Porcine genital respiratory syndrome virus-resistant animals

Abstract

To provide pigs that exhibit resistance to porcine reproductive respiratory syndrome virus (PRRSV). [Solution] This disclosure relates to a genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated and / or at least one allele of the CD163 gene is inactivated. A genetically modified pig in which both alleles of the SIGLEC-1 gene and / or both alleles of the CD163 gene are inactivated is resistant to porcine reproductive respiratory syndrome virus (PRRSV). Methods for producing such transgenic pigs are also provided.

Description

Technical Field

[0001] Government Support This invention was made with government support under contract No. (USDA / ARS) 58 - 1940 - 8 - 868 awarded by the United States Department of Agriculture. The government has certain rights in this invention.

[0002] Sequence Listing This application includes a sequence listing entitled "UMO 11053, WO SEQ_ST25" created on May 15, 2012, which is hereby incorporated by reference in its entirety.

[0003] The present invention generally relates to genetically modified pigs in which at least one allele of the SIGLEC - 1 gene is inactivated and / or at least one allele of the CD163 gene is inactivated. A method for producing such transgenic pigs is also provided.

Background Art

[0004] Porcine genitourinary respiratory syndrome (PRRS) is one of the most economically important diseases in pigs. The disease was first detected in the United States in 1987 (Keffaber 1989) and in Europe in 1990 (Wensvoort et al. 1991). Molecular analysis of prototype PRRS virus (PRRSV) VR-2332 and Lelistad (US and European isolates, respectively) suggested that a wide range of evolved strains emerged almost simultaneously on the two continents, likely due to similar changes in pig management practices (Murtaugh et al. 1995, Nelsen et al. 1999). Since its initial appearance, the virus has spread worldwide, and the European genotype of PRRSV has been detected in US pig herds (Ropp et al. 2004). PRRS is characterized by severe, sometimes fatal, respiratory disease and reproductive failure, but it also makes infected pigs more susceptible to bacterial and other viral pathogens (Benfield et al. 1992) and is a significant component of economically significant porcine respiratory complex disease (PRDC). The most consistent pathological lesions caused by PRRSV during acute infection are interstitial pneumonia and mild lymphocytic encephalitis (Plagemann 1996). After the acute phase of PRRSV infection, typically characterized by viremia and clinical disease, many pigs recover completely but still have low levels of viral infection for a long period. These “carrier” pigs remain persistently infected with PRRSV and shed the virus either intermittently or continuously, and can infect naive pigs after direct or indirect contact. Under experimental conditions, persistent infection by PRRSV has been well documented (Albina et al. 1994, Allende et al. 2000, Benfield et al. 1998, Christopherhennings et al. 1995, Sur et al. 1996, Yoon et al. 1993). Most notably, infectious virus has been recovered up to 157 days after infection (Wills et al. 1997).While tissue macrophages and monocytes are primary target cells during both acute and persistent infections (Molitor et al. 1997), lung cells and testicular epithelial germ cells have also been shown to be infected (Sur et al. 1996, Sur et al. 1997).

[0005] PRRS is an enveloped positive-sense RNA virus belonging to the family Arteriviridae and order Nidovirales. Other members of the Arterivirus family include mouse lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV), and monkey hemorrhagic fever virus (SHFV). Analysis of genome sequence data reveals not only extensive diversity among PRRSV strains but also well-conserved domains (Andreyev et al. 1997, Meng 2000, Meng et al. 1995). The genomic structure of PRRSV is similar to that of other Arteriviruses, and its genomic RNA functions as messenger RNA for the ORF1a replicase protein (Plagemann 1996). ORF1a and 1b constitute approximately 80% of the viral genome and encode polyproteins that are processed into RNA-dependent RNA polymerase and other non-structural proteins (Snijder and Meulenberg 1998). Using Relistat virus, ORF2-7 were determined to encode viral structural proteins. The proteins encoded by ORF5 (GP5) and M (Van Breedam et al. 2010b) may play a role in the induction of apoptosis by PRRSV (Suarez et al. 1996, Sur et al. 1997) and are thought to be viral adhesion proteins in closely related lactate dehydrogenase-elevated viruses. The trace envelope glycoproteins GP2a and GP4 of PRRSV interact with CD163 (Das et al. 2010). There is data suggesting that binding to SIGLEC-1 (sialic acid-bound Ig-like lectin 1) is necessary for cell entry, and in fact, double binding to both SIGLEC-1 and CD163 appears to be necessary for viral infection (Van Gorp et al. 2008).

[0006] Many features of PRRSV pathogenesis (particularly at the molecular level) and zoonotic epidemiology are not well understood, thus hindering control efforts. To gain a better understanding, infectious clones of PRRSV have been developed (Nielsen et al. 2003). Today, the procedure often involves vaccinating pigs against PRRSV with modified live attenuated strains or dead virus vaccines. However, current vaccines often fail to provide satisfactory protection due to both strain mutations and inadequate stimulation of the immune system. Protective immune responses are possible, as previous exposure has been shown to provide complete protection when pigs are challenged with homologous strains of PRRSV (Lager et al. 1999). However, protective immunity has not been consistently demonstrated with challenge using heterologous strains. In addition to concerns regarding the efficacy of available PRRSV vaccines, the modified live vaccines currently in use can persist in individual pigs and swine herds and accumulate mutations (Mengeling et al. 1999), as demonstrated using viral field isolates after experimental infection of pigs (Rowland et al. 1999). Furthermore, the vaccine virus has been shown to be excreted in the semen of vaccinated mature males (Christopherhennings et al. 1997). As an alternative to vaccination, several experts have proposed a "test and elimination" strategy in herd rearing (Dee and Molitor 1998). The successful use of this strategy depends on the elimination of all pigs infected with PRRSV, either acutely or persistently, followed by strict control to prevent reintroduction of the virus. The difficulties and high costs associated with this strategy are that little is known about the pathogenesis of persistent PRRSV infection, and therefore there is no reliable technique for identifying persistently infected pigs.

[0007] The putative cellular receptor for PRRSV was identified, purified, and sequenced using monoclonal antibodies (Vanderheijden et al. 2003, Wissink et al. 2003), and named SIGLEC-1. This molecule is similar to sialoadhesin and has been shown to mediate the entry of PRRSV into insensitive cells; however, recombinant cell lines expressing this receptor could not support productive replication of PRRSV (Vanderheijden et al. 2003). Importantly, the sialic acid molecule present on the surface of PRRSV was shown to be required for infection of alveolar macrophages. Following viral binding to this receptor, PRRSV entry occurs via receptor-mediated endocytosis (Nauwynck et al. 1999). The crucial role of sialoadhesin in PRRSV entry was established by experiments demonstrating viral uptake in PK15 cells transfected with cloned porcine sialoadhesin (a cell line intolerant to PRRSV replication), but not in untransfected control PK15 cells (Vanderheijden et al. 2003). Further research by the same group demonstrated that the interaction between sialic acid on the surface of PRRSV virions and sialoadhesin molecules is essential for PRRSV infection of alveolar macrophages (Delputte and Nauwynck 2004). The reverse strategy, namely removal of sialic acid from the surface of PRRSV or pre-incubation with sialic acid-specific lectins, also resulted in the blockade of infection (Delputte et al. 2004, Delputte and Nauwynck 2004, Van Breedam et al. 2010b). Independent of specific studies on PRRSV entry, we used site-directed mutagenesis to identify six key amino acid residues required for sialic acid binding by mouse sialoadhesin (Vinson et al. 1996), which are 69% identical to those of porcine sialoadhesin. Importantly, all six amino acids identified in mouse sialoadhesin are also conserved in the porcine molecule.

[0008] SIGLEC-1 is a transmembrane receptor belonging to the family of sialic acid-binding immunoglobulin-like lectins. It was first described as a sheep erythrocyte-binding receptor in mouse macrophages (Crocker and Gordon 1986). It is expressed on macrophages in hematopoietic and lymphoid tissues. SIGLEC consists of an N-terminal V-set domain containing the sialic acid-binding site, followed by a number of C2-set domains, then a transmembrane domain and a cytoplasmic end. In contrast to other SIGLECs, SIGLEC-1 lacks a tyrosine-based motif at its cytoplasmic end (Oetke et al. 2006). The sialic acid-binding site was located in the N-terminal immunoglobulin-like V-set domain (Nath et al. 1995). The R116 residue appears to be one of the amino acids important for sialic acid binding (Crocker et al. 1999, Delputte et al. 2007). Therefore, the intact N-terminal domain is necessary and sufficient for PRRSV binding to SIGLEC-1 (Van Breedam et al. 2010a). SIGLEC-1 knockout in mice has been reported, resulting in viable and reproductive mice with no developmental abnormalities (Oetke et al. 2006). However, these mice showed slight changes in the B cell and T cell populations, as well as reduced immunoglobulin M levels.

