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CN117987465B - Construction method of ten-gene editing xenogeneic organ transplantation donor pig - Google Patents

Construction method of ten-gene editing xenogeneic organ transplantation donor pig Download PDF

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CN117987465B
CN117987465B CN202410034211.0A CN202410034211A CN117987465B CN 117987465 B CN117987465 B CN 117987465B CN 202410034211 A CN202410034211 A CN 202410034211A CN 117987465 B CN117987465 B CN 117987465B
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魏红江
赵恒�
赵红业
王娇祥
角德灵
郭建雄
魏太云
徐凯祥
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Yunnan Agricultural University
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Abstract

The invention relates to a construction method of a ten-gene editing xenogeneic organ transplantation donor pig, belonging to the technical field of animal biology. In a wild type pig fetal fibroblast line, GGTA1, beta 4GalNT2 and CMAH genes are knocked out, hCD39, hCD46, hCD47, hCD55, hCD59, hTBM and hECR humanized genes are transferred, a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing cloned pig is constructed, and the genotype identification, mRNA and protein expression analysis of the cloned pig are further carried out, so that ten-gene editing xenogenic organ transplantation donor pigs are obtained. The invention solves the problems of hyperacute immune rejection reaction, antigen-antibody mediated immune rejection reaction, receptor immune cell activation, coagulation disorder and the like in the process of transplanting a xenogeneic organ by knocking out the pig antigen gene and transferring the gene into a humanized gene for combined modification, overcomes the problems of low productivity, low survival rate, low gene editing success rate and the like of the existing cloned pig, and has important application value for promoting preclinical research of the transplanting of the xenogeneic organ of a pig-non-human primate and clinical application of the transplanting of the xenogeneic organ of the pig-human.

Description

Construction method of ten-gene editing xenogeneic organ transplantation donor pig
Technical Field
The invention belongs to the technical field of animal biology, and particularly relates to a construction method of a ten-gene editing xenogeneic organ transplantation donor pig.
Background
Xenografts have been recognized as an effective way to address the gap between supply and demand for global human organ transplantation, and pig-human xenograft vegetation is known as the next major biomedical revolution.
The xenograft has the problems of rejection, inflammation control, coagulation disturbance regulation and the like, and in order to overcome the problems, the gene operation for the xenograft mainly comprises two purposes of (I) inactivating pig-derived xenogeneic antigens; the transgene (II) expresses human protection proteins including coagulation regulating factors, complement regulating factors, cellular immune response factors, anti-apoptosis and anti-inflammatory factors, etc. With the progress of gene editing technology in recent years, various gene editing donor pigs have been reported successively. The sub-clinical research results of pig-human xenograft at present also effectively prove that adding some other xenogeneic antigen gene knockouts and humanized gene expression on the basis of GGTA1 gene knockouts is beneficial to reducing immune rejection reaction generated by xenograft. Aiming at the main heterologous antigen genes and a large number of humanized gene expression modifications, the common problems of high difficulty in simultaneous editing of multiple gene combinations, low efficiency, low survival rate of cloned pigs subjected to multiple gene editing, uneven expression of target genes and the like are commonly existed in the application of the existing gene editing technology.
Disclosure of Invention
Aiming at the problems, the invention provides a construction method of a ten-gene editing xenogeneic organ transplantation donor pig, which is used for constructing a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing xenogeneic organ transplantation donor pig by utilizing a CRISPR/Cas9 gene editing technology and a piggyBac transposon technology and combining a somatic cell cloning technology aiming at the problem of immune rejection reaction commonality existing in pig-human xenogeneic organ transplantation, and has important value for promoting clinic application of pig-human xenogeneic organ transplantation.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
the first aspect of the invention provides a GGTA1, beta 4GalNT2 and CMAH gene sgRNA targeting vector, which is constructed by using CRISPR/Cas9 gene editing technology, wherein the sgRNA acting site is positioned at the 3 rd exon of the pig GGTA1 gene, the 2 nd exon of the beta 4GalNT2 gene and the 4 th exon of the CMAH gene.
Further, the above GGTA1, beta 4GalNT2, CMAH gene sgRNA targeting vector comprises: GGTA1-sgRNA1, GGTA1-sgRNA2, beta 4GalNT2-sgRNA1, beta 4GalNT2-sgRNA2, beta 4GalNT2-sgRNA3, CMAH-sgRNA1 and CMAH-sgRNA2; the nucleotide sequence of GGTA1-sgRNA1 is shown as SEQ ID NO. 1; the nucleotide sequence of GGTA1-sgRNA2 is shown as SEQ ID NO. 2; the nucleotide sequence of the beta 4GalNT2-sgRNA1 is shown as SEQ ID NO. 5; the nucleotide sequence of the beta 4GalNT2-sgRNA2 is shown as SEQ ID NO. 6; the nucleotide sequence of the beta 4GalNT2-sgRNA3 is shown as SEQ ID NO. 7; the nucleotide sequence of CMAH-sgRNA1 is shown as SEQ ID NO. 8; the nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO. 9.
Furthermore, the GGTA1, beta 4GalNT2 and CMAH gene sgRNA targeting vector is connected with a skeleton vector which is PX458-sgRNA-EGFP (Addgene no: 112220).
In a second aspect, the invention provides a recombinant plasmid for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome, wherein the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 10.
