Abstract: A reagent, a kit, and a method for detecting a pig economic trait-associated mutation site are provided, belonging to the technical field of molecular markers. The reagent for detecting a pig economic trait-associated mutation site is provided, and the reagent and an endonuclease are prepared into the kit for detecting a pig economic trait-associated mutation site. The method for detecting a pig economic trait-associated mutation site is also provided. After PCR amplification, gene fragments of WIP1, TRIM55, and GAS7 of a pig show site differences between different individuals. Restriction endonucleases are used for digestion, and PCR or multiplex PCR analysis is conducted to identify single nucleotide polymorphisms (SNPs) of the pig, such that the wild type, the homozygous mutant type, and the heterozygous mutant type that are difficult to differentiate in morphology are differentiated from the molecular biology level.

Abstract: A kit for breeding a pig breed with resistance to the porcine transmissible gastroenteritis virus infection and application thereof. The systems includes a genetically edited protein, pAPN-sgRNA-1, pAPN-sgRNA-2, and a donor DNA. Effectively enzymatic cleavage can be made in two target sites of a pAPN gene by the gene editing protein. By replacing the fragment to be site-directed modified located between two target sites with donor DNA, a codon encoding tryptophan at position 737 in pAPN protein can be mutated to a codon encoding alanine, thereby achieving precise mutation of tryptophan to alanine at position 737 in a pAPN protein. The systems can avoid disruption or alteration of the normal expression of other amino acids in pAPN protein, therefore, the present invention maximally retains the physiological activity function of pAPN protein on the basis of resisting TGEV infection.

Abstract: The present invention provides a pAPN mutant and a composition for site-directed modification of pAPN gene and application thereof, involving the field of gene editing technology. The mutated pAPN mutant with an alanine at position 734 can maintain its normal expression, while reducing the ability of a host expressing the pAPN mutant to specifically bind to TGEV, which has important scientific and practical significance in pig disease resistance breeding.

Abstract: This description relates to a substance causing a mutation at position 727 in an amino acid residue of the pAPN protein in any of the following: 1) constructing a cell line with site-directed modification of the pAPN gene for non-disease diagnostic and therapeutic purposes; 2) preparing a cell with resistance to the porcine transmissible gastroenteritis virus; and so on. A system for site-directed modification of the pAPN gene may effectively cleave two target sites of the pAPN gene, thereby achieving the precise mutation at position 727 in an amino acid of pAPN. Based on the precise modification of the pAPN gene while capable of avoiding disruption or alteration of the normal expression of other amino acids in the pAPN, the physiological activity function of pAPN protein on the basis of resisting TGEV infection is retained.

Abstract: The present invention provides a pAPN mutant and a composition for site-directed modification of pAPN gene and application thereof, involving the field of gene editing technology. The mutated pAPN mutant with an alanine at position 734 can maintain its normal expression, while reducing the ability of a host expressing the pAPN mutant to specifically bind to TGEV, which has important scientific and practical significance in pig disease resistance breeding.

Abstract: A kit for breeding a pig breed with resistance to the porcine transmissible gastroenteritis virus infection and application thereof. The systems includes a genetically edited protein, pAPN-sgRNA-1, pAPN-sgRNA-2, and a donor DNA. Effectively enzymatic cleavage can be made in two target sites of a pAPN gene by the gene editing protein. By replacing the fragment to be site-directed modified located between two target sites with donor DNA, a codon encoding tryptophan at position 737 in pAPN protein can be mutated to a codon encoding alanine, thereby achieving precise mutation of tryptophan to alanine at position 737 in a pAPN protein. The systems can avoid disruption or alteration of the normal expression of other amino acids in pAPN protein, therefore, the present invention maximally retains the physiological activity function of pAPN protein on the basis of resisting TGEV infection.

Abstract: Provided are compositions for simultaneously modifying amino acids of site 736 and site 738 of pAPN gene and application thereof. An sgRNA set specifically recognizing porcine pAPN gene can recognize sequences near amino acids of site 736 and site 738 of pAPN gene, and has strong specificity. On this basis, in combination with a composition consisting of a cleavage protein and a double-stranded donor sequence, simultaneous modification of amino acids of site 736 and site 738 of the pAPN gene can be realized. Under editing of the composition, the amino acids of site 736 and site 738 of the pAPN gene are modified precisely and effectively, which can prevent normal expression of other amino acids of the pAPN gene from being damaged or changed. Therefore, it can resist TGEV infection, and also can retain the physiological activity function of the pAPN protein to the maximum extent.

Abstract: Provided is a double-gene knockout vector system, a method for preparing porcine fibroblasts with both CD163 gene and CD13 gene being knocked-out, prepared porcine fibroblasts, and a method for preparing a gene-edited pig with both CD163 gene and CD13 gene being knocked-out. The vector system of the present disclosure comprises a CD163 gene knockout vector and a CD13 gene knockout vector. The CD163 gene knockout vector comprises a gene editing vector backbone and a DNA fragment ligated to the gene editing vector backbone, with a nucleotide sequence of the DNA fragment being shown in any one of SEQ ID NOs: 1-3. The CD13 gene knockout vector comprises a gene editing vector backbone and a DNA fragment ligated to the gene editing vector backbone, a nucleotide sequence of the DNA fragment being shown in any one of SEQ ID NOs: 4-6.

