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.