The third aspect of the invention provides a GGTA1 gene sgRNA targeting vector, wherein the sgRNA acting site is positioned on the 8 th exon of the pig GGTA1 gene, and the nucleotide sequence of GGTA1-sgRNA3 is shown as SEQ ID NO. 3; the nucleotide sequence of GGTA1-sgRNA4 is shown as SEQ ID NO. 4, and is respectively connected to a skeleton vector PX458-sgRNA-EGFP, and the gene sequence number of the skeleton vector is (Addgene No. 112220).
In a fourth aspect, the invention provides a recombinant plasmid for knocking out GGTA1 gene in pig genome, wherein the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 19.
In a fifth aspect, the invention provides an hCD39, hCD46, hCD55, hCD59 and hTBM humanized gene transfer expression vector, the nucleotide sequence of which is shown as SEQ ID NO. 18.
Further, the nucleotide sequence of hCD39 is shown as SEQ ID NO. 11; the nucleotide sequence of hCD46 is shown as SEQ ID NO. 12; the nucleotide sequence of hCD55 is shown as SEQ ID NO. 14; the nucleotide sequence of hCD59 is shown as SEQ ID NO. 15; hTBM has the nucleotide sequence shown in SEQ ID NO. 16.
The sixth aspect of the invention provides a hCD47, hEPCR humanized gene transfer expression vector, the nucleotide sequence of which is shown as SEQ ID NO. 20.
Further, the nucleotide sequence of hCD47 is shown as SEQ ID NO. 13; hEPCR has the nucleotide sequence shown in SEQ ID NO. 17.
In a seventh aspect the invention provides a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten gene-edited porcine fetal fibroblast line.
1) The GGTA1, beta 4GalNT2 and CMAH gene sgRNA targeting vector or recombinant plasmid for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome and hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes are transferred into expression vectors to be co-transfected into wild type pig fetal fibroblasts under the action of transposase, and the eight-gene editing positive monoclonal cell line obtained through screening is the GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing pig fibroblast line, and the eight-gene editing pig fetal fibroblast line is constructed by taking the recombinant plasmid as donor cells for somatic cell nuclear transplantation.
2) The GGTA1 gene sgRNA vector or the recombinant plasmid for knocking out the GGTA1 gene in the pig genome and hCD47, hEPCR humanized genes are transferred into an expression vector to be co-transfected into a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing pig fetus fibroblast line under the action of transposase, and ten-gene editing positive monoclonal cell lines are obtained through screening, namely the GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing pig fetus fibroblast line.
The eighth aspect of the present invention provides a construction method for a ten-gene editing xenogeneic organ transplantation donor pig, comprising the specific steps of:
(1) Construction of recombinant plasmid for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome based on CRISPR/Cas9 gene editing system
The sgRNA targeting vector is designed aiming at the 3 rd exon of GGTA1 gene, the 2 nd exon of beta 4GalNT2 gene and the 4 th exon of CMAH gene in pig genome and is respectively connected to a skeleton vector PX458-sgRNA-EGFP (Addgene no: 112220) to obtain recombinant plasmids for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome.
The sgRNA targeting vector comprises GGTA1-sgRNA1, GGTA1-sgRNA2, beta 4GalNT2-sgRNA1, beta 4GalNT2-sgRNA2, beta 4GalNT2-sgRNA3, CMAH-sgRNA1 and CMAH-sgRNA2; wherein the nucleotide sequence of GGTA1-sgRNA1 is shown as SEQ ID NO. 1; the nucleotide sequence of GGTA1-sgRNA2 is shown as SEQ ID NO. 2; the nucleotide sequence of the beta 4GalNT2-sgRNA1 is shown as SEQ ID NO. 5; the nucleotide sequence of the beta 4GalNT2-sgRNA2 is shown as SEQ ID NO. 6; the nucleotide sequence of the beta 4GalNT2-sgRNA3 is shown as SEQ ID NO. 7; the nucleotide sequence of CMAH-sgRNA1 is shown as SEQ ID NO. 8; the nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO. 9.
The nucleotide sequence of the recombinant plasmid for knocking out GGTA1, beta 4GalNT2 and CMAH genes in the pig genome is shown as SEQ ID NO. 10.
(2) Based on piggyBac transposon system, humanized genes of hCD39, hCD46, hCD55, hCD59 and hTBM are constructed to transfer into expression vector, and the nucleotide sequence is shown as SEQ ID NO. 18.
Further, the nucleotide sequence of hCD39 is shown as SEQ ID NO. 11; the nucleotide sequence of hCD46 is shown as SEQ ID NO. 12; the nucleotide sequence of hCD55 is shown as SEQ ID NO. 14; the nucleotide sequence of hCD59 is shown as SEQ ID NO. 15; hTBM has the nucleotide sequence shown in SEQ ID NO. 16.
(3) Based on CRISPR/Cas9 gene editing system, a secondary editing GGTA1 gene sgRNA targeting vector is constructed and connected to a skeleton vector PX458-sgRNA-EGFP (Addgene no: 112220).
The GGTA1 gene sgRNA targeting vector comprises GGTA1-sgRNA3 and GGTA1-sgRNA4, and the nucleotide sequence of the GGTA1-sgRNA3 is shown as SEQ ID NO. 3; the nucleotide sequence of GGTA1-sgRNA4 is shown as SEQ ID NO. 4, and the nucleotide sequence of the recombinant plasmid for knocking out GGTA1 gene in pig genome is shown as SEQ ID NO. 19.
