Kit for breeding a TGEV infection resistant pig and use thereof

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.

Claims

1. A method for constructing a cell with site-directed modification of a porcine aminopeptidase N (pAPN) gene, which comprises the following steps: introducing a kit comprising a genetically edited protein of Cas9, pAPN-sgRNA-1,pAPN-sgRNA-2, and a donor DNA, wherein the pAPN-sgRNA-1 and pAPN-sgRNA-2 target two different target sequences of the pAPN gene set forth in SEQ ID NO. 1 and SEQ ID NO. 2; and a fragment between the two different target sequences of SEQ ID NO. 1 and SEQ ID NO. 2 is designated as a fragment to be site-directed modified, which comprises a codon encoding tryptophan at position 737 in a pAPN protein; and the donor DNA contains a fragment with site-directed modification, which is obtained by mutating a codon encoding tryptophan at position 737 in the pAPN protein to a codon encoding alanine in the fragment to be site-directed modified or a set of vectors comprising a vector expressing the kit into porcine ileal epithelial cells or porcine fibroblasts to obtain the cell with site-directed modification of the pAPN gene; wherein the donor DNA is double stranded DNA set forth in SEQ ID NO. 3.

2. The method according to claim 1, wherein the set of vectors consists of a vector for expressing the genetically edited protein of Cas9 and the pAPN-sgRNA-1, a vector for expressing the genetically edited protein of Cas9 and the pAPN-sgRNA-2, and a vector containing the donor DNA.

3. The method according to claim 2, wherein the vector for expressing the genetically edited protein of Cas9 and the pAPN-sgRNA-1 is obtained by connecting annealed double stranded DNA fragments from single stranded DNAs set forth in SEQ ID NO. 5 and SEQ ID NO. 6 into a genetically edited backbone vector; alternatively, the vector for expressing the genetically edited protein of Cas9 and the pAPN-sgRNA-2 is obtained by connecting annealed double stranded DNA fragments from single stranded DNAs set forth in SEQ ID NO. 7 and SEQ ID NO. 8 into a genetically edited backbone vector.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a pattern diagram of the precise mutation of W737 single amino acid in pig pAPN protein, as provided in Example 1 of the present invention. The nucleotide sequence of E16 is shown in SEQ ID NO.17, and the nucleotide sequence of HDR template is shown in SEQ ID NO. 18.

(2) FIG. 2 is a pattern diagram of the precise mutation of E739 single amino acid in pig pAPN gene, as provided in Example 1 of the present invention. The nucleotide sequence of E16 is shown in SEQ ID NO. 17, and the nucleotide sequence of HDR template is shown in SEQ ID NO. 19.

(3) FIG. 3 is an expression graph showing overexpressed pAPN proteins in porcine ileal epithelial cells with precise modification of an amino acid at position 737 in pAPN protein, as provided in Example 2 of the present invention.

(4) FIG. 4 is a graph of the results of fluorescence quantitative PCR (qRT-PCR) for detection of the copy number of TGEV RNA in overexpressed porcine ileal epithelial cells infected with TGEV, in which the amino acid at position 737 in pAPN protein is precisely modified, as provided in Example 2 of the present invention.

(5) FIG. 5 is a graph of the results of Western Blot for detection of TGEV protein in overexpressed porcine ileal epithelial cells infected with TGEV, in which amino acids at position 737 in pAPN protein are precisely modified, as provided in Example 2 of the present invention.

(6) FIG. 6 is a graph of the results of indirect immunofluorescence assay (IFA) for detection of pAPN and TGEV expressions in overexpressed porcine ileal epithelial cells infected with TGEV, in which the amino acids at position 737 in pAPN protein is precisely modified, as provided in Example 2 of the present invention.

(7) FIG. 7 is an expression graph showing overexpressed pAPN proteins in porcine ileal epithelial cells with precise modification of an amino acid at position 739 in pAPN protein, as provided in Example 3 of the present invention.

(8) FIG. 8 is a graph of the results of qPCR for detection of the copy number of TGEV RNA in porcine ileal epithelial cells infected with TGEV, in which the amino acid at position 739 in pAPN protein is precisely modified, as provided in Example 3 of the present invention.

(9) FIG. 9 is a graph of the results of Western Blot for detection of TGEV protein in overexpressed porcine ileal epithelial cells infected with TGEV, in which amino acids at position 739 in pAPN protein are precisely modified, as provided in Example 3 of the present invention.

