KR102631856B1 - Pathogen-resistant animals having modified cd163 genes - Google Patents

Abstract

Non-human animals and their progeny comprising at least one altered chromosomal sequence in the gene encoding the CD163 protein are provided. Animal cells containing such modified chromosomal sequences are also provided. Animals and cells have increased resistance to pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). Animals and offspring have chromosomal variants of the CD163 gene. The invention further relates to breeding methods for producing pathogen-resistant animals and populations of animals created using such methods.

Description

Pathogen-resistant animals with modified CD163 genes {PATHOGEN-RESISTANT ANIMALS HAVING MODIFIED CD163 GENES}

The present invention relates to non-human animals and their progeny comprising at least one altered chromosomal sequence in the 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 animals and their offspring have chromosomal alterations in the CD163 gene that inhibit PRRSV entry and replication and thus render the animals resistant to diseases and syndromes caused by the virus. The invention also relates to breeding methods for producing pathogen-resistant animals and populations of animals created using these methods. The invention also relates to methods for gene editing of CD163 involving direct injection of embryos and development of animals, founder animals and lines resistant to pathogens such as PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the group of mammalian arteriviruses, which also includes murine lactate dehydrogenase-enhancing virus, simian hemorrhagic fever virus and equine arteritis virus. Arteriviruses share important characteristics associated with viral pathogenesis, including tropism for macrophages and the ability to cause severe disease and persistent infection. The clinical disease syndrome caused by infection with porcine reproductive and respiratory syndrome virus (PRRSV) was first reported in the United States in 1987 (Keffaber, 1989) and later in Europe in 1990 (Wensvoort et al., 1991). Infection with PRRSV causes respiratory illness including cough and fever, reproductive problems during the second trimester, and reduced growth rate. Viruses are also involved in various polymicrobial disease syndrome interactions, maintaining life-long asymptomatic infection (Rowland et al., 2012).

Since its emergence, PRRS has become the most important disease of commercial pigs in North America, Europe and Asia, with Australia and Antarctica being the only continents free from the disease. In North America alone, PRRSV-related losses are estimated to cost producers $664 million annually (Holtkamp et al., 2013). In 2006, a more severe form of the disease known as highly pathogenic PRRS (HP-PRRS) decimated pig populations across China. Genetic diversity has limited the development of vaccines needed to effectively control and eliminate the disease. Genetic selection for natural resistance may be an option, but results have so far been limited (Boddicker et al., 2014).

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

A lack of understanding of many features of both PRRSV pathogenesis (especially at the molecular level) and epizootiology makes control efforts difficult. Currently, producers often vaccinate pigs against PRRSV with modified-attenuated live strains or killed viruses, but current vaccines often do not provide satisfactory protection. This is due to both strain variation and inappropriate stimulation of the immune system. In addition to concerns about the efficacy of available PRRSV vaccines, the strains currently in use, as demonstrated by a virulent field isolate following experimental infection of pigs (Rowland et al., Virology, 259:262-266 (1999)) There is strong evidence that raw-live vaccines can persist in individual pigs and herds and increase mutations (Mengeling et al., Am. J. Vet. Res, 60(3): 334-340 (1999)). Additionally, it has been shown that the vaccine virus comes from the semen of vaccinated boars (Christopher-Hennings et al., Am. J. Vet. Res, 58(1): 40-45 (1997)). As an alternative to vaccination, some experts advocate a "test and removal" strategy from breeding herds (Dee and Molitor, Vet. Rec., 143:474-476 (1998)). Successful use of this strategy depends on the removal of all pigs acutely or persistently infected with PRRSV followed by stringent controls to prevent reintroduction of the virus. The difficulty and high cost associated with this strategy is that little is known about the incidence of persistent PRRSV infection and therefore there are no reliable techniques for identifying persistently infected pigs.

As can be seen, there is a need in the art to develop strategies to induce PRRSV resistance in animals.

Non-human animals, progeny thereof, and animal cells comprising at least one modified chromosomal sequence in the gene encoding the CD163 protein are provided.

Breeding methods for producing animals or bloodlines with reduced susceptibility to infection by pathogens are also provided. The method includes genetically modifying an oocyte or a sperm cell to introduce a modified chromosomal sequence in the gene encoding the CD163 protein into at least one of the oocyte and the sperm cell, and introducing the modified chromosomal sequence in the gene encoding the CD163 protein into at least one of the oocyte and the sperm cell. and fertilizing the oocyte with a sperm cell to produce a fertilized egg containing the oocyte. Alternatively, the method includes genetically modifying the fertilized egg to introduce a modified chromosomal sequence in the gene encoding the CD163 protein into the fertilized egg. The method comprises the steps of implanting a fertilized egg into a surrogate female animal, where pregnancy and full-term parturition produce progeny animals, screening the progeny animals for susceptibility to pathogens, and modifying the chromosome in the gene encoding the CD163 protein. Further comprising selecting progeny animals with reduced susceptibility to the pathogen compared to animals not comprising the sequence.

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

A method of increasing the resistance of livestock animals to infection with pathogens is also provided. The method includes copying at least one chromosomal sequence from the gene encoding the CD163 protein such that CD163 protein production or activity is reduced compared to CD63 protein production or activity in a domestic animal that does not contain the edited chromosomal sequence in the gene encoding the CD163 protein. Includes editing.

Modifications to the chromosomal sequence in the gene encoding the CD163 protein provided herein may be directed to an animal, progeny, cell, or population (e.g., a porcine animal, progeny, decreases the sensitivity of cells or populations).

In any of the porcine animals, progeny, cells, and methods provided herein, the modification of the chromosomal sequence in the gene encoding the CD163 protein includes an 11 base pair deletion from nucleotide 3,137 to nucleotide 3,147 relative to the reference sequence SEQ ID NO: 47; a 2 base pair insertion between nucleotides 3,149 and 3,150 compared to the reference sequence SEQ ID NO:47 and a 377 base pair deletion from nucleotides 2,573 to nucleotides 2,949 compared to the reference sequence SEQ ID NO:47 on the same allele; A 124 base pair deletion from nucleotides 3,024 to nucleotides 3,147 compared to the reference sequence SEQ ID NO:47; A 123 base pair deletion from nucleotides 3,024 to nucleotides 3,147 compared to the reference sequence SEQ ID NO:47; 1 base pair insertion between nucleotides 3,147 and 3,148 compared to the reference sequence SEQ ID NO:47; A 130 base pair deletion from nucleotides 3,030 to nucleotides 3,159 compared to the reference sequence SEQ ID NO:47; A 132 base pair deletion from nucleotides 3,030 to nucleotides 3,161 compared to the reference sequence SEQ ID NO:47; A 1506 base pair deletion from nucleotides 1,525 to nucleotides 3,030 compared to the reference sequence SEQ ID NO:47; 7 base pair insertion between nucleotides 3,148 and 3,149 compared to the reference sequence SEQ ID NO:47; A 1280 base pair deletion from nucleotides 2,818 to nucleotides 4,097 compared to the reference sequence SEQ ID NO:47; A 1373 base pair deletion from nucleotides 2,724 to nucleotides 4,096 compared to the reference sequence SEQ ID NO: 47; A 1467 base pair deletion from nucleotides 2,431 to nucleotides 3,897 compared to the reference sequence SEQ ID NO:47; A 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and nucleotide 7 in exon 7 compared to the reference sequence SEQ ID NO: 47 There is an additional 129 base pair deletion from nucleotides 3,044 to 3,172); A 28 base pair deletion from nucleotides 3,145 to nucleotides 3,172 compared to the reference sequence SEQ ID NO:47; A 1387 base pair deletion from nucleotides 3,145 to nucleotides 4,531 compared to the reference sequence SEQ ID NO:47; A 1382 base pair deletion from nucleotides 3,113 to nucleotides 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotides 3,113; A 1720 base pair deletion from nucleotides 2,440 to nucleotides 4,160 compared to the reference sequence SEQ ID NO: 47; Or it may include 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 to the sequence of SEQ ID NO: 47, wherein the nucleotide sequence contains 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. Isolated nucleic acids include SEQ ID NOs: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114.

Other purposes and features will become somewhat self-evident and will be mentioned to some extent hereinafter.

Figure 1. CRISPR and targeting vectors used to modify CD163. Panel A depicts wild-type exons 7, 8, and 9 of the CD163 gene targeted for modification using CRISPR. Panel B shows a targeting vector designed to replace porcine exon 7 (SRCR5, the porcine domain of CD163) with DNA encoding human SRCR8 of CD163L. This targeting vector 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 has been removed. This targeting vector was already used to target CD163 in neomycin-resistant cells. Primers used in the small deletion assay are illustrated by 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-pointing arrows on exon 7. The CRISPR number represents the number of base pairs from the intron-exon junction of intron 6 and exon 7.
Figure 2. CRISPR and targeting vectors used to modify CD1D. Panel A depicts wild-type exons 3, 4, 5, 6, and 7 of the CD1D gene targeted for modification by CRISPR. Panel B shows a targeting vector designed to replace exon 3 with the selection marker Neo . This targeting vector was used to modify CD1D in conjunction with CRISPR. PCR primers for far-field, left-arm, and right-arm assays are labeled with arrows for 3991, 4363, 7373, and 12806. Panel C shows exons targeted by CRISPR. The positions of CRISPR 4800, 5350, 5620 and 5626 are indicated by downward-pointing arrows on exon 3. Primers used in the small deletion assay are illustrated by arrows and labeled GCD1DF and GCD1DR. The CRISPR number represents the number of base pairs from the intron-exon junction of intron 6 and exon 7.
Figure 3. Reproductive process of CD163 and CD1D knockout pigs by CRISPR/Cas9 and SCNT. A) Targeted deletion of CD163 in somatic cells following transfection with CRISPR/Cas9 and donor DNA. The wild type (WT) genotype gives rise to a 6545 base pair (bp) band. Lanes 1-6 represent six different colonies from a single transfection with donor DNA containing CRISPR 10 with Cas9 and Neo. Lanes 1, 4, and 5 show a large homozygous deletion of 1500-2000 bp. Lane 2 represents the smaller homozygous deletion. Lanes 3 and 6 represent biallelic variants of both the WT allele and the small deletion or allele. The exact strain of each colony was determined by sequencing only the colonies used for 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 following transfection with CRISPR/Cas9 and donor DNA. The WT genotype gives rise to an 8729 bp band. Lanes 1-4 represent colonies with a 500-2000 bp deletion of CD1D. Lane 4 appears to be a WT colony. NTC ¼ no mold adjustment. C) Imaging of CD163 knockout pigs produced by SCNT during the study. These male piglets contain a homozygous 1506 bp deletion of CD163. D) Imaging of CD1D pigs produced during the study. These piglets contain a 1653 bp deletion of CD1D. E) Genotype of two SCNT littermates containing a 1506 bp deletion of CD163. Lanes 1-3 (Litter 63) and Lanes 1-4 (Litter 64) represent the genotype for each piglet from each litter. Sow represents recipient females of SCNT embryos, and WT represents WT controls. NTC = No mold control. F) Genotype of two SCNT littermates containing a 1653 bp deletion of CD1D. Lanes 1-7 (Litter 158) and Lanes 1-4 (Litter 159) represent the genotype for each piglet.
Figure 4. Effect of CRISPR/Cas9 system in porcine embryos. A) Frequency of blastocyst formation after injection of different concentrations of CRISPR/Cas9 system into zygotes. The toxicity of the CRISPR/Cas9 system was lowest at 10 ng/μl. B) The CRISPR/Cas9 system can successfully disrupt the expression of eGFP in blastocysts when introduced into zygotes. Original magnification X4. C) Types of mutations for eGFP generated using the CRISPR/Cas9 system: WT genotype (SEQ ID NO:16), #1 (SEQ ID NO:17), #2 (SEQ ID NO:18), and #3 (SEQ ID NO:19) .
Figure 5. Effect of the CRISPR/Cas9 system in targeting CD163 in porcine embryos. A) Examples of mutations generated on CD163 by the CRISPR/Cas9 system: WT genotype (SEQ ID NO:20), #1-1 (SEQ ID NO:21), #1-4 (SEQ ID NO:22), and #2-2 (SEQ ID NO: 23). All embryos tested by DNA sequencing showed mutations on CD163 (18/18). CRISPR 131 is highlighted in bold. B) Sequencing reads of homozygous deletions caused by the CRISPR/Cas9 system. Images show #1-4 from panel A with a 2 bp deletion of CD163.
Figure 6. Effect of the CRISPR/Cas9 system when introduced with two types of CRISPR. A) PCR amplification of CD163 in blastocysts injected with CRISPR/Cas9 as zygotes. Lanes 1, 3, 6, and 12 show indicated deletions between two different CRISPRs. B) PCR amplification of CD1D in blastocysts injected with CRISPR/Cas9 as zygotes. CD1D had a lower frequency of deletions as measured by gel electrophoresis when compared to CD163 (3/23); Lanes 1, 8, and 15 show clear deletions in CD1D. C) The CRISPR/Cas9 system successfully targeted two genes when the system was provided with two CRISPR targeting CD163 and eGFP. Modifications of CD163 and eGFP are shown: CD163 WT (SEQ ID NO: 24), CD163 #1 (SEQ ID NO: 25), CD163 #2 (SEQ ID NO: 26), CD163 #3 (SEQ ID NO: 27), eGFP WT (SEQ ID NO: 28), eGFP #1-1 (SEQ ID NO: 29), eGFP #1-2 (SEQ ID NO: 30), eGFP #2 (SEQ ID NO: 31), and eGFP #3 (SEQ ID NO: 32).
Figure 7. CD163 knockout pig generated by the CRISPR/Cas9 system injected into the zygote. A) PCR amplification of CD163 from knockout pigs; Clear signs of deletion were detected in littermates 67-2 and 67-4. B) Image of a CD163 knockout pig carrying a surrogate mother. All animals are healthy and show no abnormalities. C) Genotype of CD163 knockout pigs. The wild type (WT) sequence is indicated as SEQ ID NO:33. Two animals (from litters 67-1 (SEQ ID NO: 34) and 67-3 (SEQ ID NO: 37)) carry homozygous deletions or insertions in CD163. Two other animals (from litters 67-2 and 67-4) carry biallelic variants of CD163: #67-2 A1 (SEQ ID NO: 35), #67-2 A2 (SEQ ID NO: 36), #67-4 A1 (SEQ ID NO: 38), and #67-4 a2 (SEQ ID NO: 39). The deletion was caused by introducing two different CRISPR systems with Cas9. None of the animals from zygote injections for CD163 showed a mosaic genotype.
Figure 8. CD1D knockout pig generated by CRISPR/Cas9 system injected into zygotes. A) PCR amplification of CD1D from knockout pigs; 166-1 shows a mosaic genotype for CD1D. 166-2, 166-3, and 166-4 showed no size change in the amplicon, but sequence analysis of the amplicon showed alterations. WT FF = wild-type fetal fibroblasts. B) PCR amplification of the distant assay showed a clear deletion of one allele in piglets 166-1 and 166-2. C) Image of a CD1D knockout pig carrying a surrogate mother. D) Sequence data from CD1D knockout pigs; WT (SEQ ID NO: 40), #166-1.1 (SEQ ID NO: 41), #166-1.2 (SEQ ID NO: 42), #166-2 (SEQ ID NO: 43), #166-3.1 (SEQ ID NO: 44), #166- 3.2 (SEQID NO:45), and #166-4 (SEQ ID NO:46). The atg start codon in exon 3 is shown in bold lowercase letters.
Figure 9. Clinical signs during acute PRRSV infection. Results of daily assessment for presence of respiratory signs and fever for CD163 +/+ (n=6) and CD163 -/- (n=3).
Figure 10. Lung histopathology during acute PRRSV infection. Representative photomicrographs of H and E stained tissues from wild-type and knockout pigs. Left panel shows mononuclear cell infiltration and edema. The right panel from a knockout pig shows the lung structure of a normal lung.
Figure 11. Viremia in various genotypes. Note that CD163-/- piglet data lies along the X axis.
Figure 12. Antibody production in non-responsive, wild-type and uncharacterized allele pigs.
Figure 13. Cell surface expression of CD163 in individual pigs. Lines appearing to the right in the Uncharacterized A, Uncharacterized B, and CD163 +/+ panels represent CD163 antibodies, whereas lines appearing to the left of these panels show no antibody control (background). Note that CD163 staining in CD163-/- animals overlaps with the background control, and that CD163 staining in the uncharacterized allele is roughly intermediate between WT levels and background (this is on a logarithmic scale, and therefore less than ∼10%). Note).
Figure 14. Levels of CD169 (FITC labeled anti-CD169) on alveolar macrophages from three representative pigs and no antibody controls.
Figure 15. Viremia in various genotypes. Note that the Δ43 amino acid piglet data lies along the X-axis.
Figure 16. Genomic sequence of wild-type CD163 exons 7-10 used as reference sequence (SEQ ID NO:47). The sequence includes 3000 bp upstream of exon 7 to the last base of exon 10. The underlined regions show the positions of exons 7, 8, 9, and 10, respectively.

Provided herein are methods for producing animals and gene-edited animals that have modifications in the CD163 gene and are resistant to PRRSV and other related respiratory virus infections. The animal has a chromosomal alteration (insertion or deletion) that inactivates or otherwise modulates CD163 gene activity. CD163 is required for PRRSV entry into cells and viral replication. Therefore, non-responsive CD163 animals exhibit resistance to PRRSV infection when challenged. Such animals can be created using any of a number of protocols that utilize gene editing.

Additionally, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system, transcription activator-like effector nuclease (TALEN), ZFN (zinc finger nuclease), recombinase fusion protein, or using gene editing methods such as meganuclease to bind specifically to chromosomal surface regions of porcine animal cells or porcine embryos, causing double-stranded DNA degradation or otherwise inactivating the activity of the CD163 gene or protein therein. Methods for producing porcine animals are provided, including introducing an activating or reducing agent.

Also described herein is the use of one or more specific CD163 loci with polypeptides capable of cleavage and/or integration of specific nucleic acid sequences within the CD163 locus. Examples of uses of the CD163 gene with polypeptides or RNAs capable of performing cleavage and/or integration of the CD163 locus include zinc finger proteins, meganucleases, TAL domains, TALENs, RNA-guided CRISPR/Cas recombinase, leucine Zippers, and other polypeptides selected from the group consisting of those known in the art. Specific examples include chimeric (“fusion”) proteins comprising a site-specific DNA binding domain polypeptide and a cleavage domain polypeptide (e.g., a nuclease), such as ZFNs comprising a zinc-finger polypeptide and a FokI nuclease polypeptide. Contains protein. Described herein are polypeptides comprising a DNA-binding domain that specifically binds to the CD163 gene. Such polypeptides may also contain a nuclease (cleavage) domain or half-domain (e.g., a modified DNA-binding domain) such that the polypeptide can induce targeted double-strand degradation and/or promote recombination of the nucleic acid of interest at the site of degradation. a homing endonuclease including a homing endonuclease domain), and/or a ligase 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., an animal species) at one or more CD163 loci. The DNA-binding domain may comprise a zinc finger protein with one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers), which may be present in any of the CD163 genes. engineered (non-naturally occurring) to bind to the sequence of. Any of the zinc finger proteins described herein can bind to a target site (e.g., a promoter or other expression element) within or adjacent to the coding sequence of the target gene. Zinc finger proteins can bind to target sites in the CD163 gene.

Justice

Units, prefixes, and symbols may appear in SI-approved form. Unless otherwise indicated, nucleic acids are written from left to right in 5' to 3' orientation; Amino acid sequences are written from left to right, from amino to carboxy. Numerical ranges recited within the specification include the numbers defining the range and include each integer within the defined range. Amino acids may be represented herein by their commonly known three-letter symbols or by their one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Nucleotides can likewise be represented by generally accepted single-letter codes. Unless otherwise provided, software, electrical and electronics terms 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 by reference to the specification as a whole.

As will be understood by those skilled in the art, for any and all purposes, especially in the context of providing a written description, all ranges recited herein also include any and all possible sub-ranges and sub-ranges thereof. Includes combinations of, as well as individual values, especially integer values, that make up the range. A quoted range includes each specific value, integer, decimal, or identity within the range. It can be readily appreciated that any enumerated range sufficiently describes the same range and can be subdivided into at least equivalent 1/2, 1/3, 1/4, 1/5, or 1/10 parts. As a non-limiting example, each range discussed herein can be easily subdivided into lower third, middle third, and upper third, etc.

When introducing elements of the invention or preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean the presence of one or more elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than those listed.

The term “and/or” means any one of the items, any combination of items, or all of the items to which the term relates. The phrase “one or more” is readily understood by those skilled in the art, especially when read in the context of its usage.

A “binding protein” is a protein that can bind to other molecules. Binding proteins can bind, for example, to DNA molecules (DNA-binding proteins), RNA molecules (RNA-binding proteins), and/or protein molecules (protein-binding proteins). In the case of a protein-binding protein, it can bind to itself (binding to form a homodimer, homotrimer, etc.) and/or to one or more molecules of a different protein or proteins. Binding proteins may 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 variant” 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 the same or conservatively modified variant of an amino acid sequence. Because of the degeneracy of the genetic code, multiple functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG and GCU all code for the amino acid alanine. Accordingly, at any position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons listed without altering the encoded polypeptide. These nucleic acid variations are “silent variations” and represent a type of conservatively modified change. All nucleic acid sequences herein that encode polypeptides also account for all possible silent changes in the nucleic acid, with reference to the genetic code.

Those of ordinary skill in the art will recognize that each codon of a nucleic acid (except AUG, which is often the only codon for methionine; and UGG, which is often the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each subclinical variation in the nucleic acid encoding a polypeptide of the invention is included in each described polypeptide sequence and is within the scope of the invention.

With respect to amino acid sequences, the skilled artisan will determine 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 encoded sequence are "conservatively modified variants." ", wherein it will be recognized that the change is the substitution of an amino acid with a chemically similar amino acid. Accordingly, the number of amino acid residues selected from the integer group consisting of 1 to 15 may be changed in this way. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 changes may be made.

Conservatively modified variants typically provide biological activity similar to the unmodified polypeptide sequence from which they are derived. For example, the 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 one another: [1] Alanine (A), Serine (S), Threonine (T); [2] Aspartic acid (D), glutamic acid (E); [3] Asparagine (N), glutamine (Q); [4] Arginine (R), Rigin (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” stands for “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 the Cas9 protein, or a derivative thereof, and the CRISPR RNA (crRNA) for Cas9. and one or more non-coding RNAs capable of serving the function of a transcription-promoting RNA (tracrRNA), wherein the crRNA and tracrRNA produce a "guide RNA" (gRNA). The crRNA or gRNA provides a sequence complementary to the genomic target. The CRISPR/Cas9 system is further described below.

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

“Disease tolerance” is a characteristic of animals, where they avoid disease symptoms that are the result of animal-pathogen interactions, such as the interaction between swine animals and PRRSV. That is, preventing pathogens from causing animal disease and related disease symptoms, or alternatively, reducing the incidence and/or severity of clinical signs or reducing clinical symptoms. Those skilled in the art will recognize that the compositions and methods disclosed herein may be used in conjunction with other compositions and methods available in the art to protect animals from pathogen attack.

With reference to a specific nucleic acid, “encoding” or “encoded” means containing information for translation into a specific protein. Nucleic acids encoding proteins may include intervening sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information for which a protein is encoded is specified by the use of codons. Typically, amino acid sequences are encoded by nucleic acids using a “universal” genetic code. When a nucleic acid is prepared or modified synthetically, the known codon preferences of the intended host in which the nucleic acid is to be expressed can be utilized.

As used herein, “gene editing,” “gene edited,” “genetically edited,” and “gene editing effectors” include “molecular scissors,” Refers to the use of homing technology using naturally occurring or artificially engineered nucleases, also called “homing endonucleases” or “targeting endonucleases.” Nucleases create specific double-strand chromosome breaks (DSBs) at desired locations in the genome, which in some cases utilize the cell's endogenous mechanisms to initiate homologous recombination (HR) and/or non-homologous end-joining (NHEJ). Repairs amputations induced by natural processes. Gene editing effectors include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the Clustered Regularly Interspaced Short Palindromic Repeats/CAS9 (CRISPR/Cas9) system, and meganucleases (e.g., homing meganucleases reengineered as endonucleases). The term also encompasses the use of transgenic processes and techniques, including, for example, where the changes are deletions or relatively small insertions (typically less than 20 nt) and/or do not introduce DNA from foreign species. . The term also includes progeny animals, such as those produced by sexual or asexual breeding from an initial gene-edited animal.

As used herein, “heterologous” with respect to a nucleic acid means that it originates from an alien species, or is substantially from its native form in composition and/or genomic locus by the intent of human intervention if it is from the same species. It is a modified nucleic acid. For example, if promoters operably linked to a heterologous structural gene are from a different species than that driving the structural gene, or are from the same species, one or both are substantially modified from their original form. Heterologous proteins may originate from a foreign species or, if from the same species, have been substantially modified from their original form by intended human intervention.

As used herein, “homing DNA technology,” “homing technology,” and “homing endonuclease” refer to zinc finger (ZF) proteins, transcription activator-like effector (TALE) meganucleases, and the CRISPR/Cas9 system. This applies to any mechanism that allows targeting a specific molecule to a specific DNA sequence, including:

As used herein, the terms "increased resistance" and "reduced susceptibility" mean, but are not limited to, a statistically significant reduction in the incidence and/or severity of clinical signs or symptoms associated with infection by a pathogen. . For example, “increased resistance” or “reduced susceptibility” refers to the risk of infection by PRRSV in animals containing at least one altered chromosomal sequence in the gene encoding the CD163 protein compared to control animals with unmodified chromosomal sequences. It may indicate a statistically significant reduction in the incidence and/or severity of an associated clinical sign or clinical symptom. The term “statistically significant reduction in clinical symptoms” means that the incidence of at least one clinical symptom in the edited group of subjects is at least 10%, preferably at least 20%, after challenge with an infectious agent compared to the non-edited control group. %, 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” refers to replacement of an endogenous gene with a transgene or with an identical endogenous gene with minor structural modifications while maintaining transcriptional control of the endogenous gene.

“Knock-out” means disruption of the structure or regulatory mechanism of a gene. Knock-outs are generated through homologous recombination in targeting vectors, replacement vectors, or hit-and-run vectors, or through random insertion of gene trap vectors that result in complete, partial, or conditional loss of gene function. It can be.

The term “animal” includes any non-human animal, including farmed animals (e.g., livestock animals). The term “livestock animal” refers to any animal traditionally raised on livestock farms, such as swine animals, bovine animals (e.g. beef cattle or dairy cows), sheep animals, goat animals, equine animals (e.g. horses or donkeys), buffalo, Includes camels, or avian animals (e.g., chickens, turkeys, ducks, geese, guinea fowl, or newly hatched birds). The term “livestock animal” does not include rats, mice, or other rodents.

As used herein, the term “mutation” includes a modification in the nucleotide sequence of a polynucleotide, such as a gene or coding DNA sequence (CDS), compared to the wild-type sequence. The term includes, but is not limited to, substitution, insertion, frameshift, deletion, inversion, translocation, duplication, splice-donor site mutation, point-mutation, etc.

As used herein, “operably linked” includes reference to a functional linkage between two nucleic acid sequences, e.g., a promoter sequence and a secondary sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the secondary sequence. do. In general, operably linked means that the nucleic acid sequences being linked are contiguous and, optionally, join the two protein coding regions adjacent to each other and in the same reading frame.

As used herein, “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or conservatively modified variant; The term also refers to those having the essential properties of natural ribonucleotides in that they hybridize, under stringent hybridization conditions, to a nucleotide sequence substantially identical to the naturally occurring nucleotide and/or are translated into the same amino acid(s) as the naturally occurring nucleotide(s). Can refer to analogues. The polynucleotide may be the full length or subsequence of a native or heterologous structural gene or regulatory gene. Unless otherwise indicated, the term includes reference to a particular sequence as well as its complementary sequence. Accordingly, DNA or RNA having a backbone that has been modified for stability or other reasons is a “polynucleotide” as that term is intended herein. Moreover, DNA or RNA containing unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, 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 that serve a variety of useful purposes known to those skilled in the art.

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

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms may also apply to conservatively modified variants and amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. An essential property of these analogs of naturally occurring amino acids is that when incorporated into a protein, they are specifically reactive against antibodies directed against the same protein consisting entirely of naturally occurring amino acids.