(4) Based on piggyBac transposon system, hCD47, hEPCR humanized gene transfer expression vector is constructed, and the nucleotide sequence is shown as SEQ ID NO. 20.
Further, the nucleotide sequence of hCD47 is shown as SEQ ID NO. 13; hEPCR has the nucleotide sequence shown in SEQ ID NO. 17.
(5) Construction of GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR Ten Gene-edited pig fibroblast line
1) The recombinant plasmids for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome and hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes are transferred into an expression vector to be co-transfected into a wild type pig fetal fibroblast line under the action of transposase, and the positive pig fibroblast line is edited by screening to obtain GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight genes through monoclonal cell genotype identification, and somatic cell nuclear transplantation is carried out by taking the positive pig fibroblast line as a donor cell, so that the GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight genes are constructed to edit pig fetuses and a fetal fibroblast line is established.
2) The recombinant plasmid for knocking out GGTA1 gene in pig genome and hCD47, hEPCR humanized gene are transferred into expression vector and co-transfected into GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing pig fetus fibroblast line under the action of transposase, and through single cell cloning genotype identification, GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing pig fibroblast line is obtained.
Furthermore, the blood type of the wild type pig fetal fibroblast line is O-shaped, the O-shaped blood compatibility is good, and the immune rejection reaction caused by the blood type incompatibility of the xenogeneic organ transplantation can be reduced.
Further, the transfection method comprises liposome transfection or electrotransfection; electrotransfection is preferred.
(6) Somatic cell nuclear transfer and embryo transfer
The GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hT BM/hEPCR ten-gene editing pig fibroblast cell line is taken as donor cells, somatic cell nuclear transplantation is carried out, GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEP CR ten-gene editing pig cloned embryo is constructed, the cloned embryo is further transplanted to an oestrus surrogate sow to develop in vivo, the GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing pig is obtained by natural delivery after 114 days of gestation, or the fetus is taken out for genotyping and establishing a fibroblast cell line during gestation.
Further, the donor cells may be GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing positive monoclonal porcine fibroblasts or cloned fetal fibroblasts or gram Long Zhu fibroblasts, preferably cloned porcine fetal fibroblasts.
(7) Genotyping and phenotyping cloned pigs
The cloned pig genome DNA is extracted, the cloned pig genotype is identified by PCR, T7ENI and Sanger sequencing technology, and the gene mRNA and protein expression level in the tissues and organs of the cloned pig are detected by Westernblotting, immunofluorescence staining, flow cytometry, HE staining and cross-matching experimental molecular biology technology, so as to obtain the GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing cloned pig.
(8) The GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing cloned pigs were produced in batches by continuous cloning technology.
The GGTA1, beta 4GalNT2 and CMAH gene sgRNA targeting vector, and/or recombinant plasmids for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome, and/or hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes are transferred into expression vector, and/or GGTA1 gene sgRNA targeting vector, and/or recombinant plasmids for knocking out GGTA1 genes in genome, and/or hCD47 and hEPCR humanized genes are transferred into expression vector, and/or GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD 55/hCD59/hTBM/hEP CR ten-gene edited pig fetal fibroblast line, and/or a construction method of ten-gene edited xenogeneic organ transplantation donor pigs is applied in the field of biomedical research.
Inactivation of GGTA1 (producing α -Gal antigen), CMAH (producing Neu5Gc antigen) and β4galnt2 (producing Sda antigen) in pigs effectively reduces the occurrence of xenogeneic kidney transplantation antigen-antibody mediated hyperacute and acute rejection. Knocking out the CMAH and β4galnt2 genes based on GGTA1 gene inactivation would be more effective in alleviating immune rejection following xenografts. Compared with the GGTA1 single gene and GGTA1/CMAH double-gene deleted cells, the combination of the GGTA 1/CMAH/beta 4GALNT2 triple-gene knockout pig cells with heterologous IgM and IgG in vitro is obviously reduced, which suggests that the GGTA 1/CMAH/beta 4GalNT2 triple-gene knockout pig has lower immunogenicity and can promote the long-term survival of the xenograft.
Expression of human complement regulatory proteins (CD 55, CD59, CD 46), thrombomodulin (CD 39, TBM, EPCR), cellular immune response factors (CD 47) and anti-apoptotic modulation in xenograft donor pigs resulted in deregulation of complement, coagulation and inflammatory pathways due to species incompatibility. Compared with GTKO monogenic editing pigs, the survival time of the GTKO/hCD55 gene editing pig kidney transplanted to a non-human primate can reach 499 days, and the method suggests that when the heterologous antigen gene is knocked out, human complement regulatory proteins such as CD46, CD55 and CD59 are transferred into the pig body, so that rejection caused by complement activation can be effectively relieved, and the survival time of the heterologous kidney transplanted is prolonged.
The research result effectively proves that the knockout of the CMAH and beta 4GalNT2 xenogenic antigen genes and the over-expression of the humanized genes on the basis of the inactivation of GGTA1 are helpful for reducing the immune rejection reaction generated by the xenogenic organ transplantation and promoting the long-term survival of the xenogenic grafts.