(10) FIG. 10 is a graph of the results of IFA for detection of pAPN and TGEV expressions in overexpressed porcine ileal epithelial cells infected with TGEV, in which the amino acids at position 739 in pAPN protein is precisely modified, as provided in Example 3 of the present invention.

(11) FIG. 11 is a graph of the results of sequencing for porcine fibroblast with precise modification of an amino acid at position 737 in a pAPN protein, as provided in Example 4 of the present invention. The nucleotide sequence of PEF-WT is shown in SEQ ID NO. 20, and the nucleotide sequence of PEF-737PE is shown in SEQ ID NO. 21.

DETAILED DESCRIPTION

(12) The present invention is further described in detail below in conjunction with the detailed description. The Examples provided are only for the purpose of elucidating the present invention, and are not intended to limit its scope. The Examples provided below may serve as a guide for persons of ordinary skill in the art to further improve and do not limit the present invention in any way.

(13) The experimental methods in Examples below are conventional methods unless otherwise specified, and carried out according to the techniques or conditions described in the literature in art or in accordance with the product instruction. The materials, reagents and the like used in Examples below can be obtained commercially unless otherwise specified.

(14) The main reagents involved in the Examples below and their sources were as follows: the collagenase type IV used for isolating porcine fetal fibroblasts was a product from Sigma. DMEM, FBS, PS, NEAA, Glutamine, and Trypase used for cell culture were all products from Gibco. The DNA extraction kit for cells and ear tissues was a product from Tiangen Biotech Co., Ltd. The KOD FX PCR enzyme used for PCR was a product from TOYOBO.

(15) The primer sequences involved in the Examples below were synthesized by Beijing Tsingke Biotech Co., Ltd.

(16) The main instruments involved in the Examples below were as follows: CO2 incubator (Thermo Scientific, 3111), clean bench (AIRTECH, SW-CJ-IFD), inverted fluorescence microscope (ZEISS, observerA1), PCR instrument (BIO-RID, C1000 Touch), gel imaging system (BIO-RID, Universal Hood II), micromanipulation system (Eppendorf, Celltramvario), flow cytometric sorter (BD, Aria III).

(17) The amino acid sequence of the pAPN protein in the Examples below had GenBank No. NP_999442.1 in NCBI.

(18) The amino acid sequence of Cas9 protein in the Examples below had GenBank No. ANW61896.1 in NCBI.

(19) Porcine ileal epithelial cells with pAPN gene knockout (Immortal Pig Intestinal-2I Knock Out, IPI-2I-KO) in the Examples below could be found in the reference of Xu Changjiang, Wang Xiaopeng, XuKui et al. Establishment of a pAPN gene knockout IPI-2I cell lines Mediated by CRISPR/Cas9 System [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48 (7): 2282-2290. DOI: 10.16431/j.cnki. 1671-7236.2021.07.002..

Example 1. Design of a System for Site-Directed Modification in pAPN Gene and Construction of Expression Vector Thereof

(20) I. Design of a System for Site-Directed Modification in pAPN Gene

(21) 1. Design of System 1 for Site-Directed Modification in pAPN Gene

(22) System 1 for site-directed modification in pAPN gene in the present invention comprised Cas9 protein, pAPN-sgRNA-1, pAPN-sgRNA-2, and donor DNA1 (dsODN sequence 1):

(23) The target sequence of pAPN-sgRNA-1 was set forth in SEQ ID No.1.

(24) The target sequence of pAPN-sgRNA-2 was set forth in SEQ ID No.2.

(25) The donor DNA1 (dsODN sequence 1) was a double stranded DNA molecule set forth in SEQ ID No.3.

(26) The codon encoding the amino acid at position 737 in a pAPN protein in the pig genome could be replaced with GCC from TGG by System 1 for site-directed modification in a pAPN gene in the present invention, so as to alter the amino acid at position 737 in the pAPN protein from tryptophan to alanine, and the specific mutation pattern was shown in FIG. 1.

(27) 2. Design of System 2 for Site-Directed Modification in a pAPN Gene

(28) System 2 for site-directed modification in a pAPN gene in the present invention comprised Cas9 protein, pAPN-sgRNA-1, pAPN-sgRNA-2, and donor DNA2 (dsODN sequence 2):

(29) The target sequence of pAPN-sgRNA-1 was set forth in SEQ ID No.1.