The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. . As is well known and noted above, it will be appreciated that polypeptides are not always entirely linear. For example, polypeptides may be branched as a result of ubiquitination, and they generally have branching as a result of post-translational events, including natural processing events and events caused by human manipulation that do not occur naturally. It may be a prototype that does not have one. Circular, branched and branched circular polypeptides can be synthesized by non-translational natural processes and by purely synthetic methods. Additionally, the present invention contemplates the use of both methionine-containing and non-methionine-containing amino terminal variants of the proteins of the invention.

As used herein, “reduction in the incidence and/or severity of clinical signs” or “reduction in clinical signs” means reducing the number of infected subjects in a group, reducing or eliminating the number of subjects showing clinical signs of infection, compared to wild-type infection. , or reducing the severity of any clinical symptom present in one or more subjects. For example, these terms may refer to any clinical sign of infection, pulmonary pathology, viremic antibody production, reduction of pathogen load, pathogen shedding, reduction of pathogen transmission, or any clinical sign symptomatic of PRRSV. Includes reduction. Preferably these clinical signs are reduced by at least 10% in one or more animals of the invention compared to an infected subject without a variant in the CD163 gene. More preferably clinical signs are reduced in the subject of the invention by at least 20%, preferably by at least 30%, more preferably by at least 40% and even more preferably by at least 50%.

The terms “residue” or “amino acid residue” or “amino acid” are used interchangeably herein to refer to amino acids incorporated into a protein, polypeptide, or peptide (collectively, “protein”). Amino acids may be naturally occurring amino acids and, unless otherwise limited, may include non-natural analogs of natural amino acids that may function in a similar manner to naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization of a nucleic acid sequence to another nucleic acid sequence or to another biologic, under stringent hybridization conditions. When using a hybridization-based detection system, a nucleic acid probe complementary to a reference nucleic acid sequence is selected, and then, by selection of suitable conditions, the probe and reference sequence hybridize or bind to each other to form a duplex molecule. .

The terms “stringent conditions” or “stringent hybridization conditions” include reference to conditions under which a probe hybridizes to its target sequence to a detectably greater extent than to other sequences (e.g., at least two times greater than background). Stringent conditions are sequence-dependent and will vary in different situations. By controlling the stringency of hybridization and/or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probing).

Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequence such that lower degrees of similarity are detected (heterologous probing). Typically, probes are less than about 1000 nucleotides in length, and optionally less than 500 nucleotides in length.

Typically, stringent conditions include a salt concentration of less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salt) at pH 7.0 to 8.3, and a short probe (e.g., 10 to 50 nucleotides). Conditions will be at least about 30°C for long probes (e.g., 50 or more nucleotides) and at least about 60°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Specificity is typically a function of the post-hybridization wash, with the determining factors being the ionic strength and temperature of the final wash solution. For 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 the monovalent cation, % 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 base pairs. T _m is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Tm decreases by about 1°C for each 1% mismatch; Accordingly, the Tm, hybridization and/or washing conditions can be adjusted to achieve hybridization to the sequence of desired identity. For example, if sequences with >90% identity are sought, the Tm can be reduced by 10°C. Typically, stringent conditions are selected to be about 5°C lower than the Tm for a particular sequence and its complement at a defined ionic strength and pH. However, highly stringent conditions may utilize hybridization and/or washing at temperatures 1 to 4° C. below the Tm; Moderately stringent conditions may utilize hybridization and/or washing at temperatures 6 to 10° C. below the Tm; Low stringency conditions may utilize hybridization and/or washing at temperatures 11 to 20° C. below the Tm. Using the equations, hybridization and wash compositions, and desired Tm, those of ordinary skill in the art will understand that variations in the stringency of hybridization and/or wash solutions are essentially accounted for. An extensive guide to hybridization of nucleic acids is available in the literature [see; Tijssen, 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 one or more TALE repeat domains/units. The repeat domain is involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also called a “repeat”) is typically 33-35 amino acids long and exhibits at least some sequence homology to other TALE repeat sequences within naturally occurring TALE proteins. Zinc fingers and TALE binding domains can be “engineered” to bind to a given nucleotide sequence through manipulation (changing one or more amino acids) of the naturally occurring zinc finger or recognition helix region of the TALE protein. Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are non-naturally occurring proteins. A non-limiting example of a method for engineering DNA-binding proteins is design and selection. Engineered DNA binding proteins are proteins that do not occur in nature, the design/composition of which results primarily from rational criteria. Reasonable criteria for design include the application of computerized algorithms and assignment rules to process information from existing ZFP and/or TALE designs and database storage information of combined data. For example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; See also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Pat. Disclosure No. See 20110301073.

As used herein, “vector” includes reference to a nucleic acid that can be used for transfection of a host cell and inserted into a polynucleotide. Vectors are often replicons. Expression vectors enable transcription of nucleic acids inserted therein.

“Wild type” means an animal and a blastocyst, embryo or cell derived therefrom that has not been gene edited or otherwise genetically modified and is usually an inbred or outbred strain developed from a naturally occurring strain.

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

“Selected” zinc finger proteins, or TALEs, are proteins not found in nature that are primarily produced from experimental processes such as phage display, interaction traps, or hybrid selection. For example, U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. 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. Disclosure No. See 20110301073.

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

(a) As used herein, a “reference sequence” is a defined sequence used as a basis for sequence comparison with polynucleotides/polypeptides of the invention. A reference sequence may be a subset of a specified sequence or an entire specified sequence; For example, a full-length cDNA or segment of a gene sequence, or a complete cDNA or gene sequence.

(b) As used herein, a “comparison window” includes reference to a specific contiguous segment of a polynucleotide/polypeptide sequence, wherein the polynucleotide/polypeptide sequence can be compared to a reference sequence and the polynucleotide/polypeptide sequence in the comparison window A portion of may contain additions or deletions (i.e. gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Typically, the comparison window is at least 20 contiguous nucleotides/amino acid residues long, and can optionally be 30, 40, 50, 100, or longer. Those skilled in the art understand that to avoid high similarity to the reference sequence due to the inclusion of gaps in the polynucleotide/polypeptide sequence, a gap penalty is typically introduced and this is subtracted from the number of matches.

Methods for aligning sequences for comparison are well known in the art. The optimal alignment of sequences for comparison is the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)]; The homology alignment algorithm of 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 computerized implementations 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).

The BLAST family of programs that can be used for database similarity searches include: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. 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 analyzes is publicly available, for example, through the National Center for Biotechnology Information (ncbi.nlm.nih.gov/). These algorithms are fully described in numerous 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 (McEntyre J, Ostell J, eds., 2002), available at http://www.ncbi.nlm.nih.gov/ Please refer to [books/NBK21097/pdf/ch16.pdf]. The BLASTP program for amino acid sequences is also well described (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of similarity between two sequences (see, e.g., 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 (1993)) low-complexity filters. It can be used alone or in combination.

Unless otherwise specified, nucleotide and protein identity/similarity values provided herein are calculated using GAP (GCG Version 10) below default values. The Global Alignment Program (GAP) can also be used to compare polynucleotides or polypeptides of the invention to reference sequences. GAP uses the algorithm of Needleman and Bunsch (J. Mol. Biol. 48: 443-453, 1970) to find an alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP represents one member of the family of best alignments. This family can have multiple members, but no other member has a better quality. GAP represents four figures of merit for alignment: Quality, Ratio, Identity, and Similarity. Quality is the metric that is maximized for aligning 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. The similarity percentage is the percentage of similar symbols. Symbols directly across from the gap are ignored. Similarity is scored when the scoring matrix value for a pair of symbols is above the similarity threshold of 0.50. 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 alignment of sequences can be performed using the CLUSTAL alignment method (Higgins and Sharp (1989) CABIOS. 5: 151-153) with default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). 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, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences refers to residues in the two sequences that are identical when aligned for maximum correspondence over a specified window of comparison. Includes mention. When percent sequence identity is used for proteins, non-identical residue positions often depend on conservative amino acid substitutions, in which an amino acid residue is replaced by another amino acid residue with similar chemical properties (e.g. charge or hydrophobicity), thus forming a molecule. It is recognized as not changing the functional characteristics of . If the sequences differ in conservative substitutions, the percent sequence identity can be adjusted to correct for the conservative nature of the substitution. Sequences that differ due to these conservative substitutions are said to have “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 mismatches, thereby increasing the percent sequence identity. Thus, for example, if the same amino acid would receive a score of 1 and a non-conservative substitution would receive a score of 0, then the conservative substitution would receive a score between 0 and 1. Scoring of conservative substitutions can be performed using the Myers and Miller algorithm [Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988)].

(d) As used herein, “percent sequence identity” means a value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the polynucleotide sequence in the comparison window is identical to the two sequences. may contain additions or deletions (i.e. gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment. The percentage calculates the number of matched positions by finding the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences, dividing the number of matched positions by the total number of positions in the comparison window, and dividing the result by 100. It is calculated by multiplying by to yield the percent sequence identity.

Animals and cells with altered chromosomal sequences in the gene encoding the CD163 protein

CD163 has 17 exons and the protein consists of an extracellular region with nine scavenger receptor cysteine-rich (SRCR) domains, a transmembrane segment, and a short cytoplasmic tail. Several different variants result from differential splicing of single genes (Ritter et al. 1999a; Ritter et al. 1999b). The length of the cytoplasmic tail accounts for most of these modifications.

CD163 has a number of important functions, including acting as a haptoglobin-hemoglobin scavenger receptor. Removal of free hemoglobin from the blood is an important function of CD163 because the heme group can be very toxic (Kristiansen et al. 2001). CD163 has a cytoplasmic tail that promotes endocytosis. Mutations in this tail result in reduced haptoglobin-hemoglobin complex uptake (Nielsen et al. 2006). Other functions of C163 include erythroblast adhesion (SRCR2), TWEAK receptor (SRCR1-4 & 6-9), bacterial receptor (SRCR5), African swine virus receptor (Sanchez-Tones et al. 2003), and potential as an immune-modulator. role (discussed in the literature (Van Gorp et al. 2010a)). Given this important function, it was previously thought that complete knock-out of CD163 would produce non-viable or severely damaged animals (see, for example, PCT Publication No. 2012/158828).

CD163 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily and consists of an intracellular domain and nine extracellular SRCR domains. In humans, CD163-mediated endocytosis of hemoglobin-heme uptake through SRCR3 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), pathogen receptor (African Swine Fever Virus; bacteria: SRCR2), and erythroblast binding (SRCR2). do.

CD163 plays a role in infection by several different pathogens and therefore the present invention is not limited to animals with reduced susceptibility to PRRSV infection, but also for infection of cells or for late replication and/or persistence in cells. Animals with reduced susceptibility to any pathogen dependent on CD163 are included. The infection route of PRRSV begins with initial binding to heparan sulfate on the surface of alveolar macrophages. Before 2013, definitive binding was thought to occur to sialoadhesin (SIGLEC1, also known as CD169 or SN). The virus is then internalized through clatherin-mediated endocytosis. Another molecule, CD163, then promotes uncoating of the virus from endosomes (Van Breedam et al. 2010a). The viral genome is released and the cell is infected.

Disclosed herein are at least one modified chromosomal sequence, e.g., insertion or deletion (“INDEL”), in the gene encoding the CD163 protein that confers to the animal improved or complete resistance to infection by a pathogen (e.g., PRRSV). Including animals and their progeny and cells are described. Applicants have demonstrated that CD163 is an important gene in PRRSV infection and the creation of progenitor-resistant animals and strains.

The present invention provides genetically modified animals, their progeny, or animal cells comprising at least one modified chromosomal sequence in the gene encoding the CD163 protein. The present invention does not involve inactivation or editing of the SIGLEC1 (CD169) gene, which was previously postulated to be important for PRRSV resistance.

The edited chromosomal sequence may include integrated sequences that are (1) inactivated, (2) altered, or (3) resulting in non-responsive mutations. Inactivated chromosomal sequences are altered to impair, reduce, or eliminate CD163 protein function as it is associated with PRRSV infection. Accordingly, genetically edited animals containing inactivated chromosomal sequences may be called “knockouts” or “conditional knock outs.” Similarly, gene edited animals containing integrated sequences may be called “knock ins” or “conditional knock ins.” Additionally, genetically edited animals containing altered chromosomal sequences may contain targeted point mutation(s) or other modifications that result in the production of altered protein products. Briefly, the process may include introducing into the embryo or cell at least one RNA molecule encoding a target zinc finger nuclease and, optionally, at least one accessory polynucleotide. The method further includes culturing the embryo or cell to allow expression of the zinc finger nuclease, wherein a double-stranded break introduced into the targeted chromosome sequence by the zinc finger nuclease is an error. -Repaired by induced non-homologous end-joining DNA repair process or homology-directed DNA repair process. The method of editing chromosomal sequences encoding proteins involved in germline development using targeted zinc finger nuclease technology is rapid, accurate, and highly efficient.

Alternatively, the process may involve using the CRISPR/Cas9 system to modify the genomic sequence. Using the CRISPR/Cas9 system to modify the genomic sequence, the protein can be delivered directly into the cell, the mRNA encoding Cas9 can be delivered into the cell, or the gene that provides for the expression of the mRNA encoding Cas9 can be delivered into the cell. It can be transmitted as . Additionally, target-specific crRNA and tracrRNA can be delivered directly to the cell or target-specific gRNA(s) can be delivered to the cell (these RNAs can alternatively be produced by genes engineered to express such RNAs). . The design of crRNA/gRNA and selection of target sites 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 portions of genomic nucleic acids can modify the locus to produce a disrupted (i.e., reduced activity of the CD163 protein) CD163 gene.

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

Modification of the chromosomal sequence in the gene encoding the CD163 protein may increase the susceptibility of the animal, offspring, or cells to infection by a pathogen (e.g., a virus such as PRRSV). Reduces susceptibility compared to animals, offspring, or cells that do not contain the chromosomal sequence.

The animal or offspring may be embryonic, juvenile, or adult. Similarly, the cells may include embryonic cells, cells derived from juvenile animals, or cells derived from adult animals.

Animals or offspring may include farmed animals. Likewise, the cells may include cells derived from farmed animals. Domestic animals include domestic animals, such as pigs, bovine animals (e.g. beef or dairy cows), sheep animals, goat animals, equine animals (e.g. horses or donkeys), water buffaloes, camels, or avian animals (e.g. chickens). , turkeys, ducks, geese, guinea fowl, or newly hatched birds). The livestock animal is preferably a bovine or porcine animal, most preferably a porcine animal.

The animal or offspring may include a gene-edited animal. The cells may include gene edited cells.

Animals or cells can be gene edited using homing endonucleases. The homing endonuclease may be a naturally occurring endonuclease, but it is unlikely that the endonuclease is a rationally designed, non-naturally occurring homing endonuclease with a DNA recognition sequence designed to target the chromosomal sequence of the gene encoding the CD163 protein. desirable. Accordingly, the homing endonuclease may be a designed homing endonuclease. Homing endonucleases include, for example, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and recombinase fusion proteins. , meganuclease, or a combination thereof). The animal or cell is preferably an animal or cell that has been gene edited using the CRISPR/Cas9 system.

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

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

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

An insertion or deletion may reduce CD163 protein production or activity compared to CD163 protein production or activity in an animal, progeny, or cell without the insertion or deletion.

The insertion or deletion may result in the production of substantially non-functional CD163 protein by the animal, progeny, or cell. “Substantially non-functional CD163 protein” means that the level of CD163 protein in the animal, progeny, or cell is undetectable or, if detectable, is lower than the level observed in the animal, progeny, or cell that does not contain the insertion or deletion. That means at least about 90% lower.

If the animal, progeny, or cell includes a porcine animal, progeny, or cell, the modified chromosomal sequence is exon 7 of the gene encoding the CD163 protein, exon 8 of the gene encoding the CD163 protein, or the gene encoding the CD163 protein. It may include modifications in exon 7 or exon 8 and the adjacent intron, or a combination thereof. The modified chromosomal sequence suitably includes a modification in exon 7 of the gene encoding the CD163 protein.

Modifications in exon 7 of the gene encoding the CD163 protein may include deletions (e.g., in-frame deletions in exon 7). Alternatively, modifications in exon 7 of the gene encoding the CD163 protein may involve an insertion.

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

SEQ ID NO: 47 provides the nucleotide sequence for the region starting 3000 base pairs (bp) upstream of exon 7 of the wild-type porcine 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 Figure 16.

If the porcine animal or cell contains a 2 base pair insertion between nucleotides 3,149 and 3,150 relative to the reference sequence SEQ ID NO:47, the 2 base pair insertion may include the insertion of dinucleotide AG.

If the porcine animal or cell contains a one base pair insertion between nucleotides 3,147 and 3,148 relative to the reference sequence SEQ ID NO:47, the one base pair insertion may include the insertion of a single adenine residue.

If the porcine animal or cell contains a 7 base pair insertion between nucleotides 3,148 and 3,149 relative to the reference sequence SEQ ID NO: 47, then the 7 base pair insertion may include an insertion of the sequence TACTACT (SEQ ID NO: 115).

A porcine animal or cell comprising a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 relative to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488, and the reference sequence sequence Given the additional 129 base pair deletion of nucleotides 3,044 to 3,172 in exon 7 compared to number 47, the 12 base pair insertion may include the insertion of the sequence TGTGGAGAATTC (SEQ ID NO: 116).

11 bases, if the porcine animal or cell contains a 1382 base pair deletion from nucleotides 3,113 to nucleotides 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113. Paired insertions may include insertion of the sequence AGCCAGCGTGC (SEQ ID NO: 117).

If the modified chromosomal sequence of the gene encoding the CD163 protein comprises a deletion, the deletion preferably comprises an in-frame deletion. In-frame deletions are deletions that do not result in a change in the triplet reading frame and thus result in a protein product that has an internal deletion of one or more amino acids but is not truncated. If splicing occurs correctly, deletion of 3 base pairs or multiples of 3 base pairs within an exon can result in an in-frame mutation.

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

Accordingly, in porcine animals and cells, the insertion or deletion in the gene encoding the CD163 protein is a 1506 base pair deletion from nucleotide 1,525 to nucleotide 3,030 compared to the reference sequence SEQ ID NO: 47; A 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488 and nucleotide 7 in exon 7 compared to the reference sequence SEQ ID NO: 47 There is an additional 129 base pair deletion from nucleotides 3,044 to 3,172); A 1373 base pair deletion from nucleotides 2,724 to nucleotides 4,096 compared to the reference sequence SEQ ID NO: 47; A 123 base pair deletion from nucleotides 3,024 to nucleotides 3,146 compared to the reference sequence SEQ ID NO:47; A 1467 base pair deletion from nucleotides 2,431 to nucleotides 3,897 compared to the reference sequence SEQ ID NO:47; A deletion of nucleotides 3,145 to 4,5311387 base pairs compared to the reference sequence SEQ ID NO:47; A 1382 base pair deletion from nucleotides 3,113 to nucleotides 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotides 3,113; A 1720 base pair deletion from nucleotides 2,440 to nucleotides 4,160 compared to the reference sequence SEQ ID NO: 47; and combinations thereof.

The porcine animal or cell may contain an insertion or deletion selected from the group consisting of: a two base pair insertion between nucleotides 3,149 and 3,150 relative to the reference sequence SEQ ID NO:47 and on the same allele in the reference sequence SEQ ID NO:47 compared to a 377 base pair deletion from nucleotides 2,573 to nucleotides 2,949; A 28 base pair deletion from nucleotides 3,145 to nucleotides 3,172 compared to the reference sequence SEQ ID NO:47; and combinations thereof. For example, an animal or cell may have a 2 base pair insertion between nucleotides 3,149 and 3,150 relative to the reference sequence SEQ ID NO: 47, and a 377 base pair insertion from nucleotide 2,573 to nucleotide 2,949 relative to the reference sequence SEQ ID NO: 47 on the same allele. May include results. The animal or cell may comprise a 28 base pair deletion from nucleotide 3,145 to nucleotide 3,172 compared to the reference sequence SEQ ID NO:47.

The porcine animal or cell may comprise a 7 base pair insertion between nucleotides 3,148 and 3,149 relative to the reference sequence SEQ ID NO:47 and an 11 base pair deletion from nucleotides 3,137 to 3,147 relative to the reference sequence SEQ ID NO:47.

A porcine animal or cell containing any of the insertions or deletions described above may comprise a chromosomal sequence with a high degree of sequence identity to SEQ ID NO: 47 in addition to the insertion or deletion. Thus, for example, the 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 It may include a chromosomal sequence having 99.9%, or 100% sequence identity.

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

For example, a porcine animal or cell may comprise a chromosomal sequence comprising SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114. SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114 provides the nucleotide sequence for an 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.

A porcine animal or cell may contain an 11 base pair deletion in one allele of the gene encoding the CD163 protein and a 2 base pair insertion and a 377 base pair deletion in the other allele of the gene encoding the CD163 protein. there is.

A porcine animal or cell may contain a 124 base pair deletion in one allele of the gene encoding the CD163 protein and a 123 base pair deletion in another allele of the gene encoding the CD163 protein.

Porcine animals or cells may contain a one base pair insertion.

A porcine animal or cell may contain a 130 base pair deletion in one allele of the gene encoding the CD163 protein and a 132 base pair deletion in another allele of the gene encoding the CD163 protein.

Porcine animals or cells may contain a 1506 base pair deletion.

Porcine animals or cells may contain a 7 base pair insertion.

A porcine animal or cell may contain a 1280 base pair deletion in one allele of the gene encoding the CD163 protein and a 1373 base pair deletion in another allele of the gene encoding the CD163 protein.

Porcine animals or cells may contain a 1467 base pair deletion.

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

A porcine animal or cell may contain a 28 base pair deletion in one allele of the gene encoding the CD163 protein and a 1387 base pair deletion in another allele of the gene encoding the CD163 protein.

Pig animals or cells may contain a 1382 base pair deletion and an 11 base pair insertion 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. there is.

Any of the cells containing at least one altered chromosomal sequence in the gene encoding the CD163 protein may include sperm cells. Alternatively, any of these cells may comprise an egg cell (eg, a fertilized egg).

Any of the cells containing at least one altered chromosomal sequence in the gene encoding the CD163 protein may include somatic cells. For example, any of the cells may include fibroblasts (eg, fetal fibroblasts).

Targeted integration of nucleic acids at the CD163 locus

Site-specific integration of exogenous nucleic acid 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 a cell (e.g., an isolated cell or a cell of a tissue or organism) with a nucleic acid molecule comprising the exogenous nucleic acid. Such nucleic acid molecules may include nucleotide sequences flanking the exogenous nucleic acid that promote homologous recombination between the nucleic acid molecule and at least one CD163 locus. The nucleotide sequence flanking the exogenous nucleic acid that promotes homologous recombination may be complementary to the endogenous nucleotides of the CD163 locus. Alternatively, the nucleotide sequence flanking the exogenous nucleic acid that promotes homologous recombination may be complementary to the previously integrated exogenous nucleotide. Multiple exogenous nucleic acids can be integrated at one CD163 locus, such as in gene stacking.

Integration of nucleic acids at the CD163 locus may be facilitated (e.g., catalyzed) by the host cell's endogenous cellular machinery, such as, but not limited to, endogenous DNA and endogenous recombinase enzymes. Alternatively, integration of nucleic acid at the CD163 locus can be facilitated by one or more factors (e.g., polypeptides) presented to the host cell. For example, nuclease(s), recombinase(s), and/or ligase polypeptides can be expressed by contacting the polypeptide with a host cell, or by expressing the polypeptide within the host cell (either independently or as part of a chimeric polypeptide). ) can be provided. Accordingly, a nucleic acid comprising a nucleotide sequence encoding at least one nuclease, recombinase, and/or ligase polypeptide can be introduced into the host cell simultaneously or sequentially with the nucleic acid that site-specificly integrates into the CD163 locus. and wherein at least one nuclease, recombinase, and/or ligase polypeptide is expressed from the nucleotide sequence in the host cell.

DNA-binding polypeptide

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

DNA-binding polypeptides identified from naturally occurring proteins typically bind distinct nucleotide sequences or motifs (e.g., consensus recognition sequences), but it is difficult to modify many of these DNA-binding polypeptides to recognize different nucleotide sequences or motifs. Methods for this exist and are known in the art. DNA-binding polypeptides include, but are not limited to, a zinc finger DNA-binding domain; leucine zipper; UPA DNA-binding domain; GAL4; TAL; LexA; Tet inhibitor; LacI; and steroid hormone receptors.

For example, the DNA-binding polypeptide may 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 DNA by inserting an α-helix into the major groove of the target DNA double helix. Recognition of DNA by zinc fingers is modular; Each finger primarily contacts three conservative base pairs in the target, and several key residues in the polypeptide mediate recognition. By including multiple zinc finger DNA-binding domains in the targeting endonuclease, the DNA-binding specificity of the targeting endonuclease can be further increased (and thus the specificity of any gene regulatory effect conferred thereby can also be increased). See, for example, Urnov et al. (2005) Nature 435:646-51. Accordingly, the one or more zinc finger DNA-binding polypeptides are used to target endonucleases introduced into the host cell within the genome of the host cell. It can be engineered and used to interact with unique DNA sequences.

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

Engineered zinc finger binding domains can have novel binding specificities compared to naturally-occurring zinc finger proteins. Methods of operation include, but are not limited to, rational design and various types of selection. Rational designs include, for example, using databases containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruple nucleotide sequence is a specific triplet or quadruplicate nucleotide sequence. It relates to the sequence of one or more amino acids of a zinc finger that binds to the sequence. For example, U.S. Pat. Nos. See 6,453,242 and 6,534,261.

Exemplary selection methods including phage display and two-hybrid systems are described in the 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; Disclosed in WO 01/88197 and GB 2,338,237. Additionally, enhancement of binding specificity for zinc finger binding domains is described, for example, in WO 02/077227.

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

Selection of Target Site: Methods for the design and construction of ZFPs and fusion proteins (and polynucleotides encoding them) are known to those skilled in the art and can be found in the U.S.S. 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; It is described in detail in WO 02/016536 and WO 03/016496.

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

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

Native GAL4 consists of 881 amino acid residues with a molecular mass of 99 kDa. GAL4 contains functionally autonomous domains, the combined activity of which 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 contain 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 a divalent cation coordinated by six Cys residues present in the DNA binding domain. The coordinated cation-containing domain recognizes by interacting with the conserved CCG triplet at each end of the 17 bp UAS through direct contact with the major groove of the DNA helix [see literature; Marmorstein et al. (1992) Nature 356:408-14]. The DNA-binding function of the protein places the C-terminal transcriptional activation domain in the vicinity of the promoter so that the activation domain can carry out transcription.

Additional DNA-binding polypeptides that can be used include, but are not limited to, binding sequences from AVRBS3-inducible genes; A consensus binding sequence from an AVRBS3-inducible gene or a synthetic binding sequence engineered therefrom (e.g., UPA DNA-binding domain); TAL; LexA (see, e.g.; Brent & Ptashne (1985), supra); LacR (see, e.g., Labow et al. (1990) Mol. Cell. Biol. 10:3343-56; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88(12):5072-6); steroid hormone receptor (Ellliston et al. (1990) J. Biol. Chem. 265:11517-121); Tet suppressor (U.S. Pat. No. 6,271,341) and a mutated Tet suppressor that binds a Tet operator sequence in the presence, but not the absence, of tetracycline (Tc); DNA-binding domain of NF-kappaB; and fusions of GAL4, hormone receptor, and VP16 [see; 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. Patent Publication No. See 2011/0301073.

Alternatively, the nuclease may include the CRISPR/Cas system. This system includes a clustered regularly interspaced short palindromic repeats (CRISPR) locus that encodes the RNA component of the system, and a Cas (CRISPR-related) locus that encodes a protein (Jansen et al., 2002. Mol. Microbiol. 43: 1565- 1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput. Biol. 1: e60). In microbial hosts, the CRISPR locus contains a combination of Cas genes as well as non-coding RNA factors that can program the specificity of CRISPR-mediated nucleic acid cleavage.

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

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

Cas proteins may be “functional derivatives” of naturally occurring Cas proteins. A “functional derivative” of a native sequence polypeptide is a compound that has qualitative biological properties in common with the native sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of native sequence and derivatives of native sequence polypeptides and fragments thereof, 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 into fragments. The term “derivative” includes both amino acid sequence variants of a polypeptide, covalent modifications and fusions thereof. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, and covalent modifications of Cas proteins or fragments thereof. Cas proteins, including Cas proteins or fragments thereof, as well as derivatives of Cas proteins or fragments thereof, can be obtained from cells or synthesized chemically or by a combination of these two processes. Cells may be either cells that naturally produce Cas proteins, or cells that naturally produce Cas proteins and are adapted to produce higher expression levels of endogenous Cas proteins, or from exogenously introduced nucleic acids (the nucleic acids encode Cas that are the same or different from the endogenous Cas). These may be cells that are genetically engineered to produce Cas proteins. In some cases, cells do not naturally produce Cas proteins and are genetically engineered to produce Cas proteins.

