More preferably, clinical indications are reduced by at least 20%, preferably by at least 30%, more preferably by at least 40%, even more preferably by at least 50% in the subject of the invention.

The term "residue" or "amino acid residue" or "amino acid" is used interchangeably herein to refer to an amino acid that is incorporated into a protein, Amino acids can be naturally occurring amino acids and, unless otherwise limited, can include non-natural analogs of natural amino acids that can function in a manner similar to naturally occurring amino acids.

The term " selectively hybridizes "includes reference to hybridization of nucleic acid sequences to other nucleic acid sequences or other biological agents under stringent hybridization conditions. If a hybridization-based detection system is used, a nucleic acid probe complementary to the reference nucleic acid sequence is selected, and then the probe and the reference sequence hybridize or bind to each other to form a duplex molecule by selection of appropriate conditions .

The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe is hybridized to its target sequence to a detectably greater degree than other sequences (e.g., at least two times more than the background). Strict conditions are sequence-dependent and will vary in different situations. By adjusting the stringency of hybridization and / or washing conditions, 100% complementary target sequences can be identified in the probe (homology probing).

Alternatively, stringent conditions can be adjusted to allow some mismatch in the sequence to detect a lower degree of similarity (heterogeneous probing). Generally, probes are less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.

Typically, stringent conditions are those in which the salt concentration at pH 7.0 to 8.3 is less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salt) and the temperature is a short probe (e.g., 10 to 50 nucleotides) Lt; RTI ID = 0.0 > 30 C < / RTI > for long probes (e.g., more than 50 nucleotides). Strict conditions can also be achieved by addition of a destabilizing agent such as formamide. Specificity is typically the action of washing after hybridization, the critical factor being the ionic strength and the temperature of the final wash solution. In the case of DNA / DNA hybrids, the thermal melting point (Tm) is determined by the equation of Meinkoth and Wahl [Anal. Biochem., 138: 267-284 (1984)]: Tm [° C] = 81.5 + 16.6 (log M) + 0.41 (% GC) -0.61 (% form) -500 / L; Where M is the molar concentration of monovalent cations,% GC is the percent of guanosine and cytosine nucleotides in the DNA,% form is the percent of formamide in the hybridization solution, and L is the length of the hybrid in the base pair. T _m is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence is hybridized to a perfectly matched probe. Tm decreases to about 1 占 폚 for each 1% mismatch; Thus, the Tm, hybridization and / or washing conditions can be adjusted to hybridize to the sequence of the desired identity. For example, if a sequence with> 90% identity is sought, the Tm can be reduced by 10 ° C. Generally, stringent conditions are selected to be about 5 ° C lower than the T m for a particular sequence and its complement at defined ionic strength and pH. However, severely stringent conditions can utilize hybridization and / or washing at temperatures 1 to 4 캜 lower than Tm; Moderately stringent conditions may utilize hybridization and / or washing at a temperature that is 6 to 10 < 0 > C lower than Tm; Low stringency conditions can utilize hybridization and / or washing at a temperature 11 to 20 ° C lower than Tm. Using equations, hybridization and cleaning compositions, and the desired Tm, one of ordinary skill will understand that variations in the stringency of hybridization and / or wash solutions are essentially described. An extensive guide to the hybridization of nucleic acids can be found in the literature (cf. Tijssen, < / RTI > Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2, " Overview of principles of hybridization and the strategy of nucleic acid probe assays ", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).

A "TALE DNA binding domain" or "TALE" is a polypeptide comprising at least one TALE repeat domain / unit. The repeat domain is involved in the binding of TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as an "iterator") is typically 33-35 amino acids long and exhibits at least some sequence homology with other TALE repeat sequences within the naturally occurring TALE protein. The zinc finger and TALE binding domains can be "engineered" to bind to a given nucleotide sequence through the manipulation of a naturally occurring zinc finger or a recognition helical region of a TALE protein (one or more amino acid changes). Thus, the engineered DNA binding protein (zinc finger or TALE) is a non-naturally occurring protein. Non-limiting examples of methods for manipulating DNA-binding proteins are design and selection. Designed DNA binding proteins are proteins that do not occur in nature, where the design / composition is primarily caused by rational criteria. Rational criteria for design include application of computerized algorithms and assignment rules to process information in database storage information of existing ZFP and / or TALE designs and associated data. For example, U.S. Pat. Pat. Nos. 6,140,081; 6,453,242; And 6,534,261; Also in WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Pat. Release No. 20110301073.

As used herein, "vector" includes references to nucleic acids that are used for transfection of host cells and that can be inserted into polynucleotides. Vectors are often replicas. Expression vectors enable transcription of the inserted nucleic acid therein.

"Wild type" means an animal and its associated blastocyst, embryo or cell, which is genetically modified or otherwise untransformed, and is usually a crossbreed and crossbred strain developed from naturally occurring strains.

A "zinc finger DNA binding protein" (or binding domain) is a protein that binds to DNA in a sequence-specific manner through one or more zinc fingers that are regions of the amino acid sequence within the structure-stabilized binding domain through the coordination of zinc ions, It is a domain within a large protein. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

A "selected" zinc finger protein or TALE is a protein not found in nature that is produced primarily from experimental procedures such as phage display, interaction traps, or hybrid selection. For example, U.S. Pat. Pat. No. 5,789,538; U.S.A. Pat. No. 5,925,523; U.S.A. Pat. No. 6,007,988; U.S.A. Pat. No. 6,013,453; U.S.A. Pat. No. 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 and U.S. Pat. Release No. 20110301073.

The following terms are used to describe the sequence relationship between a polynucleotide / polypeptide of the invention and a reference polynucleotide / polypeptide: (a) a "reference sequence", (b) a "comparison window", (c) Quot; sequence identity ", and (d) "sequence identity percent."

(a) A "reference sequence" as used herein is a defined sequence that is used as a basis for sequence comparison with a polynucleotide / polypeptide of the present invention. A reference sequence may be a subset of the specified sequence or a whole of the specified sequence; For example, a full-length cDNA or a segment of a gene sequence, or a complete cDNA or gene sequence.

(b) "comparison window" as used herein includes reference to a contiguous specific segment of a polynucleotide / polypeptide sequence, wherein the polynucleotide / polypeptide sequence can be compared to a reference sequence and the polynucleotide / polypeptide sequence May include additions or deletions (i.e., gaps) relative to a reference sequence (without addition or deletion) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotide / amino acid residue lengths, and optionally can be 30, 40, 50, 100, or even longer. Those skilled in the art understand that a gap penalty is typically introduced and subtracted from the number of matches to avoid a high similarity with reference sequences due to inclusion of a gap into the polynucleotide / polypeptide sequence.

Methods for aligning sequences for comparison are well known in the art. Optimal alignment of the sequences for comparison was performed using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); The homology alignment algorithm of Needleman and Bruce [Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); The search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988); And can be performed by a computerized implementation of these algorithms, including but not limited to: CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, California; GAP, BESTFIT, BLAST, FASTA, and TFASTA, and related programs in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). The CLUSTAL program is described in Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994).

Programs of the BLAST family that can be used for database similarity search include: nucleotide query sequence for nucleotide database sequence BLASTN; For nucleotide query sequences against protein database sequences BLASTX; For protein query sequences for protein database sequences, BLASTP; For protein query sequences for nucleotide database sequences, TBLASTN; And nucleotide query sequences for nucleotide database sequences. See, for example, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Altschul et al., J. Mol. Biol., 215: 403-410 (1990); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Software for performing BLAST analysis is publicly available, for example, through the National Center for Biotechnology Information (ncbi.nlm.nih.gov/). These algorithms are fully described in a number of publications. For example, Altschul SF et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, 25 NUCLEIC ACIDs Res. 3389 (1997); National Center for Biotechnology Information, The NCBI Handbook [Internet], Chapter 16: The BLAST Sequence Analysis Tool (McEntire J, Ostell J, eds., 2002), available at http://www.ncbi.nlm.nih.gov/ books / NBK21097 / pdf / ch16.pdf]. The BLASTP program for amino acid sequences is also fully described (Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between the two sequences (see, for example, Karlin & Altschul, Proc. Nat'l Acad Sci USA 90: 5873- 5877 (1993)). Several low-complexity filter programs can be used to reduce this low-complexity alignment. For example, SEG (Wooten and Federhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17: 191-201 Alone or in combination.

Unless otherwise specified, the nucleotide and protein identity / similarity values provided herein are calculated using GAP (GCG Version 10) under default values. The Global Alignment Program (GAP) can also be used to compare polynucleotides or polypeptides of the invention with reference sequences. GAP uses the Needleman and Benshu's algorithm (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP represents one member of the family with the best alignment. There may be several members in this family, but no other member has a better quality. GAP represents four figures of merit for alignment: Quality, Ratio, Identity, and Similarity. Quality is a metric that is maximized to align sequences. The ratio is the quality divided by the number of bases in the shorter segment. Percent identity is the percentage of symbols that actually match. Similarity percentages are percentages of similar symbols. Ignore the sign just across the gap. The similarity is scored when the scoring matrix value for a pair of symbols is equal to or greater than 0.50, which is the similarity threshold. The scoring matrix used in version 10 of the Wisconsin Genetics software package is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

Multiple alignments of sequences can be performed using the default parameters (GAPPENALTY = 10, GAP LENGTH PENALTY = 10) and the CLUSTAL alignment (Higgins and Sharp (1989) CABIOS. 5: 151-153). The default parameters for pairwise alignment using the CLUSTAL method include KTUPLE 1, GAP PENALTY = 3, WINDOW = 5, and DIAGONALS SAVED = 5.

(c) As used herein, the term "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences, when aligned for maximum correspondence over a specified comparison window, Including references. Where a percentage of sequence identity is used for a protein, the non-identical residue positions often differ depending on the conservative amino acid substitution, wherein the amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) And does not change the functional properties of the polymer. If the sequence is different in conservative substitutions, the percent sequence identity can be adjusted to correct for the conservative properties of the substitution. By such conservative substitutions, different sequences are referred to as having "sequence similarity" or "similarity". Methods for making such adjustments are well known to those skilled in the art. Typically this involves scoring conservative substitutions as partial mismatches rather than full mismatch, thereby increasing the percent sequence identity. Thus, for example, conservative substitutions are scored between 0 and 1 when the same amino acid receives a score of 1 and a non-conservative substitution results in a score of zero. Scoring of conservative substitutions can be accomplished using the algorithm of Myers and Miller [Meyers and Miller, Computer Applic., ≪ RTI ID = 0.0 > Biol. Sci., 4: 11-17 (1988).

(d) As used herein, "percent sequence identity" means a value determined by comparing two optimally aligned sequences across a comparison window, wherein a portion of the polynucleotide sequence in the comparison window has two sequences (I.e., a gap) in comparison to a reference sequence (without addition or deletion) for optimal alignment of the target sequence. Percent is calculated by calculating the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences, calculating the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, Lt; / RTI > to calculate the percentage of sequence identity.

An animal and a cell having a chromosome sequence modified to a gene encoding the CD163 protein

CD163 has 17 exons and the protein consists of an extracellular domain with 9 scavenger receptor cysteine-rich (SRCR) domains, a transmembrane segment, and a short cytoplasmic tail. Several different variants result from differential splicing of a single gene (Ritter et al 1999a; Ritter et al 1999b). Most of these deformations take up the length of the cytoplasmic tail.

CD163 has a number of important functions including acting as a haptoglobin-hemoglobin scavenger receptor. Since the heme group can be highly toxic, elimination of free hemoglobin in blood is an important function of CD163 (Kristiansen et al. 2001). CD163 has a cytoplasmic tail that promotes endocytosis. Mutations in this tail result in decreased aggregation of the togoglobin-hemoglobin complex (Nielsen et al. 2006). Another function of C163 is the potential for use as anti-cell adhesion (SRCR2), TWEAK receptors (SRCR1-4 & 6-9), bacterial receptors (SRCR5), African pig virus receptors (Sanchez-Tones et al. 2003) (Discussed in Van Gorp et al. 2010a). Taking into account these important functions, it has been previously thought that the complete knock-out of CD163 will produce animals that are unable to survive or are severely damaged (see, for example, PCT Publication No. 2012/158828).

CD163 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily and consists of intracellular domains and nine extracellular SRCR domains. Intracellular entry of CD163 mediated hemoglobin-hem intake through SRCR3 in humans protects cells from oxidative stress (Schaer et al., 2006a; Schaer et al., 2006b; Schaer et al., 2006c). CD163 also acts as a receptor for tumor necrosis factor-like weak inducers of apoptosis (TWEAK: SRCR1-4 & 6-9), the African Swine Fever Virus (SRCR2), and red blood cell binding (SRCR2) do.

CD163 plays a role in infection by a number of different pathogens and thus the present invention is not limited to animals with reduced susceptibility to PRRSV infection but also for infection to cells or for late replication and / Lt; RTI ID = 0.0 > CD163. ≪ / RTI > The infection pathway of PRRSV begins with the initial binding to heparan sulfate on the surface of alveolar macrophages. Prior to 2013, it was believed that certain binding occurred later on sialoadhesin (SIGLEC1, also known as CD169 or SN). The virus is then internalized via clathrin-mediated intracellular entry. Another molecule, CD163, then promotes the uncoating of viruses in endosomes (Van Breedam et al. 2010a). The viral genome is released and the cells are infected.

("INDEL") to a gene encoding the CD163 protein that confers improved or complete resistance to infection by an animal pathogen (eg, PRRSV) in a subject, comprising administering at least one modified chromosomal sequence, eg, Described are animals and their progeny and cells. The Applicant has demonstrated that CD163 is an important gene for PRRSV infection and that it creates a resistant organism and lineage.

The present invention provides a transgenic animal, its offspring, or animal cells comprising at least one modified chromosomal sequence in a gene encoding the CD163 protein. The present invention does not include the inactivation or editing of the SIGLEC1 (CD169) gene, which was previously assumed to be important for PRRSV resistance.

The edited chromosomal sequences may include (1) inactivated, (2) modified, or (3) integrated sequences resulting in unreacted mutations. Inactivated chromosomal sequences are altered to impair, reduce or eliminate CD163 protein function as it is associated with PRRSV infection. Thus, a genetically edited animal comprising an inactivated chromosomal sequence may be referred to as "knock-out" or "conditional knock out. &Quot; Similarly, a gene-modified animal containing an integrated sequence may be referred to as " melted "or" conditional knocked in. " In addition, a genetically modified animal comprising a modified chromosomal sequence may include other modifications that allow targeted point mutation (s) or modified protein products to be produced. Briefly, the process may include introducing at least one RNA molecule encoding the target zinc finger nuclease into the embryo or cell, and, optionally, at least one accessory polynucleotide. The method further comprises culturing the embryo or cell to enable expression of zinc finger nuclease wherein the double-stranded break introduced by the zinc finger nuclease into the targeted chromosomal sequence is an error -Induced non-homologous end-junction DNA repair process or homology-directed DNA repair process. Methods of editing chromosomal sequences that encode proteins associated with reproductive system development using targeted zinc finger nuclease technology are rapid, accurate, and highly efficient.

Alternatively, the process may involve using a CRISPR / Cas9 system to alter the genomic sequence. Using the CRISPR / Cas9 system to alter the genomic sequence, the protein can be directly delivered to the cell, or the mRNA encoding Cas9 can be delivered to the cell, or the gene providing the expression of mRNA encoding Cas9 can be delivered to the cell Lt; / RTI > In addition, the target specific crRNA and tracrRNA may be delivered directly to the cell or the target specific gRNA (s) may be delivered to the cell (these RNAs may alternatively be produced by a gene constructed to express such RNAs) . The design of the crRNA / gRNA and the selection of the target site are well known in the art. Construction and cloning of gRNAs can be found at http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509.pdf.

At least one CD163 locus can be used as a target site for site-specific editing. Site-specific editing may involve insertion of an exogenous nucleic acid (e. G., A nucleic acid comprising a nucleotide sequence encoding a polypeptide of interest) or deletion of a nucleic acid from a locus. For example, integration of exogenous nucleic acids and / or deletion of a portion of a genomic nucleic acid can alter the locus to produce a CD163 gene that has been disrupted (i. E., Decreased activity of the CD163 protein).

Provided herein are non-human animals, progeny of said animals, and animal cells comprising at least one modified chromosomal sequence in a gene encoding a CD163 protein.

Modification of the chromosomal sequence in the gene encoding the CD163 protein may alter the sensitivity of an animal, offspring, or cell to infection by a pathogen (e.g., a virus such as PRRSV) to a gene encoding the CD163 protein for infection by a pathogen Relative to the sensitivity of an animal, offspring, or cell that does not contain the resulting chromosomal sequence.

The animal or offspring may be an embryo, juvenile, or adult. Similarly, the cell may comprise an embryonic cell, a cell derived from a juvenile animal, or a cell derived from an adult animal.

An animal or offspring may include a rabbit. Likewise, the cells may comprise cells derived from the rabbit. The domesticated animal may be a livestock animal such as a pig animal, a small animal (such as a beef cattle or a cow), a sheep animal, a goat animal, a horse and animal (eg horse or donkey), a water buffalo, , Turkey, duck, goose, guinea fowl, or freshly hatched bird). The livestock animal is preferably a cow or a pig animal, and most preferably a pig animal.

The animal or offspring may comprise a genetically modified animal. Cells can contain genetically modified cells.

The animal or cell can be genetically edited using homing endonuclease. While homing endonucleases may be naturally occurring endonucleases, they are rationally designed non-naturally occurring homing endonucleases with DNA recognition sequences designed to target the chromosomal sequence of the gene encoding the CD163 protein desirable. Thus, the homing endonuclease may be a designed homing endonuclease. The homing endonucleases may be, for example, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system, transcriptional activator-like effector nuclease (TALEN), ZFN (zinc finger nuclease), recombinant enzyme fusion protein , Meganuclease, or a combination thereof). The animal or cell is preferably an animal or cell genetically edited using the CRISPR / Cas9 system.

The genetically-edited animal, its offspring, or genetically edited cells preferably exhibit increased resistance to pathogens (such as viruses such as PRRSV) as compared to non-edited animals.

An animal, offspring, or cell may be heterozygous for a modified chromosomal sequence. Alternatively, the animal, offspring, or cell may be homozygous for a modified chromosomal sequence.

In any animal, offspring, or cell, the modified chromosomal sequence may include insertion of a gene encoding the CD163 protein, deletion of a gene encoding the CD163 protein, or a combination thereof. For example, a modified chromosomal sequence may include deletion (e. G., In-frame deletion) of a gene encoding the CD163 protein. Alternatively, the modified chromosomal sequence may comprise the insertion of a gene encoding the CD163 protein.

Insertion or deletion can reduce CD163 protein production or activity relative to CD163 protein production or activity in an insert, deletion-free animal, offspring, or cell.

Insertion or deletion may result in the production of uncomfortable functional CD163 protein by the animal, offspring, or cells. By "non-functional CD163 protein" is meant an amount of CD163 protein in an animal, offspring, or cell that is less than the level observed in an animal, offspring, or cell that does not contain insertions or deletions, At least about 90% lower.

If the animal, offspring, or cell contains a porcine animal, offspring, or cell, the modified chromosomal sequence may be exon 7 of the gene encoding the CD163 protein, exon 8 of the gene encoding the CD163 protein, gene encoding the CD163 protein Or an intron adjacent to exon 8 or exon 8, or a combination thereof. The modified chromosomal sequence suitably includes modifications to exon 7 of the gene encoding the CD163 protein.

Deformation in exon 7 of the gene encoding the CD163 protein may include deletion (e. G., In-frame deletion in exon 7). Alternatively, modifications in exon 7 of the gene encoding the CD163 protein may comprise insertion.

In either pig animal, offspring, or cell, the modification is 11 base pair deletion of 3,137 nucleotides 3,147 nucleotides relative to reference sequence SEQ ID NO: 47; Two base pair insertions of nucleotides 3,149 and 3,150 compared to reference sequence SEQ ID NO: 47 and 377 base pair deletions of nucleotides 2,573 to 2,949 compared to reference sequence SEQ ID NO: 47 on the same allele; Deletion of 124 base pairs of nucleotides 3,024 to 3,147 compared to reference sequence SEQ ID NO: 47; 123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47; Insertion of one base pair between nucleotides 3,147 and 3,148 compared to reference sequence SEQ ID NO: 47; 130 base pairs deletion of 3,030 nucleotides to 3,159 nucleotides relative to reference sequence SEQ ID NO: 47; 132 base pair deletions of 3,030 nucleotides to 3,161 nucleotides relative to reference sequence SEQ ID NO: 47; 1506 base pair deletions of nucleotides 1,525 to 3,030 compared to reference sequence SEQ ID NO: 47; Insertion of 7 base pairs between nucleotide 3,148 and nucleotide 3,149 compared to reference sequence SEQ ID NO: 47; A 1280 base pair deletion of the nucleotide 2,818 to 4,097 nucleotides relative to the reference sequence SEQ ID NO: 47; 1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47; 1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47; 1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides); 28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47; 1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47; 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113; Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47; Or a combination thereof.

SEQ ID NO: 47 provides a nucleotide sequence for the region starting from the 3,000 base pair (bp) upstream of exon 7 of the wild-type pig CD163 gene to the last base of exon 10 of this gene. SEQ ID NO: 47 is used herein as a reference sequence and is shown in FIG.

Where a porcine animal or cell comprises two base pair insertions between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47, the two base pair insertions may include insertion of the dinucleotide AG.

Where a pig animal or cell comprises one base pair insert between nucleotides 3,147 and 3,148 relative to reference sequence SEQ ID NO: 47, one base pair insert may comprise the insertion of a single adenine residue.

If the pig animal or cell contains seven base pair insertions between nucleotide 3,148 and nucleotide 3,149 compared to reference sequence SEQ ID NO: 47, the seven base pair insert may include insertion of the sequence TACTACT (SEQ ID NO: 115).

Wherein the pig animal or cell comprises a 1930 base pair deletion of nucleotide 488 to nucleotide 2,417 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insert beginning at nucleotide 488, If there is an additional 129 base pair deletion of nucleotides 3,044 to 3,172 in exon 7 compared to SEQ ID NO: 47, the 12 base pair insertions may include insertion of the sequence TGTGGAGAATTC (SEQ ID NO: 116).

If the pig animal or cell contains 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by 11 base pair inserts beginning at nucleotide 3,113, the 11 bases Pair insertions may involve insertion of the sequence AGCCAGCGTGC (SEQ ID NO: 117).

When the modified chromosomal sequence of the gene encoding the CD163 protein comprises deletion, deletion preferably includes in-frame deletion. In-frame deletion is a deletion that does not result in a change in the triplet reading frame, thus resulting in an internal deletion of one or more amino acids but not a truncated protein product. If splicing is to occur correctly, deletion of a multiple of three base pairs or three base pairs in the exon may result in in-frame mutagenesis.

The following INDELs described herein for pig animals and cells are expected to result in in-frame deletion because deletion in exon 7 of the porcine CD163 gene is a multiple of 3: compared to SEQ ID NO: 47 1506 base pair deletions of nucleotides 1,525 to 3,030; 1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides); 1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47; 123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47; 1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47; 1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47; 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113; And 1720 base pair deletions of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47.

Thus, in pig animals and cells, insertions or deletions in the gene encoding CD163 protein result in 1506 base pair deletions of nucleotides 1,525 to 3,030 compared to reference sequence SEQ ID NO: 47; 1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides); 1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47; 123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47; 1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47; Base pair 3,145 to nucleotide 4,5311,387 base pair deletions compared to reference sequence SEQ ID NO: 47; 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113; Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47; And an in-frame deletion selected from the group consisting of combinations thereof.

The pig animal or cell may include insertions or deletions selected from the group consisting of: two base pair insertions between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and a base pair insert between SEQ ID NO: 47 377 base pair deletions of nucleotides 2,573 to 2,949 nucleotides; 28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47; And combinations thereof. For example, an animal or cell may have two base pair insertions between nucleotides 3,149 and 3,150 compared to reference sequence SEQ ID NO: 47 and a base pairing of 377 nucleotide pairs from nucleotide 2,573 to nucleotide 2,949 over the same allele gene Deletion. An animal or cell may contain 28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47.

The pig animal or cell may contain seven base pair insertions between nucleotide 3,148 and nucleotide 3,149 as compared to reference sequence SEQ ID NO: 47 and eleven base pair deletions of nucleotide 3,137 to nucleotide 3,147 relative to reference sequence SEQ ID NO: 47.

A porcine animal or cell comprising any of the inserts or deletions described above may include a chromosomal sequence having a high degree of sequence identity to SEQ ID NO: 47, in addition to insertion or deletion. Thus, for example, a porcine animal or cell may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95% 99.9%, or 100% sequence identity.

The pig animal or cell may comprise a chromosomal sequence comprising SEQ ID NOS: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, have. As described further below in the Examples, SEQ ID NOs: 98-114 provide the nucleotide sequence for the region corresponding to the region of wild-type pig CD163 provided in SEQ ID NO: 47 and insert into the pig CD163 chromosome sequence as described herein Or deletion.

For example, the pig animal or cell may comprise a chromosomal sequence comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, SEQ ID NOS: 98, 101, 105, 109, 110, 112, 113, or 114 provide nucleotide sequences for in-frame deletion in exon 7 of the porcine CD163 chromosomal sequence.

As another example, a porcine animal or cell may comprise a chromosomal sequence comprising SEQ ID NO: 103 or 111.

The pig animal or cell may contain 11 base pair deletions in one allele of the gene encoding the CD163 protein and 2 base pair insertions and 377 base pair deletions in another allele of the gene encoding the CD163 protein have.

The pig animal or cell may contain 124 base pair deletions in one allele of the gene encoding the CD163 protein and 123 base pair deletions in another allele of the gene encoding the CD163 protein.

The pig animal or cell may contain one base pair insert.

The pig animal or cell may contain 130 base pair deletions in one allele of the gene encoding the CD163 protein and 132 base pair deletions in another allele of the gene encoding the CD163 protein.

The pig animal or cell may contain 1506 base pair deletions.

The pig animal or cell may contain seven base pair insertions.

The pig animal or cell may contain 1280 base pair deletions in one allele of the gene encoding the CD163 protein and 1373 base pair deletions in another allele of the gene encoding the CD163 protein.

The pig animal or cell may contain 1467 base pair deletions.

The pig animal or cell may contain a 1930 base pair intron 6 deletion of the nucleotide 488 to the nucleotide 2,417, a 12 base pair addition to the nucleotide 4,488, and an additional 129 base pair deletion to the exon 7.

The pig animal or cell can contain 138 base pair deletions in another allele of the gene encoding the 28 base pair deletion and CD163 protein in one allele of the gene encoding the CD163 protein.

The pig animal or cell may contain 1720 base pair deletions in one allele of the gene encoding the CD163 protein, 1382 base pair deletions, 11 base pair insertions and another allele of the gene encoding the CD163 protein have.

Any of the cells containing at least one modified chromosomal sequence in the gene encoding the CD163 protein may comprise sperm cells. Alternatively, any of these cells may contain egg cells (e. G. Fertilized eggs).

Any of the cells containing at least one modified chromosomal sequence in the gene encoding the CD163 protein may comprise somatic cells. For example, any of the cells may comprise fibroblasts (e. G., Fetal fibroblasts).

CD163 Locus  Nucleic acid Targeted  integrated

Site-specific integration of exogenous nucleic acids at the CD163 locus can be accomplished by any technique known to those skilled in the art. For example, integration of an exogenous nucleic acid at the CD163 locus can involve contacting the cell (e.g., an isolated cell or a cell of a tissue or organism) with a nucleic acid molecule comprising an exogenous nucleic acid. Such a nucleic acid molecule may comprise a nucleotide sequence on the side of an exogenous nucleic acid that promotes homologous recombination between the nucleic acid molecule and at least one CD163 locus. The nucleotide sequence on the side of the exogenous nucleic acid that promotes homologous recombination may be complementary to the endogenous nucleotide of the CD163 locus. Alternatively, the nucleotide sequence on the extrinsic nucleic acid side that promotes homologous recombination may be complementary to the previously integrated exogenous nucleotide. Many exogenous nucleic acids can be integrated at one CD163 locus as in gene stacking.

The integration of the nucleic acid at the CD163 locus can be facilitated (e.g., catalyzed) by the endogenous cellular machinery of the host cell, such as, but not limited to, endogenous DNA and endogenous recombinase. Alternatively, the integration of the nucleic acid at the CD163 locus may be facilitated by one or more factors (e. G., Polypeptides) provided in the host cell. For example, nuclease (s), recombinant enzyme (s), and / or ligase polypeptides can be produced by contacting a polypeptide with a host cell, or by expressing the polypeptide in a host cell (either independently or as part of a chimeric polypeptide ). Thus, a nucleic acid comprising a nucleotide sequence encoding at least one nuclease, recombinase, and / or ligase polypeptide can be introduced into a host cell either simultaneously or sequentially with a nucleic acid that is site-specifically integrated into the CD163 locus Wherein at least one nuclease, recombinase, and / or ligase polypeptide is expressed from a nucleotide sequence in a host cell.

