The invention relates to a method for constructing a porcine endogenous
retrovirus inactivated
xenotransplantation donor pig, and belongs to the technical field of
xenotransplantation. And the copy number of the PERVs and the
genotype of the PERV-pol are determined through ddPCR,
Sanger sequencing and second-generation PCR
deep sequencing. The method comprises the following steps: designing and synthesizing a porcine PERV-pol
gene targeting sequence based on a
CRISPR / Cas9 RNP
system, co-transfecting the porcine PERV-pol
gene targeting sequence and spCas9
protein into a porcine fetal
fibroblast line, screening to obtain positive cells, carrying out
somatic cell cloning and
embryo transplantation, taking out a
fetus during
pregnancy, identifying the PERV-pol knockout condition, and if the PERV-pol knockout condition is completely carried out, carrying out
somatic cell cloning and
embryo transplantation; and if the PERV-pol
gene is not completely knocked out, secondarily designing and synthesizing an sgRNA targeting sequence, transfecting the sgRNA targeting sequence into the fetal
fibroblast cell line, screening to obtain a
cell line in which the PERV-pol gene is completely knocked out, and carrying out
somatic cell cloning and
embryo transplantation to obtain the PERVs inactivated
xenotransplantation donor pig. The problem that the PERVs inactivation efficiency is low is solved, and the PERVs-inactivated xenotransplantation donor pig can be obtained through one round or multiple rounds.