A DNA-binding polypeptide is capable of specifically recognizing and binding to a target nucleotide sequence contained within the genomic nucleic acid of a host organism. Several distinct instances of target nucleotide sequences can, in some instances, be found in the host genome. The target nucleotide sequence may be rare within the genome of an organism (e.g., less than about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 copy(s) ) target sequence may be present in the genome). For example, the target nucleotide sequence may be located at a unique site within the organism's genome. Target nucleotide sequences may be, for example, but not limited to, randomly distributed throughout the genome relative to each other; are located in different linkage groups in the genome; or located in the same chain group; located on a different chromosome; located on the same chromosome; is located in the genome at a site that is expressed under similar conditions in an organism; They may be located closely together in the genome (e.g., target sequences may be contained within nucleic acids integrated as concatemers at a genomic locus).

Targeting endonuclease

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

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

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

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

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

Chimeric polypeptides may include DNA-binding domains (eg, zinc fingers, TAL-effector domains, etc.) and nuclease (cleavage) domains. The cleavage domain may be a DNA-binding domain, such as a zinc finger DNA-binding domain and a cleavage domain from a nuclease, or a TALEN DNA-binding domain and a cleavage domain, or a meganuclease DNA-binding domain from a different nuclease. and may be heterologous to the cleavage domain. Heterologous cleavage domains can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which cleavage domains can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 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 literature; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.

Similarly, the cleavage half-domain may be derived from any nuclease, or portion thereof, as described above, that requires dimerization for cleavage activity. Generally, if the fusion protein contains a cleavage half-domain, two fusion proteins are required for cleavage. Alternatively, a single protein containing two truncated half-domains can be used. The two cleavage half-domains may be derived from the same endonuclease (or functional fragment thereof), or each cleavage half-domain may be derived from a different endonuclease (or functional fragment thereof). Additionally, the target sites for the two fusion proteins are positioned in a spatial orientation such that binding of the two fusion proteins to their respective target sites causes the cleavage half-domains to form a functional cleavage domain, e.g., by dimerization. are placed relative to each other to locate the truncated half-domains. Therefore, near the edges of the target region may be separated by 5-8 nucleotides or by 15-18 nucleotides. However, any integer number of nucleotides, or nucleotide pairs, may be interposed between the two target sites (e.g., 2 to 50 nucleotide pairs or more). Typically, the site of cleavage is between target sites.

Restriction endonucleases (restriction enzymes) exist in many species and can bind sequence-specifically to DNA (at the recognition site), e.g. when one or more exogenous sequences (donor/transgene) are bound ( DNA can be cut at or near the binding site to allow integration at or near the target) site. Certain restriction enzymes (e.g., type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme Fok I catalyzes double-strand breaks in DNA at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. For example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as 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]. Accordingly, the fusion protein will contain a cleavage domain (or cleavage half-domain) from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered.

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

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

Exemplary type IIS restriction enzymes are described in the U.S. Patent Publication No. It is described in 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].

Cleavage domains are described, for example, in the U.S. Patent Publication Nos. 2005/0064474; It may comprise one or more engineered truncated half-domains (also called dimerization domain mutants) that minimize or prevent homodimerization, as described in 2006/0188987 and 2008/0131962.

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

zinc finger nuclease

Chimeric polypeptides may include custom-designed zinc finger nucleases (ZFNs) that can be designed to deliver targeted site-specific double-stranded DNA breaks so that exogenous nucleic acids, or donor DNA, can be incorporated (US See Patent Publication 2010/0257638). ZFNs are chimeric polypeptides containing non-specific cleavage domains from restriction endonucleases (eg, FokI) and zinc finger DNA-binding domain polypeptides. See, 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 may contain non-canonical zinc finger DNA binding domains (see US Patent Publication 2008/0182332). The FokI restriction endonuclease cleaves DNA and must dimerize through the nuclease domain to introduce double-stranded breaks. Thus, ZFNs containing the nuclease domain from these endonucleases also require dimerization of the nuclease domain to cleave the target DNA [see; Mani et al. (2005) Biochem. Biophys. Res. Commun. 334:1191-7; Smith et al. (2000) Nucleic Acids Res. 28:3361-9]. Dimerization of ZFNs can be promoted by two adjacent, oppositely oriented DNA-binding sites. Id.

A method for site-specific integration of an exogenous nucleic acid into at least one CD163 locus of a host may include introducing a ZFN into a cell of the host, wherein the ZFN recognizes and binds to a target nucleotide sequence and 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 the at least one CD163 locus. For example, a DNA-binding polypeptide of a ZFN can be engineered to recognize and bind to a target nucleotide sequence identified within at least one CD163 locus (e.g., by sequencing the CD163 locus). A method for site-specific integration of an exogenous nucleic acid into at least one CD163 performance locus of a host comprising introducing a ZFN into a cell of the host may also include introducing the exogenous nucleic acid into the cell, wherein at least Recombination of an exogenous nucleic acid into a host nucleic acid containing one CD163 locus is facilitated by site-specific recognition and binding of ZFNs to target sequences (and subsequent cleavage of the nucleic acid containing the CD163 locus).

Any exogenous nucleic acid for integration at the CD163 locus

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

Any exogenous nucleic acid molecule for site-specific integration

As noted above, insertion of an exogenous sequence (also called a “donor sequence” or “donor” or “transgene”) may be necessary, for example, for the expression of a polypeptide, for correction of a mutant gene, or for the increase of a nocturnal gene. Provided for expressed expression. It will be readily apparent that the donor sequence is typically not identical to the genomic sequence from which it was placed. The donor sequence may contain non-homologous sequences flanked by two regions of homology to allow for efficient homology-directed repair (HDR) at the position of interest. Additionally, the donor sequence may include vector molecules containing sequences that are not homologous to the region of interest in the cellular chromatin. The donor molecule may contain several discontinuous regions of homology to cellular chromatin. For example, for targeted insertion of a sequence that is not normally present in the region of interest, the sequence may be present in a donor nucleic acid molecule and flanked by sequences of homology 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 the cell linearly or circularly. For example, U.S. Patent Publication Nos. See 2010/0047805, 2011/0281361, 2011/0207221, and 2013/0326645. When introduced linearly, the ends of the donor sequence may be protected (e.g., from degradation by nucleic acid end hydrolysis) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule and/or a self-complementary oligonucleotide is ligated to one or both ends. For example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; See Nehls et al. (1996) Science 272:886-889. Additional methods to protect exogenous polynucleotides from degradation include the addition of terminal amino group(s) and modified internucleotide chains, such as phosphorothionate, phosphoramidate, and O-methyl ribose or deoxyribose. Including, but not limited to, the use of ribose residues.

Polynucleotides can be introduced into cells as part of a vector molecule with additional sequences, such as, for example, origins of replication, promoters, and genes encoding antibiotic resistance. Additionally, the donor polynucleotide can be introduced as a naked nucleic acid, complexed with an agent such as a liposome or poloxamer, or as a nucleic acid complexed with an agent such as a liposome or poloxamer, or as a nucleic acid complexed with an agent such as a liposome or poloxamer. It can be transmitted by a defective lentivirus (IDLV).

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

Moreover, although not required for expression, exogenous sequences may also contain transcriptional or translational regulatory sequences, such as sequences encoding promoters, enhancers, insulators, internal ribosome entry sites, 2A peptides, and/or polyadenylation signals. It can be included.

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 encoding a polypeptide of interest; Nucleic acids containing crop genes; A nucleic acid comprising a nucleotide sequence encoding an RNAi molecule; or a nucleic acid that cleaves the CD163 gene.

An exogenous nucleic acid can be integrated at the CD163 locus to modify the CD163 locus, wherein the nucleic acid comprises a nucleotide sequence encoding a 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 in commercial quantities from a nucleotide sequence encoding the polypeptide of interest. In these examples, the polypeptide of interest may be extracted from host cells, tissues, or biomass.

Nucleic acid molecule comprising a nucleotide sequence encoding a targeting endonuclease

The nucleotide sequence encoding the targeting endonuclease can be manipulated by manipulation (e.g., ligation) of the native nucleotide sequence encoding the polypeptide comprised within the targeting endonuclease. For example, the nucleotide sequence of a gene encoding a protein containing a DNA-binding polypeptide can be examined to identify the nucleotide sequence of a gene corresponding to the DNA-binding polypeptide, and the nucleotide sequence can be used to identify a targeting endoplasmic reticulum protein containing a DNA-binding polypeptide. It 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 infer the nucleotide sequence encoding the targeting endonuclease, for example, by degenerating the genetic code.

In an exemplary nucleic acid molecule comprising a nucleotide sequence encoding a targeting endonuclease, the last codon of the first polynucleotide sequence encoding the nuclease polypeptide, and the first codon of the second polynucleotide sequence encoding the DNA-binding polypeptide. Codons may be separated by any number of nucleotide triplets, eg, without an intron or coding for “STOP”. Likewise, the last codon of the nucleotide sequence encoding the first polynucleotide sequence encoding the DNA-binding polypeptide, and the first codon of the second polynucleotide sequence encoding the nuclease polypeptide may be separated into any number of nucleotide triplets. . The last codon (i.e., usually 3' in the nucleic acid sequence) of a first polynucleotide sequence encoding a nuclease polypeptide, and a second polynucleotide sequence encoding a DNA-binding polypeptide, is the codon of an additional polynucleotide coding sequence immediately adjacent thereto. may be fused at the first codon and phase-register, or may be separated therefrom by only a short peptide sequence, such as that encoded by a synthetic nucleotide linker (e.g., a nucleotide linker that may be used to effect the fusion). You can. Examples of such additional polynucleotide sequences include, but are not limited to, tags, targeting peptides, and enzymatic cleavage sites. Likewise, the first codon, usually 5' (in the nucleic acid sequence) of the first and second polynucleotide sequences, may be fused in topology with the last codon of an additional polynucleotide coding sequence immediately adjacent to it, or only to a short peptide sequence. It can be separated from this by .

Sequences that separate the polynucleotide sequences encoding functional polypeptides (e.g., DNA-binding polypeptides and nuclease polypeptides) from the targeting endonuclease may be defined, for example, by which the encoded amino acid sequence facilitates translation by the targeting endonuclease. It may consist of any sequence so as not to 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 functions of these structures.

Guitar Knockout Method

Various other techniques known in the art can be used to produce progenitor animals and create animal lines by inactivating genes to create knock-out animals and/or introducing nucleic acid constructs into the animals, wherein the knock-out or nucleic acid The construct is integrated into the genome. These techniques include, but are not limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191), retroviral mediated gene transfer to the germ line (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6148-1652), gene targeting to embryonic stem cells (Thompson et al. (1989) Cell 56, 313-321), electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3, 1803- 1814), sperm-mediated gene transfer (Lavitrano et al. (2002) Proc. Natl. Acad. Sci. USA 99, 14230-14235; Lavitrano et al. (2006) Reprod. Fert. Develop. 18, 19-23), and somatic cells. , for example, in vitro transformation of cumulus or mammary cells, or adult, fetal, or embryonic stem cells followed by nuclear transfer (Wilmut et al. (1997) Nature 385, 810-813; and Wakayama et al. (1998) Nature 394, 369 -374). Pronuclear microinjection, sperm-mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques. A genome-modified animal is an animal in which all of its cells, including germline cells, have genetic modification. If methods are used to produce animals that are mosaic in their genetic modification, the animals can be inbred and genome-modified progeny can be selected. For example, cloning can be used to create mosaic animals if cells are transformed in the blastocyst state, or genomic modifications can occur if single cells are transformed. Animals that are transformed and fail to reach sexual maturity may be homozygous or heterozygous for the transformation, depending on the specific approach used. If a specific gene is inactivated by a knockout variant, homozygosity would normally be required. If a particular gene is inactivated by RNA interference or a dominant negative strategy, heterozygosity is often appropriate.

Typically, in embryo/zygote microinjection, a nucleic acid construct or mRNA is introduced into a fertilized egg; A one or two cell fertilized egg is used as the nuclear structure containing genetic material from the sperm head and the egg is visible within the protoplasm. Fertilized eggs in the pronuclear state can be obtained in vitro or in vivo (i.e., surgically retrieved from the oviduct of a donor animal). In vitro fertilized eggs can be created as follows: For example, pig ovaries can be collected from the slaughterhouse and maintained at 22-28°C during transport. Ovaries can be washed and separated for follicular aspiration, and follicles measuring 4-8 mm can be aspirated under vacuum into a 50 mL conical centrifuge tube using an 18-gauge needle. Follicular fluid and aspirated oocytes can be washed through a pre-filter with commercially available TL-HEPES (Minitube, Verona, Wis.). Oocytes surrounded by a compact cumulus mass were selected and incubated with 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor, and 10% porcine follicular fluid for approximately 22 hours in humidified air at 38.7°C and 5% CO _{2 .} , TCM-199 oocytes supplemented with 50 μM 2-mercaptoethanol, 0.5 mg/ml cAMP, 10 IU/mL each of pregnant horse serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). It can be placed in MATURATION MEDIUM (Minitube, Verona, Wis.). Afterwards, oocytes can be transplanted into fresh TCM-199 maturation medium (which will not contain cAMP, PMSG or hCG) and cultured for an additional 22 hours. Mature oocytes can be stripped from their cumulus cells by vortexing for 1 minute in 0.1% hyaluronidase.

For pigs, mature oocytes can be fertilized in 500 μl Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, Wis.) in Minitube 5-well fertilization dishes. In preparation for in vitro fertilization (IVF), freshly-collected or frozen semen 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 with a final concentration of approximately 40 motile sperm/oocytes in a volume of 10 μl depending on the boar. All fertilized oocytes are incubated for 6 hours at 38.7°C in a 5.0% CO ₂ atmosphere. Six hours after semen injection, putative zygotes can be washed twice in NCSU-23 and transferred to 0.5 mL of the same medium. This system can routinely produce 20-30% blastocysts across most boars at a 10-30% polyspermic insemination rate.

Linearized nucleic acid constructs or mRNA can be injected into either the pronucleus or into the cytoplasm. The injected eggs can then be transferred to a recipient female (e.g., into the oviduct of the recipient female) and allowed to grow in the recipient female, producing transgenic or gene-edited animals. In particular, in vitro fertilized embryos can be centrifuged at 15,000 x g for 5 minutes to precipitate lipids, allowing visualization of the pronucleus. Embryos can be injected using an Eppendorf FEMTOJET syringe and cultured until blastocyst formation. The rate and quality of embryo cleavage and blastocyst formation can be recorded.

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

In somatic cell nuclear transfer, transgenic or gene-edited cells, such as embryonic blastomeres, fetal fibroblasts, adult ear fibroblasts, or granulosa cells, containing the above-described nucleic acid construct are introduced into an enucleated oocyte. Combined cells can be established. Oocytes can be enucleated by incision of a partial area near the polar body followed by squeezing out cytoplasm from the incision. Typically, an injection pipette with a sharp beveled tip is used to inject transgenic or gene-edited cells into enucleated oocytes that are arrested in meiosis 2. In some conventions, oocytes arrested in meiosis-2 are called eggs. After producing pig or bovine embryos (e.g., by fusing and activating oocytes), the embryos are implanted into the fallopian tubes of a female recipient approximately 20 to 24 hours after activation. For example, Cibelli et al. (1998) Science 280, 1256-1258 and U.S. Pat. Nos. See 6,548,741, 7,547,816, 7,989,657, or 6,211,429. In pigs, pregnancy can be confirmed for female recipients approximately 20-21 days after embryo transfer.

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

Once a gene-edited animal has been created, inactivation of endogenous nucleic acids can be assessed using standard techniques. Initial screening can be accomplished by Southern blot analysis to determine whether inactivation has occurred or not. For a description 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 the process or technique of amplifying target nucleic acids. Typically, sequence information from the end of the region of interest or beyond is used to design oligonucleotide primers that are identical or similar in sequence to the opposite strand of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers are typically 14 to 40 nucleotides in length, but can range from 10 nucleotides to several hundred nucleotides in length. PCR is described, for example; PCR Primers: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Nucleic acids can also be amplified by ligase chain reaction, strand displacement amplification, self-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, embryos can be processed individually for analysis by PCR, Southern hybridization and splinkerette PCR (see, e.g., Dupuy et al. Proc Natl Acad Sci USA (2002) 99:4495). .

Interfering RNAs

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

For example, an exogenous nucleic acid sequence can induce RNA interference with the nucleic acid encoding the polypeptide. For example, double-stranded small interfering RNA (siRNA) or small hairpin RNA (shRNA) homologous to the target DNA can be used to reduce expression of that DNA. Constructs for siRNA are described, for example, in the literature; 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 detail; It can be prepared as described in 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 directed to a specific gene is high. For example, the predictability of the specific sequence of a 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 gene edited animals, such as domesticated animals expressing RNAi directed against 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

Inducible systems can be used to inactivate the CD163 gene. A variety of inducible systems are known that allow spatial and temporal control of inactivation of genes. Some have been shown to be functional in vivo in porcine animals.

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

Among the more commonly used inducible systems are the tetracycline-inducible system and the Cre/loxP recombinase system (constitutive or inducible). The tetracycline-inducible system includes tetracycline-controlled transcription activator (tTA)/reverse tTA (rtTA). Methods for using this system in vivo involve generating two strains of gene-edited animals. An animal strain expresses an activator (tTA, rtTA, or Cre recombinase) under the control of a selected promoter. Another animal line expresses the receptor, wherein the expression of the gene of interest (or the gene to be modified) is under the control of a targeting sequence for the tTA/rtTA transactivator (or flanked by a loxP sequence). Mating of two animals provides control of gene expression.

The tetracycline-dependent regulatory system (tet system) consists of two components: a tetracycline-controlled transcriptional activator (tTA or rtTA) and a tTA/rtTA-dependent transactivator that controls the expression of downstream cDNA in a tetracycline-dependent manner. Depends on accelerants. In the absence of tetracycline or its derivatives (eg doxycycline), tTA binds to the tetO sequence, allowing transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target and transcription does not occur. The tet system using tTA is called tet-OFF because tetracycline or doxycycline enables transcriptional down-regulation. Administration of tetracycline or its derivatives allows temporal control of transgene expression in vivo. rtTA is a variant of tTA that fails to 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 transgenes encoding, for example, reporter genes, oncogenes, or proteins involved in signaling cascades.

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

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

Vectors and Nucleic Acids

Various nucleic acids can be introduced into cells for knockout purposes, inactivation of genes, obtaining expression of genes, or for another purpose. 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 the base moiety, sugar moiety, or phosphate backbone to improve the stability, hybridization, or solubility of the nucleic acid. Modifications in the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-doxycytidine for deoxycytidine. do. Modification of the sugar moiety includes modification of the 2' hydroxyl of the ribose sugar to form a 2'-O-methyl or 2'-O-allyl sugar. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a 6-atom, morpholino ring, or peptide nucleic acid, the deoxyphosphate backbone is replaced by a pseudopeptide backbone, and the four Two bases are retained. Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7(3):187; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5]. Additionally, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

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

Any type of promoter can be operably linked to a target nucleic acid sequence. Examples of accelerators include, without limitation, tissue-specific accelerators, constitutive accelerators, inducible accelerators, and accelerators that are responsive or unresponsive to a specific stimulus. Suitable tissue-specific promoters can cause preferential expression of nucleic acid transcripts in beta cells and include, for example, the human insulin promoter. Other tissue-specific promoters may result in preferential expression in, for example, hepatocytes or cardiac tissue and may include albumin or alpha-myosin heavy chain promoters, respectively. Promoters can be used that promote expression of nucleic acid molecules without significant tissue or time-specificity (i.e., constitutive promoters). For example, a beta-actin promoter, such as chicken beta-actin gene promoter, ubiquitin promoter, miniCAGs promoter, glyceraldehyde-3-phosphedite dehydrogenase (GAPDH) promoter, or 3-phosphoglycerate kinase. (PGK) promoters, as well as viral promoters such as the herpes simplex virus thymidine kinase (HSV-TK) promoter, SV40 promoter, or cytomegalovirus (CMV) promoter can be used. For example, a fusion of the chicken beta actin gene promoter and the CMV enhancer can be used as the promoter. For example, Xu et al. (2001) Hum. Gene Ther. 12:563; and Kiwaki et al. (1996) Hum. Gene Ther. 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 segments, IRES), enhancers, guidance elements, or introns. Although these regulatory regions may increase expression by influencing transcription, mRNA stability, translation efficiency, etc., this may not be essential. These regulatory regions can 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 a single peptide or selection marker can be used. A single peptide can be used to direct the encoded polypeptide to a specific cellular location (e.g., the cell surface). Non-limiting examples of selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), higro Includes mycin-B-phosphoribosyltransferase, thymidine kinase (TK), and xanthine-guanine phosphoribosyltransferase (XGPRT). These markers are useful for selecting stable transformants in culture. Other selection markers include fluorescent polypeptides, such as green fluorescent protein or yellow fluorescent protein.

The sequence encoding the selection marker may be flanked by recognition sequences for recombinase enzymes, such as Cre or Flp. For example, the selection marker may be flanked by loxP recognition sites (a 34-bp recognition site recognized by Cre recombinase) or FRT recognition sites so that the selection marker can be excised 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]. Transposons containing Cre- or Flp-activatable transgenes blocked by a selectable marker gene can also be used to obtain animals with conditional expression of the transgene. For example, promoter driven expression of a marker/transgene may be ubiquitous or tissue-specific, which would result in ubiquitous or tissue-specific expression of the marker in F0 animals (e.g., pigs). Tissue-specific activation of the transgene can be achieved, for example, by crossing a pig universally expressing a marker-blocked transgene to a pig expressing Cre or Flp in a tissue-specific manner, or by crossing the marker in a tissue-specific manner. -Can be achieved by crossing pigs expressing the blocked transgene to pigs universally expressing Cre or Flp recombinase. Controlled expression of the transgene or controlled excision of the marker allows expression of the transgene.

Exogenous nucleic acids can encode polypeptides. A nucleic acid sequence encoding a polypeptide may include a tag sequence encoding a “tag” designed to facilitate subsequent manipulation (e.g., to facilitate localization or detection) of the encoded polypeptide. The tag sequence can be inserted into the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include glutathione S-transferase (GST) and FLAG ^™ 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°C. Hypermethylation can be confirmed by incubating the construct with one unit of HinP1I endonuclease at 37°C for 1 hour and analyzing by agarose gel electrophoresis.

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

In the transposon system, the transcription unit of the nucleic acid construct, i.e., the regulatory region operably linked to the exogenous nucleic acid sequence, is flanked by inverted repeats 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); Tol2 (Kawakami (2007) Genome Biology 8(Suppl.1):S7; Minos (Pavlopoulos et al. (2007) Genome Biology 8(Suppl.1):S2); Hsmar1 (Miskey et al. (2007)) Mol Cell Biol. 27: 4589); Several transposon systems have been developed to introduce nucleic acids into cells, including mouse, human, and porcine cells. The Sleeping Beauty transposon is particularly useful. The transposase may be delivered as a protein, encoded on the same nucleic acid construct as an exogenous nucleic acid, introduced on a separate nucleic acid construct, or provided as mRNA (e.g., in vitro transcribed and capped mRNA).

Insulating elements may also be included in the nucleic acid construct to maintain expression of the exogenous nucleic acid and inhibit unwanted transcription of host genes. For example, U.S. Disclosure No. Please refer to 2004/0203158. Typically, insulator elements flank each side of the transcription unit and are internal to the inverted repeat of the transposon. Non-limiting examples of insulating elements include substrate attachment region- (MAR) type insulating elements and border-type insulating elements. For example, U.S. Pat. Nos. 6,395,549, 5,731,178, 6,100,448, and 5,610,053, and U.S. Disclosure No. See 2004/0203158.

Nucleic acids can be inserted into vectors. Vector is a broad term that includes any specific DNA fragment designed to transfer from a carrier to target DNA. Vector may refer to an expression vector, or vector system, which is a set of components necessary to effect DNA insertion into a genome or other targeted DNA sequence, such as an episome, plasmid, or even a viral/phage DNA fragment. am. 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) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and the DNA target sequence and inserts the vector within the target DNA sequence. Vectors very frequently contain one or more expression cassettes comprising one or more expression control sequences, each of which is a different DNA sequence or DNA sequence that controls and regulates the transcription and/or translation of the mRNA.

Many different types of vectors are known. For example, plasmids and viral vectors, such as retroviral vectors, are known. Mammalian expression plasmids typically have an origin of replication, suitable promoters and optional enhancers, essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non-transcribed sequences. Examples of vectors include: plasmids (which may also be carriers of other types of vectors), adenoviruses, adeno-associated viruses (AAVs), lentiviruses (e.g. modified HIV-1, SIV or FIV), retroviruses 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 includes, 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 nucleic acids. It refers to both RNA and DNA, including the skeleton. Nucleic acid molecules can be double-stranded or single-stranded (i.e., sense or antisense single stranded).

Archeopteryx, Animal Lineages, Traits, and Reproduction

Archetype animals can be produced by cloning and other methods described herein. The progenitor may be homozygous for a genetic variant, such as when the zygote or primary cell undergoes a homozygous variant. Similarly, heterozygous progenitors can also be created. In the case of animals containing at least one altered chromosomal sequence in the gene encoding the CD163 protein, the progenitor is preferably heterozygous. Progenitors can undergo genome modification, meaning that all of the cells in their genome undergo modification. The progenitor may be mosaic in terms of transformation, as can happen when a vector is introduced into one of a number of cells in the embryo, typically at the blastocyst stage. Offspring of mosaic animals can be tested to identify offspring with genome modifications. An animal line is established when it can produce a group of animals that can be reproduced sexually with heterozygous or homozygous progeny that consistently express the variant or that can be propagated by assisted reproductive techniques.

In livestock, multiple alleles are known to be associated with various traits such as production traits, type traits, workability traits, and other functional traits. Skilled practitioners are familiar with monitoring and characterizing these traits [see literature; Visscher et al., Livestock Production Science, 40 (1994) 123-137, U.S. Pat. No. 7,709,206, US 2001/0016315, US 2011/0023140, and US 2005/0153317]. The animal line may include traits selected from the group consisting of production traits, type traits, workability traits, fertility traits, mothering traits, and disease resistance traits. Additional traits include expression of recombinant gene products.

Animals with the desired trait or traits can be modified to prevent their sexual maturity. Because animals are sterile until maturity, sexual maturation can be controlled as a means to control dissemination in animals. Accordingly, animals that have been bred or modified to have one or more traits can be provided to recipients with a reduced risk of the recipients breeding the animals and assigning value to the traits to themselves. For example, the genome of an animal may be genetically modified, wherein the modification includes inactivation of a sexual maturation gene, wherein the sexual maturation gene in a wild-type animal expresses a factor that is selective for sexual maturation. Animals can be treated by administering compounds that rescue defects caused by loss of expression of genes that lead to sexual maturation in the animal.

Reproduction of animals requiring administration of compounds that induce sexual maturation can advantageously be accomplished in treatment facilities. Treatment facilities can implement standardized protocols for well-controlled strains to efficiently produce consistent animals. Animal offspring can be distributed to multiple locations where one wishes to raise them. Accordingly, farms and ranchers (a term that includes ranches and ranchers) can order any number of offspring with a specified range of ages and/or weights and/or traits and take them at any desired time and/or location. The recipients, i.e. the farm owners, can then raise the animals and deliver them to the market when they want.

Genetically modified livestock animals with inactivated sexual maturation genes may be delivered (eg, to multiple farms, to more than one location). Animals can range in age from about 1 day to about 180 days. An animal may possess one or more traits (eg, expressing a desired or high-value trait or a novel or recombinant trait).

Methods of reproduction and methods for increasing the resistance of animals to infection and populations of animals

Provided herein are breeding methods for producing animals or bloodlines with reduced susceptibility to infection by pathogens. The method includes genetically modifying an oocyte or a sperm cell to introduce a modified chromosomal sequence in the gene encoding the CD163 protein into at least one of the oocyte and the sperm cell, and introducing the modified chromosomal sequence in the gene encoding the CD163 protein into at least one of the oocyte and the sperm cell. and fertilizing the oocyte with a sperm cell to produce a fertilized egg containing the oocyte. Alternatively, the method includes genetically modifying the fertilized egg to introduce a modified chromosomal sequence in the gene encoding the CD163 protein into the fertilized egg. The method comprises the steps of implanting a fertilized egg into a surrogate female animal, where pregnancy and full-term parturition produce progeny animals, screening the progeny animals for susceptibility to pathogens, and modifying the chromosome in the gene encoding the CD163 protein. Further comprising selecting progeny animals with reduced susceptibility to the pathogen compared to animals not comprising the sequence.