(30) The target sequence of pAPN-sgRNA-2 was set forth in SEQ ID No.2.

(31) The donor DNA2 (dsODN sequence 2) was a double stranded DNA molecule set forth in SEQ ID No.4.

(32) The codon encoding the amino acid at position 739 in pAPN protein in the pig genome could be replaced with GCC from GAG by System 2 for site-directed modification in pAPN gene in the present invention, so as to alter the amino acid at position 739 in pAPN protein from glutamic acid to alanine, and the specific mutation pattern was shown in FIG. 2.

(33) II. Construction of an Expression Vector for a System for Site-Directed Modification in a pAPN Gene

(34) 1. Construction of Recombinant Vectors pX458-pAPN-sgRNA-1 and pX458-pAPN-sgRNA-2

(35) 1) Synthesis of complementary paired oligonucleotide sequences as follows for pAPN-sgRNA-1 and pAPN-sgRNA-2 sequences:

(36) TABLE-US-00003 pAPN-sgRNA-1-F: (SEQIDNo.5) caccgCTAGAAATACCTCAGGAAGC; pAPN-sgRNA-1-R: (SEQIDNo.6) aaacGCTTCCTGAGGTATTTCTAGc; pAPN-sgRNA-2-F: (SEQIDNo.7) caccgCGAGCGCCCAGAAAATCTGA; pAPN-sgRNA-2-R: (SEQIDNO.8) aaacTCAGATTTTCTGGGCGCTCGc.

(37) 2) The complementary oligonucleotides corresponding to pAPN-sgRNA-1 and pAPN-sgRNA-2 in step 1) were treated at 98 C. for 10 min, respectively, and then naturally cooled to the room temperature for annealing to obtain annealed double stranded fragments.

(38) 3) The PX458 backbone vector containing Cas9 encoding gene sequence (Addgene, #48138) was cleaved with the restrictive endonuclease Bbs I at 37 C. for 2 h, followed by recovery of the backbone of the linearized vector by gel cutting.

(39) 4) The annealed double-stranded fragments obtained in step 2) were connected with the backbone of the lincarized vector obtained in step 3) at 16 C. for 1 h, cooled in ice bath for 30 min, followed by heat shock for 45 s, and then transformed into Top10 or DH5a competent cells, which were coated and grown on LB plates containing ampicillin, and single colonies were picked on the next day for culturing and sequencing using primer GAGGGCCTATTTCCCATGATT (SEQ ID No.16).

(40) 5) After culturing the correctly sequenced single colonies, plasmids were extracted and named recombinant plasmids pX458-pAPN-sgRNA-1 and pX458-pAPN-sgRNA-2, respectively, and then frozen at 20 C. for subsequent cell transfection. The plasmid extraction was carried out using the EndoFree Plasmid Maxi Kit (CoWin Biotech, CW2104M).

(41) The recombinant plasmid pX458-pAPN-sgRNA-1 was obtained by connecting the annealed double stranded DNA fragments set forth in SEQ ID No.5 and SEQ ID No.6 into the restriction endonuclease Bbs I site of the PX458 backbone vector.

(42) The recombinant plasmid pX458-pAPN-sgRNA-2 was obtained by connecting the annealed double stranded DNA fragments set forth SEQ ID No.7 and SEQ ID No.8 to the restriction endonuclease Bbs I site of the PX458 backbone vector.

(43) 2. Construction of Donor Vector

(44) 1) Construction of Donor-737 Vector

(45) The dsODN sequence 1 was connected into between the BamHI and Mlul restriction endonuclease sites in the PUC57 vector (GenScript Biotech Corporation, SD1176) to obtain the recombinant vector was obtained for sequencing validation. After culturing the correctly sequenced single colony, the plasmid was extracted and named Donor-737 for subsequent cell transfection. The plasmid extraction was carried out using the EndoFree Plasmid Maxi Kit (CoWin Biotech, CW2104M).

(46) 2) Construction of Donor-739 Vector

(47) The dsODN sequence 2 was connected into between the BamHI and Mlul restriction endonuclease sites in the PUC57 vector (GenScript Biotech Corporation, SD1176) to obtain the recombinant vector was obtained for sequencing validation. After culturing the correctly sequenced single colony, the plasmid was extracted and named Donor-739 for subsequent cell transfection. The plasmid extraction was carried out using the EndoFree Plasmid Maxi Kit (CoWin Biotech, CW2104M).