DNA-binding polypeptides

Site-specific integration can be achieved, for example, by using factors that are capable of recognizing and binding specific nucleotide sequences in the genome of the host organism. For example, many proteins include polypeptide domains that can recognize and bind DNA in a site-specific manner. A DNA sequence recognized by a DNA-binding polypeptide can be referred to as a "target" sequence. A polypeptide domain that is capable of recognizing and binding DNA in a site-specific manner, even when expressed in a polypeptide other than the protein from which the domain was originally isolated, is generally capable of functioning independently for binding to DNA in a precisely folded, site- do. Similarly, target sequences for recognition and binding by DNA-binding polypeptides are generally located in large DNA structures (e.g., chromosomes), particularly where the site in which the target sequence is located is a soluble cell protein (e.g., a gene) Even if they are known to be accessible to such polypeptides.

DNA-binding polypeptides identified from proteins present in nature typically bind to distinct nucleotide sequences or motifs (e. G., Consensus recognition sequences), but are capable of altering these multiple DNA-binding polypeptides to recognize different nucleotide sequences or motifs Methods exist and are known in the art. DNA-binding polypeptides include, but are not limited to, for example: zinc finger DNA-binding domain; Leucine zipper; UPA DNA-binding domain; GAL4; TAL; LexA; Tet inhibitors; LacI; And steroid hormone receptors.

For example, the DNA-binding polypeptide may be a zinc finger. Individual zinc finger motifs can be designed to target and specifically bind to any of a wide variety of DNA sites. Canonical Cys ₂ His ₂ (as well as non-canonical Cys ₃ His) zinc finger polypeptides bind to DNA by inserting an a-helix into the major groove of the target DNA duplex. The recognition of DNA by zinc finger is modular; Each finger primarily contacts three conserved base pairs in the target, and several key residues in the polypeptide mediate recognition. By including a multiple zinc finger DNA-binding domain in the targeting endonuclease, the DNA-binding specificity of the targeting endonuclease can be further increased (thus the specificity of any gene control effect conferred thereby can also be increased One or more zinc finger DNA-binding polypeptides may be introduced into a host cell by, for example, introducing a targeting endonuclease, which is introduced into the host cell, into the genome of the host cell Can be engineered and used to interact with unique DNA sequences.

Preferably, the zinc finger protein is non-naturally occurring in that it is engineered to bind to the target site of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70: 313-340; Isalan et al. (2001) Nature Biotechnol. 19: 656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12: 632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416; U.S.A. Pat. Nos. 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. Pat. Patent disclosure Nos. 2005/0064474; 2007/0218528; 2005/0267061].

Engineered zinc finger binding domains may have novel binding specificities compared to the naturally-occurring zinc finger proteins. Methods of manipulation include, but are not limited to, rational design and various types of selection. Rationally, for example, involves the use of a database containing triplicate (or quadruple) nucleotide sequences and individual zinc finger amino acid sequences, wherein each 3-tuple or 4-tuple nucleotide sequence comprises a specific 3-tuple or 4-tuple nucleotide sequence, Is associated with one or more amino acid sequences of zinc finger binding to the sequence. For example, U.S. Pat. Pat. Nos. 6,453,242 and 6,534,261.

An exemplary selection method including phage display and two-hybrid systems is described in U.S. Pat. Pat. Nos. 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; WO 01/88197 and GB 2,338,237. In addition, the enhancement of binding specificity for zinc finger binding domains is described, for example, in WO 02/077227.

Also, as disclosed in these and other references, a zinc finger domain and / or a multi-fingered zinc finger protein may be any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length Can be connected together using < RTI ID = 0.0 > Also, for exemplary linker sequences of six or more amino acid lengths, see U.S. Pat. Pat. Nos. 6,479,626; 6,903,185; And 7,153,949. The proteins described herein may comprise any combination of suitable linkers between individual zinc finger of a protein.

Selection of Target Site: Methods for the design and construction of ZFPs and fusion proteins (and polynucleotides encoding them) are known to those of skill in the art and are described in U.S. Pat. Pat. Nos. 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; WO 02/016536 and WO 03/016496.

Also, as disclosed in these and other references, zinc finger domains and / or multi-fingered zinc finger proteins may be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length . Also, for exemplary linker sequences of six or more amino acid lengths, see U.S. Pat. Pat. Nos. 6,479,626; 6,903,185; And 7,153,949. The proteins described herein may comprise any combination of suitable linkers between individual zinc finger of a protein.

Alternatively, the DNA-binding polypeptide is a DNA-binding domain from GAL4. GAL4 is a modular transactivator in Saccharomyces cerevisiae , but it also acts as a transcriptional activator in several other organisms. See, for example, Sadowski et al. (1988) Nature 335: 563-4. In this regulatory system, the expression of the gene encoding the enzymes of the galactose mesa pathway in S. cerevisiae is strictly regulated by the available carbon sources [see literature; Johnston (1987) Microbiol. Rev. 51: 458-76]. The transcriptional regulation of these metabolic enzymes is mediated by the interaction between the positive control protein, GAL4, and the 17 bp symmetric DNA sequence (upstream activation sequence (UAS)) to which GAL4 specifically binds.

Native GAL4 consists of 881 amino acid residues with a molecular weight of 99 kDa. GAL4 includes domains that are functionally autonomous, and their combined activity accounts for the activity of GAL4 in vivo (see literature; 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 include the GAL4 DNA-binding domain [see literature; Keegan et al. (1986) Science 231: 699-704; Johnston (1987) Nature 328: 353-5]. Sequence-specific binding requires the presence of divalent cations coordinated by six Cys residues present in the DNA binding domain. The coordinated cation-containing domain is recognized by interacting with conserved CCG triplicates at each end of the 17 bp UAS through direct contact with the major groove of the DNA helix [see references; Marmorstein et al. (1992) Nature 356: 408-14]. The DNA-binding function of the protein places the C-terminal transcription activation domain near the promoter so that the activation domain can perform transcription.

Additional DNA-binding polypeptides that may be used include, for example and without limitation, a binding sequence from an AVRBS3-inducible gene; 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, for example, Brent & Ptashne (1985), supra); LacR (see for example, 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 receptors (Ellliston et al. (1990) J. Biol. Chem. 265: 11517-121); A mutated Tet inhibitor that binds to a Tet operative sequence in the absence of a Tet suppressor (U.S. Pat. No. 6,271,341) and the absence of tetracycline (Tc); DNA-binding domain of NF-kappaB; And GAL4, a hormone receptor, and the fusion of VP16 [cf. Wang et al. (1994) Proc. Natl. Acad. Sci. USA 91 (17): 8180-4.

The DNA-binding domain of one or more nucleases used in the methods and compositions described herein may comprise a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain. For example, U.S. Pat. Patent No. 2011/0301073.

Alternatively, the nuclease may comprise a CRISPR / Cas system. These systems include clustered regularly interspaced short palindromic repeats (CRISPR) loci encoding the RNA components of the system and Cas (CRISPR-related) loci encoding the proteins (Jansen et al., 2002. Mol. Microbiol. 43: 1565- 1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput. In the microbial host, the CRISPR locus contains a combination of Cas genes as well as non-coding RNA factors capable of programming 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 virtually four successive steps. First, two non-coding RNAs, pre-crRNA arrays and tracrRNAs are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of pre-crRNA, mediating the processing of pre-crRNA into mature crRNA containing individual spacer sequences. Third, the Wastson-Crick base-pairing between the prototypes on the target DNA immediately adjacent to the spacer and the spacer-spacer adjacent motif (PAM) on the crRNA, the mature crRNA: tracrRNA complex is an additional requirement for target recognition. Lt; RTI ID = 0.0 > Cas9 < / RTI > Finally, Cas9 mediates cleavage of the target DNA to produce double-strand breaks in the proto-spacer.

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

Cas proteins can be "functional derivatives" of naturally occurring Cas proteins. A "functional derivative " of a native sequence polypeptide is a compound having qualitative biological properties in common with native sequence polypeptides. "Functional derivatives" include, but are not limited to, fragments of native sequences and derivatives of native sequence polypeptides and fragments thereof, so long as they have biological activity in common with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of a functional derivative to hydrolyze a DNA substrate to a fragment. The term "derivative" encompasses amino acid sequence variants, covalent modifications, and fusion thereof, of polypeptides. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to mutants, fusion, covalent modifications of the Cas protein or fragment thereof. Cas proteins, including Cas proteins or fragments thereof, as well as derivatives of Cas proteins or fragments thereof can be obtained from cells, or can be synthesized chemically or by a combination of these two processes. Cells can be produced from naturally-occurring cells, or caspases, that naturally produce Cas proteins and produce endogenous Cas proteins at higher expression levels or from exogenously introduced nucleic acids (the nucleic acid encodes the same or different Cas as the endogenous Cas) Lt; RTI ID = 0.0 > cas < / RTI > protein. In some cases, cells do not naturally produce Cas proteins and are genetically engineered to produce Cas proteins.

The DNA-binding polypeptide can specifically recognize and bind to the target nucleotide sequence contained in the genomic nucleic acid of the host organism. Several distinct examples of the target nucleotide sequence can be found in the host genome in some instances. A target nucleotide sequence may be rare in the genome of an organism (e.g., about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, ) May be present in the genome). For example, the target nucleotide sequence may be located at a site that is unique within the genome of the organism. The target nucleotide sequences may be randomly distributed throughout the genome, for example, without limitation; Located in different linkage groups in the genome; Located in the same chain group; Located on different chromosomes; Located on the same chromosome; Located in the genome at a site that is expressed under similar conditions in an organism; (E. G., The target sequence may be contained within the integrated nucleic acid as a < / RTI > concatemer at the genomic locus).

Targeting Endonuclease

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 chimeric polypeptides can include, without limitation, nuclease, recombinase, and / or ligase polypeptides, and these polypeptides are as described above. Chimeric polypeptides comprising DNA-binding polypeptides and nuclease, recombinant enzyme, and / or ligase polypeptides may also include other functional polypeptide motifs and / or domains including, without limitation, the following: A spacer sequence located between the chimeric protein and the functional polypeptide; Leading peptides; Peptides that target the fusion protein to the organelle (e.g., the nucleus); Polypeptides that are 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 chimeric polypeptides can be operatively linked. The functional polypeptide of the chimeric polypeptide can be operatively linked by their expression from a single polynucleotide encoding at least a functional polypeptide ligated to each other in-frame to produce a chimeric gene encoding the chimeric protein. Alternatively, the functional polypeptides of the chimeric polypeptides can be operatively linked by other means, such as by bridging-binding of independently expressed polypeptides.

A DNA-binding polypeptide, or guide RNA, that specifically recognizes and binds to a target nucleotide sequence may be contained within a naturally isolated protein (or mutant thereof), wherein the naturally isolated protein or mutant thereof is (And may also contain recombinant enzymes and / or ligase polypeptides). Examples of such isolated proteins include TALENs, recombinant enzymes (e.g., Cre, Hin, Tre, and FLP recombinase), RNA-guided CRISPR / Cas9, and meganuclease.

As used herein, the term "targeting endonuclease" refers to a natural or engineered isolated protein and its mutants, including DNA-binding polypeptides or guide RNA and nuclease polypeptides, as well as DNA-binding polypeptides or guide RNAs Quot; refers to a chimeric polypeptide comprising a nuclease. Any target endonuclease (s), including DNA-binding polypeptides or guide RNAs that specifically recognize and bind to the target nucleotide sequence contained within the CD163 locus (e.g., because the target sequence is contained within the native sequence at the locus, Since the sequence has been introduced into the locus by, for example, recombination) 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 polypeptides; TALE domain; Leucine zipper; Transcription factor DNA-binding motif; And for example, without limitation, TALEN, recombinant enzymes (e.g., Cre, Hin, RecA, Tre, and FLP recombinase), RNA-guided CRISPR / Cas9, DNA recognition isolated from meganuclease / Or cleavage domain; And others known in the art. Particular examples include chimeric proteins comprising site-specific DNA binding polypeptides and nuclease polypeptides. Chimeric polypeptides can be engineered by methods known to those skilled in the art to alter the recognition sequence of a DNA-binding polypeptide contained within a chimeric polypeptide so as to target the chimeric polypeptide to a particular nucleotide sequence of interest.

Chimeric polypeptides can include DNA-binding domains (e.g., zinc finger, TAL-effector domains, etc.) and nuclease (cleavage) domains. The cleavage domain can be a DNA-binding domain, for example a zinc finger DNA-binding domain and a truncation domain or a TALEN DNA-binding domain and a truncation domain from a nuclease, or a meganuclease DNA-binding domain from a different nuclease And may be heterologous to the cleavage domain. The heterologous cleavage domain can be obtained from any endonuclease or exonuclease. Exemplary endonuclease agents from which cleavage domains can be derived include, but are not limited to, limiting endonuclease and homing endonucleases. See, e.g., 2002-2003 Catalog, New England Biolabs, Beverly, Mass .; and Belfort et al. (1997) Nucleic Acids Res. 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 Linn et al. Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.

Similarly, cleavage half-domains can be derived from any nuclease as described above, or a portion thereof, that requires dimerization for cleavage activity. Generally, if the fusion protein comprises a cleavage half-domain, two fusion proteins are required for cleavage. Alternatively, a single protein comprising two truncated half-domains can be used. The two cleavage half-domains can be derived from the same endonuclease (or functional fragment thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragment thereof). In addition, the target site for the two fusion proteins is defined by the fact that the binding of the two fusion proteins to their respective target sites results in a spatial orientation that allows the cleavage half-domain to form functional cleavage domains by, for example, Are positioned relative to one another to position the cut half-domains. Thus, near the edge of the target site can be separated by 5-8 nucleotides or by 15-18 nucleotides. However, any integer number of nucleotides, or pairs of nucleotides, can be interposed between two target sites (e.g., 2 to 50 nucleotide pairs or more). Generally, the site of cleavage is between the target sites.

Restriction endonuclease (restriction enzyme) is present in many species (at the recognition site) and can be sequence-specific to DNA, for example, one or more exogenous sequences (donor / transgene) The DNA can be cleaved at or near the binding site to be incorporated at or near the target site. Certain restriction enzymes (e. G., Type IIS) cleave DNA at sites removed from recognition sites and have detachable binding and cleavage domains. For example, type IIS enzyme Fok I catalyzes double-strand cleavage of DNA at nine nucleotides from its recognition site on one strand and thirteen nucleotides from its recognition site on the other strand. For example, U.S. Pat. Pat. Nos. 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. 269: 31, 978-31, 982). Thus, the fusion protein may comprise a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and at least one zinc finger binding domain, which may or may not be engineered.

An exemplary type IIS restriction enzyme from which a cleavage domain can be separated from a binding domain is Fok I. This particular enzyme is active as a dimer (cf. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575]. Thus, for purposes of the present invention, some of the Fok I enzymes used in the disclosed fusion proteins are considered to be cleavage half-domains. Thus, for targeted substitution and / or targeted double-strand cleavage of cell sequences using zinc finger-Fok I fusion, two fusion proteins, each comprising a FokI cleavage half-domain, Can be used for reconstruction. Alternatively, a single polypeptide molecule containing a DNA binding domain and two Fok I cleavage half-domains may also be used.

The truncation domain or cleavage half-domain can be any part of a protein that retains the cleavage activity or possesses the ability to form a functional cleavage domain by multiple trimerization (e. G., Dimerization).

Exemplary Type IIS Restriction Enzymes are U.S. Pat. Patent No. 2007/0134796. Additional restriction enzymes also contain separable binding and cleavage domains, which are contemplated by the present invention. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31: 418-420.

The cleavage domain may be, for example, as disclosed in U.S. Pat. Patent disclosure Nos. 2005/0064474; One or more engineered cleavage half-domains (also referred to as dimerization domain mutants) that minimize or prevent homodimerization as described in US Patent Application Publication No. 2006/0188987 and 2008/0131962.

Alternatively, the nuclease may be assembled in vivo at the nucleic acid target site using so-called "split-enzyme" techniques (see for example U.S. Patent Publication No. 20090068164). The components of such a split enzyme may be expressed on separate expression constructs or individual components may be sequenced in one open reading frame separated by, for example, a self-truncated 2A peptide or 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 can include an exogenous nucleic acid, or a custom-designed zinc finger nuclease (ZFN) that can be designed to integrate donor DNA, by delivering targeted site-specific double-stranded DNA cuts Patent publication 2010/0257638). ZFN is a chimeric polypeptide and a zinc finger DNA-binding domain polypeptide containing a non-specific cleavage domain from a restriction endonuclease (e. G., Fokl). 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. 27: 674-81. ZFNs can include non-canonical zinc finger DNA binding domains (see US Patent Publication No. 2008/0182332). FokI restriction endonuclease must be dimerized through the nuclease domain to cleave DNA and introduce double-strand breaks. Thus, a ZFN containing a nucleotide domain from such an endonuclease also requires dimerization of the nuclease domain to cleave the target DNA (cf. Mani et al. (2005) Biochem. Biophys. Res. Commun. 334: 1191-7; Smith et al. (2000) Nucleic Acids Res. 28: 3361-9). The dimerization of ZFN can be facilitated by two adjacent, oppositely directed DNA-binding sites. Id.

A method for site-specific integration of an exogenous nucleic acid into at least one CD163 locus of a host may include introducing a ZFN into the host's cells wherein the ZFN recognizes and binds to the target nucleotide sequence and binds to the target 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 a location other than at least one CD163 locus. For example, DNA-binding polypeptides of ZFN can be engineered to recognize and bind to target nucleotide sequences identified within at least one CD163 locus (e. G., By sequencing the CD163 locus). The method for site-specific integration of an exogenous nucleic acid into at least one CD163 performance locus of a host, including introducing ZFN into the host's cells, may also include introducing an exogenous nucleic acid into the cell, Recombination of the exogenous nucleic acid into the host nucleic acid containing 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).

CD163 Locus  Any exogenous nucleic acid for integration

Exogenous nucleic acids for integration at the CD163 locus include: an exogenous nucleic acid for site-specific integration at at least one CD163 locus, such as, without limitation, an ORF; A nucleic acid comprising a nucleotide sequence encoding a targeting endonuclease; And at least one or both of the foregoing. Thus, a particular nucleic acid comprises a nucleotide sequence encoding a polypeptide, a structural nucleotide sequence, and / or a DNA-binding polypeptide recognition and binding site.

Any exogenous nucleic acid molecule for site-specific integration

As noted above, the insertion of an exogenous sequence (also referred to as a "donor sequence" or "donor" or "graft gene") can be used, for example, for expression of a polypeptide, for the correction of a mutant gene, Lt; / RTI > expression. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence in which it is placed. Donor sequences may contain non-homologous sequences with two regions of homology in the side to enable efficient homologous-direct repair (HDR) at the site of interest. In addition, the donor sequence may comprise a vector molecule containing a sequence that is not homologous to the region of interest of the cell chromatin. The donor molecule may contain several discontinuous regions of homology to the cell chromatin. For example, for targeted insertion of a sequence that does not normally exist in the region of interest, the sequence may be present in the donor nucleic acid molecule and may have a homologous sequence to the sequence of the region of interest.

The donor polynucleotide may be DNA or RNA, single-stranded or double-stranded, and may be introduced into cells in a linear or circular manner. For example, U.S. Pat. Patent disclosure Nos. 2010/0047805, 2011/0281361, 2011/0207221, and 2013/0326645. When introduced linearly, the terminus of the donor sequence may be protected in a manner known to those skilled in the art (e. G., From degradation by nucleic acid end hydrolysis). 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 of the ends. For example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84: 4959-4963; Nehls et al. (1996) Science 272: 886-889. Additional methods for protecting extrinsic polynucleotides from degradation include addition of a modified amino group (s) and modified internucleotide sequences, such as phosphorothionate, phosphoramidate, and O-methyl ribose or deoxy But are not limited to, the use of ribose residues.

Polynucleotides may be introduced into cells as part of a vector molecule having additional sequences such as, for example, a gene encoding a replication origin, an enhancer and an antibiotic resistance. In addition, donor polynucleotides can be introduced as naked nucleic acids as nucleic acids complexed with agents such as liposomes or poloxamers, or as viruses (e.g., adenovirus, AAV, herpes virus, retrovirus, lentivirus, RTI ID = 0.0 > IDLV). ≪ / RTI >

The donor is generally integrated so that its expression is effected by an endogenous promoter at the integration site, i. E., By an accelerator (e. G., CD163) driving expression of the endogenous gene such that the donor is incorporated therein. It will be appreciated, however, that the donor may comprise a promoter and / or enhancer, such as a constitutive promoter or an inducible or tissue specific promoter.

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

Exogenous nucleic acids that can be integrated into at least one CD163 locus in a site-specific manner to modify the CD163 locus include, but are not limited to, nucleic acids comprising a nucleotide sequence that encodes a polypeptide of interest; A nucleic acid comprising a crop gene; A nucleic acid comprising a nucleotide sequence encoding an RNAi molecule; Or a nucleic acid that cleaves the CD163 gene.

The exogenous nucleic acid may be integrated at the CD163 locus to modify the CD163 locus, wherein the nucleic acid comprises a nucleotide sequence encoding the polypeptide of interest such that the nucleotide sequence is expressed from the CD163 locus in the host. In some instances, a polypeptide of interest (e. G., A foreign protein) is expressed from a nucleotide sequence that encodes a polypeptide of interest in a commercial amount. In this example, the polypeptide of interest may be extracted from a host cell, tissue, or biomass.

Targeting Endonuclease Password  A nucleic acid molecule comprising a nucleotide sequence

The nucleotide sequence encoding the targeting endonuclease may be manipulated by manipulation (e. G., Ligation) of a native nucleotide sequence encoding a polypeptide contained within the targeting endonuclease. For example, the nucleotide sequence of the gene corresponding to the DNA-binding polypeptide can be confirmed by examining the nucleotide sequence of the gene encoding the protein comprising the DNA-binding polypeptide, and the nucleotide sequence can be identified by the targeting end including the DNA- Can be used as a component of a nucleotide sequence encoding a nuclease. Alternatively, the amino acid sequence of the targeting endonuclease can be used to deduce a nucleotide sequence encoding a targeting endonuclease, for example, upon degradation of the genetic code.

In an exemplary nucleic acid molecule comprising a nucleotide sequence encoding a targeting endonuclease, the final codon of the first polynucleotide sequence encoding the nuclease polypeptide and the first codon of the second polynucleotide sequence encoding the DNA-binding polypeptide The codons can be separated by any number of nucleotide triplets, for example without coding for introns 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 can be separated into any number of nucleotide triplets . The last polynucleotide sequence encoding the nuclease polypeptide and the last polynucleotide sequence of the second polynucleotide sequence encoding the DNA-binding polypeptide (i. E., 3 ' in the nucleic acid sequence) are immediately adjacent to the polynucleotide sequence of the additional polynucleotide Can be fused at the first codon with a phase-register or separated from it by only a short peptide sequence, such as that encoded by a synthetic nucleotide linker (e. G., A nucleotide linker that can be used to achieve fusion) . Examples of such additional polynucleotide sequences include, by way of example and without limitation, tags, targeting peptides, and enzyme cleavage sites. Likewise, the first codon of the 5 ' (in the nucleic acid sequence) of the first and second polynucleotide sequences may be fused in the phase-register with the last codon of the immediate further polynucleotide coding sequence immediately adjacent thereto, or only in the short peptide sequence As shown in FIG.

Sequences that separate polynucleotide sequences encoding functional polypeptides (e. G., DNA-binding polypeptides and nuclease polypeptides) in a targeting endonuclease may, for example, be those in which the encoded amino acid sequence translates to a target endonuclease But may be constructed of any sequence that does not significantly modify it. Due to the autonomous nature of known nuclease polypeptides and known DNA-binding polypeptides, intervening sequences will not interfere with the respective function of these structures.

Other knockout methods

Various other techniques known in the art can be used to inactivate and / or introduce a nucleic acid construct into an animal to produce a protozoan animal and make an animal line to make a knock-out animal, wherein the knockout or nucleic acid The construct is integrated into the genome. These techniques include, but are not limited to, pronuclear microinjection (US Pat. No. 4,873,191), retrovirus-mediated gene transfer into germline lines (Van der Putten et al. (1985) Proc. Natl. Acad. (1983) Mol. Cell. Biol. 3, 1803-321), embryonic stem cell-specific gene targeting (Thompson et al. (1989) Cell 56, 313-321) 1814), sperm-mediated gene transfer (Lavitrano et al. (2002) Proc Natl Acad Sci USA 99, 14230-14235 Lavitrano et al 2006 Reprod. Fert. (Wilmut et al. (1997) Nature 385, 810-813; and Wakayama et al. (1998) Nature 394, 369 (1986)) after in vitro transformation of adult stem cells, for example cumulus or breast cells, or adult, fetal, or embryonic stem cells -374). Prokaryotic microinjection, sperm-mediated gene delivery, and somatic cell nuclear transfer are particularly useful techniques. Genome-modified animals are all animals whose genes, including their germline cells, are genetically modified. When a method of producing an animal that is mosaic in its genetic modification is used, the animal may be inbreeded and a genomic modified offspring may be selected. For example, cloning can be used to make a mosaic animal if the cell is transformed in the blastocyst, or genetic modification can occur if the single cell is transformed. Modified, sexually mature animals may be homozygous or heterozygous for the strain according to the particular approach used. If a particular gene is inactivated by a knockout strain, homozygosity will normally be required. If a particular gene is inactivated by RNA interference or a dominant negative strategy, heterozygosity is often appropriate.

Typically, in embryo / zona microinjection, the nucleic acid construct or mRNA is introduced into the embryo; One or two cell embryos are used as the nuclear construct containing the genetic material from the sperm heads and the oocytes appear in the protoplasm. Progenitor embryos can be obtained in vitro or in vivo (i.e., surgically recovered from the fallopian tubes). In vitro fertilization can be generated as follows. For example, pig ovaries can be collected at slaughterhouses and maintained at 22-28 ° C during transport. The ovaries can be cleaned and separated for follicular aspiration, and follicles 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 washed through a pre-filter with commercial TL-HEPES (Minitube, Verona, Wis.). Oocytes surrounded by compact cumulus mass were selected and cultured in humidified air at 38.7 ° C and 5% CO ₂ for approximately 22 hours with 0.1 mg / mL cysteine, 10 ng / mL epidermal growth factor, 10% porcine follicular fluid , TCM-199 oocytes supplemented with 50 μM 2-mercaptoethanol, 0.5 mg / ml cAMP, 10 IU / ml of each pregnancy terminated serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) MATURATION MEDIUM (Minitube, Verona, Wis.). After this, oocytes can be transplanted into fresh TCM-199 matured medium (which will not contain cAMP, PMSG or hCG) and incubated for an additional 22 hours. Matured oocytes can be stripped from their cumulus cells by vortexing for 1 minute in 0.1% hyaluronidase.

For pigs, mature oocytes can be modified in a 500 μl Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, Wis.) In a Minitube 5-well fertilizing dish. In preparation for in vitro fertilization (IVF), freshly-collected or frozen sludge can be washed and resuspended to 400,000 sperm in PORCPRO IVF Medium. Sperm concentration can be analyzed by computer assisted semen analysis (SPERMVISION, Minitube, Verona, Wis.). Final in vitro In vitro insemination can be performed at a final concentration of approximately 40 motile sperm / oocytes at a volume of 10 μl depending on the wort. All fertilized oocytes are cultured in a 5.0% CO ₂ atmosphere for 6 hours at 38.7 ° C. Six hours after sperm injection, the estimated zygote can be washed twice in NCSU-23 and transferred to 0.5 mL of the same medium. The system can routinely produce 20-30% blastocysts across most fowels with a 10-30% polyspermic insemination rate.

Linearized nucleic acid constructs or mRNA may be injected into one of the nuclei or into the cytoplasm. The injected eggs can then be transferred to recipient females (eg, into the oviduct of a recipient female) and allowed to grow in recipient females to produce transgenic or genetically modified animals. In particular, in vitro fertilized embryos can be centrifuged at 15,000 x g for 5 minutes to sediment lipids to enable visualization of the nucleus. The embryos can be injected using an Eppendorf FEMTOJET syringe and cultured until blastocyst formation. The rate and quality of embryo cutting and blastocyst formation can be recorded.

The embryo can be implanted surgically into the uterus of an asynchronous recipient. Typically, 100-200 (eg, 150-200) embryos can be placed in ampulla-isthmus junctions of the fallopian tube using a 5.5-inch TOMCAT® catheter. After surgery, real-time ultrasound testing of pregnancy can be performed.

In somatic cell nuclear transfer, gene transfer or genetically edited cells such as embryonic blastomere, fetal fibroblast, adult ear fibroblast, or granulosa cell containing the above-mentioned nucleic acid construct are introduced into the nucleus oocytes A combined cell can be established. The oocytes can be nucleated by suturing the cytoplasm in the incision following a partial incision in the vicinity of the polar body. Typically, a scanning pipette with a sharp beveled tip is used to inject genetically-engineered or genetically-edited cells into amniocytes that are inhibited by meiosis 2. In some practices, oocytes blocked by meiosis-2 are called eggs. (For example, by fusing and activating oocytes) to produce pigs or small embryos and then transplanting them into the fallopian tubes of the recipient females approximately 20 to 24 hours after activation of the embryos. See, for example, Cibelli et al. (1998) Science 280, 1256-1258 and U.S. Pat. Pat. Nos. 6,548,741, 7,547,816, 7,989,657, or 6,211,429. In the case of pigs, approximately 20 to 21 days after embryo implantation, pregnant women can be confirmed for pregnant females.

Standard breeding procedures can be used to create animals that are homozygous for the inactivated gene from the early heterozygote progenitor animal. However, homozygosity may not be necessary. The genetically modified pigs described herein can be propagated with other pigs of interest.