[0009] The PRRSV infection process begins with the initial binding of heparan sulfate to the surface of alveolar macrophages. Subsequently, secure binding to sialoadhesin occurs (also known as SIGLEC-1, CD169, or SN). The virus then moves internally via clathrin-mediated endocytosis. Another molecule, CD163, then facilitates the decoating of the virus in endosomes (Van Breedam et al. 2010a). The viral genome is released and the cell becomes infected.

[0010] CD163 has 17 exons, and the protein consists of an extracellular region with nine scavenger receptor cysteine-rich (SPCR) domains, a transmembrane segment, and a short cytoplasmic end. Several different variants are obtained by different splicing of a single gene (Ritter et al. 1999a, Ritter et al. 1999b). Many of these variations are explained by the length of the cytoplasmic end.

[0011] CD163 has many important functions, including acting as a haptoglobin-hemoglobin scavenger receptor. Because the heme group can be highly toxic, the removal of free hemoglobin from the blood is a key function of CD163 (Kristiansen et al. 2001). CD163 has a cytoplasmic end that facilitates endocytosis. Mutations at this end reduce the uptake of the haptoglobin-hemoglobin complex (Nielsen et al. 2006). Other functions of CD163 include erythroblast adhesion (SRCR2), being a TWEAK receptor (SRCR1-4 and 6-9), a bacterial receptor (SRCR5), an African swine virus receptor (Sanchez-Torres et al. 2003), and a potential role as an immunomodulator (discussed in Van Gorp et al. 2010a). [Overview of the project] [Means for solving the problem]

[0012] In one embodiment, the present invention relates to a genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated and / or at least one allele of the CD163 gene is inactivated, wherein the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

[0013] Another aspect of the present invention is a genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated, produced by a method comprising enucleating a porcine oocyte, fusing the oocyte with a donor porcine fibroblast (the genome of which fibroblast contains at least one inactivated SIGLEC-1 allele), and activating the oocyte to produce an embryo.

[0014] The present invention also relates to a genetically modified pig, produced by a method comprising enucleating a porcine oocyte, fusing the oocyte with a donor porcine fibroblast (the genome of which contains at least one inactivated CD163 allele), and activating the oocyte to produce an embryo, wherein at least one allele of the CD163 gene is inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

[0015] In another embodiment, the present invention relates to a genetically modified pig in which both alleles of the SIGLEC-1 gene are inactivated, produced by a method comprising crossing a genetically modified male pig in which at least one allele of the SIGLEC-1 gene is inactivated with a genetically modified female pig in which at least one allele of the SIGLEC-1 gene is inactivated to produce F1 offspring, and then screening the F1 offspring to identify a genetically modified pig in which both alleles of the SIGLEC-1 gene are inactivated.

[0016] Another aspect of the present invention provides genetically modified pigs in which both alleles of the CD163 gene are inactivated, produced by a method comprising crossing a genetically modified male pig in which at least one allele of the CD163 gene is inactivated with a genetically modified female pig in which at least one allele of the CD163 gene is inactivated to produce F1 offspring, and then screening the F1 offspring to identify genetically modified pigs in which both alleles of the CD163 gene are inactivated.

[0017] The present invention also relates to genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat the porcine genital respiratory syndrome virus (PRRSV), which is produced by one of three methods. The first such method involves crossing a genetically modified pig having at least one inactivated SIGLEC-1 allele with a genetically modified pig having at least one inactivated CD163 allele to produce F1 offspring, and then screening the F1 offspring to identify genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated. This method further includes crossing genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated with each other to produce F2 offspring, and then screening the F2 offspring to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

[0018] A second such method involves crossing genetically modified pigs in which both alleles of the SIGLEC-1 gene are inactivated with genetically modified pigs in which both alleles of the CD163 gene are inactivated to produce F1 offspring, crossing the F1 offspring to produce F2 offspring, and screening the F2 offspring to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

[0019] A third such method involves crossing a genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated with another genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated to produce F1 offspring, and then screening the F1 offspring to identify a genetically modified pig in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

[0020] The present invention also relates to offspring of genetically modified pigs in any of the above-mentioned ways, where (1) at least one allele of the SIGLEC-1 gene is inactivated, (2) at least one allele of the CD163 gene is inactivated, (3) at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated, or (4) both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated. In such offspring in which one or both alleles of the CD163 gene are inactivated, the inactivation results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

[0021] The present invention also relates to a method for producing genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated. The method comprises enucleating a porcine oocyte, fusing the oocyte with a donor porcine fibroblast (the genome of which contains at least one inactivated SIGLEC-1 allele), and activating the oocyte to produce an embryo.

[0022] In yet another embodiment, the present invention relates to a method for producing genetically modified pigs in which at least one allele of the CD163 gene is inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV). The method comprises enucleating a porcine oocyte, fusing the oocyte with a donor porcine fibroblast (the genome of which contains at least one inactivated CD163 allele), and activating the oocyte to produce an embryo.

[0023] The present invention also relates to a method for producing genetically modified pigs in which both alleles of the SIGLEC-1 gene are inactivated. The method comprises crossing a female genetically modified pig having at least one inactivated SIGLEC-1 allele with a male genetically modified pig having at least one inactivated SIGLEC-1 allele to produce F1 offspring, and then screening the F1 offspring to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene are inactivated.

[0024] The present invention also relates to a method for producing a genetically modified pig in which both alleles of the CD163 gene are inactivated, and inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or uncoat porcine reproductive and respiratory syndrome virus (PRRSV). This method involves mating a genetically modified female pig having at least one inactivated CD163 allele with a genetically modified male pig having at least one inactivated CD163 allele to produce F1 progeny, and screening the F1 progeny to identify a genetically modified pig in which both alleles of the CD163 gene are inactivated.

[0025] Another aspect of the present invention is a method for producing a genetically modified pig in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or uncoat porcine reproductive and respiratory syndrome virus (PRRSV). This method involves mating a genetically modified pig having at least one inactivated SIGLEC-1 allele with a genetically modified pig having at least one inactivated CD163 allele to produce F1 progeny, and screening the F1 progeny to identify a genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated. This method further involves mating genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated with each other to produce F2 progeny, and screening the F2 progeny to identify a genetically modified pig in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

[0026] The present invention also relates to another method for producing a genetically modified pig in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and inactivation of the CD163 gene results in a CD163 protein that cannot bind and / or uncoat porcine reproductive and respiratory syndrome virus (PRRSV). This method involves mating a genetically modified pig in which both alleles of the SIGLEC-1 gene are inactivated with a genetically modified pig in which both alleles of the CD163 gene are inactivated to produce F1 progeny, mating the F1 progeny to produce F2 progeny, and screening the F2 progeny to identify a genetically modified pig in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

[0027] The present invention also relates to yet another method for producing a genetically modified pig in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and inactivation of the CD163 gene results in a CD163 protein that cannot bind and / or uncoat porcine reproductive and respiratory syndrome virus (PRRSV). This method involves mating a genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated with another genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated to produce F1 progeny, and screening the F1 progeny to identify a genetically modified pig in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

[0028] In other embodiments, the present invention relates to genetically modified pig offspring produced by any of the above methods, wherein one or both alleles of the SIGLEC-1 gene are inactivated, and / or one or both alleles of the CD163 gene are inactivated, and the inactivation of CD163 results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

[0029] Other purposes and features are, in part, self-evident, and in part, will be noted hereafter in this specification. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows the structure of the sialoadhesin gene and the design of the targeting vector. Figures 1A and 1B illustrate that the human (Figure 1A) and mouse (Figure 1B) sialoadhesin genes each consist of 21 exons and are approximately 20 kb in length. Figure 1C shows the mouse mutation analysis of exon 2 (the DNA sequence of exon 2 as shown in Figure 1C is SEQ ID NO: 7, and the amino acid sequence encoded by exon 2 as shown in Figure 1C is SEQ ID NO: 8). Mutation analysis identified six amino acids that contributed to the binding of sialoadhesin to the ligand (shown in squares / bold text). Figure 1D shows the targeting vector design used to substitute a portion of exon 1, as well as exons 2 and 3 of SIGLEC-1, using stop codons. The vector also contained a neomycin (neo) selector cassette driven by a PGK promoter. [Figure 2] Figure 2 shows the targeting vector design, the structure of the sialoadhesin gene, and the structure of the altered sialoadhesin gene. [Figure 3] Figure 3 is a photograph of a gel showing PCR screening to identify targets for the sialoadhesin gene. [Figure 4]Figure 4 is a photograph of the gel showing PCR screening to identify the equivalent presence of both wild-type and targeted sialoadhesin alleles. [Figure 5] Figure 5 shows the structural domain configuration of wild-type CD163 (left), which includes nine extracellular SRCR domains, two proline, serine, and threonine (PST) rich domains, a transmembrane region, and an intracellular cytoplasmic terminal. Genetically modified CD163 is shown on the right. The structural domain configuration remains the same except that SRCR domain 5 is replaced with SRCR domain 8 of the CD163 ligand (CD163L). [Figure 6] Figure 6 shows a CD163-targeted vector, where the vector arms are DNA segments with the same sequence as native or wild-type CD163, thus the vector can anneal to CD163 already present in the cell. The modified DNA lying between the two arms of CD163 can then be inserted into the cell's DNA by homologous recombination. [Modes for carrying out the invention]

[0031] definition A "knockout pig" is a genetically modified pig in which the function of one or both alleles of a gene is altered, for example, by partial or complete deletion of the gene. If one allele of a gene is knocked out, the pig is heterozygous for that gene knockout; if both alleles are knocked out, the pig is homozygous for the gene knockout.

