A method of constructing a pig endogenous retrovirus-inactivated xenotransplant donor pig

By designing specific sgRNAs in porcine fetal fibroblasts and combining them with a multi-round screening strategy, and using CRISPR/Cas9 gene editing technology, the complete knockout of the porcine endogenous retrovirus PERV-pol gene was achieved. This solves the problem of the difficulty in efficiently removing PERVs in existing technologies and improves the safety of xenotransplantation.

CN120866423BActive Publication Date: 2026-05-29YUNNAN AGRICULTURAL UNIVERSITY
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Patent Information

Application Number
CN202511372426.4
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2025-09-24
Publication Date
2026-05-29
Estimated Expiration
2045-09-24

AI Technical Summary

Technical Problem

Existing technologies are insufficient to efficiently remove porcine endogenous retroviruses (PERVs), especially gene editing techniques, which cannot completely knock out PERVs, leading to a high risk of infection in xenotransplantation.

Method used

Specific sgRNA sequences were designed in porcine fetal fibroblasts using CRISPR/Cas9 gene editing technology. Combined with a multi-round screening strategy, the PERV-pol gene was gradually knocked out through somatic cell cloning and embryo transfer. PCR and deep sequencing were used for initial screening and identification.

Benefits of technology

The complete knockout of the porcine endogenous retrovirus PERV-pol gene was achieved, reducing the risk of infection in xenotransplantation and improving the safety and reliability of xenotransplantation donor pigs.

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Abstract

The present application relates to a method for constructing a pig endogenous retrovirus inactivated xenotransplant donor pig, and belongs to the technical field of xenogenic organ transplantation. The copy number and PERV-pol genotype of PERVs are determined by ddPCR, Sanger sequencing and second-generation sequencing. Based on the RNP system of CRISPR / Cas9, the pig PERV-pol gene targeting sequence is designed and synthesized, and is co-transfected into a pig fetal fibroblast cell line with spCas9 protein, positive cells are screened, somatic cell cloning and embryo transfer are carried out, and the PERV-pol knockout condition of the fetus is identified when pregnancy occurs. If not completely knocked out, the sgRNA targeting sequence needs to be designed and synthesized again, and the cell line with completely knocked out PERV-pol gene is screened. If completely knocked out, somatic cell cloning and embryo transfer are directly carried out; and the xenotransplant donor pig with inactivated PERVs is obtained.
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