The invention has the beneficial effects that:
1. the invention designs a plurality of different sgRNA nucleotide series, is used for knocking out a targeting vector of genes, constructs an expression vector for editing a plurality of genes simultaneously, and overcomes the technical problems of low efficiency of multi-gene editing, low birth efficiency of cloned pigs after multi-gene modification, difficult survival and the like commonly existing in the CRISPR/Cas9 technology while improving the success rate of gene targeting.
2. The invention constructs a secondary editing gene targeting vector, overcomes the problem of low efficiency of the existing multi-gene editing, carries out secondary editing on the basis of eight-gene editing, constructs a ten-gene editing fibroblast line, improves the success rate of gene editing, and lays a theoretical foundation for multi-gene combination modification.
3. The invention discloses a construction method of a ten-gene editing xenogeneic organ transplantation donor pig, which solves the problems of immune rejection reaction, inflammatory reaction, coagulation disorder and the like in the xenogeneic organ transplantation process by inactivating three saccharide antigen genes in a pig genome and over-expressing seven humanized genes in the pig genome. The GTKO/beta 4GalNT2KO/CMAHKO/hCD46/hCD55/hCD59/hCD39/hTBM ten-gene editing xenogeneic organ transplantation donor pigs constructed by the invention furthest solve the common problems of hyperacute immune rejection reaction, antigen-antibody mediated immune rejection reaction, coagulation disorder and recipient immune cell activation in the xenogeneic organ transplantation process, lays a foundation for further developing donor pigs suitable for xenogeneic transplantation of different tissues such as heart, kidney, liver and skin, and has important prospects for preclinical research of developing pig-non-human primate xenogeneic organ transplantation and clinical application of pig-human xenogeneic organ transplantation.
Drawings
FIG. 1 is a schematic diagram showing construction of GTKO/CMAHKO/beta 4GalNT2KO/hCD39/hCD46/hCD55/hCD59/hTBM gene editing vector;
in FIG. 1, A is a schematic diagram of GGTA1 gene targeting;
FIG. 1B is a schematic diagram of CMAH gene targeting;
C in FIG. 1 is a schematic diagram of beta 4GalNT2 gene targeting;
d in FIG. 1 is a schematic diagram of the construction of hCD39/hCD46/hCD55/hCD59/hTBM gene editing vector;
FIG. 2 shows the sequencing results of the single cell clone GGTA1 gene Sanger;
FIG. 3 shows the results of Sanger sequencing of the single cell clone CMAH gene;
FIG. 4 shows the results of Sanger sequencing of the single cell clone beta 4GalNT2 gene;
FIG. 5 shows the PCR identification result of single cell clone transferred hCD39/hCD46/hCD55/hCD59/hTBM gene;
FIG. 6 shows a GTKO/CMAHKO/beta 4GalNT2KO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing cloned pig fetus;
FIG. 7 shows the result of PCR identification of cloned pig fetus hCD39/hCD46/hCD55/hCD55/hTBM gene transfer;
FIG. 8 shows the results of PCR identification of cloned pig fetal GTKO/CMAHKO/beta 4GalNT2KO gene knockout;
FIG. 9 shows the results of the cloned porcine fetal GTKO/CMAHKO/beta 4GalNT2KO gene knockout Sanger sequencing;
FIG. 10 is a schematic diagram of the construction of a GTKO/hCD47/hEPCR gene-editing vector;
FIG. 10A is a schematic diagram of secondary targeting of GGTA1 gene;
FIG. 10B is a schematic diagram of the construction of hCD47/hEPCR gene editing vector.
FIG. 11 shows the PCR identification result of single cell clone secondary editing GGTA1 gene;
FIG. 12 shows the results of Sanger sequencing of the PCR product of the single cell clone double edited GGTA1 gene;
FIG. 13 shows the PCR identification result of the single cell clone transferred hCD47/hEPCR gene;
FIG. 14 shows the PCR identification results of ten gene editing cloned pig fetuses of GTKO/CMAHKO/beta 4GalNT2KO/hCD39/hCD46/hCD47/hCD55/hCD 59/hTBM/hEPCR;
FIG. 14A is a photograph of 5 surviving cloned pig fetuses;
FIG. 14B shows the result of PCR identification of GGTA1 gene;
FIG. 14C shows the results of PCR identification of the CMAH gene;
FIG. 14D shows the result of PCR identification of the beta 4GalNT2 gene;
FIG. 14E shows the results of PCR identification of hCD47 gene;
F in FIG. 14 is hEPCR gene PCR identification results;
FIG. 14G shows the results of PCR identification of hCD55/hCD59 gene;
H in FIG. 14 is the PCR identification result of hCD46 gene;
FIG. 14I shows the results of PCR identification of hCD39/hTBM gene;
FIG. 15 shows the results of Sanger sequencing identification of 5 foetal secondary editing GGTA1 genes;
FIG. 16 shows the ten genes GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR edited into cloned pigs;
FIG. 17 shows the results of PCR identification of GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing cloned pigs;
FIG. 17A shows the result of PCR identification of GGTA1 gene;
FIG. 17B shows the results of PCR identification of the CMAH gene;
C in FIG. 17 is the result of PCR identification of the beta 4GalNT2 gene;
d in FIG. 17 is the result of PCR identification of hCD47 gene;
e in FIG. 17 is hEPCR gene PCR identification results;
F in FIG. 17 is the PCR identification result of hCD55/hCD59 gene;
g in FIG. 17 is the result of PCR identification of hCD46 gene;
FIG. 17 shows the result of PCR identification of hTBM/hCD39 gene;
FIG. 18 shows the results of Sanger sequencing identification of cloned pig GGTA1 gene editing;
FIG. 19 shows the expression level of hCD39 gene mRNA in cloned pig heart, kidney, liver and lung tissues;
FIG. 20 shows the expression level of hCD46 gene mRNA in cloned pig heart, kidney, liver and lung tissues;
FIG. 21 shows the expression level of hCD55 gene mRNA in cloned pig heart, kidney, liver and lung tissue;
FIG. 22 shows the hCD59 gene mRNA expression levels in cloned pig heart, kidney, liver and lung tissues;
FIG. 23 shows the expression level of hCD47 gene mRNA in cloned pig heart, kidney, liver and lung tissues;
FIG. 24 shows the level of hEPCR gene mRNA expression in cloned porcine heart, kidney, liver, lung tissue;
FIG. 25 shows the expression level of hTBM gene mRNA in cloned pig heart, kidney, liver, lung tissue;
FIG. 26 shows immunofluorescence assay results of the expression level of the GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hTBM gene in cloned pig tissue;
FIG. 27 shows immunofluorescence assay results of hCD46/hCD47/hCD55/hCD59/hEPCR gene expression levels in cloned pig tissues.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding by the skilled person.