Once genetically modified animals are created, the inactivation of endogenous nucleic acids can be assessed using standard techniques. Initial screening can be accomplished by Southern blot analysis to determine if inactivation has occurred or not. For an explanation of Southern analysis, see sections 9.37-9.52 of Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview; N.Y]. Polymerase chain reaction (PCR) techniques can also be used for initial screening. PCR refers to a process or technique for amplifying a target nucleic acid. Generally, sequence information from the extremities of or beyond the region of interest is used to design the same or similar oligonucleotide primers in the sequence for the opposite strand of the template to amplify. PCR can be used to amplify specific sequences from RNA as well as DNA, including sequences from whole genomic DNA or whole cell RNA. Primers are typically 14 to 40 nucleotides in length, but can range from 10 nucleotides to several hundred nucleotides in length. PCR can be performed, for example, PCR Primer: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. The nucleic acid can also be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplification. See, for example, Lewis (1992) Genetic Engineering News 12,1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874; and Weiss (1991) Science 254: 1292. At the blastocyst stage, the embryos can be individually processed for analysis by PCR, Southern hybridization and splinkerette PCR (see, for example, Dupuy et al., Proc Natl Acad Sci USA (2002) 99: 4495) .

Interference RNAs

A variety of interfering RNA (RNAi) systems are known. Double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous gene transcripts. RNA-induced silencing complex (RISC) metabolizes dsRNA to small 21-23-nucleotide small interfering RNAs (siRNAs). RISC contains double strand RNAse (dsRNase, e.g., Dicer) and ssRNase (e.g., Argonaut 2 or Ago2). RISC uses antisense strands as a guide for finding cleavable targets. Both siRNAs and microRNAs (miRNAs) are known. Methods of deactivating genes in genetically modified animals include inducing RNA interference against the target gene and / or nucleic acid so that the expression of the target gene and / or nucleic acid is reduced.

For example, an exogenous nucleic acid sequence can induce RNA interference against a 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 the expression of the DNA. Constructs for siRNAs can be found, for example, in the literature (cf. 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 are described in the literature (see; McIntyre and Fanning (2006) BMC Biotechnology 6: 1. Generally, shRNAs are transcribed as single-stranded RNA molecules containing complementary regions, which can anneal to form short hairpins.

The probability of finding a single, individual functional siRNA or miRNA indicated by a specific gene is high. For example, the predictability of the specific sequence of an siRNA is about 50%, but multiple interfering RNAs can be made with good confidence that at least one of them will be effective.

In vitro cells, in vivo cells, or animal compost animals, for example livestock animals expressing the indicated RNAi for the gene encoding CD163, can be used. RNAi may be selected from the group consisting of, for example, siRNA, shRNA, dsRNA, RISC and miRNA.

Inductive system

An inductive system can be used to inactivate the CD163 gene. A variety of inductive systems are known that enable spatial and temporal control of gene inactivation. Some have been found to be functional in vivo in pigs.

An example of an inductive system is a tetracycline (tet) -on promoter system, which can be used to regulate the transcription of nucleic acids. In such a system, a mutated Tet repressor (TetR) is fused to the activation domain of a simple herpes virus VP 16 transcription-activator protein to produce a tetracycline-regulated transcriptional activator (tTA), which is tet or doxycycline ). Transcription is minimal in the absence of antibiotics, whereas transcription is induced in the presence of tet or dox. Alternative inductive systems include exdysone or rapamycin systems. Exdysone is an insect releasing hormone whose production is controlled by a heterodimer between the exdison receptor and the product of the ultrapiracle gene (USP). Expression is induced by treatment with exdysone or an analog of exdysone such as multisteon A. An agent to be administered to an animal to trigger an inducible system is referred to as an inducer.

Among the more commonly used inductive systems are tetracycline-inducible systems and Cre / loxP recombinase systems (constitutive or inducible). The tetracycline-inducible system comprises a tetracycline-controlled transcriptional activator (tTA) / reverse tTA (rtTA). A method for in vivo use of such a system involves generating two lines of genetically modified animals. One animal line expresses the active factor (tTA, rtTA, or Cre recombinase) under the control of the selected promoter. Another animal line expresses the receptor where expression of the gene of interest (or the gene to be modified) is under the control of the target sequence for the tTA / rtTA transcriptional activator (or the loxP sequence is on the side). Mating of two animals provides control of gene expression.

The tetracycline-dependent regulatory system (tet system) is composed of two components: a tetracycline-controlled transcriptional activator (tTA or rtTA) and a tTA / rtTA-dependent It depends on accelerators. In the absence of tetracycline or a derivative thereof (e.g., doxycycline), tTA binds to the tetO sequence to allow transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA can not interact with its target and transcription does not occur. The tet system using tTA is called tet-OFF because tetracycline or doxycycline enables transcription down-regulation. Administration of tetracycline or derivatives thereof allows transient control of transgene expression in vivo. rtTA is a mutant of tTA that does not function in the absence of doxycycline, but requires the presence of a ligand for transcriptional activation. Therefore, this tet system is called tet-ON. The tet system has been used in vivo for the inducible expression of several transgene genes, eg, encoding reporter genes, oncogenes, or proteins involved in signal cascades.

The Cre / lox system uses Cre recombinase, which catalyzes site-specific recombination by crossing between two separate Cre recognition sequences, the loxP site. The DNA sequence (called floxed DNA) introduced between the two loxP sequences is cleaved by Cre-mediated recombination. Controlling Cre expression in transgenic and / or transgenic animals using spatial control (by tissue- or cell-specific promoters) or temporal control (by inductive systems), DNA ablation between the two loxP sites Respectively. One application is for conditional gene inactivation (conditional knockout). Another approach is for protein overexpression in which the floxed stop codon is inserted between the promoter sequence and the DNA of interest. Genetically modified animals do not express the transgene until Cre is expressed, resulting in ablation of the floxed stop codon. This system has been applied to tissue-specific tumor formation and controlled antigen receptor expression in B lymphocytes. Inducible Cre recombinase was also developed. The inducible Cre recombinase is activated only by administration of an exogenous ligand. Inducible Cre recombinase is a fusion protein containing the original Cre recombinase and a specific ligand-binding domain. The functional activity of the Cre recombinase depends on the external ligands capable of binding to such specific domains in the fusion protein.

In vitro cells, in vivo cells, or genetically modified animals, such as livestock animals, that contain the CD163 gene under the control of an inductive system may be used. The genetic modification of an animal can be genomic or mosaic. The inductive system may be selected from the group consisting of, for example, Tet-On, Tet-Off, Cre-lox, and Hif1 alpha.

Vector and nucleic acid

A variety of nucleic acids can be introduced into the cell for knockout purposes, for gene inactivation, to obtain expression of the gene, or for other purposes. As used herein, the term nucleic acid includes DNA, RNA, and nucleic acid analogs, and double-stranded or single-stranded (i.e., sense or antisense single stranded) nucleic acids. Nucleic acid analogs can be modified, for example, in base moieties, sugar moieties, or phosphate backbones to improve nucleic acid stability, hybridization, or solubility. Deformations in the base moiety include deoxyuridine for deoxythymidine and 5-methyl-2'-deoxycytidine and 5-bromo-2'-doxcytidine for deoxycytidine do. Modifications of the sugar moiety include modifications of the 2 ' hydroxyls per ribose to form 2 ' -O-methyl or 2 ' -O-allyl sugars. Deoxyribose phosphate skeleton to produce a morpholino nucleic acid wherein each base moiety is linked to a six atom, a morpholino ring, or a peptide nucleic acid, the deoxyphosphate skeleton is replaced by a pseudo-peptide skeleton, The number of bases is retained. Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7 (3): 187; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4: 5]. In addition, the deoxyphosphate skeleton can be replaced with, for example, a phosphorothioate or phosphorodithioate skeleton, a phosphoramidite, or an alkylphosphorus ester skeleton.

The target nucleic acid sequence may be operatively linked to a regulatory region, such as an enhancer. The control region may be a pig control region or may be from other species. As used herein refers to positioning the regulatory region for a nucleic acid sequence in a manner that allows or facilitates the transcription of the operatively linked target nucleic acid.

Any type of promoter can be operatively linked to a target nucleic acid sequence. Examples of accelerators include, but are not limited to, tissue-specific promoters, constitutive promoters, inducible promoters, and accelerators that are reactive or non-reactive with a particular stimulus. Suitable tissue-specific promoters may cause preferential expression of nucleic acid transcripts in beta cells and include, for example, human insulin promoters. Other tissue specific promoters may, for example, cause preferential expression in hepatocytes or heart tissue, and may each include albumin or alpha-myosin heavy chain promoters. Promoters that promote the expression of nucleic acid molecules without significant tissue or time-specificity can be used (i. E., Constitutive promoters). For example, a beta-actin promoter such as a chicken beta-actin gene promoter, a ubiquitin promoter, a miniCAGs promoter, a glyceraldehyde-3-phosphatidase enzyme (GAPDH) promoter, or a 3-phosphoglycerate kinase (HSV-TK) promoter, an SV40 promoter, or a cytomegalovirus (CMV) promoter may be used as the promoter, as well as a viral promoter, such as a herpes simplex virus (PGK) promoter. For example, fusions of chicken beta actin gene promoter and CMV enhancer can be used as promoters. See, for example, Xu et al. (2001) Hum. Gene Ther. 12: 563; and Kiwaki et al. (1996) Hum. Gene Ther. 7: 821].

Additional regulatory regions that may be useful in nucleic acid constructs include, but are not limited to, polyadenylation sequences, translational control sequences (e.g., internal ribosome entry segment, IRES), enhancers, inducing elements, or introns. These regulatory regions may increase expression by affecting transcription, mRNA stability, translation efficiency, etc., but this may not be essential. Such regulatory regions may be included in the nucleic acid construct as desired to obtain optimal expression of the nucleic acid in the cell (s). However, sufficient expression can sometimes be obtained without these additional elements.

Nucleic acid constructs encoding single peptides or selectable markers may be used. A single peptide may be used that allows the encoded polypeptide to be directed at a particular cell location (e.g., cell surface). Non-limiting examples of selectable markers include puromycin, hepcyclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR) Methyl-B-phosphotransferase, thymidine kinase (TK), and xanthine-guanine phosphoribosyltransferase (XGPRT). Such markers are useful for selecting stable transformants in cultures. Other selectable markers include fluorescent polypeptides, e. G., Green fluorescent protein or yellow fluorescent protein.

Sequences encoding the selectable marker may, for example, have a recognition sequence for a recombinase such as Cre or Flp. For example, the selectable marker may be flanked by a loxP recognition site (a 34-bp recognition site recognized by Cre recombinase) or a FRT recognition site so that the selection marker can be ablated from the construct. Orban, et al., Proc. Natl. Acad. Sci. (1992) 89: 6861, for a review of Cre / lox technology, and Brand and Dymecki, Dev. Cell (2004) 6: 7]. A transposon containing a Cre- or Flp-activatable transgene that is blocked by a selectable marker gene may also be used to obtain an animal having the conditional expression of the transgene. For example, promoter-driven expression of a marker / transgene gene may be ubiquitous or tissue-specific, which will result in universal or tissue-specific expression of the marker in F0 animals (eg, pigs). Tissue-specific activation of the transgene can be achieved, for example, by crossing pigs that universally express the marker-blocked transgene gene into pigs expressing Cre or Flp in a tissue-specific manner, or in a tissue- - crossing the pig expressing the blocked transgene gene into a pig that commonly expresses Cre or Flp recombinase. Regulated expression of the transgene gene or controlled resection of the marker allows expression of the transgene.

The exogenous nucleic acid can encode the polypeptide. The nucleic acid sequence encoding the polypeptide may comprise a tag sequence encoding a "tag" designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate ubiquitination or detection). The tag sequence may be inserted into a 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 encrypted tags include glutathione S-transferase (GST) and FLAG ^TM tags (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 < 0 > C. And methylation can be confirmed by incubating the construct with one unit of HinP1I endonuclease at 37 ° C for 1 hour and analysis by agarose gel electrophoresis.

Nucleic acid constructs can be produced using a variety of techniques including, for example, germ cells, such as oocytes or eggs, progenitor cells, adult or embryonic stem cells, primitive germ cells, , An islet cell, a beta cell, a liver cell, or any type of embryo, fetus, or adult animal cell, including fibroblasts, for example, dermal fibroblasts. Non-limiting examples of techniques include, but are not limited to, transposon systems, recombinant viruses capable of infecting cells or liposomes, or other non-viral methods capable of delivering nucleic acids to cells, such as electroporation, microinjection, Includes the use of precipitation.

In a transposon system, the transcription unit of the nucleic acid construct, i. E., The regulatory region operatively linked to the exogenous nucleic acid sequence, is on the transversons repeatable side of the transposon. See, for example, Sleeping Beauty (U.S. Pat. No. 6,613,752 and U.S. Publication No. 2005/0003542); Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31: 6873); 27: Genome Biology 8 (Suppl.1): S2); Hsmar1 (Miskey et al. (2007)) Mol Cell Biol. 27: 4589); And Passport have been developed to introduce nucleic acids into cells, including mouse, human, and porcine cells. The Sleeping Beauty transposon is especially useful. The translocation enzyme may be delivered as a protein, enciphered as an exogenous nucleic acid on the same nucleic acid construct, introduced on a separate nucleic acid construct, or provided as an mRNA (e. G., In vitro transcription and capped mRNA).

An insulator element may also be included in the nucleic acid construct to maintain expression of the exogenous nucleic acid and inhibit unwanted transcription of the host gene. For example, U.S. Pat. Release No. 2004/0203158. Typically, the insulating element is on the sides of each side of the transcription unit and is inside the transposon inversion repeats. Non-limiting examples of insulating elements include a substrate attachment region - (MAR) type insulating element and an edge-type insulating element. For example, U.S. Pat. Pat. Nos. 6,395,549, 5,731,178, 6,100,448, and 5,610,053, and U.S. Pat. Release No. 2004/0203158.

The nucleic acid can be inserted into the vector. The vector is a broad term that encompasses any specific DNA fragment designed to migrate from the carrier to the target DNA. A vector may refer to an expression vector, or a vector system, which is a set of elements necessary to cause DNA insertion into a genome or other targeted DNA sequence, such as an episome, a plasmid, or even a viral / phage DNA fragment to be. Vector systems such as viral vectors (e.g., retroviruses, adeno-associated viruses and integrated phage viruses), or non-viral vectors (e.g., transposons) used for gene transfer in animals have two basic components: 1 ) A vector consisting of DNA (or RNA reverse transcribed into cDNA) and 2) any other integrase enzyme that recognizes both a translocation enzyme, a recombinant enzyme, or both a vector and a DNA target sequence, and inserts the vector into the target DNA sequence. The vector contains one or more expression cassettes that contain one or more expression control sequences very frequently, wherein the expression control sequences are DNA sequences that control and regulate the transcription and / or translation of different DNA sequences or mRNA, 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 a cloning start point, a suitable promoter and optional enhancer, an essential ribosome binding site, a polyadenylation site, a donor donor and acceptor site, a transcription termination sequence, and a 5 'lateral non-transcription sequence. Examples of vectors include plasmids (which may be carrier of other types of vectors), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1, SIV or FIV) Viruses (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 refers to, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, as well as naturally occurring and chemically modified nucleic acids such as synthetic bases or alternative It refers to both RNA and DNA, including the skeleton. The nucleic acid molecule may be a double-stranded or single-stranded (i. E., A sense or antisense single strand).

Ancestral animals, animal systems, traits, and breeding

The progenitor animal may be produced by cloning and other methods described herein. The progenitor may be homozygous for genetic modification, such as when the zygote or primary cell undergoes homozygous transformation. Similarly, a heterozygote can be made. In the case of animals containing at least one modified chromosomal sequence in the gene encoding the CD163 protein, the progeny are preferably heterozygous. Sijo can be transformed into a genome, which means that all of the cells in the genome undergo transformation. The progenitor may be a mosaic of transformations as can occur when the vector is typically introduced into one of a plurality of cells in the embryo at a blastocyst stage. Children of mosaic animals can be tested to identify genomic modified offspring. The animal line is established when it is possible to produce a flock of animals that can be sexually reproduced with heterozygous or homozygous offspring that continue to express deformation or that can be propagated by assisted reproduction techniques.

In livestock, several alleles are known to be involved in various traits such as production traits, type traits, workability traits, and other functional traits. Experts are accustomed to monitoring and characterizing these traits [cf. Visscher et al., Livestock Production Science, 40 (1994) 123-137, U.S. Pat. Pat. No. 7,709,206, US 2001/0016315, US 2011/0023140, and US 2005/0153317]. Animal lines can include traits selected from the group consisting of production traits, type traits, working traits, reproductive traits, mothering traits, and disease resistant traits. Additional traces include expression of the recombinant gene product.

Animals with the desired traits or traits can be modified to prevent their sexual maturation. Since the animal is infertile until maturity, sexual maturation can be regulated as a means to control the dissemination of the animal. Thus, animals that have been reared or modified to have more than one trait can be provided to the prisoner as a reduced risk of prisoners breeding the animals and setting the value of traits on their own. For example, the genome of an animal can be genetically modified, wherein the modification comprises inactivation of a sexual maturation gene, wherein the sexual maturation gene in a wild-type animal expresses a selective factor for sexual maturation. An animal can be treated by administering a compound that alleviates defects caused by loss of expression of a gene that induces sexual maturation in the animal.

Reproduction of an animal in need of administration of a compound that induces sexual maturation can be advantageously achieved in a treatment facility. The treatment facility can implement standardized protocols for well-controlled strains to efficiently produce consistent animals. They can be distributed in many places where they want to grow animal offspring. Thus, farms and farmers (including ranches and pastures) can order a desired number of offspring having a specified range of age and / or weight and / or traits and take them to a desired time and / or place. Afterwards, detainees, eg, farmers, can raise animals and deliver them to the market when they want.

Genetically modified livestock animals with inactivated sexual maturation genes (eg, to more than one place, to multiple farms) can be delivered. The animal may have an age of about 1 day to about 180 days. An animal may have more than one trait (e. G., Expressing a desired trait or high-value trait or a new trait or recombinant trait).

 Methods for breeding methods and methods for increasing animal resistance to infection and animal populations

Provided herein are breeding methods for producing an animal or lineage having reduced susceptibility to infection by pathogenic bacteria. The method comprises genetically transforming the oocyte or sperm cell to introduce a modified chromosomal sequence into the gene encoding the CD163 protein into at least one of the oocyte and sperm cell and selecting a chromosomal sequence modified to the gene encoding the CD163 protein And modifying the oocyte with sperm cells to produce a fertilized egg containing the fertilized egg. Alternatively, the method includes genetically transforming the embryo to introduce a modified chromosomal sequence into the gene encoding the CD163 protein into the fertilized egg. The method comprises the steps of transplanting an embryo into a surrogate female animal (where the pregnancy and termination produce a progeny animal), screening offspring for susceptibility to pathogens, and transforming the chromosome of the gene encoding the CD163 protein Further comprising the step of selecting offspring having reduced susceptibility to pathogens compared to animals not comprising the sequence.

The pathogen preferably comprises a virus, e.g., PRRSV.

The animal or offspring may be an embryo, a juvenile, or an adult.

An animal or offspring may include a rabbit. The domesticated animal may be a livestock animal such as a pig animal, a small animal (such as a beef cattle or a cow), a sheep animal, a goat animal, a horse and animal (eg horse or donkey), a water buffalo, , Turkey, duck, goose, guinea fowl, or freshly hatched bird). The livestock animal is preferably a cow or a pig animal, and most preferably a pig animal.

The step of genetically transforming oocytes, sperm cells, or embryos may include genetic editing of oocytes, sperm cells, or embryos. Genetic editing may involve the use of homing endonucleases. While homing endonucleases may be naturally occurring endonucleases, they are rationally designed non-naturally occurring homing endonucleases with DNA recognition sequences designed to target the chromosomal sequence of the gene encoding the CD163 protein desirable. Thus, the homing endonuclease may be a designed homing endonuclease. Thus, the homing endonuclease may be a designed homing endonuclease. The homing endonucleases may be, for example, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system, transcriptional activator-like effector nuclease (TALEN), ZFN (zinc finger nuclease), recombinant enzyme fusion protein , Meganuclease, or a combination thereof). The gene editing preferably involves the use of a CRISPR / Cas9 system.

Oocytes, sperm cells, or embryos may be heterozygous for a modified chromosomal sequence. Alternatively, oocytes, sperm cells, or embryos may be homozygous for a modified chromosomal sequence.

The modified chromosomal sequence may include insertion of a gene encoding the CD163 protein, deletion of a gene encoding the CD163 protein, or a combination thereof. For example, a modified chromosomal sequence may include deletion of a gene encoding the CD163 protein (e. G., In-frame deletion). Alternatively, the modified chromosomal sequence may comprise the insertion of a gene encoding the CD163 protein.

Insertion or deletion can reduce CD163 protein production or activity relative to CD163 protein production or activity in an animal without insertion or deletion.

Insertion or deletion can lead to the production of non-functional functional CD163 protein by the animal. By "non-functional CD163 protein" is meant an amount of CD163 protein in an animal, offspring, or cell that is less than the level observed in an animal, offspring, or cell that does not contain insertions or deletions, At least about 90% lower.

When the animal is a swine animal, the modified chromosomal sequence may include exon 7 of the gene encoding the CD163 protein, exon 8 of the gene encoding the CD163 protein, exon 7 or exon 8 of the gene encoding the CD163 protein, Combinations may include variations. The modified chromosomal sequence suitably includes modifications to exon 7 of the gene encoding the CD163 protein.

Deformation in exon 7 of the gene encoding the CD163 protein may include deletion (e. G., In-frame deletion in exon 7). Alternatively, modifications in exon 7 of the gene encoding the CD163 protein may comprise insertion.

In the case where the animal is a pig animal, the modification is 11 base pair deletion of 3,137 nucleotides 3,147 nucleotides compared to reference sequence SEQ ID NO: 47; Two base pair insertions of nucleotides 3,149 and 3,150 compared to reference sequence SEQ ID NO: 47 and 377 base pair deletions of nucleotides 2,573 to 2,949 compared to reference sequence SEQ ID NO: 47 on the same allele; Deletion of 124 base pairs of nucleotides 3,024 to 3,147 compared to reference sequence SEQ ID NO: 47; 123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47; Insertion of one base pair between nucleotides 3,147 and 3,148 compared to reference sequence SEQ ID NO: 47; 130 base pairs deletion of 3,030 nucleotides to 3,159 nucleotides relative to reference sequence SEQ ID NO: 47; 132 base pair deletions of 3,030 nucleotides to 3,161 nucleotides relative to reference sequence SEQ ID NO: 47; 1506 base pair deletions of nucleotides 1,525 to 3,030 compared to reference sequence SEQ ID NO: 47; Insertion of 7 base pairs between nucleotide 3,148 and nucleotide 3,149 compared to reference sequence SEQ ID NO: 47; A 1280 base pair deletion of the nucleotide 2,818 to 4,097 nucleotides relative to the reference sequence SEQ ID NO: 47; 1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47; 1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47; 1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides); 28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47; 1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47; 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113; Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47; Or a combination thereof.

Where a pig animal comprises two base pair insertions between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47, the two base pair inserts may include insertion of a dinucleotide AG.

Where a pig animal comprises one base pair insert between nucleotides 3,147 and 3,148 compared to reference sequence SEQ ID NO: 47, one base pair insert may include the insertion of a single adenine residue.

If the pig animal contains seven base pair insertions between nucleotide 3,148 and nucleotide 3,149 compared to reference sequence SEQ ID NO: 47, the seven base pair insert may include insertion of the sequence TACTACT (SEQ ID NO: 115).

Wherein the pig animal comprises a 1930 base pair deletion of 488 nucleotides to 2,417 nucleotides relative to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion beginning at nucleotide 488, and a reference sequence SEQ ID NO: 47 12 base pair insertions may include insertion of the sequence TGTGGAGAATTC (SEQ ID NO: 116), if there is an additional 129 base pair deletion of 3,044 nucleotides 3,172 nucleotides in exon 7 as compared to exon 7.

When a pig animal contains 1382 base pair deletions of 3,113 to 4,494 nucleotides relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by 11 base pair inserts beginning at nucleotide 3,113, the 11 base pair insert May include insertion of the sequence AGCCAGCGTGC (SEQ ID NO: 117).

When the modified chromosomal sequence of the gene encoding the CD163 protein comprises deletion, deletion preferably includes in-frame deletion. Thus, when the animal is a porcine animal, insertions or deletions in the gene encoding the CD163 protein result in 1506 base pair deletions of 1,525 nucleotides to 3,030 nucleotides relative to reference sequence SEQ ID NO: 47; 1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides); 1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47; 123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47; 1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47; 1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47; 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113; Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47; And an in-frame deletion selected from the group consisting of combinations thereof.

In the case where the animal is a pig animal, insertion or deletion has two base pair insertions between nucleotides 3,149 and 3,150 as compared to reference sequence SEQ ID NO: 47 and 377 nucleotides and 2,949 nucleotides on the same allele as compared to reference sequence SEQ ID NO: 47 Base pair deletion; 28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47; And combinations thereof. For example, oocytes, sperm cells, or fertilized eggs have two base pair insertions between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and two base pair insertions on the same allele with nucleotide 2,573 to nucleotides 2,949 377 base pair deletions. Oocytes, sperm cells, or embryos may contain 28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47.

The oocytes, sperm cells, or embryos contain seven base pair insertions between nucleotide 3,148 and nucleotide 3,149 relative to reference sequence SEQ ID NO: 47 and eleven base pair deletions of nucleotide 3,137 to nucleotide 3,147 relative to reference sequence SEQ ID NO 47 .

An oocyte, sperm cell, or embryo, including any of the insertions or deletions described above, may include a chromosomal sequence having a high degree of sequence identity to SEQ ID NO: 47, as well as insertion or deletion. Thus, for example, oocytes, sperm cells, or fertilized eggs may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 95% 99%, at least 99.9%, or 100% sequence identity.

The oocyte, sperm cell or fertilized egg may be a chromosomal sequence comprising SEQ ID NOS: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, . ≪ / RTI > As described further below in the Examples, SEQ ID NOs: 98-114 provide the nucleotide sequence for the region corresponding to the region of wild-type pig CD163 provided in SEQ ID NO: 47 and insert into the pig CD163 chromosome sequence as described herein Or deletion.

For example, an oocyte, sperm cell, or embryo may comprise a chromosomal sequence comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, SEQ ID NOS: 98, 101, 105, 109, 110, 112, 113, or 114 provide nucleotide sequences for in-frame deletion in exon 7 of the porcine CD163 chromosomal sequence.

As another example, the oocyte, sperm cell, or embryo may comprise a chromosomal sequence comprising SEQ ID NO: 103 or 111.

The oocyte, sperm cell, or embryo is infected with two base pair insertions and 377 base pair deletions in another allele of the gene encoding the 11 base pair deletion and CD163 protein encoding one allele of the gene encoding the CD163 protein . ≪ / RTI >

Oocytes, sperm cells, or embryos may contain 123 base pair deletions in one allele of the gene encoding the CD163 protein and 123 base pair deletions in another allele of the gene encoding the CD163 protein.

Oocytes, sperm cells, or fertilized eggs may contain one base pair insert.

Oocytes, sperm cells, or embryos may contain 130 base pair deletions in one allele of the gene encoding the CD163 protein and 132 base pair deletions in another allele of the gene encoding the CD163 protein.

Oocytes, sperm cells, or embryos can contain 1506 base pair deletions.

Oocytes, sperm cells, or embryos can contain seven base pair insertions.

Oocytes, sperm cells, or embryos may contain 1280 base pair deletions in one allele of the gene encoding the CD163 protein and 1373 base pair deletions in another allele of the gene encoding the CD163 protein.

Oocytes, sperm cells, or embryos can contain 1467 base pair deletions.

Oocytes, sperm cells, or embryos may contain 1930 base pair intron 6 deletions of nucleotides 488 to 2417, 12 base pair additions to nucleotides 4,488, and an additional 129 base pair deletions to exon 7.

An oocyte, sperm cell, or embryo can contain 138 base pair deletions in another allele of the gene encoding the 28 base pair deletion and the CD163 protein in one allele of the gene encoding the CD163 protein.

Oocytes, sperm cells, or embryos were cloned into an allele of the gene encoding the CD163 protein in 1720 base pair deletions, 11 base pair insertions and another allele of the gene encoding the CD163 protein . ≪ / RTI >

In any of the breeding methods, the selected animal can be used as a progeny animal.

In any of the breeding methods, the fertilization step can include artificial fertilization.

Populations of animals made by any of the breeding methods are also provided. The population of animals is preferably resistant to infection by viruses such as pathogens, e.g. PRRSV.

Methods of increasing the resistance of livestock animals to infection with pathogens are also provided. The method comprises encoding at least one chromosomal sequence from a gene encoding the CD163 protein such that CD163 protein production or activity is reduced relative to CD63 protein production or activity in a livestock animal that does not contain an edited chromosomal sequence in the gene encoding the CD163 protein Editing. The pathogen preferably comprises a virus (e.g., PRRSV).

Isolated  Nucleic acid

Isolated nucleic acids are provided. The isolated nucleic acid molecule may comprise a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 47; (b) a nucleotide sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion or deletion relative to SEQ ID NO: 47; And (c) the cDNA sequence of (a) or (b).

For example, the isolated nucleic acid may comprise a nucleotide sequence comprising SEQ ID NO: 47.