Pathogens preferably include viruses, such as PRRSV.

The animal or offspring may be embryonic, juvenile, or adult.

Animals or offspring may include farmed animals. Domestic animals include domestic animals, such as pigs, bovine animals (e.g. beef or dairy cows), sheep animals, goat animals, equine animals (e.g. horses or donkeys), water buffaloes, camels, or avian animals (e.g. chickens). , turkeys, ducks, geese, guinea fowl, or newly hatched birds). The livestock animal is preferably a bovine or porcine animal, most preferably a porcine animal.

Genetically modifying an oocyte, sperm cell, or fertilized egg may include gene editing the oocyte, sperm cell, or fertilized egg. Gene editing may include the use of homing endonucleases. The homing endonuclease may be a naturally occurring endonuclease, but it is unlikely that the endonuclease is a rationally designed, non-naturally occurring homing endonuclease with a DNA recognition sequence designed to target the chromosomal sequence of the gene encoding the CD163 protein. desirable. Accordingly, the homing endonuclease may be a designed homing endonuclease. Accordingly, the homing endonuclease may be a designed homing endonuclease. Homing endonucleases include, for example, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and recombinase fusion proteins. , meganuclease, or a combination thereof). Gene editing preferably involves the use of the CRISPR/Cas9 system.

The oocyte, sperm cell, or fertilized egg may be heterozygous for the altered chromosomal sequence. Alternatively, the oocyte, sperm cell, or fertilized egg may be homozygous for the modified chromosomal sequence.

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

The insertion or deletion may reduce CD163 protein production or activity compared to CD163 protein production or activity in animals without the insertion or deletion.

Insertions or deletions may result in the production of substantially non-functional CD163 protein by the animal. “Substantially non-functional CD163 protein” means that the level of CD163 protein in the animal, progeny, or cell is undetectable or, if detectable, is lower than the level observed in the animal, progeny, or cell that does not contain the insertion or deletion. That means at least about 90% lower.

If the animal is a porcine animal, the modified chromosomal sequence is exon 7 of the gene encoding the CD163 protein, exon 8 of the gene encoding the CD163 protein, exon 7 or an intron adjacent to exon 8 of the gene encoding the CD163 protein, or an intron thereof. Combinations may include variations. The modified chromosomal sequence suitably includes a modification in exon 7 of the gene encoding the CD163 protein.

Modifications in exon 7 of the gene encoding the CD163 protein may include deletions (e.g., in-frame deletions in exon 7). Alternatively, modifications in exon 7 of the gene encoding the CD163 protein may involve an insertion.

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

If the porcine animal contains a 2 base pair insertion between nucleotides 3,149 and 3,150 relative to the reference sequence SEQ ID NO:47, the 2 base pair insertion may include the insertion of dinucleotide AG.

If the porcine animal contains a one base pair insertion between nucleotides 3,147 and 3,148 relative to the reference sequence SEQ ID NO:47, then the one base pair insertion may include the insertion of a single adenine residue.

If the porcine animal contains a 7 base pair insertion between nucleotides 3,148 and 3,149 relative to the reference sequence SEQ ID NO: 47, then the 7 base pair insertion may include an insertion of the sequence TACTACT (SEQ ID NO: 115).

The porcine animal contains a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 relative to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by a 12 base pair insertion starting at nucleotide 488, and the reference sequence SEQ ID NO: 47 Compared to there is an additional 129 base pair deletion in exon 7 from nucleotides 3,044 to 3,172, the 12 base pair insertion may include the insertion of the sequence TGTGGAGAATTC (SEQ ID NO: 116).

If the porcine animal contains a 1382 base pair deletion from nucleotides 3,113 to nucleotides 4,494 compared to the reference sequence SEQ ID NO: 47, wherein the deleted sequence is replaced by an 11 base pair insertion starting at nucleotide 3,113, then an 11 base pair insertion. may comprise the insertion of the sequence AGCCAGCGTGC (SEQ ID NO: 117).

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

If the animal is a porcine animal, the insertion or deletion is a two base pair insertion between nucleotides 3,149 and 3,150 relative to the reference sequence SEQ ID NO:47 and 377 at nucleotides 2,573 and 2,949 relative to the reference sequence SEQ ID NO:47 on the same allele. base pair deletion; A 28 base pair deletion from nucleotides 3,145 to nucleotides 3,172 compared to the reference sequence SEQ ID NO:47; and combinations thereof. For example, an oocyte, sperm cell, or fertilized egg may have a two base pair insertion between nucleotides 3,149 and 3,150 relative to the reference sequence SEQ ID NO:47 and an insertion between nucleotides 2,573 and 2,949 relative to the reference sequence SEQ ID NO:47 on the same allele. It may contain a 377 base pair deletion. The oocyte, sperm cell, or fertilized egg may contain a 28 base pair deletion from nucleotide 3,145 to nucleotide 3,172 compared to the reference sequence SEQ ID NO:47.

The oocyte, sperm cell, or fertilized egg will contain a 7 base pair insertion between nucleotides 3,148 and 3,149 relative to the reference sequence SEQ ID NO: 47 and an 11 base pair deletion from nucleotides 3,137 to 3,147 compared to the reference sequence SEQ ID NO: 47. You can.

An oocyte, sperm cell, or fertilized egg containing any of the insertions or deletions described above may contain a chromosomal sequence with a high degree of sequence identity to SEQ ID NO: 47 in addition to the insertion or deletion. Thus, for example, the oocyte, sperm cell, or fertilized egg may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least It may comprise a chromosomal sequence having 99%, at least 99.9%, or 100% sequence identity.

The oocyte, sperm cell, or fertilized egg has a chromosomal sequence comprising SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114. may include. As further described in the Examples below, SEQ ID NOs: 98-114 provide nucleotide sequences for a region corresponding to the region of wild-type porcine CD163 provided in SEQ ID NO: 47 and inserted into the porcine CD163 chromosomal sequence described herein. or includes deletion.

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

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

An oocyte, sperm cell, or fertilized egg carries an 11 base pair deletion in one allele of the gene encoding the CD163 protein and a 2 base pair insertion and a 377 base pair deletion in the other allele of the gene encoding the CD163 protein. may include.

The oocyte, sperm cell, or fertilized egg may contain a 124 base pair deletion in one allele of the gene encoding the CD163 protein and a 123 base pair deletion in another allele of the gene encoding the CD163 protein.

An oocyte, sperm cell, or fertilized egg may contain a one base pair insertion.

An oocyte, sperm cell, or fertilized egg may contain a 130 base pair deletion in one allele of the gene encoding the CD163 protein and a 132 base pair deletion in another allele of the gene encoding the CD163 protein.

An oocyte, sperm cell, or fertilized egg may contain a 1506 base pair deletion.

An oocyte, sperm cell, or fertilized egg may contain a 7 base pair insertion.

An oocyte, sperm cell, or fertilized egg may contain a 1280 base pair deletion in one allele of the gene encoding the CD163 protein and a 1373 base pair deletion in another allele of the gene encoding the CD163 protein.

An oocyte, sperm cell, or fertilized egg may contain the 1467 base pair deletion.

The oocyte, sperm cell, or fertilized egg may contain a 1930 base pair intron 6 deletion from nucleotides 488 to nucleotides 2,417, a 12 base pair addition at nucleotide 4,488, and an additional 129 base pair deletion in exon 7.

An oocyte, sperm cell, or fertilized egg may contain a 28 base pair deletion in one allele of the gene encoding the CD163 protein and a 1387 base pair deletion in another allele of the gene encoding the CD163 protein.

The oocyte, sperm cell, or fertilized egg has a 1382 base pair deletion and an 11 base pair insertion in one allele of the gene encoding the CD163 protein and a 1720 base pair deletion in the other allele of the gene encoding the CD163 protein. may include.

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

In any of the methods of reproduction, the fertilization step may include artificial insemination.

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

A method of increasing the resistance of livestock animals to infection with pathogens is also provided. The method includes copying at least one chromosomal sequence from the gene encoding the CD163 protein such that CD163 protein production or activity is reduced compared to CD63 protein production or activity in a domestic animal that does not contain the edited chromosomal sequence in the gene encoding the CD163 protein. Includes editing. Pathogens preferably include viruses (eg 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 to the sequence of SEQ ID NO: 47, wherein the nucleotide sequence contains 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 can include the nucleotide sequence comprising SEQ ID NO:47.

Alternatively, the isolated nucleic acid may comprise a nucleotide sequence having at least 80% sequence identity to 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. Contains. 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 to the sequence of SEQ ID NO:47, wherein: The nucleotide sequence contains at least one substitution, insertion, or deletion compared to SEQ ID NO: 47)

The substitution, insertion, or deletion preferably reduces or eliminates CD163 protein production or activity compared to the nucleic acid not containing the substitution, insertion, or deletion.

The isolated nucleic acid may include SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114.

For example, the isolated nucleic acid may include SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114.

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

Isolated nucleic acids can include cDNA.

Additional isolated nucleic acids are provided. The isolated nucleic acid may include SEQ ID NO: 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114. For example, the isolated nucleic acid can include SEQ ID NO: 98, 101, 105, 109, 110, 112, 113, or 114. As another example, the isolated nucleic acid may include SEQ ID NO: 103 or 111.

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

Example

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

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

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

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

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

Materials and Methods

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

Design of gRNA to construct specific CRISPR

To ensure that CRISPR results in a DSB in wild-type CD163 but not in the domain swap targeting vector, a guide RNA was designed in a region within exon 7 of CD163 that is unique to wild-type CD163 and not present in the domain swap targeting vector (described below). The targeting vector is S. pyogenes ( Spy ) There are only four positions to introduce single nucleotide polymorphisms (SNPs) that alter the protospacer 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 for each gRNA.

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

To target eGFP , two specific gRNAs targeting the eGFP coding sequence were designed within the first 60 bp of the eGFP start codon. eGFP 1 and eGFP 2 The gRNAs, both on the antisense strand, targeted eGFP1 directly to the start 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 Zhang protocol.

Synthesis of donor DNA for CD163 and CD1D genes

Both porcine 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, LA Tag (Clontech) using the forward primer CTCTCCCTCACTCTAACCTACTT (SEQ ID NO: 11), and the reverse primer TATTTCTCTCACATGGCCAGTC (SEQ ID NO: 12) was used to amplify a 9538 bp fragment of CD163. The fragment was DNA sequenced and used to construct a domain-swap targeting vector (Figure 1). This vector contained 33 point mutations within exon 7 to encode the same amino acid sequence as human CD163L from exon 11. The replaced exon was 315 bp. Additionally, normal splicing was previously demonstrated when the subsequent intron was replaced with a modified myostatin intron B that harbored a selectable marker gene that could be removed with Cre-recombinase (Cre) and harbored the retained loxP site ( Wells, unpublished results). The long arm of the construct was 3469 bp and contained a domain swap DS exon. The short arm was 1578 bp and included exons 7 and 8 (Figure 1B). This plasmid was used to replace the coding region of exon 7 in the first transfection experiment and allowed selection of targeting events through a selection marker (G418). If targeting is desired to occur, the marker can be deleted by Cre-recombinase. The CD163 DS-targeting vector was then modified for use in cell lines already containing a Neo-disrupted SIGLEC1 gene that cannot be Cre deleted. In this targeting vector, the Neo cassette, loxP and myomycin intron B are removed, and only the DS exons remain in the WT long and short arms (Figure 1c).

The genomic sequence for porcine CD1D was amplified with LA taq using the forward primer CTCTCCCTCACTCTAACCTACTT (SEQ ID NO: 13) and the reverse primer GACTGGCCATGTGAGAGAAATA (SEQ ID NO: 14), resulting in an 8729 bp fragment. The fragment was DNA sequenced and used to construct the targeting vector shown in Figure 2. The Neo cassette is under the control of the phosphoglycerol kinase (PGK) promoter and flanked by loxP sequences, which were introduced for selection. The long arm of the construct was 4832 bp and the short arm was 3563 bp, including exons 6 and 7. If successful HR occurs, exons 3, 4, and 5 will be removed and replaced with the Neo cassette. If NHEJ repair occurs incorrectly, exon 3 will be destroyed.

Fetal fibroblast collection

To generate cell lines, porcine fetal tissue was collected on day 35 of pregnancy. 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 the Neo cassette (SIGLEC1-/- genetics) were also used in this study. Fetal fibroblasts were collected as described with minor modifications; Ground tissue from each fetus was cultured in 20 ml of digestion medium (Dulbeco containing 1 g/L D-glucose [Cellgro] and L-glutamine supplemented with 200 units/ml collagenase and 25 Kunitz units/ml DNaseI). -modified Eagle's medium [DMEM]) at 38.5°C for 5 h. After digestion, fetal fibroblasts were washed and cultured with DMEM, 15% fetal bovine serum (FBS), and 40 μg/ml gentamicin. After overnight incubation, cells were trypsinized and frozen in aliquots at -80°C in FBS with 10% dimethyl sulfoxide and stored in liquid nitrogen.

Cell transfection and genotyping

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

PCR screening for DS and large and small deletions

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

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

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

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

Somatic cell nuclear transfer (SCNT)

To produce SCNT embryos, sow-derived oocytes (ART, Inc.) or naive sow (gilt)-derived oocytes from a local slaughterhouse were used. Sow-derived oocytes were cultured in maturation medium (2.9 mM Hepes, 5 μg/ml insulin, 10 ng/ml epidermal growth factor [EGF], 0.5 μg/ml porcine follicle-stimulating hormone [p-FSH], 0.91 mM pyruvate). , TCM-199 with 0.5 mM cysteine, 10% porcine follicular fluid, and 25 ng/ml gentamicin) and transferred to fresh medium after 24 h. After 40-42 h of maturation, cumulus cells were removed from the oocytes by vortexing in the presence of 0.1% hyaluronidase. Naive sow-derived oocytes were matured for in vitro fertilization (IVF) as described below. During manipulation, oocytes were cultured in manipulation medium supplemented with 7.0 μg/ml cytochalasin B (TCM- with 0.6 mM NaHCO ₃ , 2.9 mM Hepes, 30 mM NaCl, 10 ng/ml gentamicin, and 3 mg/ml BSA). 199 [Life Technologies], osmolarity 305 mOsm). The polar body along with a portion of the adjacent cytoplasm, presumed to contain the metaphase II plate, was removed, and the donor cell was placed in the perivitelline space using a fine glass capillary. Afterwards, the reconstituted embryos were cultured in fusion medium (0.3 M mannitol, 0.1 mM CaCl ₂ , 0.1 μm) with two DC pulses (1-sec interval) for 30 lsec at 1.2 kV/cm using a BTX Electro cell manipulator (Harvard Apparatus). fused in 0.5 mM MgCl ₂ , and 0.5 mM Hepes). After fusion, fused embryos were fully activated with 200 μM thimerosalo for 10 min and 8 mM dithiothreitol for 30 min in the dark. Embryos were then cultured for 14-16 h in 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 naive prepubertal sows were obtained from an abattoir (Farmland Foods Inc.). Immature oocytes were aspirated from medium-sized (3-6 mm) follicles using an 18-gauge hypodermic needle attached to a 10 ml syringe. Thereafter, oocytes with uniformly dark cytoplasm and intact surrounding cumulus cells were selected for maturation. Approximately 50 cumulus oocyte complexes were incubated in 500 μl of maturation 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 gentamicin (APP Pharm), and TCM-199 (Invitrogen) with 0.1% polyvinyl alcohol were placed for 42-44 h at 38.5°C, 5% CO ₂ in humidified air. At the end of maturation, surrounding cumulus cells were removed from the oocyte by vortexing for 3 minutes in the presence of 0.1% hyaluronidase. Afterwards, in vitro mature oocytes were incubated in groups of 25-30 oocytes in 50 μl droplets of IVF medium (113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2, 11 mM glucose, 20 mM Tris, 2 mM caffeine, 5 modified Tris-buffered medium containing mM sodium pyruvate, and 2 mg/ml bovine serum albumin [BSA]. One 100 μl frozen semen pellet was thawed in 3 ml of Dulbecco's PBS supplemented with 0.1% BSA. Frozen WT or fresh eGFP semen was washed by centrifugation at 650 3 g in 60% Percoll for 20 min and in modified Tris-buffered medium for 10 min. In some cases, freshly collected semen heterozygous for the previously described eGFP transgene was washed three times in PBS. Afterwards, the semen pellet was resuspended in IVF medium to 0.5 × 10 ⁶ cells/ml. Fifty microliters of semen suspension was introduced into the droplet with the oocytes. Gametes were co-cultured for 5 hours at 38.5°C in an atmosphere of 5% CO ₂ in air. After fertilization, embryos were cultured in PZM3-MU1 at 38.5°C and 5% CO ₂ in air.

embryo transfer

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

In vitro synthesis of RNA for the CRISPR/Cas9 system

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

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

Microinjection of engineered CRISPR/Cas9 system in zygotes

Messenger RNA encoding Cas9 and gRNA were injected into the cytoplasm of fertilized oocytes using a FemtoJet microsyringe (Eppendorf) at 14 hours post-fertilization (presumptive zygote). Microinjections were performed in engineered medium on a heated stage of a Nikon inverted 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 a modified genome was classified as 1, and the number of colonies without genome modification was classified as 0. Differences were measured using PROC GLM (SAS), and a P value of 0.05 was considered significant. The mean was calculated as the least-squares average. Data are presented as numerical mean ± SEM.

result

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

The efficiency of four different CRISPR plasmids targeting CD163 (Guide 10, 131, 256, and 282) was tested at 2 μg/μl amount of donor DNA (Table 5). CRISPR 282 resulted in significantly higher average colony formation than CRISPR 10 and 256 treatments (P < 0.05). From the distant PCR assay described above, a large deletion ranging from 503 bp to 1506 bp was found instead of DS through HR as originally intended (Figure 3A). This was unexpected because previous reports using other DNA-editing systems showed much smaller deletions of 6-333 bp using ZFNs in pigs. CRISPR 10 and a mix of all four CRISPRs resulted in a greater number of colonies with modified genomes than CRISPR 256 and 282 (Table 5, P < 0.002). Transfection with plasmids containing Neo but not homologous to CD163 and CRISPR 10 did not result in colonies showing large deletions. Interestingly, one monoallelic deletion was also detected when donor DNA was introduced without any CRISPR. This assay likely represents an underestimate of the mutation rate because transfected somatic cells were not screened for any possible small deletions by sequencing that could not be detected on agarose gels.

Table 5. Efficiency of four different CRISPR plasmids (guides 10, 131, 256, and 282) targeting CD163. Four different CRISPRs were tested on amounts of 2 μg to 1 μg of donor DNA (shown in Figure 1).

*Mix of 4+donor DNA represents an equal 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 illustrates that the large deletions observed with CRISPR treatment were present only when CD163 donor DNA was also present.

† ANOVA was performed comparing the average number of colonies/plate and the percent of colonies with modified genomes to estimate CRISPR toxicity. The P values were 0.025 and 0.0002, respectively. n/a = There were no replicates for this treatment, so no statistical analysis was performed.

‡ One colony with HR represents a partial HR event.

^ac Superscript letters indicate significant differences between treatment groups for both the average number of colonies/plate and the percent of colonies with modified genomes (P <0.05).

The initial goal was to obtain domain swap (DS)-targeting events by HR against CD163, but CRISPR did not increase targeting CD163 efficiency. It should be noted that various combinations of these targeting vectors have been used to modify CD163 by HR by conventional transfection and resulted in 0 targeting events after screening 3399 colonies (Whitworth and Prather, unpublished results). . We obtained two pigs with partial DS resulting from HR containing 16 of the 32 mutations we wanted to introduce by transfection with CRISPR 10 and DS-targeting vector as donor DNA.

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

Table 6. Efficiency of CRISPR/Cas9-directed mutagenesis without drug selection. Four different ratios of donor DNA to CRISPR 131 DNA were compared in previously transformed cell lines without the use of G418 selection.

^a Significant difference between treatment groups for the percentage of colonies with NHEJ recovery (P>0.05).

^b There was no significant difference in the number of colonies genome modified with increasing concentrations of CRISPR (P>0.33).

Based on this experiment, targeted destruction of CD1D in somatic cells was attempted. Four different CRISPRs were designed and tested in both male and female cells. We were unable to detect modifications of CD1D from three of the applied CRISPRs, but use of CRISPR 5350 did not result in modifications of CD1D with deletions large enough to be detected by agarose gel electrophoresis (Table 7) . Interestingly, we did not obtain any genetic modifications through HR even though donor DNA was provided. However, we observed large deletions similar to CD163 knockout experiments (Figure 3B). Targeted modification of CD1D with large deletions was not detected when CRISPR/Cas9 was not used with donor DNA. Modifications of CD1D from CRISPR/Cas9-guided targeting were 4/121 and 3/28 of male and female colonies of each cell. Only INDELs detectable by agarose gel electrophoresis were included in the transfection data.

Table 7. Four different CRISPRs were tested on 2 μg to 1 μg amounts of donor DNA (shown in Figure 2). Donor DNA treatment served as a non-CRISPR control.

Production of CD163 and CD1D pigs via SCNT using GE cells

Cells expressing alterations in CD163 or CD1D were used for SCNT to produce CD163 and CD1D knockout pigs (Figure 3). Male and female fetuses transfected with the CRISPR/Cas9 system for 7-embryo transfer (CD163 Table 8), 6-embryo transfer (CD163-No Neo), and 5-embryo transfer (CD1D) into naive recipient sows. Performed with SCNT embryos from fibroblasts. Of the recipient sows that had never given birth, 6 (CD163), 2 (CD163-No Neo), and 4 (CD1D) (Table 9) conceived at term, 85.7% each. Resulting in a pregnancy rate of 33.3%, and 80%. Among CD163 recipients, 5 gave birth to healthy piglets by cesarean section. One animal (O044) gave birth naturally. The litter size ranged from 1 to 8. Four pigs were euthanized due to growth failure after birth. One piglet was euthanized due to severe cleft palate. All remaining piglets appear healthy (Figure 3c). Two littermate male piglets born from fetal fibroblasts transfected with donor DNA and CRISPR 10 described in Figure 3B had a 30 bp deletion in exon 7 adjacent to CRISPR 10 and an additional 1476 bp deletion in the preceding intron, thus CD163. The intron 6/exon 7 junction was removed (Figure 3e). Genotypes and predicted translations are summarized in Table 10. One male piglet and one female litter (4 piglets) were obtained from CD163-No Neo transfection of previously modified SIGLEC1 cells. All five piglets were double knockouts for SIGLEC1 and CD163. The male piglet had a biallelic variant of CD163 with a 28 bp deletion in exon 7 on one allele and a partial deletion of exon 7 and a complete deletion of exon 8 and a 1387 bp deletion on the other allele including the preceding intron. and thus intron-exon splicing. The female piglet had a biallelic mutation in CD163, comprising a 1382 bp deletion and an 11 bp insertion in one allele and a 1720 bp deletion of CD163 in the other allele. A summary of CD163 modifications and predicted translations can be found in Table 10. A summary of CD1D modifications and predicted translations by CRISPR modifications can be found in Table 11. Briefly, one female and two male litters were born, resulting in 13 piglets. One piglet died shortly after birth. Twelve of the 13 piglets contained biallelic or homozygous deletion of CD1D (Figure 3f). One piglet was WT.

Table 8. Embryo transfer data for CD163 .

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

† MU refers to naive sow oocytes aspirated and matured at the University of Missouri as described in the IVF section of Materials and Methods. ART refers to sow oocytes purchased and matured as described in the SCNT section of Materials and Methods.

Table 9. Embryo transfer data for CD1D .

*CD1D CRISPR NT line represents embryos generated by NT with a fetal fibroblast line modified by transfection. CRISPR-injected embryos were IVF embryos injected at the 1-cell stage with CD1D guide RNA and CAS9 RNA.

† MU refers to naive sow oocytes aspirated and matured at the University of Missouri as described in the IVF section of Materials and Methods. ART refers to sow oocytes purchased and matured as described in the SCNT section of Materials and Methods.

Table 10. Genotype and translation predictions for CD163 transformed pigs. Some pigs not only contain variants of the biallelic type but also have one allele described and another variant allele that was not amplified by PCR.

*KO, knock-out

** Not included because the piglet was euthanized.

† The SEQ ID number in this column refers to the SEQ ID number for the sequence showing the INDEL relative to SEQ ID NO: 47.

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

^b The inserted sequence was AG.

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

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

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

Table 11. Genotype and translation predictions for CD1D variant pigs.

*KO, knock-out

Efficiency of the CRISPR/Cas9 system in pig zygotes

Based on the targeted destruction of CD163 and CD1D in somatic cells using the CRISPR/Cas9 system, the method was applied to porcine embryogenesis. First, we tested the effectiveness of the CRISPR/Cas9 system in developing embryos. The CRISPR/Cas9 system targeting eGFP was introduced into semen and fertilized zygotes from boars heterozygous for 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 (Figure 4a); Embryonic development after CRISPR/Cas9 injection was lower compared to the control group. However, all concentrations of CRISPR/Cas9 tested were effective in generating variants of eGFP, as no embryos with eGFP expression were found in the CRISPR/Cas9-injected group (Figure 4B); 67.7% of non-injected control embryos were green, indicating expression of eGFP. When we genotyped individual blastocysts, we were able to identify a small mutation near the CRISPR binding site (Figure 4C). Based on toxicity and effectiveness, 10 ng/μl of gRNA and Cas9 mRNA were used in the following experiments.

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

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

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

Review

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

When the CRISPR/Cas9 system was introduced into somatic cells, it successfully induced targeted destruction of our target genes by NHEJ, but did not increase the ability to target by HR. The targeting efficiency of individual CRISPR/Cas9 in somatic cells was variable, indicating that the design of the guide affects targeting efficiency. In particular, we were unable to find any targeted modification 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 remains unclear. After screening 886 colonies (both CD163 and CD1D) transfected with CRISPR and donor DNA, only one colony had evidence for a partial HR event. Our results demonstrate that the CRISPR/Cas9 system works with introduced donor DNA to induce unexpectedly large deletions in target genes but does not increase HR efficiency for these two specific targeting vectors. However, the specific mechanism for the observation of large deletions is unknown. Previous reports from our group have suggested that donor DNA can be effectively used with ZFNs to induce HR-directed repair. Similarly, we saw an increase in targeting efficiency when donor DNA was used with the CRISPR/Cas9 system, but full HR-directed repair was not observed. In our previous studies using ZFNs, we observed that targeted modifications can occur through the combination of HR and NHEJ, as we found partial recombination of the donor DNA induced after induction of DSBs by ZFNs. One explanation could be that the HR and NHEJ pathways are not independent but may act together to complete the repair process after DSBs are induced by homing endonucleases. Although higher concentrations of CRISPR may improve targeting efficiency in somatic cells, no statistical differences were found in the results of these experiments. This may suggest that CRISPR is a limiting factor in the CRISPR/Cas9 system, but further validation is needed. Targeted cells have been successfully used to produce GE pigs via SCNT, indicating that application of CRISPR/Cas9 does not affect the ability of cells to replicate. Some piglets were euthanized for health reasons; However, this is a common occurrence in SCNT-induced piglets.

When the CRISPR/Cas9 system was introduced into developing embryos by zygote injection, nearly 100% of embryos and pigs contained INDELs in the targeted genes, demonstrating that the technology is highly effective during embryogenesis. The efficiencies we observed during our study exceeded the frequencies reported in other studies using homing endonucleases during embryogenesis. The decrease in the number of embryos reaching the blastocyst stage suggested that the concentration of CRISPR/Cas9 we introduced in this study may be toxic to embryos. Further optimizing the delivery system could increase embryo viability and thus improve the overall efficiency of the process. The nearly 100% mutagenesis rate observed here differed from previous reports in CRISPR/Cas9-mediated knockout in pigs; However, differences in efficiency between studies may be due to the combination of targets and guides selected. In our study, lower concentrations of CRISPR/Cas9 (10 ng/μl each) were effective in generating mutations in developing embryos and producing GE pigs. The concentrations are lower than previously reported in porcine zygotes (125 ng/μl Cas9 and 12.5 ng/μl CRISPR). Because introducing excessive amounts of nucleic acid into a developing embryo can be toxic, lower concentrations of CRISPR/Cas9 components may be beneficial to the developing embryo. We saw some mosaic genotypes in CRISPR/Cas9-injected embryos from our in vitro assays; However, only one piglet produced through the approach had a mosaic genotype. Potentially, injection of CRISPR/Cas9 components may be more effective than introduction of other homing endonucleases, as mosaic genotypes have been considered a major obstacle to the use of CRISPR/Cas9 systems in zygotes. Another benefit of using the CRISPR/Cas9 system, demonstrated by our results, is that we lost none of the CD163 knockout pigs produced from IVF-derived zygotes injected with the CRISPR/Cas9 system, whereas several piglets resulting from SCNT He was euthanized a few days later. This suggests that this technology 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 zygotes has been optimized, future experiments will also include co-injection of donor DNA.