Example 1: construction of ten-gene editing targeting vector
(1) Construction of GTKO/beta 4GalNT2KO/CMAHKO three-gene knockout expression vector based on CRISPR/Cas9 gene editing technology
Nucleotide sequences of the pig GGTA1 (Gene ID: 396733), beta 4GalNT2 (Gene ID: 100621328) and CMAH (Gene ID: 396918) genes were found in NCBI database, and recombinant plasmids (SEQ ID NO: 10) for GGTA1, beta 4GalNT2-sgRNA and CMAH genes were obtained by designing and screening for targeting sgRNA sites using on-line software (http:// crispor. Tefor. Net /) for exon 3 (FIG. 1A), exon 2 of the beta 4GalNT2 Gene (FIG. 1C) and exon 4 of the CMAH Gene, respectively, and ligating the sgRNA sequences to the backbone vector PX-sgRNA-EGFP (Addgene NO: 112220).
(2) Construction of hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR humanized Gene transfer expression vector based on piggyBac System
Constructing an hCD39, hCD46, hCD55, hCD59 and hTBM humanized gene transfer expression vector and an hCD47 and hEPCR humanized gene transfer expression vector based on a piggyBac transposon system; the nucleotide sequence of hCD39 is shown as SEQ ID NO. 11; the nucleotide sequence of hCD46 is shown as SEQ ID NO. 12; the nucleotide sequence of hCD47 is shown as SEQ ID NO. 13; the nucleotide sequence of hCD55 is shown as SEQ ID NO. 14; the nucleotide sequence of hCD59 is shown as SEQ ID NO. 15; hTBM has the nucleotide sequence shown in SEQ ID NO. 16; hEPCR has the nucleotide sequence shown in SEQ ID NO. 17. The nucleotide sequences of the transposons respectively connected to the backbone vector PiggyBac are shown as SEQ ID No. 18.
Further constructing a secondary editing GGTA1 gene targeting vector GGTA1-sgRNA3 (SEQ ID NO: 3) and GGTA1-sgRNA4 (SEQ ID NO: 4) aiming at the 8 th exon of the pig GGTA1 gene, and connecting the secondary editing GGTA1 gene targeting vector GGTA1-sgRNA4 to a skeleton vector PX458-sgRNA-EGFP (Addgene NO: 112220), wherein the nucleotide sequence of a recombinant plasmid of the secondary editing GGTA1 gene targeting vector GGTA1-sgRNA is shown as SEQ ID NO: 19; the hCD47/hEPCR humanized gene is transferred into expression vector, and its nucleotide sequence is shown in SEQ ID NO. 20.
Example 2: construction of ten-gene editing cloned pig
The target sgRNA sites were designed and screened for the 3 rd exon (FIG. 1A) of the pig GGTA1 Gene (Gene ID: 396733), the 2 nd exon (FIG. 1C) of the. Beta. 4GalNT2 Gene (Gene ID: 100621328), the 4 th exon (FIG. 1B) of the CMAH Gene (Gene ID: 396918) using on-line software (http:// crispor.tefor.net /), GGTA1-sgRNA1(SEQ ID NO:1)、GGTA1-sgRNA2(SEQ ID NO:2)、β4GalNT2-sgRNA1(SEQ ID NO:5)、β4GalNT2-sgRNA2(SEQ ID NO:6)、β4GalNT2-sgRNA3(SEQ ID NO:7)、CMAH-sgRNA1(SEQ ID NO:8)、CMAH–sgRNA2(SEQ ID NO:9) targeting vectors (FIG. 1A-C) were obtained, respectively, and the sgRNA sequences were ligated to the backbone vector PX458-sgRNA-EGFP (Addgene NO: 112220), obtaining recombinant plasmids (SEQ ID NO: 10) for knocking out GGTA1,. Beta. 4GalNT2, CMAH genes in the pig genome. Based on the piggyBac system, in the first step, hCD39 (SEQ ID NO: 11), hCD46 (SEQ ID NO: 12), hCD55 (SEQ ID NO: 14), hCD59 (SEQ ID NO: 15), hTBM (SEQ ID NO: 16) gene nucleotide sequences were ligated to the piggyBac transposon vector (D in FIG. 1), and hCD39/hCD46/hCD55/hCD59/hTBM humanized gene was transferred into the expression vector (SEQ ID NO: 18) (D in FIG. 1); secondly, constructing a secondary editing GGTA1 gene targeting vector GGTA1-sgRNA3 (SEQ ID NO: 3) and GGTA1-sgRNA4 (SEQ ID NO: 4) aiming at an 8 th exon of a pig GGTA1 gene (A in fig. 10), and connecting the secondary editing GGTA1-sgRNA3 and the GGTA1-sgRNA4 to a skeleton vector PX458-sgRNA-EGFP (Addgene NO: 112220), wherein the nucleotide sequence of a recombinant plasmid of the secondary editing GGTA1 gene targeting vector GGTA1-sgRNA is shown as SEQ ID NO: 19; hCD47 (SEQ ID NO: 8), hEPCR (SEQ ID NO: 12) humanized gene was transferred into an expression vector, the nucleotide sequence of which is shown as SEQ ID NO:20 (B in FIG. 10).