Alternatively, the isolated nucleic acid may comprise a nucleotide sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 47, wherein the nucleotide sequence has at least one substitution, insertion, or deletion compared to SEQ ID NO: 47 . The isolated nucleic acid may comprise a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.9% sequence identity with the sequence of SEQ ID NO: 47, The nucleotide sequence contains at least one substitution, insertion or deletion relative to SEQ ID NO: 47)

Substitution, insertion, or deletion preferably reduces or eliminates CD163 protein production or activity relative to a nucleic acid that does not comprise substitution, insertion, or deletion.

The isolated nucleic acid may comprise SEQ ID NOS: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,

For example, the isolated nucleic acid may comprise SEQ ID NO: 98, 101, 105, 109, 110, 112, 113,

For example, the isolated nucleic acid may comprise SEQ ID NO: 103 or SEQ ID NO: 111.

The isolated nucleic acid may comprise cDNA.

Additional isolated nucleic acids are provided. The isolated nucleic acid may comprise SEQ ID NOS: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, For example, the isolated nucleic acid may comprise SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, As another example, the isolated nucleic acid may comprise SEQ ID NO: 103 or SEQ ID NO: 111.

It will be apparent that modifications and variations can be made therein 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 present invention.

Example  One: In vitro  Use of the CRISPR / Cas9 system to produce genetically engineered pigs from induced oocytes and embryos

Homing endogens, such as the components in the zinc-finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR- Recent reports describing cleavage suggest that genetic engineering (GE) in pigs can now be more efficient. Targeted homing endonucleases can induce double-strand breaks (DSBs) at specific locations in the genome and provide random mutations through homologous recombination (HR) stimulation or non-homologous end joining (NHEJ) if donor DNA is provided . Targeted modification of the genome through HR can be achieved with homing endonucleases provided that the donor DNA is provided with a targeted nuclease. After inducing specific strains in somatic cells, these cells were used to produce GE pigs for various purposes through SCNT. Therefore, homing endonucleases are a useful tool in producing GE pigs. Among different homing endogenous nucleases, the modified CRISPR / Cas9 system from prokaryotes appears to be an effective approach when used as a defense mechanism. In fact, the Cas9 system contains three components: RNA (~ 20 bases) (cis-inhibited RNA [crRNA]) containing a region complementary to the target sequence, RNA containing a complementary region to the crRNA Activated crRNA [tracrRNA]), and Cas9, an enzyme protein component in this complex. A single guide RNA (gRNA) can be constructed to perform the role of base-cloning crRNA and tracrRNA. The gRNA / protein complex can scan the genome and catalyze DSB in regions complementary to the crRNA / gRNA. Unlike other engineered nuclease, only a short oligomer needs to be designed to construct the reagents needed to target the gene of interest, while a series of cloning steps are required to assemble ZFNs and TALENs.

Unlike current standard methods for gene disruption, the use of engineered nuclease offers the opportunity to use zygotes as a starting material for GE. Standard methods for gene disruption in livestock include subsequent reconstitution of embryos by HR and somatic cell nuclear transfer (SCNT) in cultured cells. Since cloned animals produced from SCNT sometimes show signs of developmental defects, offspring of the SCNT / GE tribe are typically used in the study to avoid perturbed SCNT abnormalities and phenotypes that may occur when protozoans are used in experiments. Taking into account the longer pregnancy period and the higher housing costs of the pigs compared to rodents, there is a time and cost advantage over the reduced demand for breeding. Recent reports have demonstrated that direct infusion of ZFNs and TALENs into porcine zygotes can destroy endogenous genes and produce piglets with the desired mutations. However, only about 10% of the piglets showed double allelic variation of the target gene, and some showed the mosaic genotype. Recent articles have demonstrated that the CRISPR / Cas9 system can induce mutations in developmental embryos and produce GE pigs with higher efficiency than ZFNs or TALENs. However, GE pigs produced from the CRISPR / Cas9 system also possessed the mosaic genotype. In addition, all of the above studies used in vivo induced zygotes for experiments, which require intensive labor and numerous sows to obtain a sufficient number of zygotes.

This example illustrates an efficient approach to using the CRISPR / Cas9 system in producing GE pigs via SCNT followed by injection of in vivo zygote and transformation of somatic cells. Two intrinsic genes (CD163 and CD1D) and one transgene gene (eGFP) were targeted, and only in vitro induced oocytes or zygotes were used for SCNT or RNA injection, respectively. CD163 appears to be required for proliferative infections caused by the swine respiratory syndrome virus, a virus known to cause significant economic losses to the pig industry. CD1D is considered to be a nonclassical primary histocompatibility complex protein and is involved in the presentation of lipid antigens to invariant natural killer T cells. Pigs lacking these genes were designed as agricultural and biological models. The eGFP transgene was used as a target for optimization of preliminary concept-proof experiments and methods.

Materials and methods

Chemicals and reagents . Unless otherwise indicated, all chemicals used in the study were purchased from Sigma.

certain CRISPR  To build gRNA  design

CRISPR the wild-type CD163 within the resulting DSB domain swaps, but the targeting vector was designed to a region within or not unique, a guide RNA in the wild-type CD163 (is described below), and domain-swap targeting vector is not present in exon 7 CD163. The targeting vector is S. blood coming Ness There are only four positions that introduce a single nucleotide polymorphism (SNP) that changes the Spy proto spacer adjacent motif (PAM). All four targets were selected, including:

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

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

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

(SEQ ID NO: 4) TGTAGCCACAGCAGGGACGTcGG (CRISPR 282). PAM can be identified in bold font in each gRNA.

In the case of CD1D mutations, searches for CRISPR targets were arbitrarily limited to cryptic strands within the first 1000 bp of the primary transcript. However, the RepeatMasker [26] ("pig" repetition library) identified repeating elements beginning at base 943 of the primary transcript. The search for the CRISPR target was then limited to the first 942 bp of the primary transcript. Since the last Spy PAM was located at base 873, the search was further restricted to the first 873 bp of the primary transcript. The first target (CRISPR 4800) was chosen because it overlaps the initiation codon located at base 42 in the primary transcript (CCAGCCTCGCCCAGCGACATgGG (SEQ ID NO: 5)). Two additional targets (CRISPR 5620 and 5626) were selected because they were farthest from the first selection in our arbitrarily selected region (CTTTCATTTATCTGAACTCAgGG (SEQ ID NO: 6) and TTATCTGAACTCAGGGTCCCcGG (SEQ ID NO: 7)). These targets overlap. Regarding the initiation codon, the most proximal Spy PAM was located in a simple sequence extensively containing homologous polymer sequences, as determined by visual evaluation. A fourth target (CRISPR 5350) was chosen because, in connection with the first target selection, it was the most proximal target that did not contain a broad homopolymer domain (CAGCTGCAGCATATATTTAAgGG (SEQ ID NO: 8)). The specificity of the designed crRNA was confirmed by searching for similar pig sequences in GenBank. Oligonucleotides (Table 1) Annealing and two expression cassettes, human codon were cloned into the vector containing the optimized p330X S. Ness bring blood (h Spy) Cas9 and 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 initiation codon. eGFP 1 and eGFP 2 Both gRNAs are on the antisense strand and eGFP1 directly targeted the initiation codon. The eGFP 1 gRNA sequence was CTCCTCGCCCTTGCTCACCAtGG (SEQ ID NO: 9) and the eGFP 2 gRNA sequence was GACCAGGATGGGCACCACCCcGG (SEQ ID NO: 10).

Table 1 . Designed crRNAs. Primer 1 and primer 2 were annealed according to the Zhang protocol.

CD163 and CD1D  Synthesis of donor DNA for genes

Both pig CD163 and CD1D were amplified by PCR from DNA isolated from fetal fibroblasts used for late transfection to ensure an isogenic match between the targeting vector and the transfected cell line. Briefly, the LA tag (Clontech) using the forward primer CTCTCCCTCACTCTAACCTACTT (SEQ ID NO: 11) and the back primer TATTTCTCTCACATGGCCAGTC (SEQ ID NO: 12) was used to amplify the 9538 bp fragment of CD163. The fragments were used for DNA sequencing and construction of domain-swapped targeting vectors (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 replacement exon was 315 bp. In addition, subsequent introns have been replaced with modified myostatin intron B that accepts a selectable marker gene that can be removed with Cre-recombinase (Cre), and normal splicing has previously been demonstrated when latent loxP sites are latent ( Wells, private results). The long arm of the construct was 3469 bp and contained the domain swap DS exon. The short arm was 1578 bp and contained exons 7 and 8 (FIG. 1B). These plasmids were used to replace the coding region of exon 7 in the first transfection experiment and enabled the selection of targeting events via selection marker (G418). If targeting is desired, markers can be deleted by Cre-recombinant enzymes. The CD163 DS-targeting vector was then transformed for use in cell lines already containing the SIGLECl gene disrupted by Neo that can not be Cre deleted. In this targeting vector, the Neo cassette, loxP and myomycin intron B are removed and only the DS exon remains in the WT long and short arms (Fig. 1C).

The genomic sequence for the pig CD1D was amplified with LA taq using the forward primer CTCTCCCTCACTCTAACCTACTT (SEQ ID NO: 13) and the back primer GACTGGCCATGTGAGAGAAATA (SEQ ID NO: 14), resulting in a 8729 bp fragment. The fragment was used for DNA sequencing and construction of the targeting vector shown in Fig. The Neo cassette is under the control of a phosphoglycerol kinase (PGK) promoter and has a loxP sequence on its side, which was introduced for selection. The long arm of the construct was 4832 bp, 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 Neo cassettes. If NHEJ recovery occurs incorrectly, exon 3 will be destroyed.

Fetal fibroblast collection

Porcine fetal tissues were harvested at 35 days of gestation to generate cell lines. Two wild type (WT) male and female fetal fibroblast cell lines were established from a large white domestic cross. Male and female fetal fibroblasts previously modified to contain Neo cassettes (SIGLEC1 - / - genetics) were also used in the study. Fetal fibroblasts were slightly modified and collected as described; The pulverized tissue from each fetus was suspended in 20 ml of digestion medium (1 g / L D-glucose [Cellgro] supplemented with 200 units / ml collagenase and 25 Kunitz units / ml DNase I and Dulbecco's -Modified Eagle's medium [DMEM]) at 38.5 [deg.] C for 5 h. After digestion, fetal fibroblasts were washed and incubated with DMEM, 15% fetal bovine serum (FBS), and 40 [mu] g / ml gentamycin. After overnight incubation, cells were trypsinized and stored in liquid nitrogen frozen at-80 C in aliquots in FBS with 10% dimethyl sulfoxide.

Cell Transfection and Genotyping

Transfection conditions were essentially as previously reported. The donor DNA was always used at a constant dose of 1 μg with various amounts of CRISPR / Cas9 plasmids (listed below). Donor DNA was linearized with MLUI (CD163) (NEB) or AFLII (CD1D) (NEB) before transfection. Gender of established cell lines was determined by PCR as previously described before transfection. Both male and female cell lines were transfected and genomic transformation data were analyzed together between transfection. Fetal fibroblast cell lines of similar subculture (2-4) were cultured for 2 days and cultured for 1 day with 1 g / L D-glucose supplemented with 15% FBS, 2.5 ng / ml basic fibroblast growth factor, and 10 mg / ml gentamycin (Cellgro) and L-glutamine in 75% -85% confluency. Fibroblasts were washed with phosphate-buffered saline (PBS) (Life Technologies) and trypsinized. As soon as the cells were removed, cells were resuspended in electroporation medium (75% cytosalts [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) . The cell concentration was quantitated using a hemocytometer. Cells were pelleted at 600 x g for 5 min and resuspended at a concentration of 1 x 10 ⁶ in electroporation medium. Each electroporation used 200 [mu] l cells in a 2 mm gap cuvette with three (1 msec) square wave pulses administered via BTX ECM 2001 at 250 volts. After electroporation, the cells were resuspended in the DMEM described above. For selection, 600 / / ml G418 (Life Technologies) was added after 24 hours of transfection and the medium was replaced on day 7. Colonies were selected at 14 days after transfection. Fetal fibroblasts were plated at 10,000 cells / plate if G418 selection was used and at 50 cells / plate if G418 selection was not used. Fetal fibroblast colonies were collected by applying a 10 mm autoclave cloning cylinder sealed around each colony by autoclave vacuum grease. The colonies are washed with PBS and harvested through trypsin; And resuspended in DMEM culture medium. A portion (1/3) of resuspended colonies was transferred to 96-well PCR plates and the remaining (2/3) cells were cultured in wells of 24-well plates. The cell pellet was resuspended in 6 μl of lysis buffer (40 mM Tris, pH 8.9, 0.9% Triton X-100, 0.4 mg / ml Proteinase K [NEB]) and incubated at 65 ° C for 30 min And then incubated at 85 ° C for 10 min to inactivate proteinase K. [

DS  And for large and small fruiting PCR  Screening

Detection of HR-directed recovery . Mutations to CD163 or CD1D were confirmed using remote PCR. Three different PCR assays were used to confirm HR events: PCR amplification of the region from the CD163 or CD1D sequence of the donor DNA to the left or right intrinsic CD163 or CD1D sequence and the large region of CD163 or CD1D containing the designed donor DNA Lt; / RTI > amplification. An increase in the size of the 1.8 kb (CD1D) or 3.5 kb (CD163) PCR product resulting from the addition of the 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 min followed by 33 cycles at 94 ° C for 30 sec, 50 ° C for 30 sec, and 68 ° C for 7-10 min. LA taq was used for all assays according to the manufacturer's recommendations. The primers are shown in Table 2.

Table 2. Primers used to confirm HR directed repair of CD163 and CD1D

Small deletion assay ( NHEJ ) . Small deletions were detected by PCR amplification of CD163 or CD1D with protruding cleavage sites introduced by the CRISPR / Cas9 system on the side. The size of the amplicon was 435 bp and 1244 bp for CD163 and CD1D, respectively. The lysates from both the embryo and fetal fibroblasts were PCR amplified with LA taq. The PCR conditions for the assay were 33 cycles of 94 ° C for 30 sec, 56 ° C for 30 sec, and 72 ° C for 1 min, followed by an initial denaturation at 95 ° C for 2 min. For genotyping of transfected cells, insertion and deletion (INDEL) were identified by isolating the PCR amplicon by agarose gel electrophoresis. For embryonic genotype analysis, the resulting PCR products were subsequently sequenced by DNA sequencing and the forward primers used in the PCR were used to identify small deletions. The primer information is shown in Table 3.

Table 3. Primers used to identify mutations through NHEJ on CD163 and CD1D

Somatic cell nuclear transfer ( SCNT )

To produce SCNT embryos, sow-induced oocytes (ART, Inc.) or gilt-induced oocytes from a local slaughterhouse were used. The sow-induced oocytes were cultured in mature medium (2.9 mM Hepes, 5 / / ml insulin, 10 ng / ml epidermal growth factor [EGF], 0.5 / / ml porcine follicle stimulating hormone [p-FSH], 0.91 mM pyruvate , 0.5 mM cysteine, 10% porcine follicular fluid, and TCM-199 with 25 ng / ml gentamycin) overnight and transferred to fresh medium after 24 h. After maturation of 40-42 h, cumulus cells were removed from oocytes by vortexing in the presence of 0.1% hyaluronidase. Sow-induced oocytes were matured as described below for in vitro fertilization (IVF). During operation, the operation I medium supplemented with a cell to 7.0 ㎍ / ml Saito new color _{B (0.6 mM NaHCO 3, 2.9} mM Hepes, 30 mM NaCl, 10 ng / ml gentamicin, and TCM- having a 3 mg / ml BSA 199 [Life Technologies], osmotic concentration 305 mOsm). Polar bodies were removed with a portion of the proximal cytoplasm presumed to contain medium II plates and donor cells were placed in the space around the yolk sac using a thin glass capillary. The reconstituted embryos were then seeded onto fusion media (0.3 M mannitol, 0.1 mM CaCl ₂ , 0.1 (1-sec intervals) at 1.2 kV / cm for 30 ls using a BTX Electro cell manipulator (Harvard Apparatus) mM MgCl ₂ , and 0.5 mM Hepes). After fusion, the fused embryos were fully activated with 200 [mu] M Timerosilo for 10 min in the dark and 8 mM dithiothreitol for 30 min. The embryos were then incubated for 14-16 h in a modified porcine zygote medium PZM3-MU1 with 0.5 μM Scriptaid (S7817; Sigma-Aldrich), a histone deacetylase inhibitor as described above.

In Vitro Fertilization IVF )

For IVF, ovaries from pre-adolescent sows, which have not been breed, were obtained from the slaughterhouse (Farmland Foods Inc.). Immature oocytes were aspirated from mid-sized (3-6 mm) follicles using an 18-gauge hypodermic needle attached to a 10 ml syringe. Thereafter, oocytes with uniformly dark cytoplasm and undigested surrounding cumulus cells were selected for maturation. Approximately 50 cumulus cells were incubated with 500 μl of mature medium, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng / ml EGF, 0.5 μg / ml luteinizing hormone (LH), 0.5 μg / ml FSH , 10 ng / ml gentamycin (APP Pharm), and TCM-199 (Invitrogen) with 0.1% polyvinyl alcohol at 38.5 ° C, 5% CO ₂ for 42-44 h in humidified air. At the end of maturation, surrounding cumulus cells were removed from oocytes by vortexing for 3 minutes in the presence of 0.1% hyaluronidase. Thereafter, mature oocytes in vitro were grown in a group of 25-30 oocytes in 50 쨉 l 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 a modified Tris-buffered medium containing 2 mg / ml bovine serum albumin [BSA]). One 100 [mu] l frozen semen pellet was thawed in 3 ml of Dulbecco's PBS supplemented with 0.1% BSA. Frozen WT or fresh eGFP semen were washed by centrifugation at 650 3 g for 20 min in 60% Percoll and for 10 min in modified Tris-buffered medium. In some cases, freshly collected semen heterozygous for the previously described eGFP transgene was washed three times in PBS. The semen pellet was then resuspended in IVF medium to 0.5 x 10 ⁶ cells / ml. 50 microliters of the semen suspension was introduced into the oocyte-bearing spot. Spouses were co-cultured for 5 hours at 38.5 ° C in an atmosphere of 5% CO ₂ in air. After fertilization, the embryos were cultured in PZM3-MU1 at 38.5 ° C and 5% CO ₂ in air.

Embryo implantation

Embryos produced to produce GE CD163 or CD1D pigs were transplanted into surrogate mothers on the first day (SCNT) or on the sixth day (injected sow) after a first standing stern. For transplantation at day 6, the zygote was cultured for another 5 days in PZM3-MU1 in the presence of 10 ng / ml ps48 (Stemgent, Inc.). The embryos were surgically implanted into the bulbous-shoulder-to-abutment of the surrogate moth.

CRISPR / Cas9  RNA for the system In vitro  synthesis

Template DNA for in vitro transcription was amplified using PCR (Table 4). The CRISPR / Cas9 plasmid used for all transfection experiments served as a template for PCR. Cas9 mRNA was produced using MMESSAGE mMACHINE Ultra kit (Ambion) to express Cas9 in the zygote. The polyA signal was then added to Cas9 mRNA using a poly (A) tailing kit (Ambion). The CRISPR guide RNA was produced by MEGAshortscript (Ambion). The quality of the synthesized RNA was visualized on a 1.5% agarose gel and then diluted to a final concentration of 10 ng / [mu] l (both gRNA and Cas9) and dispensed in 3 [mu] l aliquots.

Table 4. Primers used to amplify templates for in vitro transcription.

Designed at the zygote CRISPR / Cas9  Trace injection of system

Messenger RNAs encoding Cas9 and gRNA were injected into the cytoplasm of fertilized oocytes using a FemtoJet microinjector (Eppendorf) at 14 hours post-fixation (presumptive zygote). Microinjection was performed in the operating medium on a heating stage of a Nikon on-line microscope (Nikon Corporation; Tokyo, Japan). The injected zygotes were then transplanted into PZM3-MU1 with 10 ng / ml ps48 until further use.

Statistical analysis

The number of colonies with the modified genome was classified as 1 and the colonies without genomic variation were classified as zero. Differences were measured using PROC GLM (SAS) and a P value of 0.05 was considered significant. The mean was calculated as the minimum-squared equilibrium. Data are expressed as the mean of the mean ± SEM.

result

In somatic cells, CD163 and Of CD1D CRISPR / Cas9 - Mediated  knockout

The efficiency of the four different CRISPR plasmids (guides 10, 131, 256, and 282) targeting CD163 was tested in the donor DNA in the amount of 2 ug / ㎕ (Table 5). CRISPR 282 resulted in significantly higher mean colony formation than CRISPR 10 and 256 treatments (P <0.05). From the above remote PCR assays, large deletions were found, ranging from 503 bp to 1506 bp, instead of the DS through HR as originally intended (Fig. 3a). Previous reports using other DNA-editing systems were unexpected because they showed a much smaller deletion of 6-333 bp using ZFN in pigs. The mix of CRISPR 10 and all four CRISPRs resulted in a larger number of colonies with modified genomes than CRISPR 256 and 282 (Table 5, P < 0.002). Plasmid containing Neo but not homologous to CD163 and transfection with CRISPR10 did not result in colonies representing large deletions. Interestingly, when a donor DNA was introduced without any CRISPR, a single allelic deletion was also detected. This assay appears to underestimate the mutation rate because no possible small deletions by sequencing that could not be detected on the agarose gel in the transfected somatic cells were screened.

Table 5. Efficiency of four different CRISPR plasmids (guides 10, 131, 256, and 282) targeting CD163. Four different CRISPRs were tested in donor DNA in the amount of 2 [mu] g to 1 [mu] g (as shown in FIG. 1).

* 4 + The mix of donor DNA represents an equivalent mix of 0.5 μg of each CRISPR and 1 μg of donor DNA. Donor DNA treatment serves as a non-CRISPR control and 10 + Neo treatment demonstrates that the large deletion observed in CRISPR treatment was only present if CD163 donor DNA was also present.

† ANOVA was performed by comparing the average number of colonies / plates to estimate CRISPR toxicity and the percentage of colonies with modified genomes. P values were 0.025 and 0.0002, respectively. n / a = Statistical analysis was not performed because there was no replicate for this treatment.

• One cluster with HR represents a partial HR event.

^{The ac} superscript character represents a significant difference between the treatment groups for both the average number of colonies / plates and the percentage of colonies with strained genomes (P < 0.05).

The initial goal was to obtain domain swap (DS) -targeting events by HR for CD163, but the CRISPR did not increase the efficiency of the targeted CD163. It should be noted that various combinations of these targeting vectors have been used to modify CD163 by HR by conventional transfection and have resulted in zero targeting events after screening of 3399 colonies (Whitworth and Prather, unpublished results) . We obtained two pigs with a partial DS resulting from HR containing 16 of the 32 mutations for which CRISPR 10 and DS-targeting vectors were to be introduced by transfection into donor DNA.

Thereafter, the efficiency of CRISPR / Cas9-induced mutations was tested without drug selection; The fetal fibroblast cell line used in this study already had integration of Neo-resistant cassette and SIGLEC1 knockout. We also examined whether the ratio of CRISPR / Cas9 and donor DNA increased genomic modification or caused toxic effects at high concentrations. CRISPR 131 was selected for this test because in previous experiments it resulted in an increased percentage of colonies with high numbers of total colonies and strains of genomes. Increasing amounts of CRISPR 131 DNA from 3: 1 to 20: 1 did not have a significant effect on the viability of fetal fibroblast cells. The percentage of colonies with genomes modified by NHEJ was not significantly different between the various CRISPR concentrations, but had the highest number of NHEJs in the 10: 1 ratio (Table 6, P = 0.33). Even with the highest ratio of CRISPR DNA versus donor DNA (20: 1), HR was not observed.

Table 6. Efficiency of CRISPR / Cas9-induced mutations without drug selection. Four different non-donor DNA versus CRISPR 131 DNA were compared in previously transformed cell lines without the use of G418 selection.

^a significant difference (P> 0.05) between the treatment groups for the colonization percent with NHEJ restoration.

^b in the number of the increased variations in the genome of the CRISPR concentration colonies no significant difference (P> 0.33).

Based on this experiment, we attempted targeted destruction of CD1D in somatic cells. Four different CRISPRs were designed and tested in both male and female cells. We were unable to detect CD1D deformation from three of the applied CRISPRs, but the use of CRISPR 5350 did not result in the deformation of CD1D with deletions that were detectable by agarose gel electrophoresis (Table 7) . Interestingly, we were able to obtain donor DNA but not obtain any genetic modification through HR. However, we observed a large deletion similar to the CD163 knockout experiment (Figure 3b). When CRISPR / Cas9 was not used with the donor DNA, no targeted mutation of CD1D with large deletion was detected. Modifications of CD1D from the CRISPR / Cas9-guided targeting were 4/121 and 3/28 of the male and female colonies of each embryonic cell. Only INDELs detectable by agarose gel electrophoresis were included in the transfection data.

Table 7. Four different CRISPRs were tested in donor DNA in the amount of 2 [mu] g to 1 [mu] g (as shown in FIG. 2). Donor DNA treatment served as a non-CRISPR control.

Using GE cells SCNT  Through CD163 and CD1D  Production of pigs

Cells expressing CD163 or CD1D variants were used in SCNT to produce CD163 and CD1D knockout pigs (Figure 3). (CD163), 6 embryos (CD163-No Neo), and 5 embryos (CD1D) were transplanted into CRISPR / Cas9 system transfected male and female embryos SCNT embryos from fibroblasts. Six (CD163), two (CD163-No Neo), and four (CD1D) (Table 9) of the uninfected recipient sows were term pregnancies, 85.7% each. 33.3%, and 80%, respectively. Of the CD163 recipients, 5 gave birth to healthy piglets by caesarean section. One (O044) was naturally delivered. Litter size ranged from 1 to 8. Four pigs were euthanized due to postnatal growth restriction. One piglet was euthanized by a severe palatal rupture. All remaining piglets appear healthy (Figure 3c). Two pig male piglets born from donor DNA and CRISPR 10 transfected fibroblasts described in Figure 3b had a 30 bp deletion in exon 7 adjacent to CRISPR 10 and an additional 1476 bp deletion of the preceding intron, To remove the intron 6 / exon 7 junction (Figure 3e). Genotype and predicted translations are summarized in Table 10. One male piglet and one female bull (4 piglets) were obtained from CD163-No Neo transfection of previously modified SIGLEC1 cells. All five piglets were double knockout for SIGLEC1 and CD163. The male piglet had a 28 bp deletion in exon 7 and a partial deletion of exon 7 and a complete deletion of exon 8 and a double allelic modification of CD163 with a 1387 bp deletion on another allele containing the preceding intron on one allele Thus, intron exon junctions. The female piglet had a double allele mutation of CD163, including 1382 bp deletion and 11 bp insertion in one allele and 1720 bp deletion of CD163 in the other allele. A summary of the CD163 variants and predicted translations can be found in Table 10. A summary of CD1D variants and predicted translations by CRISPR variants can be found in Table 11. Briefly, one female and two male cubs were born and 13 piglets were produced. One piglet died shortly after birth. Twelve of the 13 piglets contained a double allele or homozygous deletion of CD1D (Fig. 3F). One piglet was WT.

Table 8. Embryo transfer data for CD163 .

* CD163 CRISPR NT line represents an embryo produced by NT as a fetal fibroblast line transformed by transfection. The CRISPR injected embryos were IVF embryos injected at 1 cell stage with CD163 guide RNA and CAS9 RNA. The CD163 CRISPR NT-no Neo fetal line represents an embryo generated by NT with previously transformed fetal fibroblasts that were already Neo- resistant transformed by transfection without the use of selectable markers.

† MU refers to a sperm oocyte that has not been sucked and matured by the University of Missouri, as described in the IVF section of Materials and Methods. ART refers to sperm oocytes that have been purchased and matured as described in the SCNT section of Materials and Methods.

Table 9. Embryonic transplantation data for CD1D .

* CD1D CRISPR NT line represents an embryo produced by NT as a fetal fibroblast line transformed by transfection. CRISPR injected embryos were IVF embryos injected with CD1D guide RNA and CAS9 RNA at one cell stage.

† MU refers to a sperm oocyte that has not been sucked and matured by the University of Missouri, as described in the IVF section of Materials and Methods. ART refers to sperm oocytes that have been purchased and matured as described in the SCNT section of Materials and Methods.

Table 10. Genotype and translation predictions for CD163 modified pigs. Some pigs contain variants of the double allele type as well as one allele described and another modified allele that was not amplified by PCR.

* KO, green-out

** Not included because the piglet was euthanized.

† The sequence numbers in this column refer to the sequence numbers for sequences showing INDEL in relation to SEQ ID NO: 47.

^The inserted sequence was TACTACT (SEQ ID NO: 115).

^{b The} inserted sequence was AG.

^The inserted sequence was a single adenine (A) residue.

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

^{e The} inserted sequence was AGCCAGCGTGC (SEQ ID NO: 117).