We demonstrated that introducing two CRISPRs and Cas9 into a zygote can induce chromosomal deletions in the developing embryo and produce pigs with the intended deletion, i.e., a specific deletion between the two CRISPR guides. These designed deletions can be advantageous because we can specify the size of the deletion rather than relying on random events caused by NHEJ. In particular, if there is an insertion/deletion of nucleotides in multiples of 3 than that caused by the homing endonuclease, the mutation may actually cause a hypomorphic mutation because no frame shift occurs. However, by introducing two CRISPRs, we can result in larger deletions with a higher chance of generating non-functional proteins. Interestingly, CD1D CRISPR was designed to span a larger region of the genome than CD163; There was a 124 bp gap between CD163 CRISPR 10 and 131, while for CD1D there was a 550 bp gap between CRISPR 4800 and 5350. Longer intervals between CRISPR were not as effective in generating deletions as shown in the study. However, since we only have a limited number of observations and need to consider the efficiency of individual CRISPRs, which are not pointed out here, further studies are needed to verify the relationship between the distance between CRISPRs and their ability to cause the intended deletion. .

The CRISPR/Cas9 system was also effective in targeting two genes simultaneously in the same medium, where the only additional step was introducing one additional CRISPR and crRNA. This demonstrates that it easily destroys multiple genes compared to other homing endonucleases. These results suggest that this technique can be used to target gene clusters or gene families that may have complementary effects, and thus determining the role of individual genes proves difficult unless all genes are disrupted. Our results demonstrate that CRISPR/Cas9 technology can be applied to produce GE pigs by increasing the efficiency of gene targeting in somatic cells and by direct zygote injection.

references

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

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

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

Washington, D.C.: U.S. Government Printing Office; December 2010.

4. Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 1985; 315:680-683.

5. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295:1089-1092.

6. Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, et al. Targeted disruption of the alpha1,3-galactosyltransferase genes in cloned pigs. Nat Biotechnol 2002; 20:251-255.

7. Gaj T, Gersbach CA, Barbas CF III. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013; 31: 397-405.

8. Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer R, Cost GJ, Niemann.

H. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108:12013-12017.

9. Yang D, Yang H, Li W, Zhao B, Ouyang Z, Liu Z, Zhao Y, Fan N, Song J, Tian J, Li F, Zhang J, et al. Generation of PPARgamma mono-allelic

knockout pigs via zinc-finger nucleases and nuclear transfer cloning. Cell Res 2011; 21:979-982.

10. Whyte JJ, Zhao J, Wells KD, Samuel MS, Whitworth KM, Walters EM, Laughlin MH, Prather RS. Gene targeting with zinc finger nucleases to

produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78:2.

11. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012; 482:331-338.

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

13. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. Efficient genome editing in zebrafish using a

CRISPR-Cas system. Nat Biotechnol 2013; 31:227-229.

14. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, et al. Generation of gene-modified Cynomolgus monkey via Cas9/RNA -mediated gene targeting in one-cell embryos. Cell 2014; 156:836-843.

15. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013; 153: 910-918.

16. Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M. Heritable gene targeting in the mouse and rat using a CRISPR- Cas system. Nat Biotechnol 2013; 31:681-683.

17. Hai T, Teng F, Guo R, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 2014; 24: 372-375.

18. Carter DB, Lai L, Park KW, Samuel M, Lattimer JC, Jordan KR, Estes DM, Besch-Williford C, Prather RS. Phenotyping of transgenic cloned piglets. Cloning Stem Cells 2002; 4:131-145.

19. Shimozawa N, Ono Y, Kimoto S, Hioki K, Araki Y, Shinkai Y, Kono T, Ito M. Abnormalities in cloned mice are not transmitted to the progeny. Genesis 2002; 34:203-207.

20. Lillico SG, Proudfoot C, Carlson DF, Stverakova D, Neil C, Blain C, King TJ, Ritchie WA, Tan W, Mileham AJ, McLaren DG, Fahrenkrug SC, et al. Live pigs produced from genome edited zygotes. Sci Rep 2013; 3:2847.

21. Van Breedam W, Delputte PL, Van Gorp H, Misinzo G, Vanderheijden N, Duan X, Nauwynck HJ. Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. J Gen Virol 2010; 91:1659-1667.

22. Prather RS, Rowland RR, Ewen C, Trible B, Kerrigan M, Bawa B, Teson JM, Mao J, Lee K, Samuel MS, Whitworth KM, Murphy CN, et al. An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/ internalization of the porcine reproductive and respiratory syndrome virus. J Virol 2013; 87:9538-9546.

23. Neumann EJ, Kliebenstein JB, Johnson CD, Mabry JW, Bush EJ, Seitzinger AH, Green AL, Zimmerman JJ. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J Am Vet Med Assoc 2005; 227:385-392.

24. Borg NA, Wun KS, Kjer-Nielsen L, Wilce MC, Pellicci DG, Koh R, Besra GS, Bharadwaj M, Godfrey DI, McCluskey J, Rossjohn J. CD1d-lipid-antigen recognition by the semi-invariant NKT T -cell receptor. Nature 2007; 448:44-49.

25. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339:819-823.

26. Smit, AFA, Hubley, R, Green, P. RepeatMasker Open-3.0. 1996-2010.

Current Version: open-4.0.5 (RMLib: 20140131 and Dfam: 1.2). http://www.repeatmasker.org&gt. CD163: accessed July 25, 2014; CD1D: accessed August 27, 2013.

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

28. Ross JW, Whyte JJ, Zhao J, Samuel M, Wells KD, Prather RS. Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts. Transgenic Res 2010; 19:611-620.

29. Whitworth KM, Spate LD, Li R, Rieke A, Sutovsky P, Green JA, Prather RS. Activation method does not alter abnormal placental gene expression and development in cloned pigs. Mol Reprod Dev 2010; 77:1016-1030.

30. van den Hoff MJ, Moorman AF, Lamers WH. Electroporation in 'intracellular' buffer increases cell survival. Nucleic Acids Res 1992; 20:2902.

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

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

33. Machaty Z, Wang WH, Day BN, Prather RS. Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal. Biol Reprod 1997; 57:1123-1127.

34. Yoshioka K, Suzuki C, Tanaka A, Anas IM, Iwamura S. Birth of piglets 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, Rieke A, 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, Rieke A, 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 of 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, Rieke A, 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.

40. Lee K, Redel BK, Spate L, Teson J, Brown AN, Park KW, Walters E, Samuel M, Murphy CN, Prather RS. Piglets produced from cloned blastocysts cultured in vitro with GM-CSF. Mol Reprod Dev 2013; 80: 145-154.

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

42. Lee K, Kwon DN, Ezashi T, Choi YJ, Park C, Ericsson AC, Brown AN, Samuel MS, Park KW, Walters EM, Kim DY, Kim JH, et al. Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency. Proc Natl Acad Sci U S A 2014; 111:7260-7265.

43. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci U S A 1985; 82:4438-4442.

Example 2: Increased resistance to PRRSV in pigs with altered chromosomal sequences in the gene encoding the CD163 protein

Porcine reproductive and respiratory syndrome virus (PRRSV) has been ravaging the swine industry for over a quarter of a century. Speculations on the mode of viral entry included both SIGLEC1 and CD163. Although knockout of SIGLEC1 did not affect the response to viral challenge, we show here that CD163 unresponsive animals show no clinical signs of infection, lung lesions, viremia, or antibody production (all characteristic of PRRSV infection). . Not only were PRRSV entry vectors identified; Strategies are described to prevent significant economic loss and animal suffering if similarly produced animals are allowed to enter the food supply.

Materials and Methods

Genotyping . Genotyping was based on both DNA sequencing and mRNA sequencing. The sire's genotype had an 11 bp deletion in one allele, which when translated predicted 45 amino acids into domain 5, resulting in a premature stop codon at amino acid 64. Another allele had a 2 bp addition in exon 7 and a 377 bp deletion in the intron preceding exon 7, which, when translated, predicted the first 49 amino acids of domain 5, resulting in a premature stop codon at amino acid 85. One sow had a 7 bp addition to one allele, resulting in a premature stop codon at amino acid 70 when translationally predicted for the first 48 amino acids of domain 5. The other allele was uncharacterized because there was no band from exon 7 by PCR or the distant 6.3 kb PCR (A). The other three sows were clones and had a 129 bp deletion in exon 7, which was predicted to result in a deletion of 43 amino acids from domain 5. The other allele was uncharacterized (B).

Growth of PRRSV in culture and production of viral inoculum for infection of pigs are under approved IBC support 973. The reference strain of PRRSV, isolate NVSL 97-7895 (GenBank # AF325691 2001-02-11), was grown as described in the approved IBC protocol 973. These laboratory isolates have been used in experimental studies for approximately 20 years (Ladinig et al., 2015). Secondary isolates were used for the ^2nd 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 of PRRS virus administered by intramuscular (IM) and intranasal (IN) routes. Pigs are monitored daily and pigs showing disease symptoms are treated according to the recommendations of CMG veterinarians. Pigs showing severe distress and at risk of succumbing to infection are humanely euthanized and samples collected. To eliminate bias in evaluation or treatment, staff and veterinarians were blinded to the pigs' genetic status. PRRSV is present in body fluids during infection; Therefore, blood samples were collected and stored at -80°C until measured to determine the amount or degree of viremia in each pig. At the end of the experiment, pigs were weighed and humanely euthanized, and tissues were collected, fixed in 10% buffered formalin, embedded in paraffin, and processed for histopathology by a licensed pathologist.

Phenotypic scoring of challenge-infected pigs . The pigs' phenotype was assessed daily with a blind score as follows: What is the pig's attitude? Attitude score: 0: BAR, 1: QAR, 2: slightly depressed, 3: depressed, 4: moribund. How is the pig's health? Health Status Score: 1: Emaciated, 2: Thin, 3: Ideal, 4: Fat, 5: Overweight/Obese. What is the pig's rectal temperature ? Normal body temperature 101.6-103.6 (fever is considered ≥ 104). Are you lame (level)? Which leg? Assess the leg for joint swelling and hoof lesions (check bottom and side of hoof). Limping score: 1: no limping, 2: slightly uneven gait, some joints appear stiff but no limping, 3: mild limping, slight limping while walking, 4: moderate limping, toes. Apparent limping, including toe touching lame, 5: Severe limping, unable to support weight on legs, requires encouragement to stand/walk. Do you have difficulty breathing (grade)? Do you breathe with your mouth open? Is there any nasal discharge (color of discharge, amount of discharge: mild/moderate/severe)? Have you noticed an animal coughing? Is there eye discharge? Respiratory score: 0: normal, 1: mild dyspnea and/or tachypnea when stressed (when handled), 2: mild dyspnea and/or tachypnea at rest, 3: moderate when stressed (when handled). Dyspnea and/or tachypnea, 4: Moderate dyspnea and/or tachypnea at rest, 5: Severe dyspnea and/or tachypnea when stressed (handled), 6: Severe dyspnea and/or tachypnea at rest. . Are there any signs of diarrhea (grade) or vomiting? Is there blood or mucus? Diarrhea score: 0: no visible feces, 1: normal stool, 2: soft but shaped stool (consisting of soft-serve yogurt consistency, cow dung), 3: brown/tan liquid diarrhea with particulate fecal material, 4: Brown/tan liquid diarrhea without particulate fecal material, 5: Liquid diarrhea that appears watery.

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

Measurement of PRRSV viremia . Viremia was determined using two approaches. Virus titration was performed by adding serial 1:10 dilutions of serum to confluent MARC-145 cells in 96 well-plates. Serum was diluted in Eagle's minimal essential medium supplemented with 8% fetal bovine serum, penicillin, streptomycin, and amphotericin B, as previously described ( Prather et al., 2013 ). Cells were examined for the presence of cytopathic effect after 4 days of culture using a microscope. The highest dilution showing a cytopathic effect was scored as the titration endpoint. Total RNA was isolated from serum using the Life Technologies MagMAX-96 viral RNA isolation kit to measure viral nucleic acids. Reverse transcription polymerase chain reaction was performed using the EZ-PRRSV MPX 4.0 kit from Tetracore on a CFX-96 real-time PCR system (Bio-Rad) according to the manufacturer's instructions. Each reaction (25 μl) contained 5.8 μl of RNA from serum. A standard curve was created by preparing serial dilutions of the RNA control supplied in the kit (Tetracore). Record the number of templates per PCR.

SIGLEC1 and CD163 staining of PAM cells . Porcine alveolar macrophages (PAM) were collected by dissecting lungs and filling them with ∼100 ml cold phosphate buffered saline. After recovering the phosphate-buffered saline wash, cells were pelleted, resuspended in 5 ml cold phosphate-buffered saline, and stored on ice. Approximately 10 ⁷ PAM was incubated with 5 ml of various antibodies (anti-porcine CD169 (clone 3B11/11; AbD Serotec); anti-porcine CD163 (clone) 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 washed and resuspended in a 1/100 dilution of fluorescein isothiocyanate (FITC) conjugated to goat anti-mouse IgG (life Technologies) diluted in staining buffer and incubated on ice for 30 min. At least 10 ⁴ cells were analyzed using a FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson).

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

result

Mutations of CD163 were generated using CRISPR/Cas9 technology as described in Example 1 above. Several progenitor animals were produced from zygote injection and from somatic cell nuclear transfer. Some of these ancestral animals were mated to produce offspring and studied. A single progenitor male was mated to females of two genotypes. The progenitor male (67-1) had an 11 bp deletion in exon 7 of one allele and a 2 bp addition in exon 7 of the other allele (and a 377 bp deletion in the preceding intron) and was an unresponsive animal ( CD163 ^{−/ -} ) was predicted to be ). One progenitor female (65-1) had a 7 bp addition in exon 7 in one allele and the corresponding uncharacterized allele, and was therefore predicted to be heterozygous for the knockout ( CD163 ^−/? ). . The second progenitor female genotype (three clonal animals) contained an as yet uncharacterized allele and an allele with a 129 bp deletion in exon 7. This deletion is predicted to result in a deletion of 43 amino acids in domain 5. Mating between these animals resulted in piglets inheriting both an unresponsive allele from the boar and either a 43 amino acid deletion or an uncharacterized allele from the sow. In addition to wild-type piglets, which served as positive controls for virus challenge, this produced four additional genotypes (Table 8).

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

At weaning, gene-edited piglets and wild-type same-age piglets were transported to Kansas State University for PRRSV challenge. PRRSV challenge was performed as previously described (Prather et al., 2013). Piglets were sent to the challenge facility at 3 weeks of age and maintained in single groups. All experiments were initiated after approval by institutional animal use and biosafety committees. After acclimation, pigs were challenged with PRRSV isolate, NVSL 97-7895 (Ladinig et al., 2015) and seeded on MARC-145 cells (Kim et al., 1993). Pigs were challenged with approximately 10 ⁵ TCID ₅₀ viruses. One-half of the inoculum was delivered intramuscularly and the remainder intranasally. All infected pigs were maintained in a single group, which allowed serial exposure to virus from infected pen mates. Blood samples were collected at various days up to and at the end of day 35 post infection. Pigs were necropsied and tissues were fixed in 10% buffered formalin, embedded in paraffin, and processed for histopathology. PRRSV-related clinical signs recorded during the course of infection included difficulty breathing, anorexia, lethargy, and fever. Results for clinical signs over the study period are summarized in Figure 9. As expected, Wild Type ( CD163 +/+ ) pigs showed early signs of PRRSV infection, which peaked between days 5 and 14 and persisted in the group for the remainder of the study. The percentage of febrile pigs peaked around day 10. In contrast, non-responsive ( CD163 −/−) piglets showed no signs of clinical signs throughout the entire study period. Respiratory signs during acute PRRSV infection are reflected in significant histopathological changes in the lungs (Table 9). Infection of wild-type pigs showed histopathology consistent with PRRS, including interstitial edema and mononuclear cell infiltration ( Fig. 10 ). In contrast, there were no signs of lung changes in non-responding ( CD163 −/−) pigs. Sample sizes for various genotypes are small; Nevertheless, the mean scores were 3.85 (n=7) for wild type, 1.75 (n=4) for uncharacterized A, 1.33 (n=3) for uncharacterized B, and no response (CD163-/-). In the case of 0 (n=3).

Table 13. Lung evaluation using microscopy.

Maximal clinical signs correlated with the level of PRRSV in the blood. Measurement of viral nucleic acid was performed by isolation of total RNA from serum followed by amplification of PRRSV RNA using a commercial reverse transcriptase real-time PRRSV PCR test (Tetracore, Rockville, MD). A standard curve was created by preparing serial dilutions of the PRRSV RNA control provided in the RT-PCR kit, and the results were normalized to the number of templates per 50 μl PCR reaction. PRRSV isolation followed the procedure for PRRSV viremia in wild-type CD163 +/+ pigs (Figure 11). Viremia was evident on day 4, peaked on day 11, and then decreased 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 uncharacterized allele pigs was detectable by day 14 and increased by day 28. There was no antibody production in non-responsive animals (Figure 12). Together, these data show that wild-type pigs support PRRSV replication and the development of clinical signs consistent with PRRS. In contrast, knockout pigs did not develop viremia and clinical signs even when inoculated and exposed to infected pen mates.

At the end of the study, porcine alveolar macrophages were removed by lung lavage and assayed for surface expression of SIGLEC1 (CD169, clone 3B11/11) and CD163 (clone 2A10/11) as previously described (Prather et al., 2013). dyed for. Relatively high levels of CD163 expression were detected in CD163 +/+ wild-type animals (Figure 13). In contrast, CD163 −/− pigs showed only background levels of anti-CD163 staining, thus confirming the knockout phenotype. Expression levels for another macrophage marker CD169 were similar for both wild-type and knockout pigs (Figure 14). Other macrophage surface markers, including MHC II and CD172, were identical for both genotypes (data not shown).

Although the sample size is small, wild-type pigs were significantly different from pigs of three other genotypes (non-characterized A 1.32 kg ± 0.17, n = 4; non-characterized B 1.20 kg ± 0.16, n = 3; unresponsive 1.21 kg ± 3) over the course of the experiment. There was a tendency for body weight to increase less (average daily gain 0.81 kg ± 0.33, n=7) compared to 0.16, n=3).

In a second attempt, 6 wild type, 6 Δ43 amino acid, and 6 pigs carrying the uncharacterized allele (B) were challenged as described above except that piglets were inoculated using KS06-72109. Similar to the NVSL data, wild-type and uncharacterized B piglets developed viremia. However, KS06 did not cause viremia in Δ43 amino acid pigs (Figure 15; Table 7).

Results and Conclusion

The disease of most clinical relevance to the swine industry is PRRS. Although vaccination programs have been successful in preventing or ameliorating most swine pathogens, PRRSV has proven to be challenging. Here, CD163 is identified as an entry mediator for this virus strain. The founder male pig was produced by injecting CRISPR/Cas9 into the zygote (Whitworth et al., 2014) and therefore contains no transgene. Additionally, one of the alleles from the sow (also produced using CRISPR/Cas9) does not contain the transgene. Therefore, piglet #40 has a 7 bp addition on one allele and an 11 bp deletion on the other allele, but no transgene. This virus-resistant allele of CD163 represents minimal genome editing, considering that the pig genome is approximately 2.8 billion bp (Groenen et al., 2012). If similarly produced animals were introduced into the food supply, significant economic losses could be avoided.

References

Boddicker, N.J., Bjorkquist, A., Rowland, R.R., Lunney, J.K., Reecy, J.M., Dekkers, J.C., 2014. Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection. Genetics, selection, evolution: GSE 46, 18.

Etzerodt, A., Kjolby, M., Nielsen, M.J., Maniecki, M., Svendsen, P., Moestrup, S.K., 2013. Plasma clearance of hemoglobin and haptoglobin in mice and effect of CD163 gene targeting disruption. Antioxidants & redox signaling 18, 2254-2263.

Etzerodt, A., Moestrup, S.K., 2013. CD163 and inflammation: biological, diagnostic, and therapeutic aspects. Antioxidants & redox signaling 18, 2352-2363.

Graversen, J.H., Svendsen, P., Dagnaes-Hansen, F., Dal, J., Anton, G., Etzerodt, A., Petersen, M.D., Christensen, P.A., Moller, H.J., Moestrup, S.K., 2012. Targeting the hemoglobin scavenger receptor CD163 in macrophages highly increases the anti-inflammatory potency of dexamethasone. Molecular therapy: the journal of the American Society of Gene Therapy 20, 1550-1558.

Groenen, M.A., Archibald, A.L., Uenishi, H., Tuggle, C.K., Takeuchi, Y., Rothschild, M.F., Rogel-Gaillard, C., Park, C., Milan, D., Megens, H.J., Li, S. ., Larkin, D.M., Kim, H., Frantz, L.A., Caccamo, M., Ahn, H., Aken, B.L., Anselmo, A., Anthon, C., Auvil, L., Badaoui, B., Beattie. , C.W., Bendixen, C., Berman, D., Blecha, F., Blomberg, J., Bolund, L., Bosse, M., Botti, S., Bujie, Z., Bystrom, M., Capitanu, B., Carvalho-Silva, D., Chardon, P., Chen, C., Cheng, R., Choi, S.H., Chow, W., Clark, R.C., Clee, C., Crooijmans, R.P., Dawson, H.D. , Dehais, P., De Sapio, F., Dibbits, B., Drou, N., Du, Z.Q., Eversole, K., Fadista, J., Fairley, S., Faraut, T., Faulkner, G.J.; Fowler, K.E., Fredholm, M., Fritz, E., Gilbert, J.G., Giuffra, E., Gorodkin, J., Griffin, D.K., Harrow, J.L., Hayward, A., Howe, K., Hu, Z.L.; Humphray, S.J., Hunt, T., Hornshoj, H., Jeon, J.T., Jern, P., Jones, M., Jurka, J., Kanamori, H., Kapetanovic, R., Kim, J., Kim, J.H., Kim, K.W., Kim, T.H., Larson, G., Lee, K., Lee, K.T., Leggett, R., Lewin, H.A., Li, Y., Liu, W., Loveland, J.E., Lu, Y. ., Lunney, J.K., Ma, J., Madsen, O., Mann, K., Matthews, L., McLaren, S., Morozumi, T., Murtaugh, M.P., Narayan, J., Nguyen, D.T., Ni. , P., Oh, S.J., Onteru, S., Panitz, F., Park, E.W., Park, H.S., Pascal, G., Paudel, Y., Perez-Enciso, M., Ramirez-Gonzalez, R., Reecy, J.M., Rodriguez-Zas, S., Rohrer, G.A., Rund, L., Sang, Y., Schachtschneider, K., Schraiber, J.G., Schwartz, J., Scobie, L., Scott, C., Searle. , S., Servin, B., Southey, B.R., Sperber, G., Stadler, P., Sweedler, J.V., Tafer, H., Thomsen, B., Wali, R., Wang, J., Wang, J. ., White, S., Xu, , 2012. Analyzes of pig genomes provide insight into porcine demography and evolution. Nature 491, 393-398.

Halbur, P.G., Paul, P.S., Frey, M.L., Landgraf, J., Eernisse, K., Meng, X.J., Lum, M.A., Andrews, J.J., Rathje, J.A., 1995. Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Veterinary pathology 32, 648-660.

Holtkamp, D.J., Kliebenstein, J.B., Neumann, E.J., Zimmerman, J.J., Rotto, H.F., Yoder, T.K., Wang, C., Yeske, P.E., Mowrer, C.L., Haley, C.A., 2013. Assessment of the economic impact of porcine reprodutive and respiratory syndrome virus on United States pork producers. Journal of Swine Health and Production 21, 72-84.

Keffaber, K.K., 1989. Reproductive failure of unknown etiology. American Association of Swine Practitioners Newsletter 1, 1-9.

Kim, H.S., Kwang, J., Yoon, I.J., Joo, H.S., Frey, M.L., 1993. Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch Virol 133, 477-483.

Ladinig, A., Detmer, S.E., Clarke, K., Ashley, C., Rowland, R.R., Lunney, J.K., Harding, J.C., 2015. Pathogenicity of three type 2 porcine reproductive and respiratory syndrome virus strains in experimentally inoculated pregnant gilts. . Virus Res 203, 24-35.

Merck, The Merck Veterinary Manual. http://www.merckmanuals.com/vet/appendixes/reference_guides/normal_rectal_temperature_ranges.html.

Miao, Y.L., Sun, Q.Y., Zhang, Environmental and molecular mutagenesis 50, 666-671.

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

Prather, R.S., Rowland, R.R., Ewen, C., Trible, B., Kerrigan, M., Bawa, B., Teson, J.M., Mao, J., Lee, K., Samuel, M.S., Whitworth, K.M.; Murphy, C.N., Egen, T., Green, J.A., 2013. An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus. J Virol 87, 9538-9546.

Rowland, R.R., Lunney, J., Dekkers, J., 2012. Control of porcine reproductive and respiratory syndrome (PRRS) through genetic improvements in disease resistance and tolerance. Frontiers in genetics 3, 260.

Schaer, C.A., Schoedon, G., Imhof, A., Kurrer, M.O., Schaer, D.J., 2006a. Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin. Circulation research 99, 943-950.

Schaer, D.J., Schaer, C.A., Buehler, P.W., Boykins, R.A., Schoedon, G., Alayash, A.I., Schaffner, A., 2006b. CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin. Blood 107, 373-380.

Schaer, D.J., Schaer, C.A., Schoedon, G., Imhof, A., Kurrer, M.O., 2006c. Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis. European journal of haematology 77, 432-436.

Trible, B.R., Suddith, A.W., Kerrigan, M.A., Cino-Ozuna, A.G., Hesse, R.A., Rowland, R.R., 2012. Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2. J Virol 86, 13508-13514.

Van Breedam, W., Delputte, P.L., Van Gorp, H., Misinzo, G., Vanderheijden, N., Duan, X., Nauwynck, H.J., 2010. Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage. J Gen Virol 91, 1659-1667.

Van Gorp, H., Delputte, P.L., Nauwynck, H.J., 2010. Scavenger receptor CD163, a Jack-of-all-trades and potential target for cell-directed therapy. Mol Immunol 47, 1650-1660.

Wensvoort, G., Terpstra, C., Pol, J.M.A., ter Laak, E.A., Bloemrad, M., de Kluyer, E.P., Kragten, C., van Buiten, L., den Besten, A., Wagenaar, F. , Broekhuijsen, J.M., Moonen, P.L.J.M., Zetstra, T., de Boer, E.A., Tibben, H.J., de Jong, M.F., van't Veld, P., Groenland, G.J.R., van Gennep, J.A., Voets, M.T.H., Verheijden. , J.H.M., Braamskamp, J., 1991. Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Veterinary Quarterly 13, 121-130.

Whitworth, KM, Lee, K., Benne, JA, Beaton, BP, Spate, LD, Murphy, SL, Samuel, MS, Mao, J., O'Gorman, C., Walters, EM, Murphy, CN, Driver. , J., Mileham, A., McLaren, D., Wells, KD, Prather, RS, 2014. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro -derived oocytes and embryos. Biol Reprod 91, 78.