[0032] The term “donor cells” refers to cells from which nuclear or chromatin material has been obtained for use in nuclear transplantation. As discussed elsewhere in this specification, nuclear transplantation may include transplantation of nuclear or chromatin only, such as isolated from donor cells, or transplantation of whole donor cells containing such nuclear or chromatin material.

[0033] The term "genetic modification" refers to one or more changes in a gene sequence (including coding and non-coding sequences, such as introns, promoter sequences, and 5' and 3' untranslated sequences) that alter gene expression or activity. Examples of such modifications include insertions (e.g., heterologous sequences, e.g., selection markers and / or termination signals), deletions, frameshift mutations, nonsense mutations, missense mutations, point mutations, or combinations thereof.

[0034] The term "recipient cells" refers to donor cells, donor cell nuclei, or cells into which donor cell chromatin has been introduced. Recipient cells are properly enucleated before transplantation into the nucleus. Examples of recipient cells include oocytes, zygotes, and two-cell stage embryos.

[0035] Small interfering RNAs (siRNAs) are double-stranded RNA molecules that have the ability to specifically interfere with protein expression. siRNAs are typically about 10 to 30 nucleotides long. The length of an siRNA molecule is based on the length of its antisense strand.

[0036] Description of Preferred Embodiments This invention relates to genetically modified pigs that are resistant to infection with porcine respiratory-genital syndrome virus (PRRSV). Infectivity to PRRSV arises from three specific entry mediators: (1) initial binding with heparan sulfate, (2) binding / internal translocation by sialoadhesin (SIGLEC-1), and (3) internal translocation / decoating of the virus by CD163. Therefore, by defending against the interaction between PRRSV and SIGLEC-1 and / or PRRSV and CD163, PRRSV is unable to establish infection in the host. Thus, this invention relates to genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated and / or at least one allele of the CD163 gene is inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat porcine respiratory-genital syndrome virus (PRRSV). These pigs may also be called pig knockouts for SIGLEC-1 and / or CD163.

[0037] The present invention includes pigs in which one allele of a targeted gene (SIGLEC-1 and / or CD163) is inactivated, while the other allele remains unaffected. These animals are referred to herein as “heterozygous” or “hemizygous” animals, but they can be used in breeding approaches to generate homozygous mutants. The present invention also includes homozygous mutant pigs in which both alleles of the targeted gene are inactivated by either the same or different approaches. Accordingly, the present invention includes genetically modified pigs in which (1) one allele of the SIGLEC-1 gene is inactivated, (2) one allele of the CD163 gene is inactivated, (3) both alleles of the SIGLEC-1 gene are inactivated, (4) both alleles of the CD163 gene are inactivated, (5) both alleles of the SIGLEC-1 gene and one allele of the CD163 gene are inactivated, (6) one allele of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, (7) one allele of the SIGLEC-1 gene and one allele of the CD163 gene are inactivated, or (8) both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated. In each of these cases, and generally as described in this application, inactivation of the CD163 allele(s) results in a CD163 protein that cannot bind to and / or decoat porcine genital respiratory syndrome virus (PRRSV).

[0038] Gene targeting performed to produce the animals of the present invention can result in the inactivation of a target gene by disruption, removal, modification, or substitution of the target gene sequence. Methods for gene inactivation are well known in the art. For example, a target gene can be inactivated by insertion of a heterologous sequence (e.g., a selection marker and / or stop codon) into the target gene, deletion of part or all of the gene, modification of the gene (e.g., by frameshift mutation, nonsense mutation, missense mutation, point mutation, substitution of part or all of the gene with another nucleic acid sequence), or any combination of the above.

[0039] The insertion sequence can replace or append to a previously existing sequence in the gene, depending on the design of the targeted construct. The design of the targeted construct can be varied depending on whether it is desirable to completely knock out the gene's function or to maintain a somewhat reduced level of function. In the case of SIGLEC-1, complete knockout of function is desirable. As an example, and not limiting, the SIGLEC-1 gene can be knocked out by deletion of part of exon 1 and all of exons 2 and 3, for example, by replacing part of exon 1 and all of exons 2 and 3 using a neomycin selective cassette. In some embodiments, the modification may also involve adding a LoxP site to either side of the sequence of the SIGLEC-1 gene to be mutated. In some cases, the targeted construct may include both a loxP site adjacent to the sequence to be inserted and CRE recombinase. In other cases, a two-vector system can be used, where the targeted construct includes a loxP site adjacent to the sequence to be inserted, and the second vector includes a transgene encoding CRE recombinase. The transgene for CRE recombinase can be placed under the influence of a tissue-specific promoter, and therefore, its expression will render the SIGLEC-1 gene non-functional in specific cell lineages. Similarly, an antibiotic selection cassette can be placed adjacent to the loxP site, thus allowing deletion at a later stage using Cre recombinase.

[0040] In the case of CD163, it is desirable to inactivate only its function related to PRRSV binding and / or decoating, with minimal or no impact on other functions of CD163. While we do not want to be bound to any particular theory, complete knockout of CD163 is thought to be potentially unviable or severely vulnerable to infection due to CD163's role in hemoglobin-haptoglobin complex binding and internal translocation. Therefore, CD163 can be inactivated by disrupting the fifth N-terminal scavenger receptor cysteine-rich (SRCR) domain of CD163, which has been previously shown to play a major role in PRRSV infection (Van Gorp et al. 2010), while leaving other domains unaffected. SRCR domain 5 can be genetically modified, for example, by introducing point mutations that alter the structure of this domain, or by replacing this domain with another domain. For example, SRCR domain 5 can be replaced with SRCR domain 8 derived from the CD163 ligand (CD163L). This is because this domain "exchange" has been shown to reduce relative PRRSV infectivity to 0% in cultured cells.

[0041] In other approaches, the coding sequence of the target gene is either left unchanged or minimally altered, and instead, a sequence that affects the target gene's sequence, such as the promoter sequence, is targeted. In either case, selective marker insertion is often desirable to facilitate the identification of cells in which targeting has occurred. If desired, such markers or other insertion sequences can later be removed, for example, by Cre-Lox or a similar system.

[0042] Targeted gene modification uses a nucleic acid construct that has homology to a target gene (e.g., SIGLEC-1 or CD163) or a region adjacent to the target gene, and thus, the integration of the construct into the genome alters gene expression either by altering the gene sequence and / or by altering the gene expression level. Therefore, in order to alter a gene, a targeted construct is generally designed to contain three main regions: (i) a first region homologous to the locus to be targeted (e.g., the SIGLEC-1 or CD163 gene, or its adjacent sequence); (ii) a second region which is a heterologous polynucleotide sequence (e.g., encoding a selection marker, e.g., an antibiotic resistance protein) that specifically replaces a portion of the targeted locus or is inserted into the target locus; and (iii) a third region which, like the first region, is homologous to the target locus but is typically not contiguous with the first region of the construct. Homologous recombination between the target construct and the target wild-type locus results in deletion of any locus sequence between two homologous regions shown in the target vector, and replacement of that sequence with a heterologous sequence (e.g., a heterologous sequence encoding a selection marker), or insertion of a heterologous sequence into that sequence. Exemplary constructs and vectors for carrying out such targeted modification are described in Example 1; however, other vectors that can be used in such an approach are known in the art and can be readily adapted for use in the present invention.

[0043] To facilitate homologous recombination, the first and third regions of the targeting vector (see above) contain sequences that exhibit substantial sequence identity with respect to the gene (or adjacent region) to be targeted. For example, the first and third regions of the targeting vector may contain sequences that are at least approximately 80%, at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or approximately 100% identical to the target gene or adjacent region. Sequence identity is typically measured using BLAST® (Basic Local Alignment Search Tool) or BLAST® 2 with the default parameters specified therein (Altschul et al, J.Mol.Biol.215:403-410, 1990; Tatiana et al, FEMS Microbiol.Lett.174:247-250, 1999). Therefore, homologous recombination can be facilitated by using sequences having at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or even about 100% sequence identity with respect to the target gene locus in the present invention.

[0044] The total size of the two homology regions (i.e., the first and third regions described above) could be, for example, about 2 kilobases (kb) to about 25 kb (e.g., about 4 kb to about 20 kb, about 5 kb to about 15 kb, or about 6 kb to about 10 kb). The size of the region that replaces part of the target locus or inserts into the target locus (the second region described above) could be, for example, about 0.5 kb to about 5 kb (e.g., about 1 kb to about 4 kb or about 3 to about 4 kb).