Table 11. CD1D Genotype and translation prediction for transformed pigs

* KO, green-out

From the porcine zipper CRISPR / Cas9  System efficiency

Based on the targeted destruction of CD163 and CD1D in somatic cells using the CRISPR / Cas9 system, the method was applied to embryogenesis. First, we tested the effectiveness of the CRISPR / Cas9 system in developing embryos. A CRISPR / Cas9 system targeting eGFP was introduced into semen and fertilized zygotes from heterozygous adult worms against the eGFP transgene. After injection, subsequent embryos expressing eGFP were monitored. We tested various concentrations of the CRISPR / Cas9 system and observed the cytotoxicity of the delivered CRISPR / Cas9 system (FIG. 4A); Embryo development after CRISPR / Cas9 injection was lower than in the control group. However, all concentrations of CRISPR / Cas9 tested were effective in generating a variant of eGFP, since no embryo with eGFP expression was found in the CRISPR / Cas9-injected group (Fig. 4b); 67.7% of the non-injected control embryos were green, indicating the expression of eGFP. When individual blastocysts were genotyped, we were able to identify small mutations near the CRISPR binding site (Fig. 4C). Based on toxicity and efficacy, 10 ng / [mu] g of gRNA and Cas9 mRNA were used in the following experiments.

When the CRISPR / Cas9 component designed to target CD163 was introduced into the putative zygote, we observed the targeted transformation of the gene in the subsequent blastocyst. When individual blastocysts were genotyped for CD163 mutations, specific mutations were found in all embryos (100% GE efficiency). More importantly, we were able to find embryos with homozygous or double allelic variants (8/18 and 3/18 respectively) (Fig. 5), and also with mosaic (single allelic variant) genotypes (4/18 embryos) Can be detected. Some embryos (8/10) from the pool were injected with 2 ng / Cas Cas9 and 10 ng / CR CRISPR and no difference in mutagenic efficiency was found. Next, based on the in vitro results, we introduced two CRISPRs that represent different gRNAs that destroy CD163 or CD1D during embryogenesis to induce a specific deletion of the target gene. As a result, we were able to successfully achieve the designed deletion of CD163 and CD1D by introducing two guides. Design deletions are defined as the deletion of the genomic sequence between the introduced two guides. Among the embryos provided with two CRISPRs targeting CD163, all but one embryo induced targeted transformation of CD163. We also found a 5/13 embryo (Fig. 6a) with deletions designed on CD163 and a 10/13 embryo appears to have a variant of CD163 in a homozygous or double allele manner. Targeting CD1D with two CRISPRs was also effective because all embryos (23/23) showed alterations in CD1D. However, we were able to find the designed deletion of CD1D in only two embryos (2/23) (Fig. 6b). We also found 5/23 embryos with the mosaic genotype, but the remaining embryos had homozygosity or dual allelic variability of CD1D. Finally, we tested whether multiple genes could be targeted by the CRISPR / Cas9 system in the same embryo. For this purpose, targeting of both CD163 and eGFP was performed in heterozygous eGFP semen and modified zygote. When the blastocysts from the injected embryos were genotyped for CD163 and eGFP, we found that CD163 and eGFP were successfully targeted during embryogenesis. Sequence analysis results demonstrated that multiple genes could be targeted by introducing multiple CRISPRs and Cas9 (FIG. 6C).

CRISPR / Cas9 CD163 &lt; / RTI &gt; and &lt; RTI ID = CD1D  Production of mutants

Based on our successes from our preliminary in vitro studies, we produced several CRISPR / Cas9-injected zygotes and grafted 46-55 blastocysts per recipient (this figure was derived from our in vitro- Since it has been shown to be effective in producing. We performed four embryo transfers of two each for CD163 and CD1D, and obtained pregnancy for each strain. We produced four healthy piglets with deformation on CD163 (Table 8). All cubs, ie, littermate 67 from recipient sperm ID O083, exhibited homozygosity or dual allelic modification of CD163 (FIG. 7). Two piglets exhibited the designed deletion of CD163 by the two CRISPRs delivered. All young pigs were healthy. In the case of CD1D, a single pregnancy also produced four piglets (166 pups from the recipient calf identification O165): one female and three males (Table 9). One piglet (166-1) had a mosaic mutation of CD1D including a 362 bp deletion in which exon 3 containing the initiation codon was completely removed (Fig. 8). One piglet contains 2 bp mismatches and 6 bp insertions in one allele and a large deletion in the other allele. Two additional piglets had a double allele single bp insertion. No mosaic mutations were detected for CD163.

Review

The increased efficiency of GE pig production can have widespread impact by providing more GE pigs for agriculture and biomedicine. Using the CRISPR / Cas9 system, the above data show that GE pigs with certain mutations can be produced with high efficiency. The CRISPR / Cas9 system has been successfully applied to transform genes in somatic and pre-implantation embryos.

When the CRISPR / Cas9 system was introduced into somatic cells, it successfully induced targeted destruction of our target gene 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 guidance influenced the targeting efficiency. In particular, we could not find any targeted variation of CD1D when CRISPR 5350 and Cas9 were introduced into somatic cells. This suggests that it may be advantageous to design multiple gRNAs and demonstrate their efficiency before producing pigs. The reason for the lack of HR-directed repair in the presence of donor DNA is still unclear. After screening 886 colonies (both CD163 and CD1D) transfected with CRISPR and donor DNA, only one colony had evidence for partial HR events. Our results demonstrate that the CRISPR / Cas9 system works in conjunction with the donor DNA introduced to cause an unexpected large deletion in the target gene, but does not increase HR efficiency for these two specific targeting vectors. However, no specific mechanism for large deletion observations is known. Previous reports from our group suggested that donor DNA could be used effectively with ZFN to induce HR-directed repair. Similarly, we observed an increase in targeting efficiency when donor DNA was used with the CRISPR / Cas9 system, but no complete HR directed repair was observed. In our previous study using ZFN, we observed that partial transformation of the donor DNA was induced after induction of DSB by ZFN, so we observed that the targeted transformation could occur through the combination of HR and NHEJ. One explanation might be that the HR and NHEJ pathways are not independent, but that the DSBs can work together to complete the repair process after being induced by homing endonuclease. Higher concentrations of CRISPR could improve targeting efficiency in somatic cells, but no statistical difference was found in these experimental results. This suggests that CRISPR is a limiting factor in the CRISPR / Cas9 system, but additional verification is required. Targeted cells have been successfully used to produce GE pigs via SCNT, indicating that the application of CRISPR / Cas9 does not affect the ability of cells to replicate. Some young pigs euthanized for health reasons; However, this is common in SCNT-induced piglets.

When the CRISPR / Cas9 system is introduced into an embryo developed by a zygote injection, nearly 100% embryos and pigs contain INDEL in the targeted gene, demonstrating that the technique is highly effective during embryonic development. The efficiency we observed during our study surpassed the frequency reported in other studies using homing endonuclease during embryogenesis. The reduction in the number of embryos reached at the blastocyst stage suggests that the CRISPR / Cas9 concentration we introduced in this study may be toxic to the embryo. Further optimization of the delivery system can increase the viability of the embryo and thus improve the overall efficiency of the procedure. The nearly 100% mutagenic rates observed here were different from the previous reports in CRISPR / Cas9-mediated knockout of pigs; However, the difference in efficiency between studies can be a combination of selected targets and guides. In our study, lower concentrations of CRISPR / Cas9 (10 ng / ㎕ each) were effective in producing mutants and producing GE pigs in developing embryos. Concentrations are lower (125 ng / l of Cas9 and 12.5 ng / l of CRISPR) than previously reported in porcine zygotes. Introducing excess nucleic acids into developing embryos can be toxic, which may be advantageous for embryos in which lower concentrations of CRISPR / Cas9 components are developed. We have seen some mosaic genotypes in CRISPR / Cas9-injected embryos from our in vitro assay; However, only one piglet produced through the approach had the mosaic genotype. Potentially, injections of the CRISPR / Cas9 component may be more effective than introduction of other homing endonuclease agents since the mosaic genotype was considered to be a major hurdle to using the CRISPR / Cas9 system in the zygote. Another benefit of using the CRISPR / Cas9 system demonstrated by our results is that we did not lose any of the CD163 knockout pigs produced from IVF-induced zygotes injected with the CRISPR / Cas9 system, whereas some of the piglets Was euthanized a few days later. This suggests that the technique can avoid the need for SCNT in producing knockout pigs as well as overcome common health problems associated with SCNT. Since injection of CRISPR / Cas9 mRNA into the zygote has been optimized, future experiments will also include co-injection of donor DNA.

We have demonstrated that introduction of two CRISPRs and Cas9 into the zygote can induce chromosomal deletions in the developing embryo and produce pigs with the intended deletion, that is, specific deletions between the two CRISPR guides. This designed fate can be advantageous because we can specify the size of the deletion rather than relying on a random event caused by NHEJ. In particular, if there is insertion / deletion of nucleotides in multiples of 3 of that caused by homing endonuclease, the mutation may cause a hypomorphic mutation, since no frame shift occurs. However, by introducing two CRISPRs, we can cause larger deletions with higher chances of producing nonfunctional proteins. Interestingly, CD1D CRISPR was designed over a wider region of the genome than CD163; There was a 124 bp spacing between CD163 CRISPR 10 and 131, while a 550 bp spacing between CRISPR 4800 and 5350 for CD1D. Longer intervals between CRISPRs were not as effective in producing deletions as shown in the study. However, further study is needed to verify the relationship between the distance between the CRISPRs and the ability to cause the intended loss, since we only need to consider the efficiency of individual CRISPRs with a limited number of observations and not mentioned here .

The CRISPR / Cas9 system was also effective at simultaneously targeting two genes in the same medium, where the only additional step is to introduce one additional CRISPR and crRNA. This illustrates the ease of destroying multiple genes compared to other homing endogenous nucleases. These results suggest that this technique can be used to target gene clusters or gene families that may have a complementarity effect, thus proving difficult to determine the role of individual genes unless all genes are destroyed. Our results demonstrate that CRISPR / Cas9 technology can be applied to increase the efficiency of gene targeting in somatic cells and to produce GE pigs by direct zygospore injection.

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34. Yoshioka K, Suzuki C, Tanaka A, Anas IM, Iwamura S. Pigments derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 2002; 66: 112-119.

35. Bauer BK, Spate LD, Murphy CN, Prather RS. Arginine supplementation in vitro increases porcine embryo development and affects mRNA transcript expression. Reprod Fertil Dev 2011; 23: 107.

36. Zhao J, Hao Y, Ross JW, Spate LD, Walters EM, Samuel MS, Riekeee, Murphy CN, Prather RS. Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell Reprogram 2010; 12: 75-83.

37. Zhao J, Ross JW, Hao Y, Spate LD, Walters EM, Samuel MS, Riekeee, Murphy CN, Prather RS. Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biol Reprod 2009; 81: 525-530.

38. Whitworth KM, Zhao J, Spate LD, Li R, Prather RS. Scriptaid corrects gene expression in a few aberrantly reprogrammed transcripts in nuclear transfer pig blastocyst stage embryos. Cell Reprogram 2011; 13: 191-204.

39. Whitworth KM, Li R, Spate LD, Wax DM, Riekeee, Whyte JJ, Manandhar G, Sutovsky M, Green JA, Sutovsky P, Prather RS. Method of oocyte activation affects cloning efficiency in pigs. Mol Reprod Dev 2009;

76: 490-500.

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41. Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs, such as Kwon DN, Lee K, Kang MJ, Choi YJ, Park C, Whyte JJ, Brown AN, Kim JH, Samuel M, Mao J, Park KW and Murphy CN. Sci Rep 2013; 3: 1981.

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Example  2: increased resistance to PRRSV in pigs with a chromosome sequence modified to the gene encoding the CD163 protein

The pig genital respiratory syndrome virus (PRRSV) has been infecting the pig industry for the last four and a half decades. The speculation about how to enter the virus included both SIGLEC1 and CD163. SIGLEC1 knockout did not affect response to viral test infection, but we show here that CD163 unreactive animals do not show clinical signs of infection, lung lesions, viremia or antibody production (all of which are characteristic of PRRSV infection) . Not only confirmed the PRRSV entry parameters; If similarly produced animals are allowed to enter the food supply, a significant economic loss and strategies to prevent animal suffering are described.

Materials and methods

Genotypic analysis . Genotype analysis was based on both DNA sequencing and mRNA sequencing. The genotype of the sire had an 11 bp deletion in one allele, which, when translated, predicted 45 amino acids to domain 5, resulting in an immature termination codon in amino acid 64. Another allele had a 2 bp deletion in exon 7 and a 377 bp deletion in the intron preceding exon 7 and predicted the first 49 amino acids of domain 5 when translated, resulting in an immature termination codon in amino acid 85. One sow had a 7 bp addition to one allele, resulting in an immature termination codon in amino acid 70 when translation of the first 48 amino acids of domain 5 was predicted. Other alleles were undeclared because they did not have any band from exon 7 by PCR or by remote 6.3 kb PCR (A). The other three sows were clones and had 129 bp deletion in exon 7 predicted to result in deletion of 43 amino acids from domain 5. Other alleles were undifferentiated (B).

The growth of PRRSV in culture and the production of virus inoculum for swine infection are under approved IBC support 973 . The reference strain, isolate NVRS 97-7895 (GenBank # AF325691 2001-02-11) of PRRSV was grown as described in approved IBC protocol 973. These laboratory isolates have been used for experimental research for about 20 years (Ladinig et al., 2015). The secondary isolate was used for the 2 ^nd test, KS06-72109 as previously described (Prather et al., 2013).

Infection of pigs with PRRSV . A standardized infection protocol for PRRSV was used for infection of pigs. Three week old piglets were inoculated with approximately 10 ^ 4 TCID50 PRRS virus administered intramuscularly (IM) and intranasal (IN) routes. Pigs are monitored daily and pigs that exhibit disease symptoms are treated according to the recommendations of the CMG veterinarian. Pigs who are in severe pain and at risk of falling due to infection are humanely euthanized and collect samples. In order to eliminate prejudice in evaluation or treatment, the step and veterinarians did not know about the heredity of the pigs. PRRSV is present in body fluids during infection; Therefore, blood samples were collected and stored at -80 ° C. until they were measured to determine the amount or severity of viremia in each pig. At the end of the experiment, the pigs were weighed and humanely euthanized, tissues collected, fixed in paraffin-embedded 10% buffered formalin, and licensed pathologists treated for histopathology.

Assessment of Phenotype Score in Infected Pigs . The pig phenotype was assessed 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 health condition of the pig? Health Status Score: 1: Wedge, 2: Dry, 3: Ideal, 4: Fat, 5: Excess Obesity / Obesity. What is the rectal temperature of the pig? Normal body temperature 101.6-103.6 (heat is considered ≥ 104). Do I have legs? Which bridge? Evaluate legs for joint swelling and foot lesion (bottom and side check of hoof). Limping flicker Score: 1: No flickering limping, 2: Do a little choppy when walking, some of the joints are not flicker limping but stiff, 3: flickering light limp, something flickering flew slightly limping, 4: flicker limping moderate, toes Apparent limp, including toe touching lame, 5: severe limping, unable to support weight with legs, and encouragement. Difficulty breathing (grade)? Do you breathe with open mouth? Is there nasal secretion (color of secretion, amount of secretion: weak / medium / severe)? Have you noticed animal coughing? Is there an eye discharge? Respiratory Score: 0: normal, 1: mild respiratory distress and / or respiratory distress when treated (when treated), 2: mild respiratory distress and / or respiratory distress at rest, 3: Severe breathing difficulties and / or shortness of breath, 4: moderate shortness of breath and / or shortness of breath at rest, 5: severe breathing difficulty and / or shortness of breath (when treated), 6: severe breathing difficulty and / . Are there signs of diarrhea (grade) or vomiting? Is there blood or mucus? Diarrhea score: 0: no visible fecal matter, 1: normal stool, 2: soft but shaped stool (soft ice cream yogurt illumination, cow dung), 3: brown / tan liquid diarrhea with particulate fecal matter, Brown / tan liquid diarrhea with no particulate matter, 5: liquid diarrhea that looks like water.

These scoring systems were developed by Dr. Megan Niederwerder of KSU and are based on the following publications (Halbur et al., 1995; Merck; Miao et al., 2009; Patience and Thacker, 1989; Winckler and Willen, 2001 ). Scores and temperatures were analyzed using separate ANOVA based on genotype as treatment.

PRRSV Measurement of viremia . Viralemia was determined through two approaches. Viral titration was performed by adding serial 1:10 dilutions of serum to fusogenic MARC-145 cells in 96-well plates. Serum was diluted in Eagle 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 a cytopathic effect after 4 days of culture using a microscope. The highest dilution showing cytopathic effect was scored as the appropriate endpoint. Total RNA was isolated from the serum using Life Technologies MagMAX-96 viral RNA isolation kit for the determination of viral nucleic acid. Reverse transcription polymerase chain reaction was performed using the EZ-PRRSV MPX 4.0 kit from Tetracore in the CFX-96 real-time PCR system (Bio-Rad) according to the manufacturer's instructions. Each reaction (25 [mu] l) contained RNA from 5.8 [mu] l of serum. The standard curve was created by preparing serial dilutions of the RNA counterpart supplied to the kit (Tetracore). Record the number of templates per PCR.

SIGLEC1 and CD163 staining of PAM cells . Pig alveolar macrophages (PAM) were harvested by incising the lungs and filling them with ~ 100 ml cold phosphate buffered saline. After the phosphate buffered saline was collected, the cells were pelleted and resuspended in 5 ml cold phosphate buffered saline and stored on ice. Approximately 10 ⁷ PAMs were incubated with 5 ml of various antibodies (anti-pig CD169 (clone 3B11 / 11; AbD Serotec), anti-pig CD163 diluted in phosphate buffered saline with 5% fetal bovine serum and 0.1% sodium azide 2A10 / 11; AbD Serotec)) for 30 minutes on ice. Cells were resuspended in 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 determine 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 2500 beads / 50 μl in phosphate buffered saline containing 10% goat serum and placed in the wells of 96-well round bottom polystyrene plates. Serum was diluted 1: 400 in phosphate buffered saline containing 10% goat serum and 50 를 was added to the duplicate well and incubated for 30 minutes with gentle shaking at room temperature. The plate was then washed (3X) with phosphate buffered saline containing 10% goat serum and 50 [mu] l biotin-SP-conjugated affinity diluted to 2 [mu] g / ml in phosphate buffered saline containing 10% Purified goat anti-pig secondary antibody (IgG, Jackson ImmunoResearch) or biotin-labeled affinity purified purified goat anti-pig IgM (KPL) was added. After 30 min incubation the plates were washed (3X) and 50 μl of streptavidin-conjugated picoeritrine (2 μg / ml in phosphate buffered saline containing 10% goat serum (Moss, Inc.)) was added . Plates were washed after 30 minutes and the microspheres were resuspended in phosphate buffered saline containing 100 [mu] l of 10% goat serum and analyzed using MAGPIX and Luminex xPONENT 4.2 software. Record the average fluorescence intensity (MFI).

result

Mutations of CD163 were generated using the CRISPR / Cas9 technology as described above in Example 1. [ Several protozoans were produced from zygote injection and from somatic cell nuclear transfer. Some of these progenitor animals were mated to produce chicks. A single primordial male was mated to females with two genotypes. The male founders (67-1) was added to a 2 bp 11 bp deletion in exon 7 and the other allele in exon 7 alleles were held (and 377 bp deletion in the preceding intron) no reaction Animals (CD163 ^{- / -} ). One progeny female (65-1) had a 7 bp addition and an uncharacterized corresponding allele in exon 7 in one allele and was therefore expected to be heterozygous for knockout ( CD163 ^{- /?} ) . The second protozoan female genotype (3 animals in the clone) contained an alleles with a nonspecific allele and a 129 bp deletion in exon 7. This deletion is predicted to result in the deletion of 43 amino acids in domain 5. The mating between these animals resulted in all of the piglets inherited from one of the 43 amino acid deletions or non-characterized alleles from the unreacted allele and sows from the poultry. In addition to wild-type piglets serving as positive controls for virus challenge infections, this produced four additional genotypes (Table 8).

Table 12. Genotypes tested for resistance to PRRSV test infections (NVSL and KS06 strains).

Genetically modified piglets and wildtype equine-age piglets were transported to Kansas State University for PRRSV test infections at the time of weaning. PRRSV test infection was performed as previously described (Prather et al., 2013). The piglets were sent to the test infections at 3 weeks of age and maintained as a single group. All experiments were initiated following approval of the animal use and biosafety committees. After acclimation, pigs were tested for infection with PRRSV isolate, NVSL 97-7895 (Ladinig et al., 2015) and seeded on MARC-145 cells (Kim et al., 1993). The pigs were infected with a virus of approximately 10 ⁵ TCID ₅₀ . One half of the inoculum was delivered into the muscle and the remainder was delivered into the nasal cavity. All infected pigs were kept in a single group, which allowed continuous exposure of the virus from infected penmates. Blood samples were collected at various days up to 35 days after infection and at the end of the 35th day. Pigs were autopsied and tissues were fixed in paraffin-embedded 10% buffered formalin and processed for histopathology. Clinical signs associated with PRRSV recorded during the infection course included dyspnea, anorexia, lethargy and heat. The results for clinical indications over the course of the study are summarized in FIG. As expected, wild-type Wild ( CD163 + / +) pigs showed early signs of PRRSV infection, peaking between days 5 and 14, and persisted in the group for the remainder of the study. The percentage of febrile pigs peaked at about 10 days. In contrast, unreacted ( CD163 - / -) piglets showed no sign of clinical signs throughout the study period. Respiratory signs during acute PRRSV infection are reflected in significant histopathological changes in the lungs (Table 9). Infection with wild-type pigs showed histopathology consistent with PRRS, including interstitial edema and mononuclear cell infiltration (Figure 10). On the other hand, there was no sign of lung change in unreacted ( CD163 - / -) pigs. Sample sizes for various genotypes are small; Nonetheless, the mean score was 3.85 (n = 7) for wild type, 1.75 (n = 4) for unspecified A, 1.33 (n = 3) for unspecified B, (N = 3).

Table 13. Microscopic Lung Evaluation

The maximum clinical signs correlated with the level of PRRSV in the blood. Measurement of viral nucleic acid was performed by amplification of PRRSV RNA using a commercial reverse transcriptase real-time PRRSV PCR test (Tetracore, Rockville, Md.) Followed by isolation of total RNA from serum. Standard curves were generated by preparing serial dilutions of the PRRSV RNA counterparts provided in the RT-PCR kit, and the results were normalized to 50 [mu] l as template per PCR reaction. PRRSV isolation followed the process for PRRSV viremia in wild-type CD163 + / + pigs (Figure 11). The viremia was evident at 4 days, peaking at 11 days and decreasing 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 unresponsive and nonspecific allele pigs was detectable up to 14 days and increased by 28 days. There was no antibody production in unreacted animals (Figure 12). Together, these data show that wild-type pigs support the occurrence of clinical signs consistent with PRRS and the replication of PRRSV. In contrast, knockout pigs did not develop viremia or clinical signs, even when exposed to infected penmates inoculated with pigs.

At the end of the study, pig alveolar macrophages were removed by lung lavage and surface expression of SIGLEC1 (CD169, clone 3B11 / 11) and CD163 (clone 2A10 / 11) as previously described (Prather et al. Lt; / RTI &gt; Relatively high levels of CD163 expression were detected in CD163 + / + wild-type animals (Figure 13). In contrast, CD163 - / - pigs only showed background-level anti-CD163 staining, thus confirming the knockout phenotype. Expression levels for the other macrophage marker CD169 were similar for both wild type and knockout pigs (Fig. 14). Other macrophage surface markers including MHC II and CD172 were identical for both genotypes (data not shown).

Although the sample size is small, the wild pigs were not affected by the other three genotypic pigs (1.32 kg ± 0.17, n = 4; nonspecific B 1.20 kg ± 0.16, n = 3; 0.16, n = 3) (0.81 kg ± 0.33, n = 7).

In the second trial, six pigs with six wild types, six 43 amino acids, and the non-characterized allele (B) were infected as described above except that piglets were inoculated with KS06-72109. Similar to NVSL data, wild-type and non-wild-type B pigs developed viremia. However, in the? 43 amino acid pig, KS06 did not cause viremia (FIG. 15; Table 7).

Results and conclusions

The most clinically relevant disease in the pig industry is PRRS. Although vaccination programs have been successful in preventing or ameliorating most swine pathogens, PRRSV has proved to be rather infectious. Where CD163 is identified as an entry mediator for these viral strains. Shijo pigs were produced by injecting CRISPR / Cas9 into the zygote (Whitworth et al., 2014) and thus have no transgene. In addition, one of the alleles from sows (also produced using CRISPR / Cas9) contains no transgene. Thus, piglet # 40 has a 7 bp deletion in one allele and an 11 bp deletion in the other allele, but no transgene. The virus-resistant allele of this CD163 represents the minimum genomic editing considering that the porcine genome is approximately 2.8 billion bp (Groenen et al., 2012). If similarly produced animals are introduced into the food supply, significant economic losses can be avoided.

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

In view of the foregoing, it will be appreciated that several objects of the invention are achieved and other advantageous results are obtained.

As various changes may be made in the above products and methods without departing from the scope of the invention, all of the problems contained in the above description and shown in the accompanying drawings are to be construed as illustrative rather than limiting.

