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Building method of donor pig suitable for xenotransplantation of tissue like islet cells and skin
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A technology for islet cell and xenotransplantation, which is applied in the fields of botanical equipment and methods, chemical instruments and methods, biochemical equipment and methods, etc., and can solve the problems of immunological incompatibility, disease infection, functional disorder, etc.
Pending Publication Date: 2019-10-25
YUNNAN AGRICULTURAL UNIVERSITY
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[0004] Although pigs have been recognized as a source of human organ transplantation, xenogeneic tissue transplantation still faces the risk of cross-species disease infection, immunological incompatibility and functional impairment, making pig islet cells and skin tissue as xenografts Faced with severe challenges, it is not enough to transform into clinical application of medicine, making xenotransplantation stagnant for a long time
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Embodiment 1
[0022] A construction method suitable for islet cells, skin and other tissue xenotransplantation donor pigs, the specific operation process is carried out according to the following steps:
[0024] On the basis of porcine fibroblast cell line, three antigen genes, GGTA1, CMAH and B4GalNT2, which can cause antigen-antibodyimmune rejection, were knocked out by CRISPR / Cas9 gene editing technology;
[0025] 2) Site-directed insertion of three humanized modified genes hCD55, hCD59 and hCD46
[0026] On the basis of the above-mentioned three-gene modification of GTKO / CMAHKO / B4GalNT2KO, three humanized modified genes, hCD55, hCD59 and hCD46, were inserted into specific sites to obtain a porcine fibroblast cell line modified with GTKO / CMAHKO / B4GalNT2KO / hCD55 / hCD59 / hCD46 six genes. ;
[0031] A construction method suitable for islet cells, skin and other tissue xenotransplantation donor pigs, the specific operation process is carried out according to the following steps:
[0032] A construction method suitable for tissue xenotransplantation donor pigs such as islet cells and skin, the specific steps are:
[0033] 1) Knock out the three main antigen genes that cause antigen-antibody immune rejection
[0034] On the basis of porcine fibroblast cell line, three antigen genes, GGTA1, CMAH and B4GalNT2, which can cause antigen-antibody immune rejection, were knocked out by CRISPR / Cas9 gene editing technology;
[0035] 2) Site-directed insertion of three humanized modified genes hCD55, hCD59 and hCD46
[0036] On the basis of the above-mentioned three-gene modification of GTKO / CMAHKO / B4GalNT2KO, three humanized modified genes, hCD55, hCD59 and hCD46, were inserted into specific sites to obtain a porcine fibroblast cell line modified with GTKO / CMAHKO / B4GalNT2KO / hC...
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Abstract
The invention discloses a building method of a donor pig suitable for xenotransplantation of tissue like islet cells and skin and belongs to the technical field of animal biology. The building methodincludes: on the basis of porcine fetal fibroblast, utilizing CRISPR / Cas9 gene editing technology to knock out three genes GGTA1, CMAH and B4GalNT2 at the same time, and inserting three humanized modifying genes hCD55, hCD59 and hCD46 in a fixed point manner; combining somatic cellnuclear transfer technology to build an xenotransplantation donor base pig modified by the above six genes; on the basis of the base pig, knocking out gene B2M, and inserting three modifying genes hCD47, A20 and CTLA4-Ig in a fixed point manner at the same time; utilizing the somatic cellnuclear transfer technologyto build a isletcell and skinxenotransplantation donor pig modified by multiple genes; performing functional verifying on the donor pig to obtain the donor pig suitable for isletcell and skin xenotransplantation. A brand-new research concept and method are provided for developing the donor pig suitable for xenotransplantation of the islet cells and the skin, and clinical conversion and application of pigs serving in heterogeneous islet cell and skin transplantation are promoted.
Description
technical field [0001] The invention belongs to the field of animal biotechnology, in particular to a construction method suitable for islet cells and skin tissue xenotransplantation donor pigs. Background technique [0002] Isletcell transplantation is an effective and important approach for diabetic patients with complete pancreatic failure. At present, the isolation technology of pancreatic islets is relatively mature, but the problems of immune rejection and transplantation dysfunction have not been well solved, which has led to the lack of widespread application of this technology. [0003] When it comes to xenotransplantation, because pigs are easy to raise and breed, their organ sizes match those of humans, and their physiology and metabolism are very similar to those of humans, the possibility of comorbidities between humans and pigs is low, and there are few ethical and moral disputes involved. genetic modification to enhance the matching of donor tissues and orga...
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