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 impro-ving 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 CAG-KR-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 spe-cifically expressed in germ cells.KR-PGCs were microinjected into recipient chicken embryos,and were hatched to pro-duce 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 frag-mented.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 ir-radiation;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 microinjec-tion,chimeric offspring with KR-PGCs in the gonads and capable of producing KR sperm are successfully obtained.
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