[0045] Various types of targeted construct delivery methods can be used. Targeted constructs can be delivered using cell transfection methods, including calcium phosphate, lipofection, electroporation, and nuclear injection. If the gene is transcriptionally active in the cell type used, a promoter-free selective marker strategy can then be used, so that antibiotic resistance is observed only in cells that have undergone a recombination event at the transcriptional unit level. Alternatively, if the gene is transcriptionally inactive in the cell type used, promoters that are inducible, tissue-specific, or contain insulators such as matrix-binding regions (MARs) can be used.

[0046] Alternatively, SIGLEC-1 transcripts can be "silenced" using siRNA technology. Antisense technology is well known in the art. Briefly, it uses a nucleotide sequence that typically contains about 19 to about 29 nucleotides complementary to the sense mRNA sequence of SIGLEC-1. The degree of complementarity is generally in the range of about 70% to about 100%. Preferably, the complementarity is greater than about 80%, more preferably greater than about 90%, and even more preferably greater than about 95%. Regions of SIGLEC-1 mRNA suitable for targeting with siRNA can be easily determined by comparing the efficacy of several antisense sequences designed to be complementary to various regions of SIGLEC-1 mRNA to prevent the production of SIGLEC-1 protein. Such experiments can be easily carried out without excessive experimentation using any of the techniques known in the art.

[0047] Vectors used for siRNA expression are well known in the art. A vector can be any circular or linear DNA, either integrated into the host genome or maintained in episomal form. Generally, an siRNA expression cassette can be ligated to a DNA delivery vector, such as a plasmid, or to a lentivirus, adenovirus, alphavirus, retrovirus, or other viral vector. Exemplary mammalian viral vector systems include adenovirus vectors, adeno-associated virus type 1 ("AAV-1") or adeno-associated virus type 2 ("AAV-2") vectors, hepatitis delta vectors, bio-attenuated deltavirus, herpesvirus vectors, alphavirus vectors, or retrovirus vectors (including lentivirus vectors).

[0048] Transformation of mammalian cells can be carried out according to standard techniques known in the art. Any well-known procedure for introducing an exogenous nucleotide sequence into a host cell can be used, insofar as at least the siRNA construct is successfully introduced into the host cell. These procedures include viral transduction (e.g., by retroviral infection, using any of the viral vectors enumerated in the previous paragraph, possibly in the presence of polybrenes to enhance infection efficiency), calcium phosphate transfection, electroporation, biolytic particle delivery systems (i.e., gene guns), liposomes, microinjection, and the use of any of the other known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other exogenous genetic material into a host cell. In the present invention, siRNA directed to SIGLEC-1 is introduced into donor pig fibroblasts, which are subsequently used to produce the transgenically modified pigs of the present invention.

[0049] The transgenic animals of the present invention can be produced using the following general somatic cell nuclear transfer procedure. Briefly, donor cells are created by genetically modifying the genome of porcine somatic cells (e.g., fetal fibroblasts) by gene targeting as described above. The nuclei of such genetically modified donor cells (or the whole donor cell containing the nucleus) are then transplanted into recipient cells, for example, enucleated oocytes. The donor cells can be fused with the enucleated oocytes, or the donor nuclei or the donor cells themselves can be injected into the recipient cells, or they can be injected into the perivitelline space adjacent to the oocyte membrane.

[0050] Therefore, nuclear transplantation can be performed once somatic cells with the target gene targeted (one or both alleles as described above) are obtained. In some cases, genetically modified donor cells can be cryopreserved before nuclear transplantation. Recipient cells that can be used include oocytes, fertilized zygotes, or 2-cell stage embryos, all of which may or may not be enucleated.

[0051] Recipient oocytes can be obtained using methods known in the art or purchased from commercial sources (e.g., BoMed Inc., Madison, Wis.). Oocytes can be obtained from "gilts" (sows) that have never produced offspring or "sows" (sows) that have previously produced offspring.

[0052] Therefore, genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated can be produced by enucleating porcine oocytes, fusing the oocytes with donor porcine fibroblasts (the genome of these fibroblasts contains at least one inactivated SIGLEC-1 allele), and activating the oocytes to produce embryos. Similarly, genetically modified pigs in which at least one allele of the CD163 gene is inactivated, resulting in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV) due to the inactivation of the CD163 allele, can be produced by enucleating porcine oocytes, fusing the oocytes with donor porcine fibroblasts (the genome of these fibroblasts contains at least one inactivated CD163 allele), and activating the oocytes to produce embryos. Both methods further include implanting an embryo into the reproductive system of a surrogate sow, the surrogate sow initiating estrus but not yet having completed ovulation, and the embryo becoming pregnant and giving birth at full term, thereby producing a genetically modified pig whose genome contains at least one inactivated SIGLEC-1 and / or CD163 allele. The present invention also relates to offspring of such a genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated and / or at least one allele of the CD163 gene is inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat porcine genital respiratory syndrome virus (PRRSV).

[0053] Methods for enucleating porcine oocytes are known in the art, and enucleation can be achieved by any of the standard methods. For example, enucleation of oocytes can be performed using a micropipette in a micromanagement medium.

[0054] The introduction of a membrane-bound nucleus from a donor pig cell into an enucleated recipient pig oocyte to form an oocyte containing the donor nucleus can be carried out by fusing the membrane of the membrane-bound nucleus from the donor mammalian cell with the membrane of the enucleated recipient mammalian oocyte to form an oocyte containing the nucleus from the donor mammalian cell. Alternatively, such introduction can be carried out by microinjecting a membrane-bound nucleus from a mammalian donor cell into an enucleated recipient mammalian oocyte to form an oocyte containing the nucleus from the donor mammalian cell. For example, a donor cell (or nucleus) can be introduced into the space beneath the zona pellucida or into the periuterine space of an enucleated recipient oocyte, followed by membrane fusion to produce an oocyte containing the donor nucleus in its cytoplasm. All means of introducing donor nuclear material into enucleated recipient mammalian oocytes that are known to those skilled in the art are useful in the methods disclosed herein.

[0055] For example, the fusion process can be carried out in a fusion medium. Alternatively, fusion can be promoted using an inactivated virus or a membrane fusion inducer, such as polyethylene glycol (PEG). For example, see Graham (1969) Wistar Inst.Symp.Monogr.9:19-33 and McGrath et al. (1983) Science220:1300-1302 for the use of viruses, Fisher et al. (1981) Tech.Cell.Physiol.1:1-36 for chemically induced cell fusion, and Berg (1982) Bioelectrochem.Bioenerg.9:223-228 and Robl et al. (1987) J.Anim.Sci.64:642-647 for electrically induced cell fusion.

[0056] U.S. Patent No. 6,211,429B1 (the contents of which are incorporated herein by reference) describes a method for the development of activated oocytes in vitro and in vivo. The terms “activated” or “activated” refer to the ability of unfertilized oocytes to develop at least to the pronuclear stage or beyond after treatment with oocyte modifiers and reducing agents. Generally speaking, the pronuclear stage is achieved about 3 to 7 hours after such treatment. The term “oocyte modifier” refers to an agent that can react with a substrate on or in an oocyte, such as a thiol (-SH) group (which may be a thiol group of a protein), and the effect of this reaction results in the activation of mammalian oocytes when the oocyte is subsequently treated with a reducing agent according to the method disclosed in U.S. Patent No. 6,211,429B1.

[0057] The combined use of a -SH or oocyte modifier (e.g., thimerosal) and a -SH reducing agent (e.g., dithiothreitol) can induce complete activation of mammalian oocytes. A short treatment with thimerosal, followed by treatment with a reducing agent such as DTT, combined with just one calcium permeabilizer, is particularly effective in achieving activation. Thimerosal is responsible for a series of calcium permeabilization processes in mammalian oocytes. 2+ Triggering a spike followed by incubation with a reducing agent such as DTT can stimulate pronuclear formation. Therefore, after washing away thimerosal, DTT is added to reverse the action of thimerosal and wash it away, allowing the embryo to continue developing. The thimerosal / DTT combination treatment also induces cortical granule exocytosis, subsequent zona pellucida hardening, and the development of activated oocytes to the blastocyst stage.

[0058] In addition to thimerosal, other oocyte modifiers can also be used, such as t-butyl hydroperoxide, thiourea, phenylephrine, N-alkylmaleimide (e.g., N-ethylmaleimide), oxidized glutathione, α-halo acids (e.g., iodoacetic acid, chloroacetate, and bromoacetate), iodoacetamide, p-mercurybenzoic acid, p-chlorodic mercurybenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), (2-trimethylammonium)ethylmethanethiosulfonate (MTSET), and (2-sulfonatoethyl)methanethiosulfonate (MTSES). Useful reducing agents, such as thiol (-SH) group reducing agents, in addition to DTT, include, but are not limited to, dithioerythritol (DTE), β-mercaptoethanol, cysteine, reduced glutathione, reduced thiourea, thioglycolates, and ascorbic acid.