Sequence table

<110> Curators of the University of Missouri          Prather, Randall          Wells, Kevin          Whitworth, Kristin <120> PATHOGEN-RESISTANT ANIMALS HAVING MODIFIED CD163 GENES <130> UMO 16001.USP <160> 117 <170> KoPatentin 3.0 <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 tttttttatt cctaatgtaa 180 ttagttcaaa acaaagtgca gaaatttaaa atattcaatt caacaacagt atataagtca 240 atattccccc cttaaatttt tacaaatctt tagggagtgt ttctcaattt ctcaatttct 300 ttggttgttt catgtcccat atggaagaaa acatgggtgt gaaagggaag cttactcttt 360 tgattacttc ccttttctgg ttgactccac ctccattatg aagcctttct gtatttttgt 420 ggaagtgaaa tgatttttag aattcttagt ggttctcttc ttcaggagaa catttctagg 480 taataataca agaagattta aatggcataa aaccttggaa tggacaaact cagaatggtg 540 ctacatgaaa actctggatc tgcaggtaaa atcttctcat ttattctata tttacctttt 600 aaatagagtg tagcaatatt ccgacagtca atcaatctga tttaatagtg attggcatct 660 ggagaagaag taacagggaa aaaggcaata agctttataa ggggaacttt tatcttccat 720 agactcaaaa ttgaagacgt gactagaaga ttgctagatt tggcatcagt tttgtaaaat 780 tgctgaggtg aaattaagta agggatgaaa attaactaaa ttgtgttgag tatgaaacta 840 gtagttgtta gaaaagatag aacatgaagg aatgaatatt gattgaaagt tgatgaccta 900 gaggacattt agactaacac ctctgagtgt caaagtctaa tttatgattt acatcgatgc 960 gttaaactca tttaacattc ttactttttt cccctcaagc atttaagctg aagtataaca 1020 tttcacatga aagcctggat tataaatgca cagttcagtg acctatctca gaggagtgac 1080 tgccatagca ttttttttgt ctttttgcct tcagagccac agcaacgcgg gatccgaagc 1140 cgcgtctgcg acccacacca cagctcacgg caatgccgga tctttaaccc actgagcgag 1200 gccggggatc gaacccgcag tctcatggtt cctagtagga ttcgttaacc actgcgccac 1260 gacgggaact cctaccatag catttttact tttaagttac tgttggttta gagtaagaag 1320 gagaaatgag agtgatggag cgtttgctat atttggagac aaggtcctat attggaggtt 1380 ctcaaatata aattttgtcg ctttttcctc caatgtattg ttcaactact atttagcagg 1440 ccactgtgcc aggtactggt gaaactggtg aacatgatag atgtaattca ttccctcatg 1500 gaactttcca tctaacaatg tggatcaggt aggcttggag atgagaatgc cagtggttga 1560 ctatgactct gtggctgaag ggagagctac tcacttcgta gtttcatcaa tgtctttttg 1620 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 atgtgggttc 1860 aatctctggc ctcattcagt gagtgggtta aggatctggt gtcgctgcaa gctgtggcta 1920 agatcccaca ttgccatgtc tgtggtgtag actggcacct ggagctctga tttgaccaca 1980 atcttaggaa cttcagatgt ggccataaaa aggaaaaaaa agttaggaag 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 gacaagaaac 2280 actgaaaagc aaataattgc tcctgaagtc tagatagcat ctaaaaacat gcttcatggt 2340 ttcaaggatc atatattgaa accccaggga tcctctagag tcgacctgca gcatgcaggg 2400 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 2460 gggggggggg gggggggggg gggggggggg gggggggggg gtgcataagg aaagactatc 2520 tcaacgtctt attcctcagc ttacattaga tttgaaactc tagtcaccta aaatgcaaat 2580 ctcatttact taccatcaga gatattaatg acctatagaa ttcagcataa ataaagtttc 2640 atgtatggat attagcttat ggttctagtc actgctaatt gaaacctgtg atattgctgt 2700 ttgttttgac tcctatgaaa taacattctc ccattgtacc atggatgggt ccagaaacat 2760 ttctcaaatc ctggcttgaa aaaataaata agtaatctaa agaataataa ttctctactt 2820 gctctttgaa tcttgaccaa ttgctgcatt tacctattgt tacaggagga aaagacaagg 2880 agctgaggct aacgggtggt gaaaacaagt gctctggaag agtggaggtg aaagtgcagg 2940 aggagtgggg aactgtgtgt aataatggct gggacatgga tgtggtctct gttgtttgta 3000 ggcagctggg atgtccaact gctatcaaag ccactggatg ggctaatttt agtgcaggtt 3060 ctggacgcat ttggatggat catgtttctt gtcgagggaa tgagtcagct ctctgggact 3120 gcaaacatga tggatgggga aagcataact gtactcacca acaggatgct ggagtaacct 3180 gctcaggtaa gacatacaca aataagtcaa gcctatacat gaaatgcttt gtgggaaaaa 3240 atgtatagat gagttaaaaa caaaaaggaa ccagttttct ataagtcatc tagtccatgt 3300 ataaaattac ccaatccatt actaaaagac cacttctggt attttacaca tgacaaagcc 3360 catattaaaa aaaaaaaatt cagaagagat tctgaatgct ataataaatg agcaagtgac 3420 tagcttcaat tttatattag gtcattctac cttctacttc tacatgaaaa tatcataatg 3480 tctaagttaa ttccttgtcc cctttcccaa taaagcactg ctttcatgca ctggcctatg 3540 aatcatgaac tttttgccct ttaactgatg atcaacttac caaatcaaga aataaatatt 3600 cttagcactg atcctttttt gttgttgttg gaggaagaat gttttgcaaa gtagaattgc 3660 ttttttctgt ttaacagtgc tattcatttc atttacatgg tcgttttaat ttataaaaca 3720 tttcataagt ttcacctcat atgcccttac aataactcag gaagttatat gttagacctt 3780 tctgctgaca aatcccagag tcatgtttct gacccagttc agattccttg gcttcccatt 3840 tctctttgct catgtcattg acctttatgc agccctctta cctcccacct ttctattaca 3900 gccatctcc tccataggac tggtgttaga aagtactaat ctctacccag gcattgtggt 3960 gcaatgtggg cagcacaggc tggtatctag aaaaatgctg aagtgaattc cagctcagct 4020 gctcgttaat actatcgttt taagtaagct gttcaatcct ttgaaattca ctttctgagc 4080 actcagtgat ataataaatg tagagctact ggtacactgt ctggtatgta ataggtgtta 4140 ccaattaacc ttagtttcct catgggtcac tggttctcat tacctagaca actcatttct 4200 ctttcttcct ctttctcttt ctccattctc ctcctccttc ttcctcttct tcttgtctgt 4260 tattgttata tcattttgct gagaaagtta agaaataaca actctaacct ctacatcgac 4320 cacctagagc aaagttaaaa ataataata accttgccag actcttacta taattgttgc 4380 tgtctataga gttgactgtt taagttaaga catcagtata tatttttaat ttttgtgttt 4440 tttttttcat acttttacat gaggatcctt tatataagga tgagttaaac aaacttgatt 4500 tttgaagttt atacccctga ggctcaactg cataataata gaaagggatc catagcctct 4560 caaggactta actagtttca tgagttttca gaatctgaat ttctgagatt ctccacccca 4620 attaaagctc aagcctcaga acatatatcc ttctcttggt aaattctatt cttatcacat 4680 gcgtaataat aaaaaagaga gatgttggag acagattttt ttcctcacat tctgtctcta 4740 ctgttttcta ggtgtttgat tctgtgttat ttaacctcag tttgcttatc tgtgaagtag 4800 ggattatggt aataacatat aatgctttat gttgtaaaga ctaaagaaga tagcatatgt 4860 aacacatttg gaacagggaa tgcatatttt gattgtgagc tcttattatt attaccattc 4920 agccctaata aaaatcttgg taagtggaag gctttggatt tcagaacttt taaaatctaa 4980 ttactttttc aaaaaagaac ttcttagggt tttttttttt taaccacaaa gtgtttctat 5040 tttttaggtg tcccaaaatt tcgttccaaa tatctttttc tcagatattt tagtcctcat 5100 agaacaccta gggatagtgg atagagaaaa ttttctttat taaaaagctg ttctttgcta 5160 aaaattgtag caggtacttt tgggaggggg gaaaactttg attcagaaac tgctaagaca 5220 tggagtgttt tgactaattt ttcctcaatt tttaatgttt tttataccat agggtacttt 5280 tgcaaactat tatgcatact tatatatttt tacttttttc ctgtctttta acttccaaat 5340 tcaacttcag acaattattc atgcactaaa ctgtttgtag taagaaagat taaaattaaa 5400 aaattaacca ttcaacaaat gactggtttg ccatttttac tactttgttg tatgaacaat 5460 ttttttttct acaaatgaat actttgagtc tgatttatcc attcctacat 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 tctttctttt tttttttttt gaattttagt 5760 ttctaaactc acttctgaat aaatacaact tctaaattct cgtcttttct ctactctaga 5820 tggatctgat ttagagatga ggctggtgaa tggaggaaac cggtgcttag gaagaataga 5880 agtcaaattt caaggaacgt ggggaacagt gtgtgatgat cacttcaaca taaatcatgc 5940 ttctgtggtt tgtaaacaac ttgaatgtgg aagtgctgtc agtttctctg gttcagctaa 6000 ttttggagaa ggttctggac caatctggtt tgatgatctt gtatgcaatg gaaatgagtc 6060 agctctctgg aactgcaaac atgaaggatg gggaaagcac aattgcgatc atgctgagga 6120 tgctggagtg atttgcttaa gtaaggactg acctgggttt gttctgttct ccatgagagg 6180 gcaaaaaaag gggagtaaaa gtcttaaaag ctcaaactgt taaaaacata atgatgattg 6240 cttcttttat catcttatta ttatctaatt tcaggtcgaa attctagtac ctgtgcagtt 6300 ttttacctta actgaaatta agataaatag gatagggagg aaggatgagc agtgacattt 6360 aggtccaagt catgaggtta gaaggaaatg ttcagagaat agcccattcc ctcagccctc 6420 aaagaaagaa agaaagaaaa agaaaaaaaa aaagaaagct taactagaaa attttgttct 6480 ctggatgttt tagaggcaaa ccatcccttt atcattccta cctacaaagc cttctcttaa 6540 tcacattacc caccctttcc tactatagtc aggggggggg gggggggggg gggggggggg 6600 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 6660 gggggggggg gtgaaaaaag aaccaaacaa tttcaacaaa aaaccaaaca attccaacaa 6720 aattggtcca ataagcaaac ctctagataa atttcagtgc cctggatgtt ttgttaggaa 6780 ctcttcctac aatgcgtgct ttccattctg aaaagtccta tctacttgcc tgatccactt 6840 ctccttccat cctaaacgat tttcagtggt agtatattac tgttgtctct gtctctactt 6900 atatatcttc cccttttcac tcactcctct caggtacagc tcttcagttt gcccttattc 6960 ttgtttcctt gtcaatgact tgttttgtgt ccctcttaca gatggagcag acctgaaact 7020 gagagtggta gatggagtca ctgaatgttc aggaagattg gaagtgaaat tccaaggaga 7080 atggggaaca atctgtgatg atggctggga tagtgatgat gccgctgtgg catgtaagca 7140 actgggaggt ccaactgctg tcactgccat tgtcgagtta acgccagtga gggactggac 7200 acattggctc acacacatac agccatgaca cgatctgctc tatggtccga tgattaaagg 7260 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 7320 gggggggggg gggggggggg gggggggggg ggggggggag aagagctggt ggacatttct 7380 ggaaaggaac caaaacccgg aagggccttg ttcttcagga tttgggatgg attggggag 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 7860 gtatgtttat tttgcagatg gatcagatct ggaactgaga cttaaaggtg gaggcagcca 7920 ctgtgctggg acagtggagg tggaaattca gaaactggta ggaaaagtgt gtgatagaag 7980 ctggggactg aaagaagctg atgtggtttg caggcagctg ggatgtggat ctgcactcaa 8040 aacatcatat caagtttatt ccaaaaccaa ggcaacaaac acatggctgt ttgtaagcag 8100 ctgtaatgga aatgaaactt ctctttggga ctgcaagaat tggcagtggg gtggacttag 8160 ttgtgatcac tatgacgaag ccaaaattac ctgctcaggt aagaatttca atcaatgtgt 8220 taggaaattg cattctactt tcttttacat gtagctgtcc agttttccca gcaccacttg 8280 ttgaagagac tgtcttttct tcatcatata gtcctacatc ctttgtcata aattaattga 8340 ccataggtgt gtgggtttat atctgggctc tctattctgt tcctttgatc tatatgtctg 8400 tttttatgcc agcaccatgc tgttttgatt actatagctt tgtagtatca tctgaagtca 8460 ggaaacatga ttcctccagc tttgttcttc tttctcaaga ttgttttgtc tattcagagt 8520 ttatgttccc atgcagattt aatttttaaa tttatttaat ttttattttt tatttttaat 8580 ttaaattaat ttaaattttt tatttcccaa cgtacagcca agggggccag ggtaaccttt 8640 acatgtatac attaaaaatt tcaggttttt cccccaccca tttctttctg ttggcaagta 8700 aatttttgaa caaagtttcc caatgctttt taaggggaat tcccttgggg gggggggggg 8760 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 8820 gggggggggg gggggggggg gggggggggg gggggggggg agacgaaatt gactatattt 8880 tctttgttgg gaatctttta cagttgtgac aaatctttca catattcttc atttgagtag 8940 tttggagggt tgtctgactg ttttctataa taaatgtccc aagtgctatg aggtaccaca 9000 tttcaaattc taattctacc tgaagctcca aaaagacaaa atgttatagg tcttttcttt 9060 atatctaatt tgcttatggt ttttagccat tgacaatttt tttttcttaa ctcttgaaac 9120 tataacccta tttctaacca aattcatgtt ctatactggc tcttcaaaaa cccaggagat 9180 gggaaagcca gaatctccag tgtttcagct tctgggaagg agcaagtttt taaaaatacc 9240 ctctgggagc taaattccac atgtatctat ggcctaagtg tatgtttatt ttgcagatgg 9300 atcagatctg gaactgagac ttaaaggtgg aggcagccac tgtgctggga cagtggaggt 9360 ggaaattcag aaactggtag gaaaagtgtg tgatagaagc tggggactga aagaagctga 9420 tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa acatcatatc aagtttattc 9480 caaaaccaag gcaacaaaca catggctgtt tgtaagcagc tgtaatggaa atgaaacttc 9540 tcttagggac tgcaagaatt ggcagtgggg tggacttagt tgtgatcact atgacgaaac 9600 caaaattacc tgctcaggta agaatttcaa tcaatgtgtt aggaaaattg cattctactt 9660 tcttttacat gtagctgtcc agttttccca gcaccacttg ttgaaaaaac tgtctttttc 9720 ttcatcatat agtcctacat cccttggcca taaattaatt gaccataagg ggtgtgggtt 9780 taatatccgg ggctcctcaa ttcgggtccc ttggatccta aaagccggtt ttataacccg 9840 acacatggcc tgtttttgac taaataaaac ctttggaaaa caatcccgaa ggtcgaggaa 9900 catggaatcc ccccaacaaa ggaccttctt tccccaaaaa tgcggctcag ccaactcaaa 9960 aagattttat gaatcacaaa ccgcacatta tcttcctaaa attactattc ctatgtttta 10020 atttgcaaag tcattccgat atagttggcg cagagtaact catttagata tccaccccac 10080 cagttcctca ctcaagtaag gggggggggg gggggggggg gggggggggg gggggggggg 10140 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggc 10200 ccccatgtga gattttgtgt gtcctttaag agtggagtct ctatttccca ctgctctctg 10260 gttctcccca aagtaagccc tgctggcttt caaaacttct gggagcttgc cttcttggta 10320 taggactcct gggctaggga gtctaatgtt tggcttagac cccttactgc ttgggaagaa 10380 tctctgcaac tgtaatgaat tatcttccta tttgtgggtt gctgaggata tggtcttaac 10440 tgttctgtgt tctacccctc ctatccatct tgttgtggtt ccttctttat atctttagtt 10500 gtagaaaagt ttttcttatc aacagttgct ctgtaaattg taacttgggt gtacacctag 10560 taggaggtga gctcagggtc ttcctactct gccatcttgg ccatgtcctc taaacatttt 10620 ggtgtatttc actgcaacct ttttaaaaat ctcaaaagtg agctgtgatt ggctagtctt 10680 gtggataatc tctagcattt gatgctaatc atatttatac aaatactttg ttgaaaagtg 10740 atgccttttt aactattatt aaaaaacgta ttgacataac tattgctatt atactgaaaa 10800 gaaagacctt agagaaaata gcataagagc aaaaccatta aacatggaga catctagtca 10860 tagggtggaa attttatgtg gtccatatcc cctaaccagt ggctttacac caggcacatc 10920 ctaactaaga tctgctccca agtgtcttcc ctgatgcttt aaattgtgtt acatggaaac 10980 tatcctttga tgaagaaatg caacctttta aaatacaaca ttgaaacttt tgtgctttaa 11040 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 cagagtattc acatggttta aacttcaaaa tattaaagtg taaactcttt ccccaccact 11340 gtccccagct caccaactct acttaccaca gacaactgat gtggttaggg tatttaaata 11400 gtaaatccaa gaaaatataa acaaatccgt atatataggt ttcaccccat tttattatcc 11460 taatgttgca tatcatataa actatactgt cccttgggta ttcacttagt aaaatatttt 11520 gatcataatt tcctatcagt atttaaagag ctttctgaaa ttatttctgt ataacatttc 11580 ttttctcatc ggtagggggg gggggggggg gggggggggg gggggggggg gggggggggg 11640 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggaatggg 11700 aagaaaaaac caccatggtt aatttttttt atccctctac acccgggaaa attacccttg 11760 gggccacact tttctataga aaggggatta tttaaaaggg tctgaaaaag aatttttttt 11820 tcgaaagggg aaatatttgg cctaacttag tcacataagc catgttctct ggcaagttag 11880 gtaacataca tttttgtcat 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 atggccacat ctgtggcatg tgaagttccc aggctagggg tcgaatagga 12180 gctacagctg ccagcttgca ccacagccac aacaatgcca gagccaagcc tcatctgcga 12240 cctataccac aactcatggc aatgctggtt ccttaacccc ctgagtgagg cctggggtca 12300 aacccacatc ctcatggata ctaaccggct ttgttaccgc tgagccatga gggaaactcc 12360 ctttttctca ttgaaaataa gtcaaataga taagcagctt aaggctgttt gggtgattct 12420 gtggtccagt aattatcaaa tcctactgga caagaataga gaatgtgcaa atgagggaac 12480 gtgttggtga gatcaggctc tgcccactga gctatcctct gtcatgggcc ctgtgctgtt 12540 ctcagagctg tacttcctag ggcattgttc tcatttcaat tctgagttca gtgtggagag 12600 tatacgtgtg tgggggctgc acgcttttca caacccactt tctgctgata ctgatttagg 12660 gatccttgga ttgctttaca gttgagtcat cattaactag tgtcacttgc cttcaaagtc 12720 agcaaaataa ttgtctccaa actagtaggc ttctagtgta tttgctttaa tccaatgcca 12780 tgtgaaagta acatggtcaa agaataagtt atataccttg acctaccctg tgaccaggct 12840 cttcctctta atttattgac cactgcctta aggtcatttg aaaccatggg tttgggagga 12900 aggcaaggcc taaatcccgt ctttgttgga aggctcactg tccttgtctt tagagcatca 12960 ttttttttta aactggggta cagtttattt acagtgttgt gtcaatttct gctgtacagc 13020 acagtgaccc agtcatacac atacatacat tctttttctc atactatctt caattttatt 13080 ttctgctaag tctgccattt tatcatcacc tcagtttgaa ggacaggata tttagagttt 13140 gttttttttt tccccccaat 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 ggacatggtt ttcttttcaa 13440 ggtggtattt ctctaatgtc acttgaataa caagactcgt tagttctcca ggctacaata 13500 tcctagtctg agtatattct gcatgttaat tctattcagc cacatccata atttaggttt 13560 tattcctgga acacctcact tttttttttt tttttggtct 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 atgataacac 13860 attctgaatt atttaggatt ctattatact gcatgtaata gaaatcccaa atagcaaaat 13920 ttgcaactta aggcaggttc ctgtctttac aaaatcatgt tttcctttgc tatatgtgca 13980 ctttgctttc ctctgtgaat tccctttttt gttatatttc tatagctttt 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 14280 gggaagatga gaaagataat ggcaagaatt tatccctgaa gtgtagtttt gacaaaccag 14340 tcacaaagag gtctaagaaa ttttggtcac aaagttgttt tgaatcccag gcattttatt 14400 tgcaatgatt gcatatgttc tggaaaggac atctgaacct aagaaatagt tcatttgcat 14460 tgtgttatat tttactaagg tctgagaaat aatcttgaga tgagaatgaa ctctacttct 14520 tcagagtctg gaaggaataa attatgaaaa tgtattaatg cttctttaaa ccatattgta 14580 tatttatcta ttactaaaca aaaagaagta gctctattta tttatttatt tatttattta 14640 tttatgtctt ttgtctcttt agggccacac ctgtggcata tggaggttcc caggctagag 14700 gtccaattgg agatgtagca gccagcctat gccagagcca ccgcaacacg ggatctgagc 14760 cacgtctgtg acttacacca cagctcacag caacgcctga tcctcaaccc actgagcgag 14820 gccagggatc gaacccatgt cctcatggat gctagttggg ttcgttaact gctgagccat 14880 gatgggaact ccaaattaat tatttcttat atttgttctt catatattca tttctataga 14940 aagaaataaa tacagattca gttaatgatg gcaggtaaaa gcttaactta ttaatcaaag 15000 gagttaatcc aggcacaaaa attcaattca tggctctctg ttaaaattta ggtataggtt 15060 tagcaggaag aaaaggttag tagatgcaga ctattacatt tagaatggat ggacaatgaa 15120 gtcctactat acagcacagg gaactatatc caatctcttg ggatagaata tgatggaaga 15180 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 gagaaatttg tgggagttcc 15600 catcgtggct cagtggaaac aaatccaact aggaaccatg aggttgtggg tttgatccct 15660 ggcctcactc agtgggttaa ggatccggtg ttgccgtgag ctgtggtgta ggttgcagac 15720 acggttctga tcctgcgttg ctgtggctgt ggctgtggtg taggccagca gcaaacagct 15780 ctgattagac ccctagcctg gaaacctcca tatgccacag gtgcagccct aaaaagacaa 15840 aaaaagagaa aagaoaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag 15900 aacccccaga ggtatttatt tgtttttgcc ttttttcact gactgttctt tgtttgtttg 15960 tttgagactg atctagaaga ctagagatta caagaaatat ggatttggct cactctaaga 16020 aactgctttc attccaaggt ttgggtctat ccaaaagtgg aatagaatca tatgaatact 16080 agtttatgag tatttagtga gaggaatttc aagctcaaat aatgattcag caagattaaa 16140 ttaaggaggg aattttcctt gtggctgagt gggttaagga cccaatgttg tctctgtgag 16200 gatgtaggtt ccatcctggg ctttgctcat taggttaagg atctggcatt gctgcagctc 16260 agacccagtg ctgccctggt tgtggcttag gccaaagctg cagctccaat tcaatctctg 16320 gcctgggaac ctccatgtgc tacaaggtgc ggccttaaaa ggaaaaaaaa aaaattaaat 16380 caaggactca agagtctttc attatttgtg ttgtggaagc tatatttgtt ttaaagtctt 16440 agttgtgttt agaaagcaag atgttcttca actcaaattt gggagggaac ttgtttcata 16500 catttttaat ggataagtgg caaaattttc atgctgaggt gatctatagt gttgtaatgc 16560 agaatatagt cagatcttga acattttagg aagttggtga gggccaattg tgtatctgtg 16620 ccatgctgat aagaatgtca agggatcaca agaattcgtg ttatttgaca 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 17280 ttgaagtaca acatggagac acgtggggca ccgtctgtga ttctgacttc tctctggagg 17340 cggccagcgt gctgtgcagg gaactacagt gcggcactgt ggtttccctc ctggggggag 17400 ctcactttgg agaaggaagt ggacagatct gggctgaaga attccagtgt gaggggcacg 17460 agtcccacct ttcactctgc ccagtagcac cccgccctga cgggacatgt agccacagca 17520 gggacgtcgg cgtagtctgc tcaagtgaga cccagggaat gtgttcactt tgttcccatg 17580 ccatgaagag ggtagggtta ggtagtcaca gacatctttt taaagccctg tctccttcca 17640 ggatacacac aaatccgctt ggtgaatggc aagaccccat gtgaaggaag agtggagctc 17700 aacattcttg ggtcctgggg gtccctctgc aactctcact gggacatgga agatgcccat 17760 gttttatgcc agcagcttaa atgtggagtt gccctttcta tcccgggagg agcacctttt 17820 gggaaaggaa gtgagcaggt ctggaggcac atgtttcact gcactgggac tgagaagcac 17880 atgggagatt gttccgtcac tgctctgggc gcatcactct gttcttcagg gcaagtggcc 17940 tctgtaatct gctcaggtaa gagaataagg gcagccagtg atgagccact catgacggtg 18000 ccttaagagt gggtgtacct aggagttccc attgtggctc agtggtaaca aactcgactg 18060 gtatccatga gggtatgggt ttgatccctg gccttgctca atgggttaag gatccagcat 18120 gt; gtgtaggctg actgctgcag cttcaatttg acccctagcc cgggaatttc cataggccac 18240 acgtgcagca ctaaggaagg aaaaaaagaa aaaaaaaaaa aaagagtggg tgtgcctata 18300 gtgaagaaca gatgtaaaag ggaagtgaaa gggattcccc cattctgagg gattgtgaga 18360 agtgtgccag aatattaact tcatttgact tgttacaggg aaagtaaact tgactttcac 18420 gt; gcctggttga cccatccccc tatctctatg gctatgttta tccagagcac atctatctaa 18540 cactccagct gatcttcctg acacagctgt ggcaaccctg gatcctttaa ccaactgtgc 18600 caggctggag atcaaaccta agcctctgca gcaacccaag ctgctgcagt cagattttta 18660 accccctgtg ccactgtggg tatctccgat attttgtatc ttctgtgact gagtggtttg 18720 ctgtttgcag ggaaccagag tcagacacta tccccgtgca attcatcatc ctcggaccca 18780 tcaagctcta ttatttcaga agaaaatggt gttgcctgca taggtgagaa tcagtgacca 18840 acctatgaaa atgatctcaa tcctctgaaa tgcattttat tcatgtttta tttcctcttt 18900 gcagggagtg gtcaacttcg cctggtcgat ggaggtggtc gttgtgctgg gagagtagag 18960 gtctatcatg agggctcctg gggcaccatc tgtgatgaca gctgggacct gaatgatgcc 19020 catgtggtgt gcaaacagct gagctgtgga tgggccatta atgccactgg ttctgctcat 19080 tttggggaag gaacagggcc catttggctg gatgagataa actgtaatgg aaaagaatct 19140 catatttggc aatgccactc acatggttgg gggcggcaca attgcaggca taaggaggat 19200 gcaggagtca tctgctcggg taagttctgc acatcacttc gggttacagt gatttaagaa 19260 acaactaagg tggggcaaag ggtagtgagg catatccatc agagcaaatt ccttgaaata 19320 cggactcaga gggaaccatt gtgagattga ggttcccaga ggtgtggatt taatgaatta 19380 gtgttacctc atgtacaagg tagtatacta ccagaaagat aaaaattcag aagcgagttt 19440 gcagcaaaac tcatagggag aacttctttt ataaataata tgaagctgga tatttagtgc 19500 accacctgat gaccacttta ttaataaata aagagttcct gttgtggcgc agcggaaatg 19560 aatccgacaa ataatcatga gtttgcgggt ttgatccctg acctcgctca gtgggttggg 19620 gatctggtgt tgccatgagc tgtggtgtag gtcgcagatg ctgcttggat cctgctttgc 19680 tgtggctgtg gtataggctt gtggctacag ctccgatttg accgctagcc tgggaacctc 19740 catatgctgc gggggtggcc ctcaaaagaa aaataaataa ataagtaaat aaataagtag 19800 tttaaaaagg acaagaagaa atatatttgg tgttatattc tacagagaca aagataatca 19860 ccatgcccga ttgatttttc aaggcatata aatgagacgt catgggagca aaaatggtca 19920 taatacaatg cccttgtttt gtgtacatgg taagatttta gaaagcattg tgaggtaaaa 19980 aagtgtactc agttataata tattggggaa aacagtacta tgagaagtaa aaaaatctac 20040 atgccggaag ttattttttt aatgtctctt ttagagtcgc acatgcggca tatggaggtt 20100 cccaggctag gggtcgaatc agagctatag ccactggctt atggcacagc cacaacaaca 20160 ctagatctga gccacatcag cgacctatac tatagctcat ggcaatgcca gatccttaac 20220 ctactgagcc aagccatggg tcaaatccag gtcctcacgg atcctaggca aattcatttc 20280 tgctgagcca cgaagggaac tcctcagaag tgattttgat gttactttct tttcatgaca 20340 aatctggtaa agtacataca catagaaact gaagtgtcag aaagggaaat atttcatttt 20400 aaggtaatgt atacaaaaca gtggttttac catctgagta tctcgctaaa ttttaactat 20460 caaggacaat tgccaaaaaa aaaaaaaaaa 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 ttttaataag ctctaccaag tcatccagaa gctattaaag 20760 aattactggt cactgacata gtgtacctgt tttcaaggcc attcttacat cagaataaag 20820 ggagagcacc ctctgaatct tcagaaaaga tgtgaaagtg ctaattctct atttcatccc 20880 agagttcatg tctctgagac tgatcagtga aaacagcaga gagacctgtg cagggcgcct 20940 ggaagttttt tacaacggag cttggggcag cgttggcaag aatagcatgt ctccagccac 21000 agtgggggtg gtatgcaggc agctaggctg tgcagacaga ggggacatca gccctgcatc 21060 ttcagacaag acagtgtcca ggcacatgtg ggtggacaat gttcagtgtc ctaaaggacc 21120 tgacacacta tggcagtgcc catcatctcc atggaagaag agactggcca gcccctcaga 21180 ggagacatgg atcacatgtg ccagtgagta tccattcttt agcgccactg ttatcttctg 21240 atctacctaa gcagaagttt tataatctgt agttaatccc tattctacct ggatgatggg 21300 attcattctg tttaatttgg tgtgcaggta ttcagcatca gtgatcattt tcccaaagac 21360 catcatgctc tgatggtctt ctcaaaagtt ctaatcagtt gcttcctccg tgaacagttg 21420 aggagcagag aatatgtaat tcagaatttg actattgaat catcccattt ttctttcaca 21480 tagtcttttg ttgcactgaa tataaggaga gaagcagtca gaaagatcaa tcctgaatta 21540 tttctccatt ctacatctgt tttaaatttc aaaaaaaaaa attgttatag gtgatttaca 21600 atgtctgtca atttctgctc tacagcaaag tgacccagtt atttacatat acattctgtt 21660 tctcatattt ttaaaccagg agatttctat ctgcctggcg gtttgaggga atttaacatt 21720 atgcatttat gttaacttta ttcacctgat gttttctaag tcatactgag attcttatgc 21780 ccaggatgga atacacctgg tttgctggaa agacatgtgc tttcataaag acgaattttg 21840 gaaaaaatat aaaatttaaa aggcccatta aataagcaaa gttttaagag atttcaaaaa 21900 aaatttcatc tctctctttt cctctttgac ctcttgggca cgttcatctt ctcaaatatg 21960 atcttggtgt ttctgacttt tcagacaaaa taagacttca agaaggaaac actaattgtt 22020 ctggacgtgt ggagatctgg tacggaggtt cctggggcac tgtgtgtgac gactcctggg 22080 accttgaaga tgctcaggtg gtgtgccgac agctgggctg tggctcagct ttggaggcag 22140 gaaaagaggc cgcatttggc caggggactg ggcccatatg gctcaatgaa gtgaagtgca 22200 aggggaatga aacctccttg tgggattgtc ctgccagatc ctggggccac agtgactgtg 22260 gacacaagga ggatgctgct gtgacgtgtt caggtgaggg cagagagtct ggattgagct 22320 tggaagctct ggcagcaaag agagggtggg cggtgacctg cattgggtaa agattggaag 22380 gtccagccta aggatctggt ggtgggggga gacatgatgt ttcagtctga agaatgatga 22440 aaacctgtgt ggttacgcat gggccttcgc cgaggaaagg gacataacta ccatgtatcc 22500 tcctgcagag ggaggaagaa ctaggggatt ctagttttgt gtgggaagga gcagtttact 22560 tggctcagga ggcactaaag gctcagatag gaaacagaga tctgttccat tcttactccc 22620 agaactgatt ctcttctctt ttctcctaca gaaattgcaa agagccgaga atccctacat 22680 gccacaggta tatcaaaaag tttaagaaca tgggacccat tgtctgcatt ttgtggaatc 22740 cctcttatta agacattctg ggtcagaagt tctgaggatt tgacatttac ttcagctatc 22800 tgttatctta cccaagagag ggatggtaac taggaaccca ggtcttttag ctaagacatt 22860 atcacctctt gtgatgttta cttgttctca ggtcgctcat cttttgttgc acttgcaatc 22920 tttggggtca ttctgttggc ctgtctcatc gcattcctca tttggactca gaagcgaaga 22980 cagaggcagc ggctctcagg tctgaacaaa attacggtct ctctaatgtt tctatgggag 23040 aagaagcctc