Generation and Propagation of Cluster of Differentiation 163 Biallelic Gene Editing Pigs
(2.Foshan University, Foshan, Guangdong, China 528000)
(3.Department of Animal BioSciences, University of Guelph, Canada Ontario N1G 2W1, Canada)
(4.Tianjin Ningheyuan Swinebreeding Farm, Tianjin, China 301504)
【Abstract】[Objective] Porcine reproductive and respiratory syndrome (PRRS), commonly known as “blue ear disease”, is a highly fatal infectious disease with porcine reproductive and respiratory syndrome virus (PRRSV) being the causative pathogen. PRRSV causes major economic losses in the pork industry world-wide. The genetic variability of PRRSV is high and an ideal vaccine to prevent the occurrence of this disease is not available. Cluster of differentiation 163 (CD163) is the important receptor for the entry of PRRSV into the porcine alveolar macrophage (PAM) cells. The aim of this study was to generate CD163 gene edited Large White pigs by using the CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. [Method] CRISPR/Cas9 vector was constructed for editing the exon 7 of the porcine CD163 gene. The constructed vectors were transfected into pig fetal fibroblasts to obtain gene edited positive cell colonies. The CD163 gene edited fibroblasts and in vitro matured porcine oocytes were employed as nuclear donors and nuclear receptors respectively to obtain reconstructed embryos. For obtaining CD163 gene edited pigs the reconstructed embryos were transferred into recipient sows and performing the subsequent propagation experiment. [Result] The designed gRNA could effectively recognize the intended site. Genotyping analysis of cloned cell showed that 21 colonies had mutations in the CD163 gene, of which 14 colonies had either a monoallelic mutation or a biallelic heterozygous mutation, and 7 colonies had a biallelic homozygous mutation. Through SCNT, we successfully obtained CD163 biallelic edited Large White pigs. Successful breeding allowed us to obtain F1 generation CD163 gene edited piglets, and they were all in good health. It was anticipated that more F1 piglets would be produced soon. [Conclusion] The CD163 biallelic edited Large White pigs that did not harbor a drug resistant gene in their genome were produced and they could thus safely and quickly serve as a gene donor for breeding of PRRSV resistant pigs.
【Keywords】 CD163; CRISPR/Cas9; pig; PRRS;
(Translated by CHEN YJ)
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