[0059] The period of contact between oocytes and the oocyte modifier is an effective period for activating the oocytes when subsequently treated with a reducing agent. Such a period may range from about 5 minutes to about 20 minutes, preferably from about 5 minutes to about 15 minutes, or more preferably from about 5 minutes to about 12 minutes. The period of contact between oocytes and the reducing agent is appropriately long enough to activate the oocytes when treated with the oocyte modifier first. Such a period may range from about 5 minutes to about 1 hour, preferably from about 10 minutes to about 45 minutes, more preferably from about 20 minutes to about 40 minutes, and even more preferably from about 30 minutes.

[0060] Contacting enucleated oocytes with a reducing agent after contact with an oocyte modifier can be done substantially immediately or within a time range of approximately 5 seconds to 5 minutes after exposure of the oocytes to the oocyte modifier. Oocytes treated with the oocyte modifier can be transferred to a medium containing the reducing agent without an intermediate washing step. Alternatively, oocytes treated with the oocyte modifier can be washed in a control medium or a medium containing the reducing agent to substantially remove the oocyte modifier before culturing the oocytes in a medium containing the reducing agent. Another alternative is to add the reducing agent directly to the oocytes, which can be done while the latter (oocytes) are still present in a medium containing the oocyte modifier.

[0061] Alternatively, oocyte activation can also be achieved using many other calcium-free chemical treatments, such as protein kinase inhibition (Mayes et al. (1995) Biol. Reprod. 53:270-275) or protein synthesis inhibition (Nussbaum et al. (1995) Mol. Reprod. Dev. 41:70-75).

[0062] After activation, oocytes are typically cultured in vitro for a short period. The resulting embryos are then implanted in a surrogate female, and embryonic development proceeds in the surrogate mother. For example, embryos can be cultured for about a week and then surgically or non-surgically implanted into the surrogate mother's reproductive system. Embryos can be implanted into the fallopian tubes through the fimbriae of the surrogate mother. Alternatively, embryos can be implanted into the fallopian tubes of the surrogate mother by using a catheter that penetrates the wall of the fallopian tube. Another method of embryo implantation involves culturing them to the blastocyst stage and then introducing them into the reproductive system of a surrogate sow. These methods are well known in the art and can be readily applied to the production of genetically modified pigs according to the present invention.

[0063] Further methods for producing genetically modified pigs and other large animals are known in the art and can also be used in the present invention (e.g., U.S. Patent No. 2005 / 0120400, U.S. Patent No. 5,995,577, International Publication No. 95 / 16670, International Publication No. 96 / 07732, International Publication No. 97 / 00669, International Publication No. 9700668, International Publication No. 2005 / 104835, Lai et al., Reproductive Biology and Endocrinology 1:82, 2003, Hao et al., Transgenic Res. 15:739-750, 2006, Li et al., Biology of Reproduction 75:226-230, 2006, Lai et al., Nature Biotechnology 24(4):435-436, 2006, Lai et al. See also: al., Methods in Molecular Biology 254(2):149-163, 2004; Lai et al., Cloning and Stem Cells 5(4):233-241, 2003; Park et al., Animal Biotechnology 12(2):173-181, 2001; Lai et al., Science 295:1089-1092, 2002; Park et al., Biology of Reproduction 65:1681-1685, 2001 (the contents of each are incorporated herein by reference).

[0064] Other viable methods for producing genetically modified pigs include injecting or transducing nucleases (zinc finger nucleases or Tal nucleases that would target the gene of interest) into somatic cells, followed by somatic cell nuclear transfer and embryo transfer to a surrogate mother. Furthermore, genetic modification mediated by sperm or intracytoplasmic sperm injection (ICSI) can also be used. Briefly, in ICSI-mediated modification, the targeted construct is mixed with sperm, and both are injected into oocytes. In sperm-mediated modification, the construct is mixed with sperm, and the surrogate mother is impregnated using in vitro fertilization (IVF) or sperm injection. Those skilled in the art can easily adapt these methods to the production of the knockout pigs of the present invention. Embryonic stem cell technology or induced pluripotent cells, as developed for mice but not fully developed for pigs, can be genetically modified and used as donor cells for either somatic cell nuclear transfer or the production of chimeric animals.

[0065] As described above, the present invention also relates to genetically modified pigs in which (1) both alleles of the SIGLEC-1 gene are inactivated, (2) both alleles of the CD163 gene are inactivated to produce a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV) due to the inactivation of the CD163 allele, or (3) both alleles of SIGLEC-1 and both alleles of CD163 are inactivated. Genetically modified pigs that are homozygous for gene inactivation can be produced by mating with pigs that are heterozygous for gene activation and screening the offspring to identify animals that are homozygous for gene(s) inactivation. The present invention also relates to offspring of such genetically modified pigs, in which one or both alleles of the SIGLEC-1 gene are inactivated, and / or one or both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

[0066] Accordingly, the present invention relates to a method for producing genetically modified pigs in which both alleles of the SIGLEC-1 gene are inactivated, by mating a genetically modified female pig in which at least one allele of SIGLEC-1 is inactivated with a genetically modified male pig in which at least one allele of SIGLEC-1 is inactivated to produce F1 offspring, and then screening the F1 offspring to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene are inactivated. Similarly, the present invention relates to a method for producing genetically modified pigs in which both alleles of the CD163 gene are inactivated, by mating a genetically modified female pig in which at least one allele of CD163 is inactivated with a genetically modified male pig in which at least one allele of CD163 is inactivated to produce F1 offspring, and then screening the F1 offspring to identify genetically modified pigs in which both alleles of the CD163 gene are inactivated.

[0067] The present invention also provides a method for producing genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated. One such method includes crossing a genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated with a genetically modified pig in which at least one allele of the CD163 gene is inactivated to produce F1 offspring, screening the F1 offspring to identify genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated, crossing genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated with each other to produce F2 offspring, and screening the F2 offspring to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

[0068] A method for producing genetically modified pigs in which at least one allele of the SIGLEC-1 gene and / or at least one allele of the CD163 gene is inactivated is disclosed in the preceding chapter. Screening of offspring can be carried out by, for example, PCR or Southern blotting, as is standard practice in the art.

[0069] Another method for producing genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated includes crossing a genetically modified pig that is homozygous for SIGLEC-1 inactivation with a genetically modified pig that is homozygous for CD163 inactivation to produce F1 offspring, crossing the F1 offspring to produce F2 offspring, and screening the F2 offspring to identify animals that are homozygous for inactivation of both SIGLEC-1 and CD163.

[0070] Another method for producing genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated includes crossing a genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated with another genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated to produce F1 offspring, and then screening the F1 offspring to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

[0071] The present invention also relates to genetically modified pig offspring produced by any of the above methods, wherein one or both alleles of the SIGLEC-1 gene are inactivated, and / or one or both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 gene results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

[0072] In addition to being obtainable through breeding approaches that include heterozygous animals, homozygous mutant animals can also be obtained using approaches that involve gene targeting by homologous recombination to achieve modification of the remaining alleles, using cells containing one mutation in one allele (e.g., fetal fibroblasts), such as cells obtained from animals produced using the methods summarized above. The resulting donor cells can then be used as a source of modified nuclei for nuclear transfer into recipient cells, such as enucleated oocytes, thereby forming homozygous mutant embryos, which, when transplanted into a surrogate female, develop into homozygous mutant animals. Genetically modified pigs in which both alleles of the SIGLEC-1 and / or cD163 gene (including multiple alleles) are inactivated can also be produced by injecting or transducing somatic cells with zinc finger nuclease or Tal nuclease (which can simultaneously target both alleles of the gene), followed by somatic cell nuclear transfer (SCNT) or embryo transfer into a surrogate mother to produce such pigs. The present invention also relates to offspring of such genetically modified pigs, in which one or both alleles of the SIGLEC-1 gene are inactivated, and / or one or both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 gene results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

[0073] Although the present invention has been described in detail, modifications and changes are possible without departing from the scope of the invention as defined in the appended claims.