Example 2 Establishment and Functional Verification of the Overexpressed Porcine Ileal Epithelial Cells with Precise Modification of the Amino Acid at Position 737 in a pAPN Gene

(48) I. Establishment of the Overexpressed Porcine Ileal Epithelial Cells with Precise Modification of the Amino Acid at Position 737 in a pAPN Gene

(49) 1. The CDS sequence of wild-type pAPN gene (SEQ ID No.9) was connected into a PLVX vector (Qincheng Biotechnology Co., Ltd., QCP0424) to obtain a PLVX-WT recombinant vector.

(50) The CDS sequence of a pAPN gene (SEQ ID No.10) with precise modification of the amino acid at position 737 was connected into a PLVX vector (Qincheng Biotechnology Co., Ltd., QCP0424) to obtain a PLVX-737 recombinant vector.

(51) 2. On the day before electrotransfection, porcine ileal epithelial cells with pAPN gene knockout (Immortal Pig Intestinal-2I Knock Out, IPI-2I-KO) were recovered into 10 cm dishes, and the cell transfection could be performed until to about 80% confluence of cells.

(52) 3. PLVX-WT recombinant vector, PLVX-737 recombinant vector, and PLVX vector were electrotransfected into IPI-2I-KO cells to obtain successfully overexpressed cells with precise modification, named IPI-2I-WTOE, IPI-2I-737OE and IPI-2I-Vector respectively, for use as donor cells in subsequent TGEV infection.

(53) II. Functional Verification

(54) IPI-2I-WTOE, IPI-2I-737OE and IPI-2I-Vector cells obtained in above step I were tested for TGEV infection with specific steps of:

(55) 1. IPI-2I-WTOE, IPI-2I-737OE and IPI-2I-Vector cells were inoculated with TGEV virus strains (MOI=1), respectively.

(56) 2. Cells were collected at 12 h after infection, followed by extraction of cell proteins for detection of the expression of pAPN by Western blot. Meanwhile, IPI-2I-KO cells without virus inoculation were used as the Mock group.

(57) The results of pAPN protein detection were shown in FIG. 3, indicating that PAPNs were normally expressed in both the IPI-2I-WTOE and IPI-2I-737OE groups after virus inoculation.

(58) 3. Cells were collected at 12 h after infection, and washed 4-5 times with PBS, followed by extraction of RNA from cells for detection of the copy number of TGEV virus in cells by qRT-PCR. Meanwhile, IPI-2I-KO cells without virus inoculation were used as the Mock group.

(59) The qRT-PCR results were shown in FIG. 4, indicating that the copy number of TGEV genomic RNA in IPI-2I-737OE cells was significantly reduced (*** P<0.001), compared with IPI-2I-WTOE cells.

(60) 4. Cells were collected at 12 h after infection, followed by extraction of cell proteins for detection of the expression of TGEV virus by Western Blot. Meanwhile, IPI-2I-KO cells without virus inoculation were used as the Mock group.

(61) The infection amount of TGEV virus was shown in FIG. 5, indicating that the amount of TGEV infection in cells of the IPI-2I-737OE group was significantly reduced, compared with cells of IPI-2I-WTOE group.

(62) 5. Cells were collected at 12 h after infection, followed by detection of TGEV infection in cells by IFA. Meanwhile, IPI-2I-KO cells without virus inoculation were used as the Mock group.

(63) The IFA detection results were set forth in FIG. 6, indicating that IPI-2I-WT cells were infected with a large number of TGEVs after virus inoculation; and the amount of TGEV infection in IPI-2I-737OE cells was significantly reduced compared with IPI-2I-WT cells.

(64) In summary, the above results showed that the overexpressed porcine ileal epithelial cells with precise modification of amino acids at position 737 in a pAPN gene could effectively resist TGEV infection, indicating that the position 737 in the pAPN gene was a key site for TGEV infection, and the precise modification of the amino acid at position 737 in pAPN gene could effectively resist TGEV infection.

Example 3. Establishment and Functional Verification of the Monoclone from Porcine Ileal Epithelial Cells with Precise Modification of the Amino Acid at Position 739 in a pAPN Gene

(65) 1. The CDS sequence of wild-type pAPN gene (SEQ ID No.9) was connected into a PLVX vector to obtain a PLVX-WT recombinant vector.

