Research on KillerRed-mediated elimination of chicken primordial germ cells

【Objective】 To construct a chicken primordial germ cell line（PGCs）that specifically expressed KillerRed （KR）in order to provide theoretical reference for preparing germ cell-eliminable recipient chicken embryos and improving the gene transfer efficiency of exogenous PGCs in chimeras. 【Method】 KR was integrated into the genome of chicken embryo fibroblast cell line（DF-1）using hyPBase transposase，and a DF-1 cell line（KR-DF-1）stably expressing CAGKR-EGFP was established. The cells were irradiated with green light，and the elimination efficiency of DF-1 was detected by Trypan blue staining. KR was site-specifically knocked into exon number 11 of the DAZL gene in PGCs at the chicken germ cell level using the CRISP/Cas9 system，and a PGCs cell line（KR-PGCs）specifically expressing DAZL-KR-EGFP in germ cells was established. After irradiating the cells with green light，the elimination efficiency of PGCs was detected by Trypan blue staining. Real-time fluorescence quantitative PCR was used to detect the relative expression of genes specifically expressed in germ cells. KR-PGCs were microinjected into recipient chicken embryos，and were hatched to produce chimeric offspring. PCR was used to detect whether the semen of chimeric offspring contained KR. 【Result】 KR-DF-1 was successfully constructed. Compared with wild-type DF-1，there was no significant difference in cell proliferation （P>0.05，the same below）. After green light irradiation，the death rate of KR-DF-1 was extremely significantly increased （P<0.01，the same below）. KR-PGCs were successfully constructed. After green light irradiation，the mortality rate of KR-PGCs was extremely significantly higher than that of other groups. After green light irradiation，the nuclei were fragmented. After green light irradiation for 12 h，the relative expression levels of SOD2 and CAS3 genes in KR-PGCs were extremely significantly increased；cell counting results showed that KR-PGCs showed negative growth after green light irradiation；the specific genes DAZL，DDX4，Pou5f3，NANOG and DNA1 of KR-PGCs were normally expressed，and they still had the ability to migrate and colonize to the gonads，and the semen of sexually mature chimeric roosters could produce semen containing KR. 【Conclusion】 The KR-PGCs cell line which expresses specifically at the DAZL gene loci is successfully constructed，and KR-PGCs can be specifically eliminated after green light irradiation. Through microinjection，chimeric offspring with KR-PGCs in the gonads and capable of producing KR sperm are successfully obtained.