[0074] Examples The following non-limiting embodiments are provided to further illustrate the present invention. [Examples]

[0075] Modification of the SIGLEC-1 gene The approach used to excise the sialoadhesin gene involved removing the protein-coding exon and using homologous recombination to introduce a termination in the remaining coding sequence of the sialoadhesin gene. The porcine sialoadhesin gene (SIGLEC-1, NCBI reference sequence NM_214346) encodes a 210 kDa protein from a 5,193 nucleotide mRNA transcript (Vanderheijden et al., 2003). Using the porcine genome sequence from the region surrounding the sialoadhesin gene (Genbank accession number CU467609), oligonucleotides were generated, and the genomic fragments were amplified by high-fidelity PCR [AccuTaq(Invitrogen)] for the generation of targeted constructs. One fragment ("upper arm") contained the first coding exon and 3304 bp upstream from the start of translation. The second ("lower arm") fragment was 4753 bp long and contained most of the intron downstream of the third coding exon, extending to the sixth intron (including the fourth, fifth, and sixth coding exons). Based on comparison with mouse and human sialoadhesin genome sequences, the porcine sialoadhesin gene was predicted to consist of 21 exons (Figure 1A, Figure 1B). Exon 2 is conserved among pigs, mice, and humans. Amino acid alignment of exon 2 revealed that six amino acids in mouse sialoadhesin, known to be associated with sialic acid binding activity, were conserved in porcine sialoadhesin (Figure 1C). The initial targeting strategy focused on creating changes in the sialoadhesin gene, and therefore, it was not expected that a functional protein would be obtained from the mutant gene. Other inactivation strategies may include targeted modification of selected residues in exon 2 of the sialoadhesin gene, or alteration of the immunoglobulin domains (possibly by altering their order or by substituting them with equivalent domains of other species) to prevent PRRS virus binding. Further modifications may include placing one of the neomycin cassettes or immunoglobulin-like domains adjacent to the loxP site to enable inducible or tissue-specific desorption, if desired.

[0076] In the current gene disruption, a portion of exon 1 and all of exons 2 and 3 were replaced using a neomycin selection cassette with a substitution vector (Figure 1D) (Mansour et al. 1988). In the plasmid construct, a phosphoglycerol kinase (PGK) promoter was used to drive the expression of the neomycin cassette, enabling positive selection of transfected colonies.

[0077] Donor cell preparation Primary fetal fibroblast cell lines from male 35-day-old gestation pigs were isolated from large commercial white pigs (Landrace). The cells were cultured and grown for 48 hours until 80% confluence in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5 mM glutamine, sodium bicarbonate (3.7 g / L), penicillin-streptomycin, and 1 g / L d-glucose, as well as 15% Hyclone fetal bovine serum, 10 μg / ml gentamicin, and 2.5 ng / ml basic fibroblast growth factor (Sigma). The medium was removed and replaced with fresh medium 4 hours prior to transfection. Fibroblasts were washed with 10 ml of phosphate-buffered saline (DPBS, Invitrogen) and transfused 75 cm³ with 1 ml of 0.05% trypsin-EDTA (Invitrogen). 2 The cells were removed from the flask. The cells were resuspended in DMEM and collected by centrifugation at 600 × g for 10 minutes. The cells were washed with Opti-MEM (Invitrogen) and again centrifuged at 600 × g for 10 minutes. The pellet was suspended using cytosalts (75% cytosalt [120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, pH 7.6, 5 mM MgCl2]) and 25% Opti-Mem (van den Hoff et al. 1992). The cells were counted using a hemocytometer, and 1 × 10⁶ cells were collected. 6 The concentration was adjusted to individual cells / ml. Electroporation of cells, 1 × 10 6Transfection was performed using 4 μg of single-stranded targeted DNA (achieved by thermal denaturation) in 200 μl of transfection medium containing 10,000 cells / ml. Cells were electroporated in a BTX ECM2001 electrocell manipulator using three 1 mSec pulses at 250 V. Electroporated cells were diluted in DMEM / FBS / FGF at a rate of 10,000 cells per 13 cm plate. Electroporated cells were cultured overnight without selective pressure. The following day, the medium was replaced with culture medium containing G418 (0.6 mg / ml). After 10 days of selection, G418-resistant colonies were isolated and transferred to 24-well plates for growth. After growth in 24-well plates, cells were divided into 6-well plates (approximately half were used for genomic DNA isolation). PCR was used to determine whether the sialoadhesin gene had been successfully targeted. The reaction used oligonucleotides that annealed to phosphoglucokinase (PGK) cassettes (including Neo), in addition to those that annealed to genomic DNA at the sialoadhesin locus beyond the region of the targeted arm (see Figure 2). Successful targeting of both “arms” was thus evaluated. Targeted fibroblast clones (4–18) were identified, and some of the cells in the culture medium were used for nuclear transfer (see below), while others were frozen for further use. Southern blotting confirmation of homologous recombination provided further confirmation of successful targeting.

[0078] Oocyte collection and in vitro maturation (IVM) Porcine oocytes were purchased from ART Inc (Madison, WI) and matured according to the manufacturer's instructions. After maturation in vitro for 42–44 hours, the oocytes were detached from their cumulus cells by gently vortexing in 0.5 mg / ml hyaluronidase. Following the removal of the cumulus cells, oocytes with good morphology and a visible polar body (metaphase II) were selected and kept in micromanagement medium at 38.5°C until nuclear transfer.

[0079] Somatic cell nuclear transfer, fusion / activation of nuclear transfer complex oocytes, and in vitro developmental culture Under a microscope, oocytes without cumulus oophorus were held in droplets of micromanaging medium supplemented with 7.5 μg / mL of cytochalasin B and coated with mineral oil using a holding micropipette. The zona pellucida was punctured near the first polar body using a microglass injection micropipette, and the first polar body and adjacent cytoplasm (possibly containing metaphase II chromosome) were aspirated into the pipette, withdrawn from the pipette, and the contents discarded. Single, round, and bright donor cells with a smooth surface were selected and transplanted into the perivitelline space adjacent to the oocyte membrane (Lai et al. 2006, Lai et al. 2002).

[0080] Nuclear transfer complexes (oocytes + fibroblasts) were fused in fusion medium using low concentrations of calcium (0.3 M mannitol, 0.1 mM CaCl22H2O, 0.1 mM MgCl26H2O, and 0.5 mM HEPES). The fused oocytes were then activated by treatment with 200 μM thimerosal in the dark for 10 minutes, followed by rinsing and treatment with 8 mM dithiothreitol (DTT) for 30 minutes. The oocytes were then rinsed again to remove the DTT (Machaty and Prather 2001, Machaty et al. 1997). After fusion / activation, oocytes were washed three times with porcine zygote medium 3 (PZM3) supplemented with 4 mg / ml BSA (Im et al. 2004), and then cultured for 30 minutes at 38.5°C in a humidified atmosphere of 5% O2, 90% N2, and 5% CO2. Such successfully fused complexes were cultured for 15–21 hours until surgical embryo transfer to a surrogate mother.

[0081] Surrogate mother preparation, embryo transfer, pregnancy diagnosis, and childbirth. Surrogate sows (gilts) were synchronized over 14 days using a estrous cycle stage-dependent scheme by administering 18-20 mg of REGU-MATE (altrenodist 0.22% solution) (Intervet, Millsboro, DE) mixed with food. After the last REGU-MATE treatment (105 hours), estrus was induced by intramuscular injection of 1000 units of hCG. Other surrogate mothers that were following their natural cycles on appropriate days were also included in the surrogate mother pool. Surrogate mothers were used on the persistent estrus day (day 0), i.e., the first day after persistent estrus (Lai et al. 2002). Surrogate mothers were prepared aseptically, and a caudal-ventral incision was made to expose the reproductive system. The embryo was implanted through the fimbriae into one fallopian tube. Pregnancy was confirmed in the surrogate mothers by abdominal ultrasound at approximately day 30, and then weekly thereafter during the pregnancy. In pigs, birth generally occurs on the 114th day of gestation.

[0082] Following transfection and screening of fetal fibroblasts, candidate donor cells were identified and used for somatic cell nuclear transfer (SCNT). 666 SCNT embryos were implanted into two surrogate mothers. One mother gave birth to six healthy male piglets at 115 days of gestation, and the other mother underwent a cesarean section at 117 days of gestation, yielding two healthy male piglets as shown in Table 1. [Table 1] Embryo transplantation results using SIGLEC-1+ / - male donor cells [Table 1]

[0083] Figure 2 shows the composition of the sialoadhesin (SIGLEC-1) gene, the targeting vector, and the expected recombinant genotype. The upper panel of Figure 2 shows the targeted construct used for homologous recombination. As mentioned above, the “upper” DNA fragment used to construct the targeted construct contained approximately 3.5 kb upstream of exon 1 and a portion of exon 1 (after the start codon). The “lower” DNA fragment began in intron 3 and contained exons 4, 5, and 6, as well as a portion of exon 7. Most of exon 1 and all of exons 2 and 3 were replaced using a neomycin (neo) cassette, and a thymidine kinase (TK) cassette was available just downstream of the forearm for use as a negative selection marker if necessary. The lower panel of Figure 2 shows the mutated sialoadhesin gene after homologous recombination. Arrows indicate oligonucleotide binding sites used for PCR-based cloning of targeted cell lines and cloned pigs. Targeting by the upper and lower arms was performed by annealing of oligonucleotides indicated by arrows showing the primers "Upper Sialo Targeting C" and "PGK Poly A Reverse" (SEQ ID NOs: 1 and 3) and "PGK Promoter Forward" and "7Rwl" (SEQ ID NOs: 4 and 6), respectively. The oligonucleotide sequences are listed in Table 2 below. Further check PCR was performed using oligonucleotides adjacent to the excised region / Neo cassette (primers "Exon 1 Terminal ck" and "Intron 3 ck Reverse" (SEQ ID NOs: 2 and 5, respectively)). There is a difference of approximately 500 bp in product size between the disrupted allele and the wild-type allele. [Table 2] Primer names and sequences used in the design [Table 2] The primers in Table 2 were assigned sequence ID numbers based on the position of the arrows in the lower panel of Figure 2, from left to right. Thus, the leftmost arrow in the lower panel of Figure 2 indicates the position of the "upper sialo-targeted C" primer (SEQ ID NO: 1), the next arrow to the right indicates the "exon 1-terminal ck" primer (SEQ ID NO: 2), the next arrow to the right of that indicates the "PGK poly A reverse direction" primer (SEQ ID NO: 3), and so on.