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

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

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

Since various changes may be made in the products and methods without departing from the scope of the invention, all matters contained in the foregoing description and shown in the accompanying drawing[s] should be construed as illustrative and not limiting.

ranking 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 tttttttat cctaatgtaa 180 ttagttcaaa acaaagtgca gaaatttaaa atattcaatt caacaacagt atataagtca 240 atattccccc cttaaatttt tacaaatctt tagggagtgt ttctcaattt ctcaatttct 300 ttggttgttt catgtcccat atggaagaaa acatgggtgt gaaagggaag cttactcttt 360 t gattacttc 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 tttacc tttt 600 aaatagagtg tagcaatatt ccgacagtca atcaatctga tttaatagtg attggcatct 660 ggagaagaag taacagggaa aaaggcaata agctttataa ggggaacttt tatcttccat 720 agactcaaaa ttgaagacgt gactagaaga ttgctagatt tggcatcagt tttgtaaaat 780 tgct gaggtg 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 aagtataac a 1020 tttcacatga aagcctggat tataaatgca cagttcagtg acctatctca gaggagtgac 1080 tgccatagca ttttttttgt ctttttgcct tcagagccac agcaacgcgg gatccgaagc 1140 cgcgtctgcg acccacacca cagctcacgg caatgccgga t ctttaaccc 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 tgtct ttttg 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 atgtgggtt c 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 gggggggg gg gggggggggg 246 0 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 tcctatga aa 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 tggat gggga aagcataact gtactcacca acaggatgct ggagtaacct 3180 gctcaggtaa gacatacaca aataagtcaa gcctatacat gaaatgcttt gtgggaaaaa 3240 atgtatagat gagttaaaaa caaaaaggaa ccagttttct ataagtcatc tagtccatgt 3300 ataaaattac ccaatccatt actaaaagac c acttctggt 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 atcaact tac caaatcaaga aataaatatt 3600 cttagcactg atcctttttt gttgttgttg gaggaagaat gttttgcaaa gtagaattgc 3660 ttttttctgt ttaacagtgc tattcatttc atttacatgg tcgttttaat ttataaaaca 3720 tttcataagt ttcacctcat atgccctt ac aataactcag gaagttatat gttagacctt 3780 tctgctgaca aatcccagag tcatgtttct gacccagttc agattccttg gcttcccatt 3840 tctctttgct catgtcattg acctttatgc agccctctta cctcccacct ttctattaca 3900 gaccatctcc tccataggac tggtgttaga aagtactaat ctctacccag gcattgtggt 3960 gcaatgtggg cagcacaggc tggtatctag aaaaatgctg aagtgaatt c cagctcagct 4020 gctcgttaat actatcgttt taagtaagct gttcaatcct ttgaaattca ctttctgagc 4080 actcagtgat ataataaatg tagagctact ggtacactgt ctggtatgta ataggtgtta 4140 ccaattaacc ttagtttcct catgggtcac tggttctcat tacctaga ca actcatttct 4200 ctttcttcct ctttctcttt ctccattctc ctcctccttc ttcctcttct tcttgtctgt 4260 tattgttata tcattttgct gagaaagtta agaaataaca actctaacct ctacatcgac 4320 cacctagagc aaagttaaaa ataataataa accttgccag actcttacta taattgttgc 4380 tgtctataga gttgactgtt taagttaaga catcagtata tatttttaat ttttgtgttt 44 40 tttttttcat acttttacat gaggatcctt tatataagga tgagttaaac aaacttgatt 4500 tttgaagttt atacccctga ggctcaactg cataataata gaaagggatc catagcctct 4560 caaggactta actagtttca tgagttttca gaatctgaat ttctgagatt ctccacccca 4620 at taaagctc 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 tagcata tgt 4860 aacacatttg gaacagggaa tgcatatttt gattgtgagc tcttattatt attaccattc 4920 agccctaata aaaatcttgg taagtggaag gctttggatt tcagaacttt taaaatctaa 4980 ttactttttc aaaaaagaac ttcttagggt tttttttttt taaccacaaa gtg tttctat 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 tg caaactat tatgcatact tatatatttt tacttttttc ctgtctttta acttccaaat 5340 tcaacttcag acaattattc atgcactaaa ctgtttgtag taagaaagat taaaattaaa 5400 aaattaacca ttcaacaaat gactggtttg ccatttttac tactttgttg tatgaacaat 5460 tttt ttttct 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 tcttt ctttt tttttttttt gaattttagt 5760 ttctaaactc acttctgaat aaatacaact tctaaattct cgtcttttct ctactctaga 5820 tggatctgat ttagagatga ggctggtgaa tggaggaaac cggtgcttag gaagaataga 5880 agtcaaattt caaggaacgt ggggaacagt gt gtgatgat cacttcaaca taaatcatgc 5940 ttctgtggtt tgtaaacaac ttgaatgtgg aagtgctgtc agtttctctg gttcagctaa 6000 ttttggagaa ggttctggac caatctggtt tgatgatctt gtatgcaatg gaaatgagtc 6060 agctctctgg aactgcaaac atgaaggatg gggaaagcac aattgcgatc atgctgagga 6120 tgctggagt g atttgcttaa gtaaggactg acctgggttt gttctgttct ccatgagagg 6180 gcaaaaaaag gggagtaaaa gtcttaaaag ctcaaactgt taaaaacata atgatgattg 6240 cttcttttat catcttatta ttatctaatt tcaggtcgaa attctagtac ctgtgcagtt 6300 t tttacctta actgaaatta agataaatag gatagggagg aaggatgagc agtgacattt 6360 aggtccaagt catgaggtta gaaggaaatg ttcagagaat agcccattcc ctcagccctc 6420 aaagaaagaa agaaagaaaa agaaaaaaaaa aaagaaagct taactagaaa attttgttct 6480 ctggatgttt tagaggcaaa ccatcccttt atcattccta cctacaaagc cttctcttaa 6540 tcacattacc caccctttcc tactatagtc aggggggg gg gggggggggg gggggggggg 6600 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 6660 gggggggggg gtgaaaaaag aaccaaacaa tttcaacaaa aaaccaaaca attccaaacaa 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 tgtttt gtgt ccctcttaca gatggagcag acctgaaact 7020 gagagtggta gatggagtca ctgaatgttc aggaagattg gaagtgaaat tccaaggaga 7080 atggggaaca atctgtgatg atggctggga tagtgatgat gccgctgtgg catgtaagca 7140 actgggaggt ccaactgctg tcactgc cat 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 attgggga gg 7440 gagaaaattg tttctaatat ttcttggtgg gaattctttt acagttgtga caaatctttc 7500 acatattctt catttgagta gtttggaggg ttgtctgact gttttctata ataaatgtcc 7560 caagtgctat gaggtaccac atttcaaatt ctaattctac ctgaagctcc aa aaagacaa 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 78 60 gtatgtttat tttgcagatg gatcagatct ggaactgaga cttaaaggtg gaggcagcca 7920 ctgtgctggg acagtggagg tggaaattca gaaactggta ggaaaagtgt gtgatagaag 7980 ctggggactg aaagaagctg atgtggtttg caggcagctg ggatgtggat ctg cactcaa 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 ttgaagag ac tgtcttttct tcatcatata gtcctacatc ctttgtcata aattaattga 8340 ccataggtgt gtgggtttat atctgggctc tctattctgt tcctttgatc tatatgtctg 8400 tttttatgcc agcaccatgc tgttttgatt actatagctt tgtagtatca tctgaagtca 8460 g gaaacatga ttcctccagc tttgttcttc tttctcaaga ttgttttgtc tattcagagt 8520 ttatgttccc atgcagattt aatttttaaa tttatttaat ttttatttt tatttttaat 8580 ttaaattaat ttaaattttt tatttcccaa cgtacagcca agggggccag ggtaaccttt 8640 acatgtatac attaaaaatt tcaggttttt cccccaccca tttctttctg ttggcaagta 8700 aatttttgaa caa agtttcc caatgctttt taaggggaat tcccttgggg gggggggggg 8760 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 8820 gggggggggg gggggggggg gggggggggg gggggggggg agacgaaatt gactatattt 8880 tctttgttgg ga atctttta 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 aattcatg tt ctatactggc tcttcaaaaa cccaggagat 9180 gggaaagcca gaatctccag tgtttcagct tctgggaagg agcaagtttt taaaaatacc 9240 ctctgggagc taaattccac atgtatctat ggcctaagtg tatgtttatt ttgcagatgg 9300 atcagatctg gaactgag ac ttaaaggtgg aggcagccac tgtgctggga cagtggaggt 9360 ggaaattcag aaactggtag gaaaagtgtg tgatagaagc tggggactga aagaagctga 9420 tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa acatcatatc aagtttattc 9480 caaaaccaag gcaacaaaca catggctgtt tgtaagcagc tgtaatggaa atgaaacttc 9540 tctttgggac tgcaagaatt ggcagtgggg tggacttagt tgtgatcact atgacgaaac 9600 caaaattacc tgctcaggta agaatttcaa tcaatgtgtt aggaaaaattg 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 tcccccaaaaa 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 gggggggg gg gggggggggg gggggg gggg ggggggggc 10200 ccccatgtga gattttgtgt gtcctttaag agtggagtct ctatttccca ctgctctctg 10260 gttctcccca aagtaagccc tgctggcttt caaaacttct gggagcttgc cttcttggta 10320 taggactcct gggctaggga gtctaatgtt tggcttagac cccttactgc ttgggaagaa 10380 tctctgcaac tgtaatgaat tatcttccta tttgtgggtt gctga ggata 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 tagggtgg aa attttatgtg gtccatatcc cctaaccagt ggctttacac caggcacatc 10920 ctaactaaga tctgctccca agtgtcttcc ctgatgcttt aaattgtgtt acatggaaac 10980 tatcctttga tgaagaaatg caacctttta aaatacaaca ttgaaacttt tgtgctttaa 110 40 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 cagagtatt c 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 caccat ggtt aatttttttt atccctctac acccgggaaa attacccttg 11760 gggccacact tttctataga aaggggatta tttaaaaggg tctgaaaaag aatttttttt 11820 tcgaaagggg aaatatttgg cctaacttag tcacataagc catgttctct ggcaagttag 11880 gtaacataca ttttt gtcat 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 atggc cacat 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 ggcatt gttc tcatttcaat tctgagttca gtgtggagag 12600 tatacgtgtg tgggggctgc acgcttttca caacccactt tctgctgata ctgatttagg 12660 gatccttgga ttgctttaca gttgagtcat cattaactag tgtcacttgc cttcaaagtc 12720 agca aaataa 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 acag tgttgt gtcaatttct gctgtacagc 13020 acagtgaccc agtcatacac atacatacat tctttttctc atactatctt caattttatt 13080 ttctgctaag tctgccattt tatcatcacc tcagtttgaa ggacaggata tttagagttt 13140 gtttttttt tccccccca at 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 ggacatgg tt ttcttttcaa 13440 ggtggtattt ctctaatgtc acttgaataa caagactcgt tagttctcca ggctacaata 13500 tcctagtctg agtatattct gcatgttaat tctattcagc cacatccata atttaggttt 13560 tattcctgga acacctcact tttttttttt tttt tggtct 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 atgataac ac 13860 attctgaatt atttaggatt ctattatact gcatgtaata gaaatcccaa atagcaaaat 13920 ttgcaactta aggcaggttc ctgtctttac aaaatcatgt tttcctttgc tatatgtgca 13980 ctttgctttc ctctgtgaat tccctttttt gttatatttc tatagct ttt 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 tggaaggac atctgaacct aagaaatagt tcatttgcat 14460 tgtgttatat tttactaagg tctgagaaat aatcttgaga tgagaatgaa ctctacttct 14520 tcagagtctg gaaggaataa attatgaaaa tgtattaatg cttctttaaa ccatattgta 14580 tatttatcta ttactaaaca aaaagaagta gctctattta tttatttat tatttattta 14640 tttatgtctt ttgtctcttt agggccacac ctgtggcata tggaggttcc caggctagag 14700 gtccaattgg agatgtag ca gccagcctat gccagagcca ccgcaacacg ggatctgagc 14760 cacgtctgtg acttacacca cagctcacag caacgcctga tcctcaaccc actgagcgag 14820 gccagggatc gaacccatgt cctcatggat gctagttggg ttcgttaact gctgagccat 14880 gatgggaact cc aaattaat 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 cttta atagg 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 gagaaattt g tgggagttcc 15600 catcgtggct cagtggaaac aaatccaact aggaaccatg aggttgtggg tttgatccct 15660 ggcctcactc agtgggttaa ggatccggtg ttgccgtgag ctgtggtgta ggttgcagac 15720 acggttctga tcctgcgtt g ctgtggctgt ggctgtggtg taggccagca gcaaacagct 15780 ctgattagac ccctagcctg gaaacctcca tatgccacag gtgcagccct aaaaagacaa 15840 aaaaagagaa aagacaaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaag 15900 aacccccaga ggtatttatt tgtttttgcc ttttttcact gactgttctt tgtttgtttg 15960 tttgagactg atctagaaga ctagagatta caagaaatat ggatttggct cactctaaga 16020 aactgctttc attccaaggt ttgggtctat ccaaaagtgg aatagaatca tatgaatact 16080 agtttatgag tatttagtga gaggaatttc aagctcaaat aatgattcag caagattaaa 16140 ttaaggaggg aattttcctt gtggctgagt gggttaagga cc caatgttg 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 tat atttgtt ttaaagtctt 16440 agttgtgttt agaaagcaag atgttcttca actcaaattt gggagggaac ttgtttcata 16500 catttttaat ggataagtgg caaaattttc atgctgaggt gatctatagt gttgtaatgc 16560 agaatatagt cagatcttga acattttagg a agttggtga 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 1 6860 catgtcttct gctttatttc tggtgtgcct ttgactccag attacagtaa atggaggact 16920 gagtataggg ctaaaaagta gagagaatgg atgcatatta tctgtggtct ccaatgtgat 16980 gaatgaagta ggcaaatact caaaggaaag agaaagcatg ctccaagaat tatgggttcc 17 040 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 gaggg gcacg 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 aacattctt g ggtcctgggg gtccctctgc aactctcact gggacatgga agatgcccat 17760 gttttatgcc agcagcttaa atgtggagtt gccctttcta tcccgggagg agcacctttt 17820 gggaaaggaa gtgagcaggt ctggaggcac atgtttcact gcactgggac tgagaagcac 17880 atgggagatt gttccgtcac tgctctgggc gcatcactct gttcttcagg gcaagtggcc 17940 tctgtaatct gctcaggtaa gagaataagg gcagccagtg atgagccact catgacggtg 18000 ccttaagagt gggtgtacct aggagttccc attgtggctc agtggtaaca aactcgactg 18060 gtatccatga gggtatgggt ttgatccctg gccttgctca atgggttaag gatccagcat 18120 tgctgtgagc tgt ggtatag gttgcagact ctgctcaggt cccatgttgc tgtgattgtg 18180 gtgtaggctg actgctgcag cttcaatttg acccctagcc cgggaatttc cataggccac 18240 acgtgcagca ctaaggaagg aaaaaaagaa aaaaaaaaaaa aaagagtggg tgtgcctata 183 00 gtgaagaaca gatgtaaaag ggaagtgaaa gggattcccc cattctgagg gattgtgaga 18360 agtgtgccag aatattaact tcatttgact tgttacaggg aaagtaaact tgactttcac 18420 ggacctccta gttacctggt gcttactata tgtcttctca gagtacctga ttcattccca 18480 gcctggttga cccatccccc tatctctatg gctatgttta tccagagcac atctatctaa 18540 cactccagct gatcttcctg acacagctgt ggca accctg gatcctttaa ccaactgtgc 18600 caggctggag atcaaaccta agcctctgca gcaacccaag ctgctgcagt cagattttta 18660 accccctgtg ccactgtggg tatctccgat attttgtatc ttctgtgact gagtggtttg 18720 ctgtttgcag ggaacca gag 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 tgt gatgaca gctgggacct gaatgatgcc 19020 catgtggtgt gcaaacagct gagctgtgga tgggccatta atgccactgg ttctgctcat 19080 tttggggaag gaacagggcc catttggctg gatgagataa actgtaatgg aaaagaatct 19140 catatttggc aatgccactc acatggttgg gggc ggcaca 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 aagcgagtt t 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 c catgcccga ttgatttttc aaggcatata aatgagacgt catgggagca aaaatggtca 19920 taatacaatg cccttgtttt gtgtacatgg taagatttta gaaagcattg tgaggtaaaa 19980 aagtgtactc agttataata tattggggaa aacagtacta tgagaagtaa aaaaatctac 2004 0 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 caaggaca at tgccaaaaaa aaaaaaaaaaa gagagagaga gagaacagaa tagggttatg 20520 aagctaaaat cacagggtta tgaagctaaa atcacagtaa tttagggaga aaaaaatcca 20580 aagcatgtaa ttgataaaag gttctgagcc tttgtttgag atttagaatt caacttagaa 20640 ataccggtgg tattttaaag cagtccataa gtataaaatc caaggctaaa aaaccagaag 20700 gtatttgtag aacaaatata tt ttaataag ctctaccaag tcatccagaa gctattaaag 20760 aattactggt cactgacata gtgtacctgt tttcaaggcc attcttacat cagaataaag 20820 ggagagcacc ctctgaatct tcagaaaaga tgtgaaagtg ctaattctct atttcatccc 20880 agagttcatg tctctgagac tgatcagt ga 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 tgacacact a tggcagtgcc catcatctcc atggaagaag agactggcca gcccctcaga 21180 ggagacatgg atcacatgtg ccagtgagta tccattcttt agcgccactg ttatcttctg 21240 atctacctaa gcagaagttt tataatctgt agttaatccc tattctacct ggatgatggg 21300 attcatt ctg 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 aa aaaaaaaa attgttatag gtgatttaca 21600 atgtctgtca atttctgctc tacagcaaag tgacccagtt atttacatat acattctgtt 21660 tctcatattt ttaaaccagg agatttctat ctgcctggcg gtttgaggga atttaacatt 21720 atgcatttat gttaacttta ttcacctgat gtt ttctaag 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 cgcatttgg c 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 t tcagtctga 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 2286 0 atcacctctt gtgatgttta cttgttctca ggtcgctcat cttttgttgc acttgcaatc 22920 tttggggtca ttctgttggc ctgtctcatc gcattcctca tttggactca gaagcgaaga 22980 cagaggcagc ggctctcagg tctgaacaaa attacggtct ctcta atgtt 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 atgtaaaacaa ataaatgggg gg gggggggg 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 gggggg gggg gggggggggg gggggggggg gggggggggg gggggggggg 23760 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23820 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg 23880 gggggggggg gg gg gggggg 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 cta aatccct 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 gctt ggacct tttttcacta 24600 aaacaagtca gtcttctcaa aggtagttta gcttttcata tatctttcac agtttcttcc 24660 attcccattt cctgccattt tcctttctct aacttttatt tattatattt tttcctaaaa 24720 gtttaaattt tctatatctt tatcccttca ga agccatcc 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 ca ggaaaacc tacataagac 25020 ataggaaaga cttgctgtgc ttcttaaacc ccactgtttg ttttcctaat tgtgaacagt 25080 atttttaaag ttaacaagag agcttctaag gcacttgagg ggagatctga tttatttccc 25140 agtaattatt ttcttccttt cagaaaattc c actgaataa gatggtttta acggatgtgg 25200 gactaatttt tgtgtctaaa tctcttccta tttctggatg aaaaaaagga gaccactctg 25260 aagtacaatg aaaaggaaaa tgggaattat aacctggtga ggtgagtagg aagaatttat 25320 tcatcattgc tgaaaacagg tacattcctt ttgaaagttg agaactcctc tggtattaga 25380 aaaaaaaaaaa gaacgtatat acacatatat ttccatgtct atgtttatgt tt gtaaatcc 25440 atattcagaa tatgcaacaa ctttttataa ctatgacttc agtccatctt ttagttacat 25500 atatattcta aacaacaact attgctaaga gaagctgggt aagtaaatgt gaataaatct 25560 tctaaagata ttacaggaag ttcctgctgc ggctcagtgg gttaaag act tgatgtcttt 25620 gtgaagatga gggctcgagc cctggcctca ctcagtgagt taaggatcta gcattgctgt 25680 aagctgcagc gtaggttgca gatagggctc agatccagtg ttgctgtggc tgtggcctca 25740 gttgcagctc tgattcaacc cttaggcgag gaacttccat atgcagcaaa tgtggccatt 25800 aaaaaaaaaa aaaacattat aggagtcatt tcataaaaga gataagacgt ttctatag tt 25860 atatagtgca tactctggta aagatagtat aggatactat aggaatatag aaagcttgcc 25920 tatgaaaatt tgggaagatt gtggaaaaga catctcaaaa tatggcatag aaaagaatca 25980 tatctttgag gaacagtaag tttttcattc aaaaccgtgt attgaacata cttgtggt ga 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 2628 0 gaagattaag ctgaggcaag acacaatcag attgcctttt ataatttact ttcaggagga 26340 aagtctaact aaaaaaagaa ttcgatatca agcttatcga taccgtcgac ctcgaggagg 26400 cccgcctgcc cttttggggg gggggggggg gggggggggg gg gg gggggg 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 gctggaaaaaa 26820 ttttagtgca cccaaccaac aaagaaaaac catgcacaaa aattcaataa atatcatctc 26880 acttt tgtgt tcatgttatt gaatataatt aaacataatg tttacatcta taaaattatc 26940 atatgtatac atgtaaagaa acattaaaac atttttaaca gactgtaaac ttgaggactg 27000 tgaatgactt ttgattgata atctcaaaca tatggatact attctgatgt aataaataat 27060 gattaaattt tttccctaaa gagtaatcac tactgaatcg ttgcctcaga atcatatgga 27120 ggtgctttta 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 2 7420 aagattacaa tacagtcctc tgtctcctgg tattccaaag actgctgttg aatttctaaa 27480 aaatagattg gtgaatgtga ctactcaaag ttgtatgtaa gactttcaag ggcattaaat 27540 aaaaaagaat attgctgatt cttgttcttg attttctgaa tttctgaatc tcttattggg 27600 cttctaattt aaaaaaaaaat atctgggcgc ccgcagatat cgaactcttg ggcagtgtga 27660 ccaaacgaag acatatccaa tcaagcatgc aaatggacca gcccactgta ctagcacgct 27720gtggcagcca atctgaccga gaaagcagac aaccgcaggg agcaacg 27767 <210> 16 <211> 55 <212> DNA <213> Sus scrofa <400> 16 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatc 55 <210> 17 <211> 54 <212> DNA <213> Sus scrofa <400> 17 ggtcgccacc atggccatga gcaagggcga ggagctgttc accggggtgg tgcc 54 <210> 18 <211> 48 <212> DNA <213> Sus scrofa <400> 18 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggt 48 <210> 19 <211> 55 <212> DNA <213> Sus scrofa <400> 19 ggtcgccacc atggttgagc aagggcgagg agctgttcac cggggtggtg cccat 55 <210> 20 <211> 43 <212> DNA <213> Sus scrofa <400> 20 tgcagggaac tacagtgcgg cactgtggtt tccctcctgg ggg 43 <210> 21 <211> 60 <212> DNA <213> Sus scrofa <400> 21 tgcagggaac tacagtgcgg cactgtaaac cactactact gtggtttccc tcctgggggg 60 60 <210> 22 <211> 41 <212> DNA <213> Sus scrofa <400> 22 tgcagggaac tacagtgcgg ctgtggtttc cctcctgggg g 41 <210> 23 <211> 46 <212> DNA <213> Sus scrofa <400> 23 tgcagggaac tacagtgcgg aactactgtg gtttccctcc tggggg 46 <210> 24 <211> 31 <212> DNA <213> Sus scrofa <400> 24 gaaacccagg ctggttggag gggacattcc c 31 <210> 25 <211> 24 <212> DNA <213> Sus scrofa <400> 25 gaaacccagg ctgggggacat tccc 24 <210> 26 <211> 13 <212> DNA <213> Sus scrofa <400> 26 aggggacatt ccc 13 <210> 27 <211> 13 <212> DNA <213> Sus scrofa <400> 27 gaaacccat ccc 13 <210> 28 <211> 31 <212> DNA <213> Sus scrofa <400> 28 ggtcgccacc atggtgagca agggcgagga g 31 <210> 29 <211> 32 <212> DNA <213> Sus scrofa <400> 29 ggtcgccacc atggctgagc aagggcgagg ag 32 <210> 30 <211> 29 <212> DNA <213> Sus scrofa <400>30 ggtcgccacc atggtgagag ggcgaggag 29 <210> 31 <211> 32 <212> DNA <213> Sus scrofa <400> 31 ggtcgccacc atggttgagc aagggcgagg ag 32 <210> 32 <211> 48 <212> DNA <213> Sus scrofa <400> 32 ggtcgccacc atggtgagca agggcgagga gaacccaggc tggttgga 48 <210> 33 <211> 49 <212> DNA <213> Sus scrofa <400> 33 tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggg 49 <210> 34 <211> 38 <212> DNA <213> Sus scrofa <400> 34 tgctgtgcag ggaactctgt ggtttccctc ctgggggg 38 <210> 35 <211> 22 <212> DNA <213> Sus scrofa <400> 35 ctgtggtttc cctcctgggg gg 22 <210> 36 <211> 23 <212> DNA <213> Sus scrofa <400> 36 actgtggttt ccctcctggg ggg 23 <210> 37 <211> 50 <212> DNA <213> Sus scrofa <400> 37 tgctgtgcag ggaactacag tgcggcaact gtggtttccc tcctgggggg 50 <210> 38 <211> 10 <212> DNA <213> Sus scrofa <400> 38 tcctgggggg 10 <210> 39 <211> 8 <212> DNA <213> Sus scrofa <400> 39 ctgggggg 8 <210> 40 <211> 52 <212> DNA <213> Sus scrofa <400> 40 agagagcaga gccagcgact cgcccagcga catggggtac ctgccgtttg tg 52 <210> 41 <211> 33 <212> DNA <213> Sus scrofa <400> 41 agagagcaga gccagcgact cgcccagcga gat 33 <210> 42 <211> 30 <212> DNA <213> Sus scrofa <400> 42 agagagcaga gccagcgact cgcccagcga 30 <210> 43 <211> 50 <212> DNA <213> Sus scrofa <400> 43 agagccagcc tcgcccagca ggggtaccat ggggtacctg ccgtttgtgt 50 <210> 44 <211> 53 <212> DNA <213> Sus scrofa <400> 44 agagagcaga gccagcgact cgcccagcga gcagtgggta cctgccgttt gtg 53 <210> 45 <211> 53 <212> DNA <213> Sus scrofa <400> 45 agagagcaga gccagcgact cgcccagcga tcagtgggta cctgccgttt gtg 53 <210> 46 <211> 53 <212> DNA <213> Sus scrofa <400> 46 agagagcaga gccagcgact cgcccagcga acatggggta cctgccgttt gtg 53 <210> 47 <211> 4990 <212> DNA <213> Sus scrofa <400> 47 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggga gctcactttg 3180 gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc 3240 tttcactctg cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg 3300 gcgtagtctg ctcaagtgag acccagggaa tgtgttcact ttgttcccat gccatgaaga 3360 gggtagggtt aggtagtcac agacatcttt ttaaagccct gtctccttcc aggatacaca 3420 caaatccgct tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt 3480 gggtcctggg ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc 3540 cagcagctta aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga 3600 agtgagcagg tctggaggca catgtttcac tgcactggga ctgagaagca catggggagat 3660 tgttccgtca ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc 3720 tgctcaggta agagaataag ggcagccagt gatgagccac tcatgacggt gccttaagag 3780 tgggtgtacc taggagttcc cattgtggct cagtggtaac aaactcgact ggtatccatg 3840 agggtatggg tttgatccct ggccttgctc aatgggttaa ggatccagca ttgctgtgag 3900 ctgtggtata ggttgcagac tctgctcagg tcccatgttg ctgtgattgt ggtgtaggct 3960 gactgctgca gcttcaattt gacccctagc ccgggaattt ccataggcca cacgtgcagc 4020 actaaggaag gaaaaaaaaga aaaaaaaaaa aaaagagtgg gtgtgcctat agtgaagaac 4080 agatgtaaaa gggaagtgaa agggattccc ccattctgag ggattgtgag aagtgtgcca 4140 gaatattaac ttcatttgac ttgttacagg gaaagtaaac ttgactttca cggacctcct 4200 agttacctgg tgcttactat atgtcttctc agagtacctg attcattccc agcctggttg 4260 acccatcccc ctatctctat ggctatgttt atccagagca catctatcta acactccagc 4320 tgatcttcct gacacagctg tggcaaccct ggatccttta accaactgtg ccaggctgga 4380 gatcaaacct aagcctctgc agcaacccaa gctgctgcag tcagattttt aaccccctgt 4440 gccactgtgg gtatctccga tattttgtat cttctgtgac tgagtggttt gctgtttgca 4500 gggaaccaga gtcagacact atccccgtgc aattcatcat cctcggaccc atcaagctct 4560 attatttcag aagaaaatgg tgttgcctgc ataggtgaga atcagtgacc aacctatgaa 4620 aatgatctca atcctctgaa atgcatttta ttcatgtttt atttcctctt tgcagggagt 4680 ggtcaacttc gcctggtcga tggaggtggt cgttgtgctg ggagagtaga ggtctatcat 4740 gagggctcct ggggcaccat ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg 4800 tgcaaacagc tgagctgtgg atgggccatt aatgccactg gttctgctca ttttggggaa 4860 ggaacagggc ccatttggct ggatgagata aactgtaatg gaaaagaatc tcatatttgg 4920 caatgccact cacatggttg ggggcggcac aattgcaggc ataaggagga tgcaggagtc 4980 atctgctcgg 4990 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 48 caccggaaac ccaggctggt tgga 24 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 49 aaactccaac cagcctgggt ttcc 24 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 50 caccggaact acagtgcggc actg 24 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 51 aaaccagtgc cgcactgtag ttcc 24 <210> 52 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 52 caccgcagta gcaccccgcc ctgac 25 <210> 53 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 53 aaacgtcagg gcggggtgct actgc 25 <210> 54 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 54 caccgtgtag ccacagcagg gacgt 25 <210> 55 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 55 aaacacgtcc ctgctgtggc tacac 25 <210> 56 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 56 caccgccagc ctcgcccagc gacat 25 <210> 57 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 57 aaacatgtcg ctgggcgagg ctggc 25 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 58 caccgcagct gcagcatata tttaa 25 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 59 aaacttaaat atatgctgca gctgc 25 <210>60 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>60 caccgctttc atttatctga actca 25 <210> 61 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 61 aaactgagtt cagataaatg aaagc 25 <210> 62 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>62 caccgttatc tgaactcagg gtccc 25 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 63 aaacgggacc ctgagttcag ataac 25 <210> 64 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 64 caccgctcct cgcccttgct cacca 25 <210> 65 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>65 aaactggtga gcaagggcga ggagc 25 <210> 66 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 66 caccggacca ggatgggcac caccc 25 <210> 67 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 67 aaacgggtgg tgcccatcct ggtcc 25 <210> 68 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 68 ttgttggaag gctcactgtc cttg 24 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 69 acaactaagg tggggcaaag 20 <210>70 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>70 ttgttggaag gctcactgtc cttg 24 <210> 71 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 71 ggagctcaac attcttgggt cct 23 <210> 72 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 72 ggcaaaattt tcatgctgag gtg 23 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 73 gcacatcact tcgggttaca gtg 23 <210> 74 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 74 cccaagtatc ttcagttctg cag 23 <210> 75 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 75 tacaggtagg agagcctgtt ttg 23 <210> 76 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 76 cccaagtatc ttcagttctg cag 23 <210> 77 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 77 ctcaaaagga tgtaaaccct gga 23 <210> 78 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 78 tgttgatgtg gtttgtttgc cc 22 <210> 79 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 79 tacaggtagg agagcctgtt ttg 23 <210>80 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>80 ggaggtctag aatcggctaa gcc 23 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 81 ggctacatgt cccgtcaggg 20 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 82 gcaggccact aggcagatga a 21 <210> 83 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 83 gagctgacac ccaagaagtt cct 23 <210> 84 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 84 ggctctagag cctctgctaa cc 22 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 85 ggacttgaag aagtcgtgct gc 22 <210> 86 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 86 taatacgact cactataggg agaatggact ataaggacca cgac 44 <210> 87 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 87 gcgagctcta ggaattctta c 21 <210> 88 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 88 ttaatacgac tcactatagg ctcctcgccc ttgctcacca 40 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 89 aaaagcaccg actcggtgcc 20 <210> 90 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400>90 ttaatacgac tcactatagg aaacccaggc tggttgga 38 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 91 aaaagcaccg actcggtgcc 20 <210> 92 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 92 ttaatacgac tcactatagg aactacagtg cggcactg 38 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 93 aaaagcaccg actcggtgcc 20 <210> 94 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 94 ttaatacgac tcactatagg ccagcctcgc ccagcgacat 40 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 95 aaaagcaccg actcggtgcc 20 <210> 96 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 96 ttaatacgac tcactatagg cagctgcagc atatatttaa 40 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 97 aaaagcaccg actcggtgcc 20 <210> 98 <211> 3484 <212> DNA <213> Sus scrofa <400> 98 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggcacattc cctgctctgg tcgtgttgaa gtacaacatg 1560 gagacacgtg gggcaccgtc tgtgattctg acttctctct ggaggcggcc agcgtgctgt 1620 gcagggaact acagtgcggc actgtggttt ccctcctggg gggagctcac tttggagaag 1680 gaagtggaca gatctgggct gaagaattcc agtgtgaggg gcacgagtcc cacctttcac 1740 tctgcccagt agcaccccgc cctgacggga catgtagcca cagcagggac gtcggcgtag 1800 tctgctcaag tgagacccag ggaatgtgtt cactttgttc ccatgccatg aagagggtag 1860 ggttaggtag tcacagacat ctttttaaag ccctgtctcc ttccaggata cacacaaatc 1920 cgcttggtga atggcaagac cccatgtgaa ggaagagtgg agctcaacat tcttgggtcc 1980 tggggggtccc tctgcaactc tcactgggac atggaagatg cccatgtttt atgccagcag 2040 cttaaatgtg gagttgccct ttctatcccg ggaggagcac cttttgggaa aggaagtgag 2100 caggtctgga ggcacatgtt tcactgcact gggactgaga agcacatggg agattgttcc 2160 gtcactgctc tgggcgcatc actctgttct tcagggcaag tggcctctgt aatctgctca 2220 ggtaagagaa taagggcagc cagtgatgag ccactcatga cggtgcctta agagtgggtg 2280 tacctaggag ttcccattgt ggctcagtgg taacaaactc gactggtatc catgagggta 2340 tgggtttgat ccctggcctt gctcaatggg ttaaggatcc agcattgctg tgagctgtgg 2400 tataggttgc agactctgct caggtcccat gttgctgtga ttgtggtgta ggctgactgc 2460 tgcagcttca atttgacccc tagcccggga atttccatag gccacacgtg cagcactaag 2520 gaaggaaaaa aagaaaaaaa aaaaaaaaga gtgggtgtgc ctatagtgaa gaacagatgt 2580 aaaaggggaag tgaaaagggat tcccccattc tgagggatg tgagaagtgt gccagaatat 2640 taacttcatt tgacttgtta cagggaaagt aaacttgact ttcacggacc tcctagttac 2700 ctggtgctta ctatatgtct tctcagagta cctgattcat tcccagcctg gttgacccat 2760 ccccctatct ctatggctat gtttatccag agcacatcta tctaacactc cagctgatct 2820 tcctgacaca gctgtggcaa ccctggatcc tttaaccaac tgtgccaggc tggagatcaa 2880 acctaagcct ctgcagcaac ccaagctgct gcagtcagat ttttaacccc ctgtgccact 2940 gtgggtatct ccgatatttt gtatcttctg tgactgagtg gtttgctgtt tgcagggaac 3000 cagagtcaga cactatcccc gtgcaattca tcatcctcgg acccatcaag ctctattatt 3060 tcagaagaaa atggtgttgc ctgcataggt gagaatcagt gaccaaccta tgaaaatgat 3120 ctcaatcctc tgaaatgcat tttattcatg ttttattcc tctttgcagg gagtggtcaa 3180 cttcgcctgg tcgatggagg tggtcgttgt gctgggagag tagaggtcta tcatgagggc 3240 tcctggggca ccatctgtga tgacagctgg gacctgaatg atgcccatgt ggtgtgcaaa 3300 cagctgagct gtggatgggc cattaatgcc actggttctg ctcattttgg ggaaggaaca 3360 gggcccattt ggctggatga gataaactgt aatggaaaaag aatctcatat ttggcaatgc 3420 cactcacatg gttgggggcg gcacaattgc aggcataagg aggatgcagg agtcatctgc 3480 tcgg 3484 <210> 99 <211> 4997 <212> DNA <213> Sus scrofa <400> 99 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggctact actactgtgg tttccctcct ggggggagct 3180 cactttggag aaggaagtgg acagatctgg gctgaagaat tccagtgtga ggggcacgag 3240 tcccaccttt cactctgccc agtagcaccc cgccctgacg ggacatgtag ccacagcagg 3300 gacgtcggcg tagtctgctc aagtgagacc cagggaatgt gttcactttg ttcccatgcc 3360 atgaagaggg tagggttagg tagtcacaga catcttttta aagccctgtc tccttccagg 3420 atacacacaa atccgcttgg tgaatggcaa gaccccatgt gaaggaagag tggagctcaa 3480 cattcttggg tcctgggggt ccctctgcaa ctctcactgg gacatggaag atgcccatgt 3540 tttatgccag cagcttaaat gtggagttgc cctttctatc ccgggaggag caccttttgg 3600 gaaaggaagt gagcaggtct ggaggcacat gtttcactgc actgggactg agaagcacat 3660 gggagatgt tccgtcactg ctctgggcgc atcactctgt tcttcagggc aagtggcctc 3720 tgtaatctgc tcaggtaaga gaataagggc agccagtgat gagccactca tgacggtgcc 3780 ttaagagtgg gtgtacctag gagttcccat tgtggctcag tggtaacaaa ctcgactggt 3840 atccatgagg gtatgggttt gatccctggc cttgctcaat gggttaagga tccagcattg 3900 ctgtgagctg tggtataggt tgcagactct gctcaggtcc catgttgctg tgattgtggt 3960 gtaggctgac tgctgcagct tcaatttgac ccctagcccg ggaatttcca taggccacac 4020 gtgcagcact aaggaaggaa aaaaagaaaa aaaaaaaaaaa agagtgggtg tgcctatagt 4080 gaagaacaga tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag 4140 tgtgccagaa tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg 4200 acctcctagt tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc 4260 ctggttgacc catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca 4320 ctccagctga tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca 4380 ggctggagat caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac 4440 cccctgtgcc actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct 4500 gtttgcaggg aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc 4560 aagctctatt atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac 4620 ctatgaaaat gatctcaatc ctctgaaatg cattttatattc atgttttatt tcctctttgc 4680 agggagtggt caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt 4740 ctatcatgag ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca 4800 tgtggtgtgc aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt 4860 tggggaaagga acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca 4920 tatttggcaa tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc 4980 aggagtcatc tgctcgg 4997 <210> 100 <211> 3710 <212> DNA <213> Sus scrofa <400> 100 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaagggaa 2820 agggattccc ccattctgag ggattgtgag aagtgtgcca gaatattaac ttcatttgac 2880 ttgttacagg gaaagtaaac ttgactttca cggacctcct agttacctgg tgcttactat 2940 atgtcttctc agagtacctg attcattccc agcctggttg acccatcccc ctatctctat 3000 ggctatgttt atccagagca catctatcta acactccagc tgatcttcct gacacagctg 3060 tggcaaccct ggatccttta accaactgtg ccaggctgga gatcaaacct aagcctctgc 3120 agcaacccaa gctgctgcag tcagattttt aaccccctgt gccactgtgg gtatctccga 3180 tattttgtat cttctgtgac tgagtggttt gctgtttgca gggaaccaga gtcagacact 3240 atccccgtgc aattcatcat cctcggaccc atcaagctct attatttcag aagaaaatgg 3300 tgttgcctgc ataggtgaga atcagtgacc aacctatgaa aatgatctca atcctctgaa 3360 atgcatttta ttcatgtttt atttcctctt tgcagggagt ggtcaacttc gcctggtcga 3420 tggaggtggt cgttgtgctg ggagagtaga ggtctatcat gagggctcct ggggcaccat 3480 ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg 3540 atgggccatt aatgccactg gttctgctca ttttggggaa ggaacagggc ccattggct 3600 ggatgagata aactgtaatg gaaaagaatc tcatatttgg caatgccact cacatggttg 3660 ggggcggcac aattgcaggc ataaggagga tgcaggagtc atctgctcgg 3710 <210> 101 <211> 3617 <212> DNA <213> Sus scrofa <400> 101 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gattgaaagg gattccccca ttctgaggga ttgtgagaag 2760 tgtgccagaa tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg 2820 acctcctagt tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc 2880 ctggttgacc catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca 2940 ctccagctga tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca 3000 ggctggagat caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac 3060 cccctgtgcc actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct 3120 gtttgcaggg aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc 3180 aagctctatt atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac 3240 ctatgaaaat gatctcaatc ctctgaaatg cattttattc atgttttatt tcctctttgc 3300 agggagtggt caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt 3360 ctatcatgag ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca 3420 tgtggtgtgc aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt 3480 tggggaaagga acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca 3540 tatttggcaa tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc 3600 aggagtcatc tgctcgg 3617 <210> 102 <211> 4979 <212> DNA <213> Sus scrofa <400> 102 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactctgt ggtttccctc ctggggggag ctcactttgg agaaggaagt 3180 ggacagatct gggctgaaga attccagtgt gaggggcacg agtcccacct ttcactctgc 3240 ccagtagcac cccgccctga cgggacatgt agccacagca gggacgtcgg cgtagtctgc 3300 tcaagtgaga cccagggaat gtgttcactt tgttcccatg ccatgaagag ggtagggtta 3360 ggtagtcaca gacatctttt taaagccctg tctccttcca ggatacacac aaatccgctt 3420 ggtgaatggc aagaccccat gtgaaggaag agtggagctc aacattcttg ggtcctgggg 3480 gtccctctgc aactctcact gggacatgga agatgcccat gttttatgcc agcagcttaa 3540 atgtggagtt gccctttcta tcccgggagg agcacctttt gggaaaggaa gtgagcaggt 3600 ctggaggcac atgtttcact gcactgggac tgagaagcac atgggagatt gttccgtcac 3660 tgctctgggc gcatcactct gttcttcagg gcaagtggcc tctgtaatct gctcaggtaa 3720 gagaataagg gcagccagtg atgagccact catgacggtg ccttaagagt gggtgtacct 3780 aggagttccc attgtggctc agtggtaaca aactcgactg gtatccatga gggtatgggt 3840 ttgatccctg gccttgctca atgggttaag gatccagcat tgctgtgagc tgtggtatag 3900 gttgcagact ctgctcaggt cccatgttgc tgtgattgtg gtgtaggctg actgctgcag 3960 cttcaatttg acccctagcc cgggaatttc cataggccac acgtgcagca ctaaggaagg 4020 aaaaaaagaa aaaaaaaaaa aaagagtggg tgtgcctata gtgaagaaca gatgtaaaag 4080 ggaagtgaaa gggattcccc cattctgagg gattgtgaga agtgtgccag aatattaact 4140 tcatttgact tgttacaggg aaagtaaact tgactttcac ggacctccta gttacctggt 4200 gcttactata tgtcttctca gagtacctga ttcattccca gcctggttga cccatccccc 4260 tatctctatg gctatgttta tccagagcac atctatctaa cactccagct gatcttcctg 4320 acacagctgt ggcaaccctg gatcctttaa ccaactgtgc caggctggag atcaaaccta 4380 agcctctgca gcaacccaag ctgctgcagt cagattttta accccctgtg ccactgtggg 4440 tatctccgat attttgtatc ttctgtgact gagtggtttg ctgtttgcag ggaaccagag 4500 tcagacacta tccccgtgca attcatcatc ctcggaccca tcaagctcta ttatttcaga 4560 agaaaatggt gttgcctgca taggtgagaa tcagtgacca acctatgaaa atgatctcaa 4620 tcctctgaaa tgcattttat tcatgtttta tttcctcttt gcagggagtg gtcaacttcg 4680 cctggtcgat ggaggtggtc gttgtgctgg gagagtagag gtctatcatg agggctcctg 4740 gggcaccatc tgtgatgaca gctgggacct gaatgatgcc catgtggtgt gcaaacagct 4800 gagctgtgga tgggccatta atgccactgg ttctgctcat tttggggaag gaacagggcc 4860 catttggctg gatgagataa actgtaatgg aaaagaatct catatttggc aatgccactc 4920 acatggttgg gggcggcaca attgcaggca taaggaggat gcaggagtca tctgctcgg 4979 <210> 103 <211> 4615 <212> DNA <213> Sus scrofa <400> 103 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aagaaggaaa 2580 atattggaat catattctcc ctcaccgaaa tgctattttt cagcccacag gaaacccagg 2640 ctggttggag gggacattcc ctgctctggt cgtgttgaag tacaacatgg agaacacgtgg 2700 ggcaccgtct gtgattctga cttctctctg gaggcggcca gcgtgctgtg cagggaacta 2760 cagtgcggca cagtgtggtt tccctcctgg ggggagctca ctttggagaa ggaagtggac 2820 agatctgggc tgaagaattc cagtgtgagg ggcacgagtc ccacctttca ctctgcccag 2880 tagcaccccg ccctgacggg acatgtagcc acagcaggga cgtcggcgta gtctgctcaa 2940 gtgagaccca gggaatgtgt tcactttgtt cccatgccat gaagagggta gggttaggta 3000 gtcacagaca tctttttaaa gccctgtctc cttccaggat acacacaaat ccgcttggtg 3060 aatggcaaga ccccatgtga aggaagagtg gagctcaaca ttcttgggtc ctgggggtcc 3120 ctctgcaact ctcactggga catggaagat gcccatgttt tatgccagca gcttaaatgt 3180 ggagttgccc tttctatccc gggaggagca ccttttggga aaggaagtga gcaggtctgg 3240 aggcacatgt ttcactgcac tgggactgag aagcacatgg gagattgttc cgtcactgct 3300 ctgggcgcat cactctgttc ttcagggcaa gtggcctctg taatctgctc aggtaagaga 3360 ataagggcag ccagtgatga gccactcatg acggtgcctt aagagtgggt gtacctagga 3420 gttcccattg tggctcagtg gtaacaaact cgactggtat ccatgagggt atgggtttga 3480 tccctggcct tgctcaatgg gttaaggatc cagcattgct gtgagctgtg gtataggttg 3540 cagactctgc tcaggtccca tgttgctgtg attgtggtgt aggctgactg ctgcagcttc 3600 aatttgaccc ctagcccggg aatttccata ggccacacgt gcagcactaa ggaaggaaaa 3660 aaagaaaaaa aaaaaaaaag agtgggtgtg cctatagtga agaacagatg taaaagggaa 3720 gtgaaaggga ttcccccatt ctgagggatt gtgagaagtg tgccagaata ttaacttcat 3780 ttgacttgtt acagggaaag taaacttgac tttcacggac ctcctagtta cctggtgctt 3840 actatatgtc ttctcagagt acctgattca ttcccagcct ggttgaccca tccccctatc 3900 tctatggcta tgtttatcca gagcacatct atctaacact ccagctgatc ttcctgacac 3960 agctgtggca accctggatc ctttaaccaa ctgtgccagg ctggagatca aacctaagcc 4020 tctgcagcaa cccaagctgc tgcagtcaga tttttaaccc cctgtgccac tgtgggtatc 4080 tccgatattt tgtatcttct gtgactgagt ggtttgctgt ttgcagggaa ccagagtcag 4140 acactatccc cgtgcaattc atcatcctcg gacccatcaa gctctattat ttcagaagaa 4200 aatggtgttg cctgcatagg tgagaatcag tgaccaacct atgaaaatga tctcaatcct 4260 ctgaaatgca ttttatcat gttttatttc ctctttgcag ggagtggtca acttcgcctg 4320 gtcgatggag gtggtcgttg tgctgggaga gtagaggtct atcatgaggg ctcctggggc 4380 acatctgtg atgacagctg ggacctgaat gatgcccatg tggtgtgcaa acagctgagc 4440 tgtggatggg ccattaatgc cactggttct gctcattttg gggaaggaac agggcccatt 4500 tggctggatg agataaactg taatggaaaa gaatctcata tttggcaatg ccactcacat 4560 ggttgggggc ggcacaattg caggcataag gaggatgcag gagtcatctg ctcgg 4615 <210> 104 <211> 4866 <212> DNA <213> Sus scrofa <400> 104 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttctgtggt ttccctcctg gggggagctc actttggaga 3060 aggaagtgga cagatctggg ctgaagaatt ccagtgtgag gggcacgagt cccacctttc 3120 actctgccca gtagcacccc gccctgacgg gacatgtagc cacagcaggg acgtcggcgt 3180 agtctgctca agtgagaccc agggaatgtg ttcactttgt tcccatgcca tgaagagggt 3240 agggttaggt agtcacagac atctttttaa agccctgtct ccttccagga tacacacaaa 3300 tccgcttggt gaatggcaag accccatgtg aaggaagagt ggagctcaac attcttgggt 3360 cctggggggtc cctctgcaac tctcactggg acatggaaga tgcccatgtt ttatgccagc 3420 agcttaaatg tggagttgcc ctttctatcc cgggaggagc accttttggg aaaggaagtg 3480 agcaggtctg gaggcacatg tttcactgca ctgggactga gaagcacatg ggagattgtt 3540 ccgtcactgc tctgggcgca tcactctgtt cttcagggca agtggcctct gtaatctgct 3600 caggtaagag aataagggca gccagtgatg agccactcat gacggtgcct taagagtggg 3660 tgtacctagg agttcccatt gtggctcagt ggtaacaaac tcgactggta tccatgaggg 3720 tatgggtttg atccctggcc ttgctcaatg ggttaaggat ccagcattgc tgtgagctgt 3780 ggtataggtt gcagactctg ctcaggtccc atgttgctgt gattgtggtg taggctgact 3840 gctgcagctt caatttgacc cctagcccgg gaatttccat aggccacacg tgcagcacta 3900 aggaaggaaa aaaagaaaaa aaaaaaaaaaa gagtgggtgt gcctatagtg aagaacagat 3960 gtaaaaggga agtgaaaggg attcccccat tctgagggat tgtgagaagt gtgccagaat 4020 attaacttca tttgacttgt tacagggaaa gtaaacttga ctttcacgga cctcctagtt 4080 acctggtgct tactatatgt cttctcagag tacctgattc attcccagcc tggttgaccc 4140 atccccctat ctctatggct atgtttatcc agagcacatc tatctaacac tccagctgat 4200 cttcctgaca cagctgtggc aaccctggat cctttaacca actgtgccag gctggagatc 4260 aaacctaagc ctctgcagca acccaagctg ctgcagtcag atttttaacc ccctgtgcca 4320 ctgtgggtat ctccgatatt ttgtatcttc tgtgactgag tggtttgctg tttgcaggga 4380 accagagtca gacactatcc ccgtgcaatt catcatcctc ggacccatca agctctatta 4440 tttcagaaga aaatggtgtt gcctgcatag gtgagaatca gtgaccaacc tatgaaaatg 4500 atctcaatcc tctgaaatgc attttatca tgttttatt cctctttgca gggagtggtc 4560 aacttcgcct ggtcgatgga ggtggtcgtt gtgctgggag agtagaggtc tatcatgagg 4620 gctcctgggg caccatctgt gatgacagct gggacctgaa tgatgcccat gtggtgtgca 4680 aacagctgag ctgtggatgg gccattaatg ccactggttc tgctcatttt ggggaaggaa 4740 cagggcccat ttggctggat gagataaact gtaatggaaa agaatctcat atttggcaat 4800 gccactcaca tggttggggg cggcacaatt gcaggcataa ggaggatgca ggagtcatct 4860 gctcgg 4866 <210> 105 <211> 4867 <212> DNA <213> Sus scrofa <400> 105 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttactgtgg tttccctcct ggggggagct cactttggag 3060 aaggaagtgg acagatctgg gctgaagaat tccagtgtga ggggcacgag tcccaccttt 3120 cactctgccc agtagcaccc cgccctgacg ggacatgtag ccacagcagg gacgtcggcg 3180 tagtctgctc aagtgagacc cagggaatgt gttcactttg ttcccatgcc atgaagaggg 3240 tagggttagg tagtcacaga catcttttta aagccctgtc tccttccagg atacacacaa 3300 atccgcttgg tgaatggcaa gaccccatgt gaaggaagag tggagctcaa cattcttggg 3360 tcctgggggt ccctctgcaa ctctcactgg gacatggaag atgcccatgt tttatgccag 3420 cagcttaaat gtggagttgc cctttctatc ccgggaggag caccttttgg gaaaggaagt 3480 gagcaggtct ggaggcacat gtttcactgc actgggactg agaagcacat gggagattgt 3540 tccgtcactg ctctgggcgc atcactctgt tcttcagggc aagtggcctc tgtaatctgc 3600 tcaggtaaga gaataagggc agccagtgat gagccactca tgacggtgcc ttaagagtgg 3660 gtgtacctag gagttcccat tgtggctcag tggtaacaaa ctcgactggt atccatgagg 3720 gtatgggttt gatccctggc cttgctcaat gggttaagga tccagcattg ctgtgagctg 3780 tggtataggt tgcagactct gctcaggtcc catgttgctg tgattgtggt gtaggctgac 3840 tgctgcagct tcaatttgac ccctagcccg ggaatttcca taggccacac gtgcagcact 3900 aaggaaggaa aaaaagaaaa aaaaaaaaaaa agagtgggtg tgcctatagt gaagaacaga 3960 tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag tgtgccagaa 4020 tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg acctcctagt 4080 tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc ctggttgacc 4140 catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca ctccagctga 4200 tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca ggctggagat 4260 caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac cccctgtgcc 4320 actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct gtttgcaggg 4380 aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc aagctctatt 4440 atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac ctatgaaaat 4500 gatctcaatc ctctgaaatg cattttatattc atgttttat tcctctttgc agggagtggt 4560 caacttcgcc tggtcgatgg aggtggtcgt tgtgctggga gagtagaggt ctatcatgag 4620 ggctcctggg gcaccatctg tgatgacagc tgggacctga atgatgccca tgtggtgtgc 4680 aaacagctga gctgtggatg ggccattaat gccactggtt ctgctcattt tggggaagga 4740 acagggccca tttggctgga tgagataaac tgtaatggaa aagaatctca tatttggcaa 4800 tgccactcac atggttgggg gcggcacaat tgcaggcata aggaggatgc aggagtcatc 4860 tgctcgg 4867 <210> 106 <211> 4991 <212> DNA <213> Sus scrofa <400> 106 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcggcaact gtggtttccc tcctgggggg agctcacttt 3180 ggagaaggaa gtggacagat ctgggctgaa gaattccagt gtgaggggca cgagtcccac 3240 ctttcactct gcccagtagc accccgccct gacgggacat gtagccacag cagggacgtc 3300 ggcgtagtct gctcaagtga gacccaggga atgtgttcac tttgttccca tgccatgaag 3360 agggtagggt taggtagtca cagacatctt tttaaagccc tgtctccttc caggatacac 3420 acaaatccgc ttggtgaatg gcaagacccc atgtgaagga agagtggagc tcaacattct 3480 tgggtcctgg gggtccctct gcaactctca ctgggacatg gaagatgccc atgttttatg 3540 ccagcagctt aaatgtggag ttgccctttc tatcccggga ggagcacctt ttgggaaagg 3600 aagtgagcag gtctgggaggc acatgtttca ctgcactggg actgagaagc acatggggaga 3660 ttgttccgtc actgctctgg gcgcatcact ctgttcttca gggcaagtgg cctctgtaat 3720 ctgctcaggt aagagaataa gggcagccag tgatgagcca ctcatgacgg tgccttaaga 3780 gtgggtgtac ctaggagttc ccattgtggc tcagtggtaa caaactcgac tggtatccat 3840 gagggtatgg gtttgatccc tggccttgct caatgggtta aggatccagc attgctgtga 3900 gctgtggtat aggttgcaga ctctgctcag gtcccatgtt gctgtgattg tggtgtaggc 3960 tgactgctgc agcttcaatt tgacccctag cccgggaatt tccataggcc acacgtgcag 4020 cactaaggaa ggaaaaaaag aaaaaaaaaaa aaaaagagtg ggtgtgccta tagtgaagaa 4080 cagatgtaaa agggaagtga aagggattcc cccattctga gggattgtga gaagtgtgcc 4140 agaatattaa cttcatttga cttgttacag ggaaagtaaa cttgactttc acggacctcc 4200 tagttacctg gtgcttacta tatgtcttct cagagtacct gattcattcc cagcctggtt 4260 gacccatccc cctatctcta tggctatgtt tatccagagc acatctatct aacactccag 4320 ctgatcttcc tgacacagct gtggcaaccc tggatccttt aaccaactgt gccaggctgg 4380 agatcaaacc taagcctctg cagcaaccca agctgctgca gtcagatttt taaccccctg 4440 tgccactgtg ggtatctccg atattttgta tcttctgtga ctgagtggtt tgctgtttgc 4500 agggaaccag agtcagacac tatccccgtg caattcatca tcctcggacc catcaagctc 4560 tattatttca gaagaaaatg gtgttgcctg cataggtgag aatcagtgac caacctatga 4620 aaatgatctc aatcctctga aatgcatttt attcatgttt tatttcctct ttgcagggag 4680 tggtcaactt cgcctggtcg atggaggtgg tcgttgtgct gggagagtag aggtctatca 4740 tgagggctcc tggggcacca tctgtgatga cagctgggac ctgaatgatg cccatgtggt 4800 gtgcaaacag ctgagctgtg gatgggccat taatgccact ggttctgctc attttgggga 4860 aggaacaggg cccatttggc tggatgagat aaactgtaat ggaaaagaat ctcatatttg 4920 gcaatgccac tcacatggtt gggggcggca caattgcagg cataaggagg atgcaggagt 4980 catctgctcg g 4991 <210> 107 <211> 4860 <212> DNA <213> Sus scrofa <400> 107 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggt cctgggggga gctcactttg gagaaggaag 3060 tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc tttcactctg 3120 cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg gcgtagtctg 3180 ctcaagtgag acccagggaa tgtgttcact ttgttcccat gccatgaaga gggtagggtt 3240 aggtagtcac agacatcttt ttaaagccct gtctccttcc aggatacaca caaatccgct 3300 tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt gggtcctggg 3360 ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc cagcagctta 3420 aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga agtgagcagg 3480 tctggaggca catgtttcac tgcactggga ctgagaagca catggggagat tgttccgtca 3540 ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc tgctcaggta 3600 agagaataag ggcagccagt gatgagccac tcatgacggt gccttaagag tgggtgtacc 3660 taggagttcc cattgtggct cagtggtaac aaactcgact ggtatccatg agggtatggg 3720 tttgatccct ggccttgctc aatgggttaa ggatccagca ttgctgtgag ctgtggtata 3780 ggttgcagac tctgctcagg tcccatgttg ctgtgattgt ggtgtaggct gactgctgca 3840 gcttcaattt gacccctagc ccgggaattt ccataggcca cacgtgcagc actaaggaag 3900 gaaaaaaaga aaaaaaaaaaa aaaagagtgg gtgtgcctat agtgaagaac agatgtaaaa 3960 gggaagtgaa agggattccc ccattctgag ggattgtgag aagtgtgcca gaatattaac 4020 ttcatttgac ttgttacagg gaaagtaaac ttgactttca cggacctcct agttacctgg 4080 tgcttactat atgtcttctc agagtacctg attcattccc agcctggttg acccatcccc 4140 ctatctctat ggctatgttt atccagagca catctatcta acactccagc tgatcttcct 4200 gacacagctg tggcaaccct ggatccttta accaactgtg ccaggctgga gatcaaacct 4260 aagcctctgc agcaacccaa gctgctgcag tcagattttt aaccccctgt gccactgtgg 4320 gtatctccga tattttgtat cttctgtgac tgagtggttt gctgtttgca gggaaccaga 4380 gtcagacact atccccgtgc aattcatcat cctcggaccc atcaagctct attatttcag 4440 aagaaaatgg tgttgcctgc ataggtgaga atcagtgacc aacctatgaa aatgatctca 4500 atcctctgaa atgcatttta ttcatgtttt atttcctctt tgcaggggagt 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 gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggc tggggggagc tcactttgga gaaggaagtg 3060 gacagatctg ggctgaagaa ttccagtgtg aggggcacga gtcccacctt tcactctgcc 3120 cagtagcacc ccgccctgac gggacatgta gccacagcag ggacgtcggc gtagtctgct 3180 caagtgagac ccagggaatg tgttcacttt gttcccatgc catgaagagg gtagggttag 3240 gtagtcacag acatcttttt aaagccctgt ctccttccag gatacacaca aatccgcttg 3300 gtgaatggca agacccccatg tgaaggaaga gtggagctca acattcttgg gtcctggggg 3360 tccctctgca actctcactg ggacatggaa gatgcccatg ttttatgcca gcagcttaaa 3420 tgtggagttg ccctttctat cccgggagga gcaccttttg ggaaaggaag tgagcaggtc 3480 tggaggcaca tgtttcactg cactgggact gagaagcaca tgggagattg ttccgtcact 3540 gctctgggcg catcactctg ttcttcaggg caagtggcct ctgtaatctg ctcaggtaag 3600 agaataaggg cagccagtga tgagccactc atgacggtgc cttaagagtg ggtgtaccta 3660 ggagttccca ttgtggctca gtggtaacaa actcgactgg tatccatgag ggtatgggtt 3720 tgatccctgg ccttgctcaa tgggttaagg atccagcatt gctgtgagct gtggtatagg 3780 ttgcagactc tgctcaggtc ccatgttgct gtgattgtgg tgtaggctga ctgctgcagc 3840 ttcaatttga cccctagccc gggaatttcc ataggccaca cgtgcagcac taaggaagga 3900 aaaaaagaaa aaaaaaaaaaa aagagtgggt gtgcctatag tgaagaacag atgtaaaagg 3960 gaagtgaaag ggattccccc attctgaggg attgtgagaa gtgtgccaga atattaactt 4020 catttgactt gttacaggga aagtaaactt gactttcacg gacctcctag ttacctggtg 4080 cttactatat gtcttctcag agtacctgat tcattcccag cctggttgac ccatccccct 4140 atctctatgg ctatgtttat ccagagcaca tctatctaac actccagctg atcttcctga 4200 cacagctgtg gcaaccctgg atcctttaac caactgtgcc aggctggaga tcaaacctaa 4260 gcctctgcag caacccaaagc tgctgcagtc agatttttaa ccccctgtgc cactgtgggt 4320 atctccgata ttttgtatct tctgtgactg agtggtttgc tgtttgcagg gaaccagagt 4380 cagacactat ccccgtgcaa ttcatcatcc tcggacccat caagctctat tatttcagaa 4440 gaaaatggtg ttgcctgcat aggtgagaat cagtgaccaa cctatgaaaa tgatctcaat 4500 cctctgaaat gcattttat catgttttat ttcctctttg cagggagtgg tcaacttcgc 4560 ctggtcgatg gaggtggtcg ttgtgctggg agagtagagg tctatcatga gggctcctgg 4620 ggcaccatct gtgatgacag ctgggacctg aatgatgccc atgtggtgtg caaacagctg 4680 agctgtggat gggccattaa tgccactggt tctgctcatt ttggggaagg aacagggccc 4740 atttggctgg atgagataaa ctgtaatgga aaagaatctc atatttggca atgccactca 4800 catggttggg ggcggcacaa ttgcaggcat aaggaggatg caggagtcat ctgctcgg 4858 <210> 109 <211> 3523 <212> DNA <213> Sus scrofa <400> 109 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gagctgtggt ataggttgca gactctgctc 2460 aggtcccatg ttgctgtgat tgtggtgtag gctgactgct gcagcttcaa tttgacccct 2520 agcccgggaa tttccatagg ccacacgtgc agcactaagg aaggaaaaaa agaaaaaaaaa 2580 aaaaaaagag tgggtgtgcc tatagtgaag aacagatgta aaagggaagt gaaagggatt 2640 cccccattct gagggattgt gagaagtgtg ccagaatatt aacttcattt gacttgttac 2700 agggaaagta aacttgactt tcacggacct cctagttacc tggtgcttac tatatgtctt 2760 ctcagagtac ctgattcatt cccagcctgg ttgacccatc cccctatctc tatggctatg 2820 tttatccaga gcacatctat ctaacactcc agctgatctt cctgacacag ctgtggcaac 2880 cctggatcct ttaaccaact gtgccaggct ggagatcaaa cctaagcctc tgcagcaacc 2940 caagctgctg cagtcagatt tttaaccccc tgtgccactg tgggtatctc cgatattttg 3000 tatcttctgt gactgagtgg tttgctgttt gcagggaacc agagtcagac actatccccg 3060 tgcaattcat catcctcgga cccatcaagc tctattattt cagaagaaaa tggtgttgcc 3120 tgcataggtg agaatcagtg accaacctat gaaaatgatc tcaatcctct gaaatgcatt 3180 ttattcatgt tttattcct ctttgcaggg agtggtcaac ttcgcctggt cgatggaggt 3240 ggtcgttgtg ctgggagagt agaggtctat catgagggct cctggggcac catctgtgat 3300 gacagctggg acctgaatga tgcccatgtg gtgtgcaaac agctgagctg tggatgggcc 3360 attaatgcca ctggttctgc tcattttggg gaaggaacag ggcccatttg gctggatgag 3420 ataaactgta atggaaaaaga atctcatatt tggcaatgcc actcacatgg ttgggggcgg 3480 cacaattgca ggcataagga ggatgcagga gtcatctgct cgg 3523 <210> 110 <211> 3603 <212> DNA <213> Sus scrofa <400> 110 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgttgt ggagaattcc acaagaattc gtgttattg acagcagtca tctttaaaag 540 gcatttgaga aagtccaatt tcaaatgcat ttcctttctt taaaagataa attgaagaaa 600 ataagtcttt atttcccaag taaattgaat tgcctctcag tctgttaaaa gaaactctta 660 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 720 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 780 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 840 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 900 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 960 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 1020 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 1080 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 1140 gagatgttgt ggagaattcc acaagaattc gtgttattg acagcagtca tctttaaaag 1200 gcatttgaga aagtccaatt tcaaatgcat ttcctttctt taaaagataa attgaagaaa 1260 ataagtcttt atttcccaag taaattgaat tgcctctcag tctgttaaaa gaaactctta 1320 ccttgatgat tgcgctctta acctggcaaa gattgtcttt aaaatctgag ctccatgtct 1380 tctgctttat ttctggtgtg cctttgactc cagattacag taaatggagg actgagtata 1440 gggctaaaaa gtagagagaa tggatgcata ttatctgtgg tctccaatgt gatgaatgaa 1500 gtaggcaaat actcaaagga aagagaaagc atgctccaag aattatgggt tccagaaggc 1560 aaagtcccag aattgtctcc agggaaggac agggaggtct agaatcggct aagcccactg 1620 taggcagaaa aaccaagagg catgaatggc ttccctttct cacttttcac tctctggctt 1680 actcctatca tgaaggaaaa tattggaatc atattctccc tcaccgaaat gctatttttc 1740 agcccacagg aaacccaggc tggttggagg ggacattccc tgctctcact ttggagaagg 1800 aagtggacag atctgggctg aagaattcca gtgtgagggg cacgagtccc acctttcact 1860 ctgcccagta gcaccccgcc ctgacgggac atgtagccac agcagggacg tcggcgtagt 1920 ctgctcaagt gagacccagg gaatgtgttc actttgttcc catgccatga agagggtagg 1980 gttaggtagt cacagacatc tttttaaagc cctgtctcct tccaggatac acacaaatcc 2040 gcttggtgaa tggcaagacc ccatgtgaag gaagagtgga gctcaacatt cttgggtcct 2100 gggggtccct ctgcaactct cactgggaca tggaagatgc ccatgtttta tgccagcagc 2160 ttaaatgtgg agttgccctt tctatcccgg gaggagcacc ttttgggaaa ggaagtgagc 2220 aggtctggag gcacatgttt cactgcactg ggactgagaa gcacatggga gattgttccg 2280 tcactgctct gggcgcatca ctctgttctt cagggcaagt ggcctctgta atctgctcag 2340 gtaagagaat aagggcagcc agtgatgagc cactcatgac ggtgccttaa gagtgggtgt 2400 acctaggagt tcccattgtg gctcagtggt aacaaactcg actggtatcc atgagggtat 2460 gggtttgatc cctggccttg ctcaatgggt taaggatcca gcattgctgt gagctgtggt 2520 ataggttgca gactctgctc aggtcccatg ttgctgtgat tgtggtgtag gctgactgct 2580 gcagcttcaa tttgacccct agcccgggaa tttccatagg ccacacgtgc agcactaagg 2640 aaggaaaaaa agaaaaaaaaa aaaaaaagag tgggtgtgcc tatagtgaag aacagatgta 2700 aaagggaagt gaaagggatt cccccattct gagggattgt gagaagtgtg ccagaatatt 2760 aacttcattt gacttgttac agggaaagta aacttgactt tcacggacct cctagttacc 2820 tggtgcttac tatatgtctt ctcagagtac ctgattcatt cccagcctgg ttgacccatc 2880 cccctatctc tatggctatg tttatccaga gcacatctat ctaacactcc agctgatctt 2940 cctgacacag ctgtggcaac cctggatcct ttaaccaact gtgccaggct ggagatcaaa 3000 cctaagcctc tgcagcaacc caagctgctg cagtcagatt tttaaccccc tgtgccactg 3060 tgggtatctc cgatattttg tatcttctgt gactgagtgg tttgctgttt gcagggaacc 3120 agagtcagac actatccccg tgcaattcat catcctcgga cccatcaagc tctattattt 3180 cagaagaaaa tggtgttgcc tgcataggtg agaatcagtg accaacctat gaaaatgatc 3240 tcaatcctct gaaatgcatt ttattcatgt tttattcct ctttgcaggg agtggtcaac 3300 ttcgcctggt cgatggaggt ggtcgttgtg ctgggagagt agaggtctat catgagggct 3360 cctggggcac catctgtgat gacagctggg acctgaatga tgcccatgtg gtgtgcaaac 3420 agctgagctg tggatgggcc attaatgcca ctggttctgc tcattttggg gaaggaacag 3480 ggcccatttg gctggatgag ataaactgta atggaaaaga atctcatatt tggcaatgcc 3540 actcacatgg ttgggggcgg cacaattgca ggcataagga ggatgcagga gtcatctgct 3600 cgg 3603 <210> 111 <211> 4962 <212> DNA <213> Sus scrofa <400> 111 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcgtcactt tggagaagga agtggacaga tctgggctga 3180 agaattccag tgtgaggggc acgagtccca cctttcactc tgcccagtag caccccgccc 3240 tgacgggaca tgtagccaca gcagggacgt cggcgtagtc tgctcaagtg agacccaggg 3300 aatgtgttca ctttgttccc atgccatgaa gagggtaggg ttaggtagtc acagacatct 3360 ttttaaagcc ctgtctcctt ccaggataca cacaaatccg cttggtgaat ggcaagaccc 3420 catgtgaagg aagagtggag ctcaacattc ttgggtcctg ggggtccctc tgcaactctc 3480 actgggacat ggaagatgcc catgttttat gccagcagct taaatgtgga gttgcccttt 3540 ctatcccggg aggagcacct tttgggaaag gaagtgagca ggtctggagg cacatgtttc 3600 actgcactgg gactgagaag cacatgggag attgttccgt cactgctctg ggcgcatcac 3660 tctgttcttc agggcaagtg gcctctgtaa tctgctcagg taagagaata agggcagcca 3720 gtgatgagcc actcatgacg gtgccttaag agtgggtgta cctaggagtt cccattgtgg 3780 ctcagtggta acaaactcga ctggtatcca tgagggtatg ggtttgatcc ctggccttgc 3840 tcaatgggtt aaggatccag cattgctgtg agctgtggta taggttgcag actctgctca 3900 ggtcccatgt tgctgtgatt gtggtgtagg ctgactgctg cagcttcaat ttgaccccta 3960 gcccgggaat ttccataggc cacacgtgca gcactaagga aggaaaaaaaa gaaaaaaaaa 4020 aaaaaagagt gggtgtgcct atagtgaaga acagatgtaa aagggaagtg aaagggattc 4080 ccccattctg agggattgtg agaagtgtgc cagaatatta acttcatttg acttgttaca 4140 gggaaagtaa acttgacttt cacggacctc ctagttacct ggtgcttact atatgtcttc 4200 tcagagtacc tgattcattc ccagcctggt tgacccatcc ccctatctct atggctatgt 4260 ttatccagag cacatctatc taacactcca gctgatcttc ctgacacagc tgtggcaacc 4320 ctggatcctt taaccaactg tgccaggctg gagatcaaac ctaagcctct gcagcaaccc 4380 aagctgctgc agtcagattt ttaaccccct gtgccactgt gggtatctcc gatattttgt 4440 atcttctgtg actgagtggt ttgctgtttg cagggaacca gagtcagaca ctatccccgt 4500 gcaattcatc atcctcggac ccatcaagct ctattatttc agaagaaaat ggtgttgcct 4560 gcataggtga gaatcagtga ccaacctatg aaaatgatct caatcctctg aaatgcattt 4620 tattcatgtt ttatttcctc tttgcaggga gtggtcaact tcgcctggtc gatggaggtg 4680 gtcgttgtgc tgggagagta gaggtctatc atgagggctc ctggggcacc atctgtgatg 4740 acagctggga cctgaatgat gcccatgtgg tgtgcaaaca gctgagctgt ggatgggcca 4800 ttaatgccac tggttctgct cattttgggg aaggaacagg gcccatttgg ctggatgaga 4860 taaactgtaa tggaaaaagaa tctcatattt ggcaatgcca ctcacatggt tgggggcggc 4920 acaattgcag gcataaggag gatgcaggag tcatctgctc gg 4962 <210> 112 <211> 3603 <212> DNA <213> Sus scrofa <400> 112 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 3120 tgctgtgcag ggaactacag tgcgattcat catcctcgga cccatcaagc tctattattt 3180 cagaagaaaa tggtgttgcc tgcataggtg agaatcagtg accaacctat gaaaatgatc 3240 tcaatcctct gaaatgcatt ttattcatgt tttattcct ctttgcaggg agtggtcaac 3300 ttcgcctggt cgatggaggt ggtcgttgtg ctgggagagt agaggtctat catgagggct 3360 cctggggcac catctgtgat gacagctggg acctgaatga tgcccatgtg gtgtgcaaac 3420 agctgagctg tggatgggcc attaatgcca ctggttctgc tcattttggg gaaggaacag 3480 ggcccatttg gctggatgag ataaactgta atggaaaaga atctcatatt tggcaatgcc 3540 actcacatgg ttgggggcgg cacaattgca ggcataagga ggatgcagga gtcatctgct 3600 cgg 3603 <210> 113 <211> 3619 <212> DNA <213> Sus scrofa <400> 113 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac agcagtcatc tttaaaaggc 2460 atttgagaaa gtccaatttc aaatgcattt cctttcttta aaagataaat tgaagaaaat 2520 aagtctttat ttcccaagta aattgaattg cctctcagtc tgttaaaaga aactcttacc 2580 ttgatgatg cgctcttaac ctggcaaaga ttgtctttaa aatctgagct ccatgtcttc 2640 tgctttattt ctggtgtgcc tttgactcca gattacagta aatggaggac tgagtatagg 2700 gctaaaaagt agagagaatg gatgcatatt atctgtggtc tccaatgtga tgaatgaagt 2760 aggcaaatac tcaaaggaaa gagaaagcat gctccaagaa ttatgggttc cagaaggcaa 2820 agtcccagaa ttgtctccag ggaaggacag ggaggtctag aatcggctaa gcccactgta 2880 ggcagaaaaa ccaagaggca tgaatggctt ccctttctca cttttcactc tctggcttac 2940 tcctatcatg aaggaaaata ttggaatcat attctccctc accgaaatgc tatttttcag 3000 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 3060 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcagccagcg 3120 tgctttgcag ggaaccagag tcagacacta tccccgtgca attcatcatc ctcggacccca 3180 tcaagctcta ttatttcaga agaaaatggt gttgcctgca taggtgagaa tcagtgacca 3240 acctatgaaa atgatctcaa tcctctgaaa tgcattttat tcatgtttta tttcctcttt 3300 gcagggagtg gtcaacttcg cctggtcgat ggaggtggtc gttgtgctgg gagagtagag 3360 gtctatcatg agggctcctg gggcaccatc tgtgatgaca gctgggacct gaatgatgcc 3420 catgtggtgt gcaaacagct gagctgtgga tgggccatta atgccactgg ttctgctcat 3480 tttggggaag gaacagggcc catttggctg gatgagataa actgtaatgg aaaagaatct 3540 catatttggc aatgccactc acatggttgg gggcggcaca attgcaggca taaggaggat 3600 gcaggagtca tctgctcgg 3619 <210> 114 <211> 3270 <212> DNA <213> Sus scrofa <400> 114 tatagatgac aaggctttgt gtctgatagg ggccagcgaa ctcagtaaag agggaagatg 60 agaaagataa tggcaagaat ttatccctga agtgtagttt tgacaaacca gtcacaaaga 120 ggtctaagaa attttggtca caaagttgtt ttgaatccca ggcattttat ttgcaatgat 180 tgcatatgtt ctggaaagga catctgaacc taagaaatag ttcatttgca ttgtgttata 240 ttttactaag gtctgagaaa taatcttgag atgagaatga actctacttc ttcagagtct 300 ggaaggaata aattatgaaa atgtattaat gcttctttaa accatattgt atatttatct 360 attactaaac aaaaagaagt agctctattt atttatttat ttatttattt atttatgtct 420 tttgtctctt tagggccaca cctgtggcat atggaggttc ccaggctaga ggtccaattg 480 gagatgtagc agccagccta tgccagagcc accgcaacac gggatctgag ccacgtctgt 540 gacttacacc acagctcaca gcaacgcctg atcctcaacc cactgagcga ggccagggat 600 cgaacccatg tcctcatgga tgctagttgg gttcgttaac tgctgagcca tgatgggaac 660 tccaaattaa ttatttctta tatttgttct tcatatattc atttctatag aaagaaataa 720 atacagattc agttaatgat ggcaggtaaa agcttaactt attaatcaaa ggagttaatc 780 caggcacaaa aattcaattc atggctctct gttaaaattt aggtataggt ttagcaggaa 840 gaaaaggtta gtagatgcag actattacat ttagaatgga tggacaatga agtcctacta 900 tacagcacag ggaactatat ccaatctctt gggatagaat atgatggaag acaaaatcag 960 aacaagagag tatatatata tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1020 tgtgtgactg ggtcaccctg cggcacagca gaaattggca gaacattgta aatcaactat 1080 actttaatag gaaaaatact tttaagggct aaatttccaa tattctaacc atgtacacag 1140 agtaaatgtc ataaggatgc cagtctgtgt agagattgat gtgttactag cagattcatg 1200 aaataaaggc tgaggatgta gtccccaagt cacttctgag tggaagaatt tctcctttgt 1260 cctggactca aatattttag gataaaggaa aaaagaagat atttatagaa gggacttgtt 1320 ttcaagtact tgacaaaatt tcaccattaa agagaaattt gtggggagttc ccatcgtggc 1380 tcagtggaaa caaatccaac taggaaccat gaggttgtgg gtttgatccc tggcctcact 1440 cagtgggtta aggatccggt gttgccgtga gctgtggtgt aggttgcaga cacggttctg 1500 atcctgcgtt gctgtggctg tggctgtggt gtaggccagc agcaaacagc tctgattaga 1560 cccctagcct ggaaacctcc atatgccaca ggtgcagccc taaaaagaca aaaaaagaga 1620 aaagacaaaa aaaaaaaaaaa aaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa gaacccccag 1680 aggtattat ttgtttttgc cttttttcac tgactgttct ttgtttgttt gtttgagact 1740 gatctagaag actagagatt acaagaaata tggatttggc tcactctaag aaactgcttt 1800 cattccaagg tttgggtcta tccaaaagtg gaatagaatc atatgaatac tagtttatga 1860 gtatttagtg agaggaattt caagctcaaa taatgattca gcaagattaa attaaggagg 1920 gaattttcct tgtggctgag tgggttaagg acccaatgtt gtctctgtga ggatgtaggt 1980 tccatcctgg gctttgctca ttaggttaag gatctggcat tgctgcagct cagacccagt 2040 gctgccctgg ttgtggctta ggccaaagct gcagctccaa ttcaatctct ggcctgggaa 2100 cctccatgtg ctacaaggtg cggccttaaa aggaaaaaaaa aaaaattaaa tcaaggactc 2160 aagagtcttt cattatttgt gttgtggaag ctatatttgt tttaaagtct tagttgtgtt 2220 tagaaagcaa gatgttcttc aactcaaatt tgggagggaa cttgtttcat acatttttaa 2280 tggataagtg gcaaaatttt catgctgagg tgatctatag tgttgtaatg cagaatatag 2340 tcagatcttg aacattttag gaagttggtg agggccaatt gtgtatctgt gccatgctga 2400 taagaatgtc aagggatcac aagaattcgt gttattgac ttgttacagg gaaagtaaac 2460 ttgactttca cggacctcct agttacctgg tgcttactat atgtcttctc agagtacctg 2520 attcattccc agcctggttg acccatcccc ctatctctat ggctatgttt atccagagca 2580 catctatcta acactccagc tgatcttcct gacacagctg tggcaaccct ggatccttta 2640 accaactgtg ccaggctgga gatcaaacct aagcctctgc agcaacccaa gctgctgcag 2700 tcagattttt aaccccctgt gccactgtgg gtatctccga tattttgtat cttctgtgac 2760 tgagtggttt gctgtttgca gggaaccaga gtcagacact atccccgtgc aattcatcat 2820 cctcggaccc atcaagctct attatttcag aagaaaatgg tgttgcctgc ataggtgaga 2880 atcagtgacc aacctatgaa aatgatctca atcctctgaa atgcatttta ttcatgtttt 2940 atttcctctt tgcagggagt ggtcaacttc gcctggtcga tggaggtggt cgttgtgctg 3000 ggagagtaga ggtctatcat gagggctcct ggggcaccat ctgtgatgac agctgggacc 3060 tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg atgggccatt aatgccactg 3120 gttctgctca ttttggggaa ggaacagggc ccatttggct ggatgagata aactgtaatg 3180 gaaaagaatc tcatatttgg caatgccact cacatggttg ggggcggcac aattgcaggc 3240 ataaggagga tgcaggagtc atctgctcgg 3270 <210> 115 <211> 7 <212> DNA <213> Sus scrofa <400> 115 tactact 7 <210> 116 <211> 12 <212> DNA <213> Sus scrofa <400> 116 tgtggagaat tc 12 <210> 117 <211> 11 <212> DNA <213> Sus scrofa <400> 117 agccagcgtg c 11

