Generation of ETV5 Knockout Pigs with CRISPR/Cas9

Background: Ets variant factor 5 (ETV5) plays an important regulatory role in mouse spermatogonial stem cells (SSCs) self-renewal. ETV5 knockout (KO) mice exhibit a progressive loss of SSCs and resulting in a sertoli cell-only phenotype. The current study was aimed to use gene editing technology to obtain ETV5-KO pigs as a model for studying the apoptosis mechanism of SSCs and further clarify the function of ETV5 gene in pigs. Methods: A gene editing plasmid for the porcine ETV5 gene was constructed, transfected into porcine fetal fibroblasts by electroporation to obtain ETV5-KO cells. ETV5-KO cells were used as donors to prepare ETV5-KO pigs by somatic cell nuclear transfer (SCNT). Testis tissues were collected for hematoxylin and eosin (HE), immunohistochemistry ( IHC), RT- PCR testing and blood for ELISA testing from ETV5-KO pig. Result: In the present study, we used the CRISPR/Cas9 system and SCNT to genrate homozygous ETV5-KO pigs. We observed 3 phenotypes in these pigs: normal testis development after birth, the SSCs in the seminiferous tubules did not show obviously extinction at sexual maturity and normal spermatogenesis.