(66) The CDS sequence of a pAPN gene (SEQ ID No.11) with precise modification of the amino acid at position 739 was connected into a PLVX vector to obtain a PLVX-739 recombinant vector.

(67) 2. On the day before electrotransfection, porcine ileal epithelial cells with pAPN gene knockout (Immortal Pig Intestinal-2I Knock Out, IPI-2I-KO) were recovered into 10 cm dishes, and the cell transfection could be performed until to about 80% confluence of cells.

(68) 3. PLVX-WT recombinant vector, PLVX-739 recombinant vector, and PLVX vector were electrotransfected into IPI-2I-KO cells to obtain successfully overexpressed cells with precise modification, named IPI-2I-WTOE, IPI-2I-739OE and IPI-2I-Vector respectively, for use as donor cells in subsequent TGEV infection.

(69) II. Functional Verification

(70) IPI-2I-WTOE, IPI-2I-739OE and IPI-2I-Vector cells obtained in above step I were tested for TGEV infection with the following specific steps

(71) 1. IPI-2I-WTOE, IPI-2I-739OE and IPI-2I-Vector cells were inoculated with TGEV virus strains (MOI=1), respectively.

(72) 2. Cells were collected at 12 h after infection, followed by extraction of cell proteins for detection of the expression of pAPN by Western Blot. Meanwhile, IPI-2I-KO cells without virus inoculation were used as the Mock group.

(73) The results of pAPN protein detection were shown in FIG. 7, indicating that PAPNs were normally expressed in both the IPI-2I-WTOE and IPI-2I-739OE groups after virus inoculation.

(74) 3. Cells were collected at 12 h after infection, and washed 4-5 times with PBS, followed by extraction of RNA from cells for detection of the copy number of TGEV virus in cells by qRT-PCR. Meanwhile, IPI-2I-KO cells without virus inoculation were used as the Mock group.

(75) The qRT-PCR results were shown in FIG. 8, indicating that the copy number of TGEV genomic RNA in IPI-2I-7390E cells was significantly reduced (*P<0.05), compared with IPI-2I-WTOE cells.

(76) 4. Cells were collected at 12 h after infection, followed by extraction of cell proteins for detection of the expression of TGEV virus by Western Blot. Meanwhile, IPI-2I-KO cells without virus inoculation were used as the Mock group.

(77) The infection amount of TGEV virus was shown in FIG. 9, indicating that there was no significant change in the amount of TGEV infection in cells of the IPI-2I-7390E group, compared with cells of IPI-2I-WTOE group.

(78) 5. Cells were collected at 12 h after infection, followed by detection of TGEV infection in cells by IFA. Meanwhile, IPI-2I-KO cells without virus inoculation were used as the Mock group.

(79) The IFA detection results were shown in FIG. 10, indicating that there was no significant change in the amount of TGEV infection in cells of the IPI-2I-7390E group, compared with cells of IPI-2I-WT group.

(80) In summary, the above results showed that the overexpressed porcine ileal epithelial cells with precise modification of amino acids at position 739 in the pAPN gene could not effectively resist TGEV infection, indicating that the position 739 in the pAPN gene was not a key site for TGEV infection, and the precise modification of the amino acid at position 739 in the pAPN gene could not effectively resist TGEV infection.

Example 4. Establishment of the Monoclone from Porcine Fibroblasts with Precise Modification of the Amino Acid at Position 737 in a pAPN Gene

(81) I. Preparation of Porcine Fetal Fibroblasts

(82) The head, tail, limbs, viscera, and bones from a pig embryo at 35 days old were removed, and the blood was cleaned. The fetus was continuously cut using an elbow ophthalmic scissor for 30 min to ensure sufficient fragmentation, and then the fetal tissue fragments were pipetted into a 15 mL centrifuge tube using the head-cut blue tip, into which 5 mL of complete culture medium were added, followed by natural settlement for a few minutes to remove the supernatant, and a few drops of fetal bovine serum were added to the lower tissue block, which was sucked out using a 15 cm curved glass Pasteur tube bent at 1 cm from the tip, and placed into two T75 culture bottles with the bottom facing upwards, followed by addition of 15 mL of complete culture medium to the opposite side. The culture bottles were carefully turned over after 6-8 h, so that the tissue block was immersed in the culture medium with refreshment every two days to obtain cells, which were frozen and stored for future use until filling the T75 culture bottles. In this process, pigs were fed in the base pig farm of the Beijing Institute of Animal Science of CAAS.