Abstract: Provided is a double-gene knockout vector system, a method for preparing porcine fibroblasts with both CD163 gene and CD13 gene being knocked-out, prepared porcine fibroblasts, and a method for preparing a gene-edited pig with both CD163 gene and CD13 gene being knocked-out. The vector system of the present disclosure comprises a CD163 gene knockout vector and a CD13 gene knockout vector. The CD163 gene knockout vector comprises a gene editing vector backbone and a DNA fragment ligated to the gene editing vector backbone, with a nucleotide sequence of the DNA fragment being shown in any one of SEQ ID NOs: 1-3. The CD13 gene knockout vector comprises a gene editing vector backbone and a DNA fragment ligated to the gene editing vector backbone, a nucleotide sequence of the DNA fragment being shown in any one of SEQ ID NOs: 4-6.

Abstract: The present invention discloses a method for identifying Wuzhishan miniature pig inbred line by using 145 SNPs. The method for auxiliarily identifying whether a pig to be tested is Wuzhishan miniature pig inbred line, provided by the present invention, comprises the following steps: testing genotypes based on 145 SNP sites of the pig to be tested; if all standards of (1) to (145) are satisfied, the pig to be tested is a candidate for Wuzhishan miniature pig inbred line; if all the standards of the above (1) to (145) are not satisfied, the pig to be tested is a candidate for non-Wuzhishan miniature pig inbred line.

Abstract: The present invention provides a method of site-directed insertion to H11 locus in pigs by using site-directed cutting system, includes the following steps: 1) identify the targeted sequence targeted by the targeted cutting system in the targeted genome sequence of pigs; 2) design and construct the targeting sequence of the corresponding cutting system according to the targeted site; 3) construction of targeting vector; 4) transfect cells, identify the efficiency of fixed-point insertion by PCR amplification. The invention is dependent on the site-directed cutting system of H11 locus in pigs, to insert the target gene into the target site, in order to solve the problems such as low efficiency of traditional shooting technique, inconvenience design of PCR detection primer, harder to detect. The invention provides a method of site-directed insertion which can stably express the foreign gene at the H11 locus, to build an efficient platform for the production of transgenic pigs.

Abstract: The present invention discloses a method for identifying Wuzhishan miniature pig inbred line by using 145 SNPs. The method for auxiliarily identifying whether a pig to be tested is Wuzhishan miniature pig inbred line, provided by the present invention, comprises the following steps: testing genotypes based on 145 SNP sites of the pig to be tested; if all standards of (1) to (145) are satisfied, the pig to be tested is a candidate for Wuzhishan miniature pig inbred line; if all the standards of the above (1) to (145) are not satisfied, the pig to be tested is a candidate for non-Wuzhishan miniature pig inbred line.

Abstract: A method for auxiliary identification of WZSP inbred line and its special primers are disclosed. The method may comprise: testing whether a deoxyribose nucleotide at site 1273 from 5? end of genomic DNA comprising SEQ ID NO: 1 in a test pig is A or G, measuring whether the test pig genotype is GG, GA or AA wherein the test pig with GG genotype is a candidate for the WZSP inbred line and the test pigs with GA genotype and AA genotype are non-WZSP inbred line. The method can be applied to breed WZSP inbred line, which can be used to preliminarily screen all the pigs in test pig group, eliminate non-WZSP inbred line, find out a candidate WZSP inbred line and make a further confirmation in combination with other methods. The method and primers can also be used to judge whether a WZSP on market is counterfeiting or not.

Abstract: A method for preparing a transgenic animal of simultaneous multiple-gene expression is provided. Additionally, a method for preparing a transgenic embryo, which introduces both phytase gene and human myxovirus resistant gene A into a target embryo, to obtain a transgenic embryo is provided. The transgenic animal of simultaneous multiple-gene expression can be achieved by transplanting the transgenic embryo into the body of a female target animal. A significant advantage of the foregoing methods, among many others, exists in that the simultaneous expression of multiple genes can be achieved in one transgenosis, which provides a convenient method for the preparation of combined-gene transferred animals.

Abstract: A method for cultivating a transgenic animal with an increased expression amount of porcine growth hormone is provided. Additionally, a method for cultivating a transgenic embryo, which introduces a recombinant expression vector of 1) TRE-GH or 2) TET-ON into a target embryo to obtain a transgenic embryo with a higher expression amount of porcine growth hormone than a target embryo without introduction is provided. A transgenic animal can be obtained by transplanting the embryo into an animal. The experiments demonstrate that growth hormone (GH) is sharply increased in blood of transgenic pigs after adding DOX.

Abstract: An auxiliary method for identification of WZSP inbred line and its special primers are provided. The method is to test if DNA at site 1273 from 5? end of genomic DNA SEQ ID NO: 1 in test pig is A or G, measure the test pig genotype is GG, GA or AA; GG or AA genotype is homozygote that DNA at site 1273 is G or A; GA genotype is their heterozygote; test pig of GG genotype is candidate for the WZSP inbred line; test pigs of GA and AA genotypes are not WZSP inbred line. The method can be applied to breed WZSP inbred line, can preliminarily screen all the pigs in test pig group, eliminate non WZSP inbred line, find out candidate WZSP inbred line, combine with other methods for further confirmation. The method can also be used to judge whether WZSP on market is counterfeiting or not.

Abstract: A method for preparing a transgenic animal of simultaneous multiple-gene expression is provided. Additionally, a method for preparing a transgenic embryo, which introduces both phytase gene and human myxovirus resistant gene A into a target embryo, to obtain a transgenic embryo is provided. The transgenic animal of simultaneous multiple-gene expression can be achieved by transplanting the transgenic embryo into the body of a female target animal. A significant advantage of the foregoing methods, among many others, exists in that the simultaneous expression of multiple genes can be achieved in one transgenosis, which provides a convenient mean for the preparation of combined-gene transferred animals etc.