[0084] Screening for SIGLEC-1 inactivation Genomic DNA was isolated from piglets and used to confirm the targeting event. Successful targeting of the SIGLEC-1 gene was determined by using PCR with oligonucleotides that anneal within the Neocassette, in conjunction with oligonucleotides that anneal to the SIGLEC-1 genomic DNA beyond the region contained within the targeting construct. In the upper panel of Figure 3, targeting by the "upper" arm was investigated using "PGK polyA reversed" and "upper sialo-targeting C oligonucleotide" (SEQ ID NOs. 3 and 1, respectively, shown in Figure 2). Products of the expected size (approximately 4500 bp) were produced. In the lower panel, successful targeting by the "lower" arm was determined using "PGK promoter forward" and "7Rwl" oligonucleotides (SEQ ID NOs. 4 and 6, respectively, shown in Figure 2). Products of the expected size (approximately 5000 bp) were produced. The "lower arm plasmid" control was a partial construct containing a Neocassette with a sialoadhesin gene fragment showing most of intron 3 and most of exon 7. The 7Rwl oligonucleotide was able to anneal to the exon 7 sequence present in the plasmid and, together with the PGK promoter-forward oligonucleotide, produced a product identical to that which would be produced from a successful targeting event. Both panels show targeted PCR reactions performed on genomic DNA extracted from eight piglet clones generated from 4–18 targeted embryonic fibroblast cell lines.

[0085] Detection of both wild-type and targeted sialoadhesin alleles was performed using PCR with oligonucleotides annealing to DNA adjacent to the targeting region of the sialoadhesin gene. "Exon 1-terminus ck" and "intron 3-terminus ck reverse direction" oligonucleotides were used (SEQ ID NOs. 2 and 5, respectively, shown in Figure 2). The resulting products were approximately 2400 bp for the wild-type allele and approximately 2900 bp for the targeted allele. In Figure 4, the left panel (Figure 4A) shows the test reaction performed on wild-type genomic DNA, the targeted plasmid used for transfection, genomic DNA from successfully targeted fibroblast clones (4-18) (two bands shown), and genomic DNA from untargeted fibroblast clones (4-3). The right panel (Figure 4B) shows the reaction performed on genomic DNA extracted from eight piglet clones generated from targeted embryonic fibroblast cell lines 4-18. Each lane contained two PCR products of the expected size, while the wild-type DNA and targeted plasmid template had only one band. Therefore, all piglets produced by SCNT were heterozygous for the intended mutation, i.e., SIGLEC+ / -.

[0086] Production of homozygous animals Genetically modified male pigs identified as heterozygous for SIGLEC-1 inactivation were used as male founders (F0 generation) and mated with wild-type females to produce male and female animals (F1) each possessing one inactivated SIGLEC-1 allele. Subsequently, F1 males were mated with F1 females to produce F2 offspring, which possess both inactivated SIGLEC-1 alleles. Such animals can be identified postnatally for SIGLEC-1 by the PCR described above or alternatively by Southern blotting. [Examples]

[0087] Generation of CD163 Targeted Structures As has already been established, deletion of the cytoplasmic domain of CD163 eliminates PRRSV infectivity, as does deletion or modification of SRCR domain 5. Since some of the SRCR domains of CD163 have functions crucial to animal survival (e.g., hemoglobin removal), gene modification that retains these other functions presents a reliable strategy for creating pigs resistant to PRRSV. Previous studies have also suggested that infectivity is blocked by substituting the SRCR5 domain with CD163L domain 8 (Van Gorp et al., 2010b). Therefore, a targeted construct can be designed, as shown in Figure 6, to replace SRCR5 of CD163 with the SRCR domain CD163L.

[0088] Once the targeted construct is created, genetically modified pigs can be produced by a method generally the same as that described in Example 1, which is heterozygous for inactivated CD163 and produces a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV) due to inactivation of the CD163 gene. First, the targeted vector can be inserted into donor cells and recombined with the endogenous CD163 gene. Then, donor cells with this specific modification are selected and used for somatic cell nuclear transfer to produce genetically modified embryos. The embryos are then transplanted into a surrogate mother for full-term pregnancy. After identifying transgenic pigs with the inactivated CD163 allele by either PCR or Southern blotting, these animals are allowed to reach sexual maturity and then used for natural mating to transfer the gene to the fetus or offspring.

[0089] References Albina E, Madec F, Cariolet R, Torrison J. 1994. Immune Response and Persistence of the Porcine Reproductive and Respiratory Syndrome Virus in Infected Pigs and Farm Units. Veterinary Record 134(22):567-573. Allende R, Laegreid WW, Kutish GF, Galeota JA, Wills RW, Osorio FA. 2000. Porcine reproductive and respiratory syndrome virus: Description of persistence in individual pigs upon experimental infection. Journal of Virology 74(22): 10834-10837. Andreyev VG, Wesley RD, Mengeling WL, Vorwald AC, Lager KM. 1997. Genetic Variation and Phylogenetic Relationships of 22 Porcine Reproductive and Respiratory Syndrome Virus (Prrsv) Field Strains Based on Sequence Analysis of Open Reading Frame 5. Archives of Virology 142(5):993-1001. Benfield DA, Nelson E, Collins JE, Harris L, Goyal SM, Robison D, Christianson WT, Morrison RB, Gorcyca D, Chladek D. 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). 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Molecular analysis of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis. Biochemical Journal 341(Part 2):355-361. Das PB, Dinh PX, Ansari IH, de Lima M, Osorio FA, Pattnaik AK. 2010. The minor envelope glycoproteins GP2a and GP4 of porcine reproductive and respiratory syndrome virus interact with the receptor CD 163. J Virol 84(4): 1731-1740. Dee SA, Molitor TW. 1998. Elimination of Porcine Reproductive and Respiratory Syndrome Virus Using a Test and Removal Process. Veterinary Record 143(17):474-476. Delputte PL, Meerts P, Costers S, Nauwynck HJ. 2004. Effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages. Vet Immunol Immunopathol 102(3): 179-188. Delputte PL, Nauwynck HJ. 2004. Porcine arterivirus infection of alveolar macrophages is mediated by sialic acid on the virus. Journal of Virology 78(15):8094-8101. 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[0090] When introducing elements of the present invention or their preferred embodiments (including multiple elements), the articles “a,” “an,” “the,” and “said” indicate that there is one or more elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and indicate that there may be further elements other than those listed.

[0091] Considering the above, it can be seen that several objectives of the present invention are achieved and other beneficial results are obtained.

[0092] Various modifications can be made to the above products and methods without departing from the scope of the present invention, and all matters included in the above description and shown in the accompanying drawings (including multiple drawings) are interpretive and not intended to limit the scope.

Claims

1. A genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated.

2. A genetically modified pig in which at least one allele of the CD163 gene is inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

3. A genetically modified pig according to claim 1 or 2, wherein both alleles of the SIGLEC-1 gene are inactivated.

4. A genetically modified pig according to any one of claims 1 to 3, wherein both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 alleles results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

5. A genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated.

6. A genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated, produced by a method comprising: Enucleation of porcine oocytes, The oocyte is fused with a donor pig fibroblast, wherein the fibroblast's genome contains at least one inactivated SIGLEC-1 allele. To activate the oocytes and produce an embryo.

7. A genetically modified pig according to claim 6, produced by a method further comprising implanting an embryo into the reproductive system of a surrogate sow, wherein the surrogate sow has begun estrus but has not yet completed ovulation, and the pregnancy and full-term birth of the embryo produces a genetically modified pig whose genome comprises at least one inactivated SIGLEC-1 allele.

8. A genetically modified pig having at least one allele of the CD163 gene inactivated, the inactivation of the CD163 allele resulting in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV), produced by a method comprising: Enucleation of porcine oocytes, The oocyte is fused with a donor pig fibroblast, wherein the genome of the fibroblast contains at least one inactivated CD163 allele. To activate the oocytes and produce an embryo.

9. A genetically modified pig according to claim 8, produced by a method further comprising implanting the embryo into the reproductive system of a surrogate sow, wherein the surrogate sow has begun estrus but has not yet completed ovulation, and pregnancy and full-term birth produce a genetically modified pig whose genome contains at least one inactivated CD163 allele.

10. A genetically modified pig according to any one of claims 1 to 9, wherein at least one of the alleles is inactivated by partial or complete deletion.