tctggataat aaaacaaaaa aattacattc aagtatcagt tggccagaaa 23100 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 atgtaaacaa ataaatgggg 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 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23760 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23820 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23880 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23940 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 24000 gggggggggg gggggggggg gggggggggg gggggggggg ggggggggtt ggcgggcccc 24060 cctcgaggtc gacggtatcg ataagcttga tatcgaattc gtgagccaga ggacgagact 24120 agagatggat gatgactacg ttatgcttgc actgctgggg aaaagcacac atagggaggg 24180 aacgttttat tatgacccag tccctaacct atgacctctg ttatcagttt tctcaggagg 24240 agagaattct gtccatcaaa ttcaataccg ggagatgaat tcttgcctga aagcagatga 24300 aacggatatg ctaaatccct caggtccgtg ggttctttga ggggctgtag ccctggggtt 24360 cagatcagca gctgcagttg aggttgaggc atgctacttt gcatagcagt agaaagaaat 24420 ctcaactgta ataggaagct tgggatgcat atgaggaaga aaggcaagaa tgaactacaa 24480 attattctta gggaagataa aaattgcagt catggggaga cctctggctg agagggccgt 24540 gattatttct gacagaggga ttatggagta gaatatgatg gcttggacct 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 cagaaaattc 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 aaaaaaaaaa gaacgtatat acacatatat ttccatgtct atgtttatgt ttgtaaatcc 25440 atattcagaa tatgcaacaa ctttttataa ctatgacttc agtccatctt ttagttacat 25500 atatattcta aacaacaact attgctaaga gaagctgggt aagtaaatgt gaataaatct 25560 tctaaagata ttacaggaag ttcctgctgc ggctcagtgg gttaaagact tgatgtcttt 25620 gtgaagatga gggctcgagc cctggcctca ctcagtgagt taaggatcta gcattgctgt 25680 aagctgcagc gtaggttgca gatagggctc agatccagtg ttgctgtggc tgtggcctca 25740 gttgcagctc tgattcaacc cttaggcgag gaacttccat atgcagcaaa tgtggccatt 25800 aaaaaaaaaaaaaacattat aggagtcatt tcataaaaga gataagacgt ttctatagtt 25860 atatagtgca tactctggta aagatagtat aggatactat aggaatatag aaagcttgcc 25920 tatgaaaatt tgggaagatt gtggaaaaga catctcaaaa tatggcatag aaaagaatca 25980 tatctttgag gaacagtaag tttttcattc aaaaccgtgt attgaacata cttgtggtga 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 26280 gaagattaag ctgaggcaag acacaatcag attgcctttt ataatttact ttcaggagga 26340 aagtctaact aaaaaaagaa ttcgatatca agcttatcga taccgtcgac ctcgaggagg 26400 cccgcctgcc cttttggggg gggggggggg gggggggggg gggggggggg gggggggggg 26460 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggcagca 26520 ccaattttat tattggcggg aataaagaga aaaatgtaat ttcaaagatt gctgttggaa 26580 atgaggggtg tggtagcttt tggagaaagc attctggaga cttctattaa tttttttttt 26640 ttaagtgctt caaagatcct ttgatccaac aattctactc ctaaaaattt cttccataca 26700 gataaagcca tttgtctgta tataacaaat agaagagaat tcctttttgc agccttgtta 26760 gtagtgcccc caaactggaa acaaagtgaa tatcagtcag tggggtagcg gctggaaaaa 26820 ttttagtgca cccaaccaac aaagaaaaac catgcacaaa aattcaataa atatcatctc 26880 acttttgtgt tcatgttatt gaatataatt aaacataatg tttacatcta taaaattatc 26940 atatgatatac atgtaaagaa acattaaaac atttttaaca gactgtaaac ttgaggactg 27000 tgaatgactt ttgattgata atctcaaaca tatggatact attctgatgt aataaataat 27060 gattaaattt tttccctaaa gagtaatcac tactgaatcg ttgcctcaga atcatatgga 27120 ggtgctttta aaaaaggcat ttctgcactg 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 tttctgaatc tcttattggg 27600 cctctaattt aaaaaaaaat atctgggcgc ccgcagatat cgaactcttg ggcagtgtga 27660 ccaaacgaag acatatccaa tcaagcatgc aaatggacca gcccactgta ctagcacgct 27720 gtggcagcca atctgaccga gaaagcagac aaccgcaggg agcaacg 27767 <210> 16 <211> 55 <212> DNA <213> Sus scrofa <400> 16 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatc 55 <210> 17 <211> 54 <212> DNA <213> Sus scrofa <400> 17 ggtcgccacc atggccatga gcaagggcga ggagctgttc accggggtgg tgcc 54 <210> 18 <211> 48 <212> DNA <213> Sus scrofa <400> 18 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggt 48 <210> 19 <211> 55 <212> DNA <213> Sus scrofa <400> 19 ggtcgccacc atggttgagc aagggcgagg agctgttcac cggggtggtg cccat 55 <210> 20 <211> 43 <212> DNA <213> Sus scrofa <400> 20 tgcagggaac tacagtgcgg cactgtggtt tccctcctgg ggg 43 <210> 21 <211> 60 <212> DNA <213> Sus scrofa <400> 21 tgcagggaac tacagtgcgg cactgtaaac cactactact gtggtttccc tcctgggggg 60                                                                           60 <210> 22 <211> 41 <212> DNA <213> Sus scrofa <400> 22 tgcagggaac tacagtgcgg ctgtggtttc cctcctgggg g 41 <210> 23 <211> 46 <212> DNA <213> Sus scrofa <400> 23 tgcagggaac tacagtgcgg aactactgtg gtttccctcc tggggg 46 <210> 24 <211> 31 <212> DNA <213> Sus scrofa <400> 24 gaaacccagg ctggttggag gggacattcc c 31 <210> 25 <211> 24 <212> DNA <213> Sus scrofa <400> 25 gaaacccagg ctggggacat tccc 24 <210> 26 <211> 13 <212> DNA <213> Sus scrofa <400> 26 aggggacatt ccc 13 <210> 27 <211> 13 <212> DNA <213> Sus scrofa <400> 27 gaaacccatt ccc 13 <210> 28 <211> 31 <212> DNA <213> Sus scrofa <400> 28 ggtcgccacc atggtgagca agggcgagga g 31 <210> 29 <211> 32 <212> DNA <213> Sus scrofa <400> 29 ggtcgccacc atggctgagc aagggcgagg ag 32 <210> 30 <211> 29 <212> DNA <213> Sus scrofa <400> 30 ggtcgccacc 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggga gctcactttg 3180 gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc 3240 tttcactctg cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg 3300 gcgtagtctg ctcaagtgag acccagggaa tgtgttcact ttgttcccat gccatgaaga 3360 gggtagggtt aggtagtcac agacatcttt ttaaagccct gtctccttcc aggatacaca 3420 caaatccgct tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt 3480 gggtcctggg ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc 3540 cagcagctta aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga 3600 agtgagcagg tctggaggca catgtttcac tgcactggga ctgagaagca catgggagat 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 gaaaaaaaga 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 gttctgcta 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggcacattc cctgctctgg tcgtgttgaa gtacaacatg 1560 ggacacgtg gggcaccgtc tgtgattctg acttctctct ggaggcggcc agcgtgctgt 1620 gcagggaact acagtgcggc actgtggttt ccctcctggg gggagctcac tttggagaag 1680 gaagtggaca gatctgggct gaagaattcc agtgtgaggg gcacgagtcc cacctttcac 1740 tctgcccagt agcaccccgc cctgacggga catgtagcca cagcagggac gtcggcgtag 1800 tctgctcaag tgagacccag ggaatgtgtt cactttgttc ccatgccatg aagagggtag 1860 ggttaggtag tcacagacat ctttttaaag ccctgtctcc ttccaggata cacacaaatc 1920 cgcttggtga atggcaagac cccatgtgaa ggaagagtgg agctcaacat tcttgggtcc 1980 tgggggtccc tctgcaactc tcactgggac atggaagatg cccatgtttt atgccagcag 2040 cttaaatgtg gagttgccct ttctatcccg ggaggagcac cttttgggaa aggaagtgag 2100 caggtctgga ggcacatgtt tcactgcact gggactgaga agcacatggg agattgttcc 2160 gtcactgctc tgggcgcatc actctgttct tcagggcaag tggcctctgt aatctgctca 2220 ggtaagagaa taagggcagc cagtgatgag ccactcatga cggtgcctta 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 aaaagggaag tgaaagggat tcccccattc tgagggattg tgagaagtgt gccagaatat 2640 taacttcatt tgacttgtta cagggaaagt aaacttgact ttcacggacc tcctagttac 2700 ctggtgctta ctatatgtct tctcagagta cctgattcat tcccagcctg gttgacccat 2760 ccccctatct ctatggctat gtttatccag agcacatcta tctaacactc cagctgatct 2820 tcctgacaca gctgtggcaa ccctggatcc tttaaccaac tgtgccaggc tggagatcaa 2880 acctaagcct ctgcagcaac ccaagctgct gcagtcagat ttttaacccc ctgtgccact 2940 gtgggtatct ccgatatttt gtatcttctg tgactgagtg gtttgctgtt tgcagggaac 3000 cagagtcaga cactatcccc gtgcaattca tcatcctcgg acccatcaag ctctattatt 3060 tcagaagaaa atggtgttgc ctgcataggt gagaatcagt gaccaaccta tgaaaatgat 3120 ctcaatcctc tgaaatgcat tttattcatg ttttatttcc tctttgcagg gagtggtcaa 3180 cttcgcctgg tcgatggagg tggtcgttgt gctgggagag tagaggtcta tcatgagggc 3240 tcctggggca ccatctgtga tgacagctgg gacctgaatg atgcccatgt ggtgtgcaaa 3300 cagctgagct gtggatgggc cattaatgcc actggttctg ctcattttgg ggaaggaaca 3360 gggcccattt ggctggatga gataaactgt aatggaaaag aatctcatat ttggcaatgc 3420 cactcacatg gttgggggcg gcacaattgc aggcataagg aggatgcagg agtcatctgc 3480 tcgg 3484 <210> 99 <211> 4997 <212> DNA <213> Sus scrofa <400> 99 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggctact actactgtgg tttccctcct ggggggagct 3180 cactttggag aaggaagtgg acagatctgg gctgaagaat tccagtgtga ggggcacgag 3240 tcccaccttt cactctgccc agtagcaccc cgccctgacg ggacatgtag ccacagcagg 3300 gacgtcggcg tagtctgctc aagtgagacc cagggaatgt gttcactttg ttcccatgcc 3360 atgaagaggg tagggttagg tagtcacaga catcttttta aagccctgtc tccttccagg 3420 atacacacaa atccgcttgg tgaatggcaa gaccccatgt gaaggaagag tggagctcaa 3480 cattcttggg tcctgggggt ccctctgcaa ctctcactgg gacatggaag atgcccatgt 3540 tttatgccag cagcttaaat gtggagttgc cctttctatc ccgggaggag caccttttgg 3600 gaaaggaagt gagcaggtct ggaggcacat gtttcactgc actgggactg agaagcacat 3660 gggagattgt tccgtcactg ctctgggcgc atcactctgt tcttcagggc aagtggcctc 3720 tgtaatctgc tcaggtaaga gaataagggc agccagtgat gagccactca tgacggtgcc 3780 ttaagagtgg gtgtacctag gagttcccat tgtggctcag tggtaacaaa ctcgactggt 3840 atccatgagg gtatgggttt gatccctggc cttgctcaat gggttaagga tccagcattg 3900 ctgtgagctg tggtataggt tgcagactct gctcaggtcc catgttgctg tgattgtggt 3960 gtaggctgac tgctgcagct tcaatttgac ccctagcccg ggaatttcca taggccacac 4020 gtgcagcact aaggaaggaa aaaaagaaaa aaaaaaaaaa 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 cattttattc atgttttatt tcctctttgc 4680 agggagtggt caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt 4740 ctatcatgag ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca 4800 tgtggtgtgc aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt 4860 tggggaagga acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca 4920 tatttggcaa tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc 4980 aggagtcatc tgctcgg 4997 <210> 100 <211> 3710 <212> DNA <213> Sus scrofa <400> 100 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaagggaa 2820 agggattccc ccattctgag ggattgtgag aagtgtgcca gaatattaac ttcatttgac 2880 ttgttacagg gaaagtaaac ttgactttca cggacctcct agttacctgg tgcttactat 2940 atgtcttctc agagtacctg attcattccc agcctggttg acccatcccc ctatctctat 3000 ggctatgttt atccagagca catctatcta acactccagc tgatcttcct gacacagctg 3060 tggcaaccct ggatccttta accaactgtg ccaggctgga gatcaaacct aagcctctgc 3120 agcaacccaa gctgctgcag tcagattttt aaccccctgt gccactgtgg gtatctccga 3180 tattttgtat cttctgtgac tgagtggttt gctgtttgca gggaaccaga gtcagacact 3240 atccccgtgc aattcatcat cctcggaccc atcaagctct attatttcag aagaaaatgg 3300 tgttgcctgc ataggtgaga atcagtgacc aacctatgaa aatgatctca atcctctgaa 3360 atgcatttta ttcatgtttt atttcctctt tgcagggagt ggtcaacttc gcctggtcga 3420 tggaggtggt cgttgtgctg ggagagtaga ggtctatcat gagggctcct ggggcaccat 3480 ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg 3540 atgggccatt aatgccactg gttctgctca ttttggggaa ggaacagggc ccatttggct 3600 ggatgagata aactgtaatg gaaaagaatc tcatatttgg caatgccact cacatggttg 3660 ggggcggcac aattgcaggc ataaggagga tgcaggagtc atctgctcgg 3710 <210> 101 <211> 3617 <212> DNA <213> Sus scrofa <400> 101 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg 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 tggggaagga acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca 3540 tatttggcaa tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc 3600 aggagtcatc tgctcgg 3617 <210> 102 <211> 4979 <212> DNA <213> Sus scrofa <400> 102 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactctgt ggtttccctc ctggggggag ctcactttgg agaaggaagt 3180 ggacagatct gggctgaaga attccagtgt gaggggcacg agtcccacct ttcactctgc 3240 ccagtagcac cccgccctga cgggacatgt agccacagca gggacgtcgg cgtagtctgc 3300 tcaagtgaga cccagggaat gtgttcactt tgttcccatg ccatgaagag ggtagggtta 3360 ggtagtcaca gacatctttt taaagccctg tctccttcca ggatacacac aaatccgctt 3420 ggtgaatggc aagaccccat gtgaaggaag agtggagctc aacattcttg ggtcctgggg 3480 gtccctctgc aactctcact gggacatgga agatgcccat gttttatgcc agcagcttaa 3540 atgtggagtt gccctttcta tcccgggagg agcacctttt gggaaaggaa gtgagcaggt 3600 ctggaggcac atgtttcact gcactgggac tgagaagcac atgggagatt gttccgtcac 3660 tgctctgggc gcatcactct gttcttcagg gcaagtggcc tctgtaatct gctcaggtaa 3720 gagaagagg 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aagaaggaaa 2580 atattggaat catattctcc ctcaccgaaa tgctattttt cagcccacag gaaacccagg 2640 ctggttggag gggacattcc ctgctctggt cgtgttgaag tacaacatgg agacacgtgg 2700 ggcaccgtct gtgattctga cttctctctg gaggcggcca gcgtgctgtg cagggaacta 2760 cagtgcggca cagtgtggtt tccctcctgg ggggagctca ctttggagaa ggaagtggac 2820 agatctgggc tgaagaattc cagtgtgagg ggcacgagtc ccacctttca ctctgcccag 2880 tagcaccccg ccctgacggg acatgtagcc acagcaggga cgtcggcgta gtctgctcaa 2940 gtgagaccca gggaatgtgt tcactttgtt cccatgccat gaagagggta gggttaggta 3000 gtcacagaca tctttttaaa gccctgtctc cttccaggat acacacaaat ccgcttggtg 3060 aatggcaaga ccccatgtga aggaagagtg gagctcaaca 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 ttttattcat gttttatttc ctctttgcag ggagtggtca acttcgcctg 4320 gtcgatggag gtggtcgttg tgctgggaga gtagaggtct atcatgaggg ctcctggggc 4380 accatctgtg atgacagctg ggacctgaat gatgcccatg tggtgtgcaa acagctgagc 4440 tgtggatggg ccattaatgc cactggttct gctcattttg gggaaggaac agggcccatt 4500 tggctggatg agataaactg taatggaaaa gaatctcata tttggcaatg ccactcacat 4560 ggttgggggc ggcacaattg caggcataag gaggatgcag gagtcatctg ctcgg 4615 <210> 104 <211> 4866 <212> DNA <213> Sus scrofa <400> 104 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttctgtggt ttccctcctg gggggagctc actttggaga 3060 aggaagtgga cagatctggg ctgaagaatt ccagtgtgag gggcacgagt cccacctttc 3120 actctgccca gtagcacccc gccctgacgg gacatgtagc cacagcaggg acgtcggcgt 3180 agtctgctca agtgagaccc agggaatgtg ttcactttgt tcccatgcca tgaagagggt 3240 agggttaggt agtcacagac atctttttaa agccctgtct ccttccagga tacacacaaa 3300 tccgcttggt gaatggcaag accccatgtg aaggaagagt ggagctcaac attcttgggt 3360 cctgggggtc cctctgcaac tctcactggg acatggaaga tgcccatgtt ttatgccagc 3420 agcttaaatg tggagttgcc ctttctatcc cgggaggagc 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 aaaaaaaaaa 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 cctcctgaca 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 attttattca tgttttattt cctctttgca gggagtggtc 4560 aacttcgcct ggtcgatgga ggtggtcgtt gtgctgggag agtagaggtc tatcatgagg 4620 gctcctgggg caccatctgt gatgacagct gggacctgaa tgatgcccat gtggtgtgca 4680 aacagctgag ctgtggatgg gccattaatg ccactggttc tgctcatttt ggggaaggaa 4740 cagggcccat ttggctggat gagataaact gtaatggaaa agaatctcat atttggcaat 4800 gccactcaca tggttggggg cggcacaatt gcaggcataa ggaggatgca ggagtcatct 4860 gctcgg 4866 <210> 105 <211> 4867 <212> DNA <213> Sus scrofa <400> 105 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttactgtgg tttccctcct ggggggagct cactttggag 3060 aaggaagtgg acagatctgg gctgaagaat tccagtgtga ggggcacgag tcccaccttt 3120 cactctgccc agtagcaccc cgccctgacg ggacatgtag ccacagcagg gacgtcggcg 3180 tagtctgctc aagtgagacc cagggaatgt gttcactttg ttcccatgcc atgaagaggg 3240 tagggttagg tagtcacaga catcttttta aagccctgtc tccttccagg atacacacaa 3300 atccgcttgg tgaatggcaa gaccccatgt gaaggaagag tggagctcaa cattcttggg 3360 tcctgggggt ccctctgcaa ctctcactgg gacatggaag atgcccatgt tttatgccag 3420 cagcttaaat gtggagttgc cctttctatc ccgggaggag caccttttgg gaaaggaagt 3480 ggcaggtct ggaggcacat gtttcactgc actgggactg agaagcacat gggagattgt 3540 tccgtcactg ctctgggcgc atcactctgt tcttcagggc aagtggcctc tgtaatctgc 3600 tcaggtaaga gaataagggc agccagtgat gagccactca tgacggtgcc ttaagagtgg 3660 gtgtacctag gagttcccat tgtggctcag tggtaacaaa ctcgactggt atccatgagg 3720 gtatgggttt gatccctggc cttgctcaat gggttaagga tccagcattg ctgtgagctg 3780 tggtataggt tgcagactct gctcaggtcc catgttgctg tgattgtggt gtaggctgac 3840 tgctgcagct tcaatttgac ccctagcccg ggaatttcca taggccacac gtgcagcact 3900 aaggaaggaa aaaaagaaaa aaaaaaaaaa 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 cattttattc atgttttatt tcctctttgc agggagtggt 4560 caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt ctatcatgag 4620 ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca tgtggtgtgc 4680 aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt tggggaagga 4740 acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca tatttggcaa 4800 tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc aggagtcatc 4860 tgctcgg 4867 <210> 106 <211> 4991 <212> DNA <213> Sus scrofa <400> 106 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggcaact gtggtttccc tcctgggggg agctcacttt 3180 ggagaaggaa gtggacagat ctgggctgaa gaattccagt gtgaggggca cgagtcccac 3240 ctttcactct gcccagtagc accccgccct gacgggacat gtagccacag cagggacgtc 3300 ggcgtagtct gctcaagtga gacccaggga atgtgttcac tttgttccca tgccatgaag 3360 caggatacc 3420 acaaatccgc ttggtgaatg gcaagacccc atgtgaagga agagtggagc tcaacattct 3480 tgggtcctgg gggtccctct gcaactctca ctgggacatg gaagatgccc atgttttatg 3540 ccagcagctt aaatgtggag ttgccctttc tatcccggga ggagcacctt ttgggaaagg 3600 aagtgagcag gtctggaggc acatgtttca ctgcactggg actgagaagc acatgggaga 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 aaaaaaaaaa 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 gcccatccc 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggt cctgggggga gctcactttg gagaaggaag 3060 tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc tttcactctg 3120 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 catgggagat 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 aaaaaaaaaa aaaagagtgg gtgtgcctat agtgaagaac agatgtaaaa 3960 gggaagtgaa agggattccc ccattctgag ggattgtgag aagtgtgcca gaatattaac 4020 ttcatttgg 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 tttttgtat 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                                                                         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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggc tggggggagc tcactttgga gaaggaagtg 3060 gacagatctg ggctgaagaa ttccagtgtg aggggcacga gtcccacctt tcactctgcc 3120 cagtagcacc ccgccctgac gggacatgta gccacagcag ggacgtcggc gtagtctgct 3180 caagtgagac ccagggaatg tgttcacttt gttcccatgc catgaagagg gtagggttag 3240 gtagtcacag acatcttttt aaagccctgt ctccttccag gatacacaca aatccgcttg 3300 gtgaggca agaccccatg tgaaggaaga gtggagctca acattcttgg gtcctggggg 3360 tccctctgca actctcactg ggacatggaa gatgcccatg ttttatgcca gcagcttaaa 3420 tgtggagttg ccctttctat cccgggagga gcaccttttg ggaaaggaag tgagcaggtc 3480 tggaggcaca tgtttcactg cactgggact gagaagcaca tgggagattg ttccgtcact 3540 gctctgggcg catcactctg ttcttcaggg caagtggcct ctgtaatctg ctcaggtaag 3600 agaataaggg cagccagtga tgagccactc atgacggtgc cttaagagtg ggtgtaccta 3660 ggagttccca ttgtggctca gtggtaacaa actcgactgg tatccatgag ggtatgggtt 3720 tgatccctgg ccttgctcaa tgggttaagg atccagcatt gctgtgagct gtggtatagg 3780 ttgcagactc tgctcaggtc ccatgttgct gtgattgtgg tgtaggctga ctgctgcagc 3840 ttcaatttga cccctagccc gggaatttcc ataggccaca cgtgcagcac taaggaagga 3900 aaaaaagaaa aaaaaaaaaa 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 caacccaagc tgctgcagtc agatttttaa ccccctgtgc cactgtgggt 4320 atctccgata ttttgtatct tctgtgactg agtggtttgc tgtttgcagg gaaccagagt 4380 cagacactat ccccgtgcaa ttcatcatcc tcggacccat caagctctat tatttcagaa 4440 gaaaatggtg ttgcctgcat aggtgagaat cagtgaccaa cctatgaaaa tgatctcaat 4500 cctctgaaat gcattttatt catgttttat ttcctctttg cagggagtgg tcaacttcgc 4560 ctggtcgatg gaggtggtcg 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gagctgtggt ataggttgca gactctgctc 2460 aggtcccatg ttgctgtgat tgtggtgtag gctgactgct gcagcttcaa tttgacccct 2520 agcccgggaa tttccatagg ccacacgtgc agcactaagg aaggaaaaaa agaaaaaaaa 2580 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 tttatttcct ctttgcaggg agtggtcaac ttcgcctggt cgatggaggt 3240 ggtcgttgtg ctgggagagt agaggtctat catgagggct cctggggcac catctgtgat 3300 gacagctggg acctgaatga tgcccatgtg gtgtgcaaac agctgagctg tggatgggcc 3360 attaatgcca ctggttctgc tcattttggg gaaggaacag ggcccatttg gctggatgag 3420 ataaactgta atggaaaaga atctcatatt tggcaatgcc actcacatgg ttgggggcgg 3480 cacaattgca ggcataagga ggatgcagga gtcatctgct cgg 3523 <210> 110 <211> 3603 <212> DNA <213> Sus scrofa <400> 110 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga 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 gtgttatttg 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 gtgttatttg 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 cagattacaga 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 agaaaaaaaa 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 tttatttcct 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcgtcactt tggagaagga agtggacaga tctgggctga 3180 agaattccag tgtgaggggc acgagtccca cctttcactc tgcccagtag caccccgccc 3240 tgacgggaca tgtagccaca gcagggacgt cggcgtagtc tgctcaagtg agacccaggg 3300 aatgtgttca ctttgttccc atgccatgaa gagggtaggg ttaggtagtc acagacatct 3360 ttttaaagcc ctgtctcctt ccaggataca cacaaatccg cttggtgaat ggcaagaccc 3420 catgtgaagg aagagtggag ctcaacattc ttgggtcctg ggggtccctc tgcaactctc 3480 actgggacat ggaagatgcc catgttttat gccagcagct taaatgtgga gttgcccttt 3540 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 gt; gcccgggaat ttccataggc cacacgtgca gcactaagga aggaaaaaaa gaaaaaaaaa 4020 aaaaaagagt gggtgtgcct atagtgaaga acagatgtaa aagggaagtg aaagggattc 4080 ccccattctg agggattgtg agaagtgtgc cagaatatta acttcatttg acttgttaca 4140 gggaaagtaa acttgacttt cacggacctc ctagttacct ggtgcttact atatgtcttc 4200 tcagagtacc tgattcattc ccagcctggt tgacccatcc ccctatctct atggctatgt 4260 ttatccagag cacatctatc taacactcca gctgatcttc ctgacacagc tgtggcaacc 4320 ctggatcctt taaccaactg tgccaggctg gagatcaaac ctaagcctct gcagcaaccc 4380 aagctgctgc agtcagattt ttaaccccct gtgccactgt gggtatctcc gatattttgt 4440 atcttctgtg actgagtggt ttgctgtttg cagggaacca gagtcagaca ctatccccgt 4500 gcaattcatc atcctcggac ccatcaagct ctattatttc agaagaaaat ggtgttgcct 4560 gcataggtga gaatcagtga ccaacctatg aaaatgatct caatcctctg aaatgcattt 4620 tattcatgtt ttatttcctc tttgcaggga gtggtcaact tcgcctggtc gatggaggtg 4680 gtcgttgtgc tgggagagta gaggtctatc atgagggctc ctggggcacc atctgtgatg 4740 acagctggga cctgaatgat gcccatgtgg tgtgcaaaca gctgagctgt ggatgggcca 4800 ttaatgccac tggttctgct cattttgggg aaggaacagg gcccatttgg ctggatgaga 4860 taaactgtaa tggaaaagaa tctcatattt ggcaatgcca ctcacatggt tgggggcggc 4920 acaattgcag gcataaggag gatgcaggag tcatctgctc gg 4962 <210> 112 <211> 3603 <212> DNA <213> Sus scrofa <400> 112 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcgattcat catcctcgga cccatcaagc tctattattt 3180 cagaagaaaa tggtgttgcc tgcataggtg agaatcagtg accaacctat gaaaatgatc 3240 tcaatcctct gaaatgcatt ttattcatgt tttatttcct ctttgcaggg agtggtcaac 3300 ttcgcctggt 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgattg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcagccagcg 3120 tgctttgcag ggaaccagag tcagacacta tccccgtgca attcatcatc ctcggaccca 3180 tcaagctcta ttatttcaga agaaaatggt gttgcctgca taggtgagaa tcagtgacca 3240 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 gtgggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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 aggaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttatttgac ttgttacagg gaaagtaaac 2460 ttgactttca cggacctcct agttacctgg tgcttactat atgtcttctc agagtacctg 2520 attcattccc agcctggttg acccatcccc ctatctctat ggctatgttt atccagagca 2580 catctatcta acactccagc tgatcttcct gacacagctg tggcaaccct ggatccttta 2640 accaactgtg ccaggctgga gatcaaacct aagcctctgc agcaacccaa gctgctgcag 2700 tcagattttt aaccccctgt gccactgtgg gtatctccga tattttgtat cttctgtgac 2760 tgagtggttt gctgtttgca gggaaccaga gtcagacact atccccgtgc aattcatcat 2820 cctcggaccc atcaagctct attatttcag aagaaaatgg tgttgcctgc ataggtgaga 2880 atcagtgacc aacctatgaa aatgatctca atcctctgaa atgcatttta ttcatgtttt 2940 atttcctctt tgcagggagt ggtcaacttc gcctggtcga tggaggtggt cgttgtgctg 3000 ggagagtaga ggtctatcat gagggctcct ggggcaccat ctgtgatgac agctgggacc 3060 tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg atgggccatt aatgccactg 3120 gttctgctca ttttggggaa ggaacagggc ccatttggct ggatgagata aactgtaatg 3180 gaaaagaatc tcatatttgg caatgccact cacatggttg ggggcggcac aattgcaggc 3240 ataaggagga tgcaggagtc atctgctcgg 3270 <210> 115 <211> 7 <212> DNA <213> Sus scrofa <400> 115 tactact 7 <210> 116 <211> 12 <212> DNA <213> Sus scrofa <400> 116 tgtggagaat tc 12 <210> 117 <211> 11 <212> DNA <213> Sus scrofa <400> 117 agccagcgtg c 11