The GGTA1, beta 4GalNT2, CMAH gene sgRNA targeting vector and hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes are transferred into an expression vector to be transfected into a wild type pig fetal fibroblast line together under the action of transposase, after the maintenance and screening of the puromycin are carried out for 48 hours, 49 monoclonal cells are obtained after extremely diluted culture (figure 5), genotype identification results show that GGTA1 gene mutation is carried out on 10 monoclonal cells (figure 2), CMAH gene mutation is carried out on 4 monoclonal cells (figure 3), beta 4GalNT2 gene mutation is carried out on 6 monoclonal cells (figure 4), 4 GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing positive monoclonal cell line is successfully obtained, somatic cell transplantation construction embryo clone is carried out on the donor cells by using C2#, C3#, C4#, C5# positive monoclonal cell line as donor cells, and the embryo clone is carried out on the pig embryo, and the embryo is taken out when the embryo is transplanted into a pregnant pig fetal embryo line (figure 33-hCD 33/hCD 39/hCD46/hCD 34) in the human embryo, and the result is shown as the result of establishing the human embryo line for the human embryo is taken out in the human embryo, and the human embryo is taken out in the human embryo.
The nucleotide sequence of hCD47, hEPCR humanized genes was further ligated to piggyBac transposon vector (FIG. 10B) using on-line software (http:// crispor.tefor.net /) to design and screen for secondary targeting of the sgRNA site of the GGTA1 Gene and ligating the sgRNA sequence to the backbone vector to obtain PX458-GGTA1-sgRNA3/4 (SEQ ID NO: 19) (FIG. 10A), based on the piggyBac system, and hCD47, hEPCR humanized genes were constructed into expression vectors (SEQ ID NO: 20).
Transferring the secondary editing GGTA1 gene sgRNA targeting vector and hCD47, hEPCR humanized genes into an expression vector, co-transfecting the expression vector to No.1 GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing cloned pig fetal fibroblasts under the action of transposase, screening puromycin for 48 hours, obtaining 58 single cell clones altogether after extremely diluting and culturing, performing GGTA1 gene PCR amplification (FIG. 11), simultaneously selecting 16 single cell clones for Sanger sequencing, showing that 4 single cell clones are double allelic mutation of GGTA1 genes (FIG. 12), successfully transferring 15 single cell clones into the gene hCD47/hEPCR (FIG. 13), further performing somatic cell nuclear transplantation by using a C3 positive single cell line as a donor cell to construct a cloned embryo, and transplanted into a oestrus surrogate sow, 5 fetuses (A in FIG. 14) are taken out for genotyping at 33 days of gestation, GGTA1 (B in FIG. 14), CMAH (C in FIG. 14), beta 4GalNT2 (D in FIG. 14) are shown to be mutated, GGTA1 genes are double allele mutations (FIG. 15), hCD47 (E in FIG. 14), hEPCR (F in FIG. 14), hCD55/hCD59 (G in FIG. 14), hCD46 (H in FIG. 14) and hCD39/hTBM (I in FIG. 14) are successfully transferred, cloned pig fetuses are successfully edited to 100%, and a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene edited pig fetal fibroblast line is established. The cloned embryo is constructed by taking a No. 3 GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing pig fetus fibroblast line as a donor cell for somatic cell nuclear transplantation, and the cloned embryo is transplanted to 49 pregnant sows together, and after 114 days, 64 ten-gene editing cloned piglets are successfully obtained after delivery, and 35 piglets survive and have the survival rate of 54.69 percent (figure 16).
Example 3: genotyping and phenotyping ten-gene editing cloned pigs
The obtained cloned pigs were subjected to genotyping, and the results showed ten-gene editing cloned pigs of GTKO (a in fig. 17, fig. 18), β4galnt2KO (C in fig. 17), CMAHKO (B in fig. 17), hCD39 (H in fig. 17), hCD46 (G in fig. 17), hCD47 (D in fig. 17), hCD55 (F in fig. 17), hCD59 (F in fig. 17), hTBM (H in fig. 17), hEPCR (E in fig. 17). The expression of 7 transgenic humanized genes in heart, kidney and liver tissues of 3-ten-gene-edited piglets was detected by q-PCR using human umbilical cord endothelial cells as a positive control, and the mRNA levels of hCD39 (fig. 19), hCD46 (fig. 20), hCD55 (fig. 21), hCD59 (fig. 22), hCD47 (fig. 23), hEPCR (fig. 24) and hTBM (fig. 25) genes in heart and kidney of ten-gene-edited piglets were significantly increased compared to wild-type pigs. Further, the knockout of 3 heterogeneous antigens and the expression of 7 humanized proteins in the cloned pig tissues are verified by 4-head ten-gene editing cloned pig immunofluorescence staining, and compared with the wild type, the three saccharide antigens GGTA1, CMAH and beta 4GalNT2 genes in the ten-gene editing pig tissues are successfully inactivated (figure 26), hTBM/hCD39 (figure 26), hCD46/hCD47/hCD55/hCD59/hEPCR (figure 27) are expressed, which indicates that GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM are expressed
The construction of hEPCR ten gene editing cloned pigs was successful.