Claims (12)

A porcine animal or its progeny or cell comprising at least one modified chromosomal sequence in the gene encoding the CD163 protein, wherein said modification
A variant involving an in-frame deletion in exon 7 of the gene encoding the CD163 protein;
Variation in exon 8 of the gene encoding the CD163 protein,
Modifications in exon 7 or exon 8 and the adjacent intron of the gene encoding the CD163 protein, and
selected from the group consisting of combinations thereof, wherein the modification
An 11 base pair deletion from nucleotide 3,337 to nucleotide 3,347 compared to the reference sequence SEQ ID NO: 47;
A 2 base pair insertion between nucleotides 3,149 and 3,150 and a 377 base pair deletion between nucleotides 2,573 and 2,949 compared to the reference sequence SEQ ID NO: 47 on the same allele;
A 129 base pair deletion from nucleotide 3,044 to nucleotide 3,172 and a 1930 base pair deletion from nucleotide 488 to nucleotide 2,417 compared to the reference sequence SEQ ID NO: 47 on the same allele, wherein the deleted sequence includes 12 bases starting at nucleotide 488. Replaced by pair insertion;
A 1467 base pair deletion from nucleotides 2,431 to nucleotides 3,897 compared to the reference sequence SEQ ID NO:47;
A 1280 base pair deletion from nucleotides 2,818 to nucleotides 4,097 compared to the reference sequence SEQ ID NO:47;
and combinations thereof,
The modification is relative to the susceptibility to infection by porcine reproductive and respiratory syndrome virus (PRRSV) of porcine animals or their progeny or cells that do not contain the altered chromosomal sequence in the gene encoding the CD163 protein. Reducing the sensitivity of porcine animals or their progeny or cells,
Pig animal or its progeny or cell. 2. The porcine animal or progeny or cell of claim 1, wherein the modification results in production of a substantially non-functional CD163 protein by the porcine animal, progeny, or cell. 2. The method of claim 1, wherein the porcine animal or progeny is an embryo, a juvenile animal, or an adult, or the cells comprise embryonic cells, cells derived from a juvenile porcine animal, or cells derived from an adult porcine animal. , porcine animals or their progeny or cells. The cell of claim 1, wherein the cell is selected from sperm cells, egg cells and somatic cells. The cell according to claim 4, wherein the egg cell is a fertilized egg. The cell according to claim 4, wherein the somatic cell is a fibroblast. The cell of claim 6, wherein the fibroblast is a fetal fibroblast. 2. The porcine animal or its progeny or cell according to claim 1, which is heterozygous for the modified chromosomal sequence of the gene encoding the CD163 protein. 2. The porcine animal or its progeny or cell according to claim 1, which is homozygous for the modified chromosomal sequence of the gene encoding the CD163 protein. delete delete delete