摘要
【目的】构建特异性表达KillerRed(KR)的鸡原始生殖细胞系(PGCs),为制备生殖细胞可消除的受体鸡胚和提高外源PGCs在嵌合体中的基因传递效率提供理论参考。【方法】利用hyPBase转座酶将KR整合到鸡胚成纤维细胞系(DF-1)基因组中,建立稳定表达CAG-KR-EGFP的DF-1细胞系(KR-DF-1)。利用绿光照射细胞,台盼蓝染色检测DF-1的消除效率。采用CRISP/Cas9系统将KR定点敲入到鸡生殖细胞水平的PGCs中DAZL基因的第11号外显子中,建立生殖细胞特异表达DAZL-KR-EGFP的PGCs细胞系(KR-PGCs)。利用绿光照射细胞,台盼蓝染色检测PGCs消除效率。实时荧光定量PCR检测生殖细胞特异性表达基因的相对表达量。显微注射KR-PGCs到受体鸡胚,孵化产生嵌合体后代,PCR检测嵌合体后代精液中是否含有KR。【结果】成功构建KR-DF-1,与野生型DF-1相比,细胞增殖无显著差异(P>0.05,下同);绿光照射后,KR-DF-1死亡率极显著升高(P<0.01,下同);成功构建KR-PGCs,绿光照射后,KR-PGCs死亡率极显著升高,细胞核呈破碎状;绿光照射12h,KR-PGCs中SOD2和CAS3基因相对表达量极显著升高;细胞计数结果显示,KR-PGCs在绿光照射后出现负增长;KR-PGCs的特异性基因DAZL、DDX4、Pou5f3、NANOG和DNA1正常表达,仍具有迁移定植到性腺的能力,且性成熟嵌合体公鸡精液能产生含KR的精液。【结论】成功构建了在DAZL基因位点特异性表达KR的KR-GCs细胞系,且绿光照射后可特异性消除KR-PGCs。通过显微注射成功获得了含有KR-PGCs且能产生含KR精液的嵌合体后代。
【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 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
作者
张嘉乐
黄振文
贾肖轩
彭煜琳
温静儿
季娜
许惠艳
刘兴廷
冉明霞
陆阳清
ZHANG Jia-le;HUANG Zhen-wen;JIA Xiao-xuan;PENG Yu-lin;WEN Jing-er;JI Na;XU Hui-yan;LIU Xing-ting;RAN Ming-xia;LU Yang-qing(College of Animal Science and Technology,Guangxi University/Guangxi Key Laboratory of Livestock and Poultry Breeding and Disease Prevention and Control,Nanning,Guangxi 530004,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2024年第10期3136-3146,共11页
Journal of Southern Agriculture
基金
国家重点研发计划项目(2021YFD1300100)。