Aiming at the common problem of immune rejection in xenogeneic organ transplantation, ten-gene editing xenogeneic organ transplantation donor pigs constructed by the invention improve the production efficiency and survival rate of cloned pigs while improving the polygene combination modification efficiency. On the basis of ten-gene editing xenograft donor pigs, the method can further aim at the scientific problems faced by different organ xenograft, adopts different gene editing strategies to carry out related gene modification, and has important application value for developing effective xenograft donor pigs through polygene combination modification.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (5)

  1. A gtko/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene edited porcine fetal fibroblast cell line, characterized in that:
    The preparation method of the ten-gene editing pig fetus fibroblast line comprises the following steps:
    1) Transferring GGTA1, beta 4GalNT2 and CMAH gene sgRNA targeting vectors or recombinant plasmids for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome and hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes into wild type pig fetal fibroblasts under the action of transposase, and screening to obtain eight-gene editing positive monoclonal cell lines, namely GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing pig fibroblast cell lines, and carrying out somatic cell nuclear transfer by taking the recombinant plasmids as donor cells to construct GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing pig fetal fibroblast cell lines;
    the GGTA1, beta 4GalNT2 and CMAH gene sgRNA targeting vector is constructed by utilizing CRISPR/Cas9 gene editing technology, and the sgRNA acting site is positioned at the 3 rd exon of the pig GGTA1 gene, the 2 nd exon of the beta 4GalNT2 gene and the 4 th exon of the CMAH gene;
    The nucleotide sequence of the recombinant plasmid for knocking out GGTA1, beta 4GalNT2 and CMAH genes in the pig genome is shown as SEQ ID NO. 10;
    The hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes are transferred into an expression vector, and based on a piggyBac transposon system, the hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes are constructed and transferred into the expression vector, and the nucleotide sequence of the humanized genes is shown as SEQ ID NO. 18;
    2) Transferring GGTA1 gene sgRNA targeting vector or recombinant plasmid for knocking out GGTA1 gene in genome and hCD47, hEPCR humanized gene into expression vector to co-transfect into GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing pig fetus fibroblast cell line under the action of transposase, screening to obtain ten-gene editing positive monoclonal cell line, namely GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD 59/hTBM/hEPCR ten-gene editing pig fetus fibroblast line;
    The sgRNA targeting vector of the GGTA1 gene, the sgRNA acting site is positioned on the 8 th exon of the GGTA1 gene of the pig, and the sgRNA targeting vector comprises: GGTA1-sgRNA3 and GGTA1-sgRNA4; the nucleotide sequence of GGTA1-sgRNA3 is shown as SEQ ID NO. 3; the nucleotide sequence of GGTA1-sgRNA4 is shown as SEQ ID NO:4, and is respectively connected to a skeleton vector PX458-sgRNA-EGFP, and the gene sequence of the skeleton vector is as follows: addgene no:112220;
    The nucleotide sequence of the recombinant plasmid for knocking out the GGTA1 gene in the genome is shown as SEQ ID NO. 19;
    the nucleotide sequences of the hCD47 and hEPCR humanized genes transferred into the expression vector are shown as SEQ ID NO. 20.
  2. 2. The GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten gene-edited porcine fetal fibroblast cell line according to claim 1, wherein: the GGTA1, beta 4GalNT2 and CMAH gene sgRNA targeting vector comprises: GGTA1-sgRNA1, GGTA1-sgRNA2, beta 4GalNT2-sgRNA1, beta 4GalNT2-sgRNA2, beta 4GalNT2-sgRNA3, CMAH-sgRNA1 and CMAH-sgRNA2; the nucleotide sequence of GGTA1-sgRNA1 is shown as SEQ ID NO. 1; the nucleotide sequence of GGTA1-sgRNA2 is shown as SEQ ID NO. 2; the nucleotide sequence of the beta 4GalNT2-sgRNA1 is shown as SEQ ID NO. 5; the nucleotide sequence of the beta 4GalNT2-sgRNA2 is shown as SEQ ID NO. 6; the nucleotide sequence of the beta 4GalNT2-sgRNA3 is shown as SEQ ID NO. 7; the nucleotide sequence of CMAH-sgRNA1 is shown as SEQ ID NO. 8; the nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO. 9.