(83) II. Cell Transfection

(84) The primary porcine fetal fibroblasts were recovered into a 10 cm plate at the day before transfection, and the cell transfection could be performed until to about 80% confluence of cells. 5 g pX458-pAPN-sgRNA-1 plasmids prepared in Example 1, 5 g pX458-pAPN-sgRNA-2 plasmids prepared in Example 1 and 5 g Donor-737 plasmids prepared in Example 1 were co-transfected into porcine fetal fibroblasts with steps following strictly to the instructions of the Basic Primary Fibroblasts Nucleofector Kit (Lonza), and then the electrotransfected cells were transferred to a 6-well plate for culture.

(85) III. Flow Cytometric Sorting and Passage of Monoclonal Cells

(86) Cells were digested and collected into a tube for flow cytometry at 48 h after electroporation. Individual GFP positive cells were sorted using a flow cytometric sorter and cultured in a 96-well plate with refreshment of the culture medium every 3 days. Cells were passaged to a 48 well plate for culture until filling the 96-well plate, and then a portion of cells were taken for genome extraction and genotype identification until filling the 48 well plate.

(87) IV. Identification of Monoclonal Cells

(88) The picked monoclonal cells were identified with specific steps of: the extracted cell genomic DNA as a template, using pAPN-TY-F2 and pAPN-TY-R2 for PCR amplification to obtain PCR products with a size of 1443 bp.

(89) TABLE-US-00004 pAPN-TY-F2: (SEQIDNo.12) 5-CAAGGATTTGTGGAGGAGAA-3; pAPN-TY-R2: (SEQIDNO.13) 5-GCTGAGCGGAGTTTGTCG-3.

(90) The amplification condition for PCR was as follows: 94 C. for 5 min; 94 C. for 30 s, 62.6 C. for 30 s, 68 C. for 1 min 40 s, 34 cycles; 72 C. for 5 min. PCR products were sequenced by Beijing TianyiHuiyuan Company. Based on the results of sequencing, porcine fibroblasts with precise modification of the amino acid at position 737 in pAPN protein were screened as donor cells for nuclear transplantation.

(91) The results of sequencing showed that multiple strains of porcine fibroblasts with precise modification of the amino acid at position 737 in pAPN protein were successfully obtained in this example, and some results of sequencing for the positive cells were shown as FIG. 11.

Example 5. Preparation of a Genetically Edited Pig with Precise Modification of the Amino Acid at Position 737 in pAPN Gene by Somatic Cell Nuclear Transplantation Technology

(92) The positive cells obtained from homozygous gene editing in Example 3 were used as donor cells for nuclear transfer, and the enucleated porcine oocytes matured in vitro for 40 h were used as recipient cells for nuclear transfer. The donor cells for nuclear transfer were transferred into the oocytes, which were electrically fused and activated to construct recombinant cloned embryos. The well-developed cloned recombinant embryos were selected and surgically transplanted into the uterus of naturally estrous multiparous white sows for pregnancy. In this process, steps of surgical embryo transfer were as follows: the recipient sow was anesthetized by intravenous injection of Zoletil with a dosage of 5 mg/kg body weight. After anesthesia, the recipient sows were moved to an operating rack for supine fixation, followed by respiratory anesthesia (with a concentration of 3% to 4% isoflurane). An about 10 cm long of surgical incision was made at the midline of the abdomen of the recipient sow to expose ovaries, fallopian tubes, and uterus. An embryo transplantation glass tube was used to enter about 5 cm along the fimbria of the fallopian tubes, and the well-developed cloned recombinant embryos were transplanted to the junction between the ampulla and isthmus of the fallopian tubes. Embryos were regularly observed by the technicians after transplantation, and the pregnancy statuses of the recipient sows were examined by B-type ultrasound. Ear tissues were cut from piglets after birth, followed by extraction of genomic DNA, which was amplified by PCR using pAPN-TY-F2 and pAPN-TY-R2, and the products from PCR amplification were sequenced for genotype detection.

(93) The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented over a wide range of parameters, concentrations, and conditions. Although the present invention provides special Examples, it should be understood that further improvements can be made to the present invention. In summary, according to the principle of the present invention, the present application intends to include any changes, uses, or improvements to the present invention, including changes made outside the disclosed scope of the present application and using conventional techniques known in the art. According to the scope of the accompanying claims, some basic features can be applied.