Claims (158)

A non-human animal or progeny or animal cell comprising at least one modified chromosomal sequence in a gene encoding the CD163 protein. The method according to claim 1, wherein said modification is selected from the group consisting of an animal, a progeny, an offspring of an infection by a pathogenic organism, an offspring of an animal, a progeny, or a cell of a cell that does not contain a chromosome sequence modified to a gene encoding the CD163 protein , Or an animal, offspring, or cell that reduces the sensitivity of the cell. The animal, offspring, or cell according to claim 1 or 2, wherein the pathogen comprises a virus. 4. The animal, progeny, or cell according to claim 3, wherein the virus comprises porcine reproductive and respiratory syndrome virus (PRRSV). The method according to any one of claims 1 to 4, wherein the animal or offspring is an embryo, adolescent, or adult and the cell is an embryo cell, a cell derived from a juvenile animal, or an animal, Or cells. An animal, offspring, or cell according to any one of claims 1 to 4, wherein said animal or offspring comprises a domesticated animal and wherein the cell comprises a cell derived from a domesticated animal. 7. The animal, offspring, or cell according to claim 6, wherein the animal is a livestock animal. The animal, offspring, or cell of claim 7, wherein the animal is selected from the group consisting of a pig animal, a small animal, a sheep animal, a goat animal, a horse and an animal, a water buffalo, a camel, or a bird animal. 9. The animal, progeny, or cell according to claim 8, wherein the small animal comprises beef cattle or cow. 9. The animal, offspring, or cell of claim 8, wherein the bird animal comprises a chicken, a turkey, a duck, a goose, a guinea fowl, or a freshly hatched bird. 9. The animal, progeny, or cell of claim 8, wherein the horse and animal comprise horses or donkeys. 9. The animal according to claim 8, wherein the animal is a cow or pig animal. The animal according to claim 12, wherein said animal is a pig animal, offspring, or cell. An animal, offspring, or cell according to any one of claims 1 to 13, wherein the animal or offspring comprises a genetically edited animal or offspring, or wherein the cell comprises genetically edited cells. 15. The animal, progeny, or cell of claim 14, wherein the animal or cell is genetically edited using a homing endonuclease. 16. The animal, progeny, or cell of claim 15, wherein the animal comprises a homing endonuclease designed with the homing endonuclease. 16. The method of claim 15 or 16, wherein the homing endonuclease is selected from the group consisting of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system, transcription factor-like effector nuclease (TALEN), zinc finger nuclease (ZFN) An animal, a progeny, or a cell comprising a fusion protein, a meganuclease, or a combination thereof. An animal, offspring, or cell according to any one of claims 14 to 17, wherein the animal or cell is genetically edited using the CRISPR / Cas9 system. An animal, offspring, or cell according to any one of claims 14 to 18, wherein said edited animal, offspring, or cell exhibits increased resistance to PRRSV relative to a non-edited animal. The animal, progeny, or cell according to any one of claims 1 to 19, wherein said animal, offspring, or cell is heterozygous for a modified chromosomal sequence. An animal, offspring, or cell according to any one of claims 1 to 19, wherein said animal, offspring, or cell is homozygous for a modified chromosomal sequence. An animal, offspring, or cell according to any one of claims 1 to 21, wherein said modified chromosomal sequence comprises the insertion of a gene encoding a CD163 protein, deletion of a gene encoding a CD163 protein, or a combination thereof. 23. The animal, progeny, or cell of claim 22, wherein the modified chromosomal sequence comprises deletion of a gene encoding the CD163 protein. 24. The animal of claim 23, wherein the deletion comprises an in-frame deletion. The animal, progeny, or cell according to any one of claims 22 to 24, wherein the modified chromosomal sequence comprises the insertion of a gene encoding the CD163 protein. The use according to any one of claims 22 to 25 wherein the insertion or deletion is selected from the group consisting of an animal, a progeny, a progeny, or an animal that reduces CD163 protein production or activity, as compared to CD163 protein production or activity in an animal, Or cells. An animal, offspring, or cell according to any one of claims 22 to 26, wherein said insertion or deletion results in the production of an insignificant functional CD163 protein by an animal, offspring, or cell. The method according to any one of claims 13 to 27, wherein the modified chromosomal sequence comprises exon 7 of a gene encoding a CD163 protein, exon 8 of a gene encoding a CD163 protein, exon 7 or exon 8 of a gene encoding a CD163 protein A porcine animal or cell comprising a modification to an adjacent intron, or a combination thereof. 29. The porcine animal or cell of claim 28, wherein the modified chromosomal sequence comprises a modification of exon 7 of a gene encoding the CD163 protein. 29. The porcine animal or cell of claim 29, wherein the strain encoding the CD163 protein comprises a deletion in the exon 7 deletion. 31. The porcine animal or cell of claim 30, wherein said deletion comprises in-frame deletion in exon 7. [ 29. The porcine animal or cell according to any one of claims 29 to 31, wherein the modification of the gene encoding the CD163 protein into exon 7 comprises insertion. 32. The method according to any one of claims 28 to 32,
11 base pair deletion of nucleotide 3,137 to nucleotide 3,147 compared to reference sequence SEQ ID NO: 47;
Two base pair insertions between nucleotides 3,149 and 3,150 compared to reference sequence SEQ ID NO: 47 and 377 base pair deletions of nucleotides 2,573 to 2,949 compared to reference sequence SEQ ID NO: 47 on the same allele;
Deletion of 124 base pairs of nucleotides 3,024 to 3,147 compared to reference sequence SEQ ID NO: 47;
123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47;
One base pair insert between nucleotides 3,147 and 3,148 compared to reference sequence SEQ ID NO: 47;
130 base pairs deletion of 3,030 nucleotides to 3,159 nucleotides relative to reference sequence SEQ ID NO: 47;
132 base pair deletions of 3,030 nucleotides to 3,161 nucleotides relative to reference sequence SEQ ID NO: 47;
1506 base pair deletions of nucleotides 1,525 to 3,030 compared to reference sequence SEQ ID NO: 47;
Insertion of 7 base pairs between nucleotide 3,148 and nucleotide 3,149 compared to reference sequence SEQ ID NO: 47;
A 1280 base pair deletion of the nucleotide 2,818 to 4,097 nucleotides relative to the reference sequence SEQ ID NO: 47;
1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47;
1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47;
1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides);
28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47;
1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47;
1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113;
Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47;
&Lt; / RTI &gt; and combinations thereof. 34. The porcine animal or cell of claim 33, wherein the two base pair insertions between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 comprise the insertion of a dinucleotide AG. 34. The porcine animal or cell of claim 33, wherein one base pair insert between nucleotides 3,147 and 3,148 relative to reference sequence SEQ ID NO: 47 comprises insertion of a single adenine residue. 34. The porcine animal or cell of claim 33, wherein the 7 base pair insert between nucleotide 3,148 and nucleotide 3,149 relative to reference sequence SEQ ID NO: 47 comprises insertion of the sequence TACTACT (SEQ ID NO: 115). 34. The method of claim 33, wherein the animal comprises a 1930 base pair deletion of 488 nucleotides to 2,417 nucleotides relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insert beginning at nucleotide 488, A porcine animal or cell wherein an additional 129 base pair deletion of 3,044 nucleotides 3,172 nucleotides in exon 7 compared to SEQ ID NO: 47 and a 12 base pair insertion comprises insertion of the sequence TGTGGAGAATTC (SEQ ID NO: 116). 34. The method of claim 33, wherein the animal comprises 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by eleven base pair inserts beginning at nucleotide 3,113, Wherein the dog base pair insert comprises insertion of the sequence AGCCAGCGTGC (SEQ ID NO: 117). 34. The method of claim 33,
1506 base pair deletions of nucleotides 1,525 to 3,030 compared to reference sequence SEQ ID NO: 47;
1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides);
1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47;
123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47;
1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47;
1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47;
1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113;
Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47;
And an exon-7 deletion selected from the group consisting of a combination thereof. 33. The method of claim 33 or 34,
Two base pair insertions between nucleotides 3,149 and 3,150 compared to reference sequence SEQ ID NO: 47 and 377 base pair deletions of nucleotides 2,573 to 2,949 compared to reference sequence SEQ ID NO: 47 on the same allele;
28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47;
&Lt; / RTI &gt; and combinations thereof. 41. The method of claim 40, wherein said animal or cell has two base pair insertions between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47, 377 nucleotides from nucleotide 2,573 to 2,949 nucleotides over the same allele gene Pig animal or cell containing paired deletion. 41. The porcine animal or cell of claim 40, wherein said animal or cell comprises 28 base pair deletions of 3,145 nucleotides 3,172 nucleotides relative to reference sequence SEQ ID NO: 47. 33. The method of claim 33 or 36, wherein the animal or cell is flanked by 7 base pairs between nucleotide 3,148 and nucleotide 3,149 relative to reference sequence SEQ ID NO: 47 and 11 base pairs deletion from nucleotide 3,137 to nucleotide 3,147 relative to reference sequence SEQ ID NO: 47 Lt; / RTI &gt; animal or cell. 47. A porcine animal or cell according to any one of claims 33 to 43, wherein said animal or cell comprises a chromosomal sequence having at least 80% sequence identity to SEQ ID NO: 47 in the region of said chromosomal sequence other than insertion or deletion. 47. The porcine animal or cell of claim 44, wherein said animal or cell comprises a chromosomal sequence having at least 85% sequence identity to SEQ ID NO: 47 in the region of said chromosomal sequence other than insertion or deletion. 44. The porcine animal or cell of claim 44, wherein said animal or cell comprises a chromosomal sequence having at least 90% sequence identity to SEQ ID NO: 47 in the region of said chromosomal sequence other than insertions or deletions. 47. The porcine animal or cell of claim 44, wherein the animal or cell comprises a chromosomal sequence having at least 95% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. 47. The porcine animal or cell of claim 44, wherein said animal or cell comprises a chromosomal sequence having at least 98% sequence identity to SEQ ID NO: 47 in the region of said chromosomal sequence other than insertions or deletions. 47. The porcine animal or cell of claim 44, wherein said animal or cell comprises a chromosomal sequence having at least 99% sequence identity to SEQ ID NO: 47 in the region of said chromosomal sequence other than insertion or deletion. 47. The porcine animal or cell of claim 44, wherein said animal or cell comprises a chromosomal sequence having at least 99.9% sequence identity to SEQ ID NO: 47 in the region of said chromosomal sequence other than insertion or deletion. 47. The porcine animal or cell of claim 44, wherein said animal or cell comprises a chromosomal sequence having 100% sequence identity to SEQ ID NO: 47 in the region of said chromosomal sequence other than insertions or deletions. The method of any one of claims 33 to 51, wherein said animal or cell is selected from the group consisting of SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, Or &lt; RTI ID = 0.0 &gt; 114. &Lt; / RTI &gt; 53. The porcine animal or cell of claim 52 comprising a chromosomal sequence comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, 53. The porcine animal or cell according to claim 52, comprising a chromosomal sequence comprising SEQ ID NO: 103 or SEQ ID NO: The method of any one of claims 33, 34, 40, 41, 44 to 52, and 54, wherein the animal or cell encodes an 11 base pair deletion and an CD163 protein in one allele of a gene encoding the CD163 protein A pig animal or cell containing two base pair insertions and 377 base pair deletions in another allele of the gene. 54. The method of any one of claims 33, 39, and 44-53, wherein said animal or cell is further characterized by the deletion of 124 bases and one allele of the gene encoding the CD163 protein in one allele of the gene encoding the CD163 protein Pigs animals or cells containing 123 base pair deletions in. The pig animal or cell according to any one of claims 33, 35, and 44 to 52, wherein the animal or cell comprises one base pairing. The method of any one of claims 33 and 44 to 52, wherein said animal or cell is selected from the group consisting of 132 alleles of the gene coding for CD163 protein and 130 alleles of the gene encoding CD163 protein and 132 alleles of the gene encoding CD163 protein Pig animal or cell containing a base pair deletion. 33. A porcine animal or cell according to any one of claims 33, 39, and 44-53, wherein said animal or cell comprises a 1506 base pair deletion. The pig animal or cell according to any one of claims 33, 36, and 44 to 52, wherein said animal or cell comprises seven base pair insertions. 33. The method of any one of claims 33, 39, and 44-53, wherein said animal or cell is selected from the group consisting of a 1280 base pair deletion in one allele of the gene encoding the CD163 protein and another allele of the gene encoding the CD163 protein A pig animal or cell containing 1373 base pair deletions in. 33. A porcine animal or cell according to any one of claims 33, 39, and 44-53, wherein said animal or cell comprises 1467 base pair deletions. The method of any one of claims 33, 37, 39, and 44-53, wherein said animal or cell is further provided with a 1930 base pair intron 6 deletion of nucleotide 488 to nucleotide 2,417, a 12 base pair attachment to nucleotide 4,488, and an exon 7 Pigs animals or cells containing 129 base pair deletions of. 33. The method of any one of claims 33, 39, 40, 42, and 44-53, wherein the animal or cell has a 28 base pair deletion in one allele of the gene encoding the CD163 protein and a gene encoding the CD163 protein A pig animal or cell containing 1387 base pair deletions in another allele. 33, 38, 39, and 44 to 53, wherein the animal or cell has 1382 base pair deletions, 11 base pair insertions, and CD163 proteins in one allele of the gene encoding the CD163 protein A pig animal or cell containing a 1720 base pair deletion in another allele of the encoding gene. 65. The cell of any one of claims 1 to 65 wherein the cell is a sperm cell. 66. The cell according to any one of claims 1 to 65, wherein the cell is an egg cell. 65. The cell of claim 67, wherein the oocyte is a fertilized egg. 65. The cell according to any one of claims 1 to 65, wherein the cell is a somatic cell. 71. The cell of claim 69, wherein the somatic cell comprises fibroblasts. 69. The cell of claim 70, wherein the fibroblast comprises fetal fibroblasts. Genetically transforming an oocyte or sperm cell to introduce a modified chromosomal sequence into a gene encoding a CD163 protein into at least one of an oocyte and sperm cell, and a step of genetically transforming the oocyte or sperm cell into a gene encoding a CD163 protein The step of modifying the oocyte with sperm cells to produce an embryo;
Genetically transforming the embryo to introduce a modified chromosomal sequence into the gene encoding the CD163 protein into the fertilized egg;
Implanting the embryo into a surrogate female female animal wherein the pregnancy and term poultry produce offspring;
Screening said offspring for susceptibility to pathogens; And
Selecting a progeny animal that has reduced sensitivity to pathogens compared to an animal that does not contain a modified chromosomal sequence in the gene encoding the CD163 protein, Lt; / RTI &gt; 73. The method of claim 72, wherein the pathogen comprises a virus. 72. The method of claim 73, wherein the virus comprises PRRSV. 72. The method of any one of claims 72 to 74, wherein the animal is an embryo, adolescent, or adult. 72. The method of any one of claims 72 to 75, wherein the animal comprises a rabbit. 75. The method of claim 76, wherein the animal comprises a livestock animal. 77. The method of claim 77, wherein the animal is selected from the group consisting of a pig animal, a small animal, a sheep animal, a goat animal, a horse and an animal, a buffalo, a camel, or an avian animal. 77. The method of claim 78, wherein the bovine animal comprises beef cattle or cow. 78. The method of claim 78, wherein the bird animal comprises a chicken, turkey, duck, goose, guinea fowl, or freshly hatched bird. 77. The method of claim 78, wherein the horse and animal comprise horses or donkeys. 77. The method of claim 78, wherein the animal is a cow or a pig animal. 83. The method of claim 82, wherein the animal is a pig animal. 72. The method of any one of claims 72 to 83, wherein the step of genetically transforming the oocyte, sperm cell, or embryo comprises genetic editing of the oocyte, sperm cell, or embryo. 84. The method of claim 84, wherein said gene editing comprises the use of a homing endonuclease. 94. The method of claim 85, wherein the homing endonuclease comprises a homing endonuclease designed. 83. The method of claim 83 or 84, wherein the homing endonuclease is selected from the group consisting of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 system, a transcriptional activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a recombinant enzyme A fusion protein, a meganuclease, or a combination thereof. 84. The method of any one of claims 84-87, wherein said gene editing comprises the use of a CRISPR / Cas9 system. 72. The method of any one of claims 72 to 88 wherein said oocyte, sperm cell, or embryo is heterozygous for a modified chromosomal sequence. 72. The method of any one of claims 72 to 88, wherein said oocyte, sperm cell, or embryo is homozygous for a modified chromosomal sequence. 72. The method of any one of claims 72 to 90, wherein said modified chromosomal sequence comprises insertion of a gene encoding a CD163 protein, deletion of a gene encoding a CD163 protein, or a combination thereof. The method of claim 91, wherein the modified chromosomal sequence comprises deletion of a gene encoding a CD163 protein. The method of claim 91, wherein the deletion includes an in-frame deletion. 92. The method of any one of claims 91 to 93, wherein said modified chromosome sequence comprises the insertion of a gene encoding a CD163 protein. 92. The method of any one of claims 91 to 94 wherein said insertion or deletion reduces CD163 protein production or activity relative to CD163 protein production or activity in an animal without insertion or deletion. 95. The method of any one of claims 91 to 95 wherein said insertion or deletion results in the production of non-functional functional CD163 protein. 83. The method of any one of claims 83 to 96, wherein the modified chromosomal sequence comprises exon 7 of the gene encoding the CD163 protein, exon 8 of the gene encoding the CD163 protein, exon 7 or exon 8 of the gene encoding the CD163 protein, An adjacent intron, or a combination thereof. The method of claim 97, wherein said modified chromosomal sequence comprises a modification to exon 7 of a gene encoding a CD163 protein. The method of claim 98, wherein the modification of the gene encoding the CD163 protein to exon 7 comprises deletion. The method of claim 99, wherein said deletion comprises an in-frame deletion in exon 7. 98. The method of any one of claims 98-100, wherein the modification of the gene encoding the CD163 protein to exon 7 comprises insertion. 97. The method according to any one of claims 97 to 101,
11 base pair deletion of nucleotide 3,137 to nucleotide 3,147 compared to reference sequence SEQ ID NO: 47;
Two base pair insertions between nucleotides 3,149 and 3,150 compared to reference sequence SEQ ID NO: 47 and 377 base pair deletions of nucleotides 2,573 to 2,949 compared to reference sequence SEQ ID NO: 47 on the same allele;
Deletion of 124 base pairs of nucleotides 3,024 to 3,147 compared to reference sequence SEQ ID NO: 47;
123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47;
One base pair insert between nucleotides 3,147 and 3,148 compared to reference sequence SEQ ID NO: 47;
130 base pairs deletion of 3,030 nucleotides to 3,159 nucleotides relative to reference sequence SEQ ID NO: 47;
132 base pair deletions of 3,030 nucleotides to 3,161 nucleotides relative to reference sequence SEQ ID NO: 47;
1506 base pair deletions of nucleotides 1,525 to 3,030 compared to reference sequence SEQ ID NO: 47;
Insertion of 7 base pairs between nucleotide 3,148 and nucleotide 3,149 compared to reference sequence SEQ ID NO: 47;
A 1280 base pair deletion of the nucleotide 2,818 to 4,097 nucleotides relative to the reference sequence SEQ ID NO: 47;
1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47;
1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47;
1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides);
28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47;
1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47;
1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113;
Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47;
And combinations thereof. 103. The method of claim 102, wherein two base pair insertions between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 comprise insertion of a denucleotide AG. 103. The method of claim 102, wherein one base pair insert between nucleotides 3,147 and 3,148 relative to reference sequence SEQ ID NO: 47 comprises insertion of a single adenine residue. 96. The method of claim 102, wherein the 7 base pair insertion between nucleotide 3,148 and nucleotide 3,149 relative to reference sequence SEQ ID NO: 47 comprises insertion of the sequence TACTACT (SEQ ID NO: 115). 99. The method of claim 102, wherein the oocyte, sperm cell, or embryo comprises a 1930 base pair deletion of nucleotides 488 to 2,417 compared to reference sequence SEQ ID NO: 47, wherein the deleted sequence comprises a base pair of 12 bases starting at nucleotide 488 (SEQ ID NO: 116), with an additional 129 base pair deletion of 3,044 nucleotides 3,172 in exon 7 compared to reference sequence SEQ ID NO: 47 and a 12 base pair insert comprising insertion of the sequence TGTGGAGAATTC . 103. The method of claim 102 wherein the oocyte, sperm cell, or embryo comprises 1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence comprises 11 base pairs starting from nucleotide 3,113 Wherein the 11 base pair insert comprises insertion of the sequence AGCCAGCGTGC (SEQ ID NO: 117). 103. The method of claim 102,
1506 base pair deletions of nucleotides 1,525 to 3,030 compared to reference sequence SEQ ID NO: 47;
1930 base pair deletion of nucleotide 488 to nucleotide 2,417 compared to reference sequence SEQ ID NO 47 wherein the deleted sequence is replaced by 12 base pair insertions starting at nucleotide 488 and nucleotide at exon 7 compared to reference sequence SEQ ID NO: There is an additional 129 base pair deletion of 3,044 to 3,172 nucleotides);
1373 base pair deletions of 2,724 nucleotides and 4,096 nucleotides relative to reference sequence SEQ ID NO: 47;
123 base pair deletion of 3,024 nucleotides to 3,146 nucleotides relative to reference sequence SEQ ID NO: 47;
1467 base pair deletions of nucleotides 2,431 to 3,897 compared to reference sequence SEQ ID NO: 47;
1387 base pair deletions of nucleotides 3,145 to 4,531 compared to reference sequence SEQ ID NO: 47;
1382 base pair deletions of nucleotides 3,113 to 4,494 relative to reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion beginning at nucleotide 3,113;
Deletion of 1720 base pairs of nucleotides 2,440 to 4,160 compared to reference sequence SEQ ID NO: 47;
And a combination thereof. &Lt; / RTI &gt; 102. The method of claim 102 or 103,
Two base pair insertions between nucleotides 3,149 and 3,150 compared to reference sequence SEQ ID NO: 47 and 377 base pair deletions of nucleotides 2,573 to 2,949 compared to reference sequence SEQ ID NO: 47 on the same allele;
28 base pair deletions of nucleotides 3,145 to 3,172 compared to reference sequence SEQ ID NO: 47;
And combinations thereof. The method of claim 109, wherein the oocyte, sperm cell, or fertilized egg has two base pair insertions between nucleotides 3,149 and 3,150 relative to reference sequence SEQ ID NO: 47 and nucleotide 2,573 to 2,949 nucleotides relative to reference sequence SEQ ID NO: 47 on the same allele. Lt; RTI ID = 0.0 &gt; 377 &lt; / RTI &gt; The method of claim 109, wherein the oocyte, sperm cell, or embryo comprises 28 base pair deletions of nucleotides 3,145 to 3,172 relative to reference sequence SEQ ID NO: 47. 103. The method of claim 102 or 105, wherein the oocyte, sperm cell, or embryo has 7 base pair insertions between nucleotide 3,148 and nucleotide 3,149 relative to reference sequence SEQ ID NO: 47 and 11 base pairs between nucleotide 3,137 and nucleotide 3,147 relative to reference sequence SEQ ID NO: 47. A method comprising an open pair deletion. 47. The method of any one of claims 102 to 112, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence having at least 80% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. The method of claim 113, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence having at least 85% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. The method of claim 113, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence having at least 90% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. The method of claim 113, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence having at least 95% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. 112. The method of claim 113, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence having at least 98% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. 112. The method of claim 113, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence having at least 99% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. 112. The method of claim 113, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence having at least 99.9% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. The method of claim 113, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence having 100% sequence identity to SEQ ID NO: 47 in the region of the chromosomal sequence other than insertion or deletion. Wherein the oocyte, sperm cell, or embryo is selected from the group consisting of SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or &lt; RTI ID = 0.0 &gt; 114 &lt; / RTI &gt; The method of claim 121, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, The method of claim 121, wherein the oocyte, sperm cell, or embryo comprises a chromosomal sequence comprising SEQ ID NO: 103 or 111. The method of any one of claims 102, 103, 109, 110, 113 to 121, and 123 wherein the oocyte, sperm cell, or embryo is deficient in 11 base pairs and CD163 A method comprising two base pair insertions and 377 base pair deletions in another allele of the gene encoding the protein. The method of any one of claims 102, 108, and 113 to 122, wherein the oocyte, sperm cell, or embryo has 124 base pair deletions in one allele of the gene encoding the CD163 protein and a gene encoding the CD163 protein A method comprising 123 base pair deletions in another allele. The method of any one of claims 102, 104, and 113 to 121, wherein the oocyte, sperm cell, or embryo comprises one base pairing. The method of any one of claims 102 and 113 to 121, wherein the oocyte, sperm cell, or fertilized egg has 130 base pair deletions in one allele of the gene encoding the CD163 protein and another in the gene encoding the CD163 protein Wherein the gene comprises 132 base pair deletions in the gene. The method of any one of claims 102, 108, and 113 to 122, wherein the oocyte, sperm cell, or embryo comprises 1506 base pair deletions. The method of any one of claims 102, 105, and 113 to 121, wherein the oocyte, sperm cell, or embryo comprises seven base pair insertions. The method of any one of claims 102, 108, and 113 to 122, wherein the oocyte, sperm cell, or embryo has 1280 basepairs deleted in one allele of the gene encoding the CD163 protein and a gene encoding the CD163 protein A method comprising 1373 base pair deletions in another allele. The method of any one of claims 102, 108, and 113 to 122, wherein the oocyte, sperm cell, or embryo comprises 1467 base pair deletions. The method of any one of claims 102,106, 108, and 113 to 122, wherein the oocyte, sperm cell, or embryo is infected with a 1930 base pair intron 6 deletion of nucleotides 488 to 2417 and a 12 base pair deletion at nucleotide 4, A method comprising an additional 129 base pair deletion in exon 7. The method of any one of claims 102, 108, 109, 111, and 113 to 122, wherein the oocyte, sperm cell, or embryo has 28 base pair deletions and CD163 proteins in one allele of the gene encoding the CD163 protein A method comprising the deletion of 1387 bases in another allele of the gene encoding the gene. The method of any one of claims 102, 107, 108, and 113 to 122, wherein the oocyte, sperm cell, or embryo has 1382 base pair deletions and 11 base pair insertions in one allele of the gene encoding the CD163 protein And a 1720 base pair deletion in another allele of the gene encoding the CD163 protein. 72. The method of any one of claims 72 to 134 wherein the selected animal is used as a founder animal. 72. The method of any one of claims 72 to 135, wherein the correcting step comprises artificial insemination. An animal population made by the method of any one of claims 72 to 136. 137. The population of claim 137, wherein the animal population is resistant to infection by pathogens. 144. The population of Claim 138, wherein the pathogen comprises a virus. 144. The population of claim 139, wherein the virus comprises PRRSV. Genetically editing at least one chromosomal sequence from a gene encoding the CD163 protein such that CD163 protein production or activity is reduced relative to CD63 protein production or activity in a livestock animal that does not contain an edited chromosome sequence in the gene encoding the CD163 protein Wherein the method comprises increasing the tolerance of a livestock animal to infection with a pathogen. (a) a nucleotide sequence comprising SEQ ID NO: 47;
(b) a nucleotide sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47; And
(c) an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the cDNA sequences of (a) or (b). 142. The isolated nucleic acid of claim 142, comprising a nucleotide sequence comprising SEQ ID NO: 47. 142. The isolated nucleic acid of claim 142, comprising a nucleotide sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47. 144. The isolated nucleic acid of claim 144, comprising a nucleotide sequence having at least 85% sequence identity to the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47. 144. The isolated nucleic acid of claim 144, comprising a nucleotide sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47. 144. The isolated nucleic acid of claim 144, comprising a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47. 144. The isolated nucleic acid of claim 144, comprising a nucleotide sequence having at least 98% sequence identity to the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47. 144. The isolated nucleic acid of claim 144, comprising a nucleotide sequence having at least 99% sequence identity with the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47. 144. The isolated nucleic acid of claim 144, comprising a nucleotide sequence having at least 99.9% sequence identity to the sequence of SEQ ID NO: 47, wherein said nucleotide sequence comprises at least one substitution, insertion, or deletion relative to SEQ ID NO: 47. 142. The nucleic acid of any one of claims 142 and 144-150 wherein the substitution, insertion, or deletion reduces or eliminates CD163 protein production or activity relative to a nucleic acid that does not comprise substitution, insertion, or deletion. The isolated nucleic acid molecule of any one of claims 142,144, or 151, wherein the isolated sequence comprises SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, Lt; / RTI &gt; 152. The isolated nucleic acid of claim 152, comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, 154. The isolated nucleic acid of claim 152, comprising SEQ ID NO: 103 or SEQ ID NO: 142. The isolated nucleic acid of claim 142, comprising the cDNA. An isolated nucleic acid comprising SEQ ID NOS: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, The isolated nucleic acid of claim 156, comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, 154. The isolated nucleic acid of claim 156, comprising SEQ ID NO: 103 or SEQ ID NO:

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