11. A genetically modified pig according to any one of claims 1 to 10, wherein at least one allele is inactivated using the Cre-lox recombinant system.

12. A genetically modified pig according to any one of claims 1, 3 to 7, 10, and 11, wherein the SIGLEC-1 allele is inactivated by deletion of part of exon 1 and all of exons 2 and 3.

13. A genetically modified pig according to any one of claims 2 to 5 and 8 to 11, wherein the CD163 allele is inactivated by substitution of exon 7 using the scavenger receptor cysteine-rich (SRCR) domain 8 of CD163L.

14. The genetically modified pig according to any one of claims 6 to 13, wherein the enucleation is performed using a micropipette in a micromanagement medium.

15. The genetically modified pig according to any one of claims 6 to 14, wherein the fusion is carried out in a fusion medium.

16. The genetically modified pig according to any one of claims 6 to 15, wherein the activation of the oocytes comprises incubating the oocytes in the presence of thimerosal.

17. The genetically modified pig according to any one of claims 7 and 9 to 16, wherein the transplantation comprises transplanting the embryo into the fallopian tube through the fimbriae of the surrogate mother.

18. A genetically modified pig in which both alleles of the SIGLEC-1 gene are inactivated, produced by a method comprising: The method involves crossing a genetically modified male pig according to any one of claims 1, 6, and 7 with a genetically modified female pig according to any one of claims 1, 6, and 7 to produce F1 offspring. The F1 offspring are screened to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene are inactivated.

19. A genetically modified pig, produced by a method comprising the following: both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 alleles results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV); The method involves crossing a genetically modified male pig according to any one of claims 2, 8, and 9 with a genetically modified female pig according to any one of claims 2, 8, or 9 to produce F1 offspring. The F1 offspring are screened to identify genetically modified pigs in which both alleles of the CD163 gene are inactivated.

20. A genetically modified pig, produced by a method comprising the following: both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV); The method involves mating a genetically modified female pig according to claim 1, 6, or 7 with a genetically modified male pig according to claim 2, 8, or 9, or mating a genetically modified male pig according to claim 1, 6, or 7 with a genetically modified female pig according to claim 2, 8, or 9 to produce F1 offspring. The F1 offspring are screened to identify genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated. The process involves crossbreeding genetically modified pigs, in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated, to produce F2 offspring. The F2 offspring are screened to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

21. A genetically modified pig, wherein both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 gene results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV), is produced by a method comprising: The method involves mating a genetically modified female pig according to claim 3 or 18 with a genetically modified male pig according to claim 4 or 19, or mating a genetically modified male pig according to claim 3 or 18 with a genetically modified female pig according to claim 4 or 19 to produce F1 offspring. The F1 offspring are crossbred to produce F2 offspring, The F2 offspring are screened to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

22. A genetically modified pig, wherein both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 gene results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV), is produced by a method comprising: The method involves crossing a genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated with another genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated to produce F1 offspring. The F1 offspring are screened to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

23. The genetically modified pig according to claim 18 or 19, wherein the screening comprises using polymerase chain reaction (PCR) or Southern blotting.

24. The offspring of a genetically modified pig according to any one of claims 6 to 23, wherein at least one allele of the SIGLEC-1 gene is inactivated.

25. The genetically modified pig offspring according to any one of claims 6 to 23, wherein at least one allele of the CD163 gene is inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

26. The offspring of a genetically modified pig according to any one of claims 6 to 23, wherein at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated, and the inactivation of the CD163 allele produces a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

27. The offspring of a genetically modified pig according to any one of claims 6 to 23, wherein both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

28. A method for producing a genetically modified pig in which at least one allele of the SIGLEC-1 gene is inactivated, comprising: Enucleation of porcine oocytes, The oocyte is fused with a donor pig fibroblast, wherein the genome of the fibroblast contains at least one inactivated SIGLEC-1 allele. To activate the oocytes and produce an embryo.

29. A method according to claim 28, further comprising implanting the embryo into the reproductive system of a surrogate sow, wherein the surrogate sow has begun estrus but has not yet completed ovulation, and pregnancy and full-term birth produce a genetically modified sow whose genome contains at least one inactivated SIGLEC-1 allele.

30. A method for producing a genetically modified pig, wherein at least one allele of the CD163 gene is inactivated, and the inactivation of the CD163 allele results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV), the method comprising: Enucleation of porcine oocytes, The oocyte is fused with a donor pig fibroblast, wherein the genome of the fibroblast contains at least one inactivated CD163 allele. To activate the oocytes and produce an embryo.

31. A method according to claim 30, further comprising implanting the embryo into the reproductive system of a surrogate sow, wherein the surrogate sow has entered estrus but has not yet completed ovulation, and pregnancy and full-term birth produce a genetically modified sow whose genome contains at least one inactivated CD163 allele.

32. A method for producing genetically modified pigs in which both alleles of the SIGLEC-1 gene are inactivated, comprising: The method involves crossing a genetically modified female pig produced by the method of claim 28 or 29 with a genetically modified male pig produced by the method of claim 28 or 29 to produce F1 offspring. The F1 offspring are screened to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene are inactivated.

33. A method for producing a genetically modified pig in which both alleles of the CD163 gene are inactivated, thereby producing a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV), the method comprising: The method involves crossing a genetically modified female pig produced by the method of claim 30 or 31 with a genetically modified male pig produced by the method of claim 30 or 31 to produce F1 offspring. The F1 offspring are screened to identify genetically modified pigs in which both alleles of the CD163 gene are inactivated.

34. The method according to any one of claims 28 to 33, wherein at least one allele is inactivated by partial or complete deletion.

35. The method according to any one of claims 28 to 34, wherein at least one allele is inactivated using a Cre-lox recombinant system.

36. The method according to any one of claims 28, 29, 32, 34, and 35, wherein the SIGLEC-1 allele is inactivated by deletion of part of exon 1 and all of exons 2 and 3.

37. The method according to any one of claims 30, 31, and 33-35, wherein the CD163 allele is inactivated by substitution of exon 7 using the scavenger receptor cysteine-rich (SRCR) domain 8 of CD163L.

38. The method according to any one of claims 28 to 37, wherein the nucleation is performed using a micropipette in a micro-operated culture medium.

39. The method according to any one of claims 28 to 38, wherein the fusion is carried out in a fusion culture medium.

40. The method according to any one of claims 28 to 39, wherein the activation of the oocytes comprises incubating the oocytes in the presence of thimerosal.

41. The method according to any one of claims 29 and 31 to 40, wherein the transfer includes transferring the embryo through the fimbriae of the surrogate mother into the fallopian tube.

42. A method for producing a genetically modified pig, wherein both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 gene results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV), the method comprising: The method involves mating a genetically modified female pig according to claim 1, 6, or 7 with a genetically modified male pig according to claim 2, 8, or 9, or mating a genetically modified male pig according to claim 1, 6, or 7 with a genetically modified female pig according to claim 2, 8, or 9 to produce F1 offspring. The F1 offspring are screened to identify genetically modified pigs in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated. The genetically modified pigs, in which at least one allele of the SIGLEC-1 gene is inactivated and at least one allele of the CD163 gene is inactivated, are crossbred to produce F2 offspring. The F2 offspring are screened to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

43. A method for producing a genetically modified pig, wherein both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 gene results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV), the method comprising: The method involves mating a genetically modified female pig according to claim 3 or 18 with a genetically modified male pig according to claim 4 or 19, or mating a genetically modified male pig according to claim 3 or 18 with a genetically modified female pig according to claim 4 or 19 to produce F1 offspring. The F1 offspring are crossbred to produce F2 offspring, The F2 offspring are screened to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

44. A method for producing a genetically modified pig, wherein both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 gene results in a CD163 protein that cannot bind to and / or decoat the porcine reproductive respiratory syndrome virus (PRRSV), the method comprising: The method involves crossing a genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated with another genetically modified pig in which at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated to produce F1 offspring. The F1 offspring are screened to identify genetically modified pigs in which both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated.

45. The method according to any one of claims 42 to 44, wherein the screening includes using polymerase chain reaction (PCR) or Southern blotting.

46. A genetically modified pig offspring produced by the method according to any one of claims 28 to 45, wherein at least one allele of the SIGLEC-1 gene is inactivated.

47. Genetically modified pig offspring produced by the method according to any one of claims 28 to 45, wherein at least one allele of the CD163 gene is inactivated, and the inactivation of the CD163 gene produces a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

48. Genetically modified pig offspring produced by the method according to any one of claims 28 to 45, wherein at least one allele of the SIGLEC-1 gene and at least one allele of the CD163 gene are inactivated, and the inactivation of the CD163 gene produces a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

49. Genetically modified pig offspring produced by the method of any one of claims 28 to 45, wherein both alleles of the SIGLEC-1 gene and both alleles of the CD163 gene are inactivated, and the inactivation of the CD163 gene produces a CD163 protein that cannot bind to and / or decoat porcine reproductive respiratory syndrome virus (PRRSV).

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