  3. 3. A construction method of a ten-gene editing xenogeneic organ transplantation donor pig is characterized by comprising the following steps: the method comprises the following specific steps:
    (1) Construction of recombinant plasmid for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome based on CRISPR/Cas9 gene editing system
    Designing sgRNA targeting vectors aiming at the 3 rd exon of GGTA1 genes, the 2 nd exon of beta 4GalNT2 genes and the 4 th exon of CMAH genes in pig genomes, and respectively connecting the sgRNA targeting vectors to skeleton vectors to obtain recombinant plasmids for knocking out the GGTA1, beta 4GalNT2 and CMAH genes in pig genomes;
    The sgRNA targeting vector comprises GGTA1-sgRNA1, GGTA1-sgRNA2, beta 4GalNT2-sgRNA1, beta 4GalNT2-sgRNA2, beta 4GalNT2-sgRNA3, CMAH-sgRNA1 and CMAH-sgRNA2; wherein the nucleotide sequence of GGTA1-sgRNA1 is shown as SEQ ID NO. 1; the nucleotide sequence of GGTA1-sgRNA2 is shown as SEQ ID NO. 2; the nucleotide sequence of the beta 4GalNT2-sgRNA1 is shown as SEQ ID NO. 5; the nucleotide sequence of the beta 4GalNT2-sgRNA2 is shown as SEQ ID NO. 6; the nucleotide sequence of the beta 4GalNT2-sgRNA3 is shown as SEQ ID NO. 7; the nucleotide sequence of CMAH-sgRNA1 is shown as SEQ ID NO. 8; the nucleotide sequence of CMAH-sgRNA2 is shown as SEQ ID NO. 9;
    The nucleotide sequences of recombinant plasmids for knocking out GGTA1, beta 4GalNT2 and CMAH genes in the pig genome are shown in SEQ ID NO. 10;
    (2) Constructing hCD39, hCD46, hCD55, hCD59 and hTBM humanized gene transfer expression vectors based on piggyBac transposon system, wherein the nucleotide sequence of the humanized gene transfer expression vectors is shown as SEQ ID NO. 18;
    (3) Based on a CRISPR/Cas9 gene editing system, constructing a secondary edited GGTA1 gene sgRNA targeting vector and connecting the targeting vector to a skeleton vector PX458-sgRNA-EGFP, wherein the gene sequence number is as follows: addgene no:112220;
    GGTA1 gene sgRNA targeting vectors include GGTA1-sgRNA3 and GGTA1-sgRNA4; the nucleotide sequence of GGTA1-sgRNA3 is shown as SEQ ID NO. 3, and the nucleotide sequence of GGTA1-sgRNA4 is shown as SEQ ID NO. 4; the nucleotide sequence of the recombinant plasmid for knocking out GGTA1 gene in pig genome is shown as SEQ ID NO. 19;
    (4) Constructing hCD47 and hEPCR humanized gene transfer expression vectors based on piggyBac transposon system, wherein the nucleotide sequence of the hCD47 and hEPCR humanized gene transfer expression vectors is shown as SEQ ID NO. 20;
    (5) Construction of GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR Ten-gene-edited porcine fetal fibroblast line
    1) Transferring recombinant plasmids for knocking out GGTA1, beta 4GalNT2 and CMAH genes in pig genome and hCD39, hCD46, hCD55, hCD59 and hTBM humanized genes into an expression vector to be co-transfected into a wild type pig fetal fibroblast line under the action of transposase, carrying out genotype identification on monoclonal cells, screening to obtain a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing positive pig fibroblast line, and carrying out somatic cell nuclear transplantation by taking the positive pig fibroblast line as a donor cell to construct a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing pig fetus and establishing a fibroblast line;
    2) Transferring recombinant plasmids for knocking out GGTA1 genes in pig genome and hCD47, hEPCR humanized genes into an expression vector, and co-transfecting the recombinant plasmids into a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD55/hCD59/hTBM eight-gene editing pig fetus fibroblast line under the action of transposase, and carrying out single-cell cloning genotype identification to obtain a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing pig fetus fibroblast line;
    (6) Somatic cell nuclear transfer and embryo transfer
    Taking GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hT BM/hEPCR ten-gene edited pig fetus fibroblast line as donor cells, performing somatic cell nuclear transfer, constructing GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene edited cloned embryo, further transplanting the cloned embryo into a pregnant sow in oestrus to develop, obtaining GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene edited cloned pig during pregnancy, taking out the fetus for genotyping and establishing a fibroblast line;
    (7) Genotyping and phenotyping cloned pigs
    Extracting cloned piglet genome DNA, identifying cloned pig genotypes by PCR, T7ENI and Sanger sequencing technologies, detecting gene mRNA and protein expression levels in cloned piglet tissues and organs by Western blotting, immunofluorescence staining, flow cytometry, HE staining and cross-matching experimental molecular biology technologies, and obtaining GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD 47/hCD 55/hCD59/hTBM/hEPCR ten-gene editing cloned pigs;
    (8) The GTKO/beta 4GalNT2KO/CMAHKO/hCD 39/hCD 46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene editing cloned pigs can be produced in batches by a continuous cloning technology.
  4. 4. A method of constructing a ten-gene-edited xenograft donor pig as claimed in claim 3, wherein: the transfection method described in step (5) includes liposome transfection or electrotransfection.
  5. 5. The construction method of a GTKO/beta 4GalNT2KO/CMAHKO/hCD39/hCD46/hCD47/hCD55/hCD59/hTBM/hEPCR ten-gene-edited porcine fetal fibroblast line according to any one of claims 1-2 or a ten-gene-edited xenograft donor pig according to claim 3 for use in biomedical research.
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