南方农业学报2023，Vol.54Issue(11)：3340-3348,9.DOI:10.3969/j.issn.2095-1191.2023.11.020
利用iCaspase9特异性诱导鸡原始生殖细胞消除的研究
Specific induction of ablation of chicken primordial germ cells using iCaspase9
摘要
Abstract
[Objective]The purpose of the study was to prepare chicken germ cell lines that express iCaspase9 specifi-cally in primordial germ cells(PGCs),to improve the reproductive transmission efficiency of exogenous PGCs,and to lay the foundation for the production of male or female receptor chicken embryos that induce infertility.[Method]The iCaspase9 fragment was integrated into the chicken genome using the piggyBac transposon,and a stable CAG-iCaspase9-mCherry DF-1 cell line transfected with the iCaspase9 fragment was constructed.Its cell ablation effect under the induc-tion of substrate B/B homodimerizer by the iCaspase9 system was identified by fluorescence microscope.Using CRISPR/Cas9 technology,the iCaspase9 fragment was inserted into the 11th exon of the DAZL gene to construct Dazl-iCaspase9-EGFP PGCs.After identification of iCaspase9 fragment insertion and PGC reproductive characteristics,its ablation effect under B/B homodimerizer induction was identified by fluorescence microscope.[Result]There was no significant differ-ence in growth and proliferation between the successfully established CAG-iCaspase9-mCherry DF-1 cell line and wild-type DF-1 cells(WT DF-1)(P>0.05,the same below).CCK-8 assay revealed that 0.25～5.00 nmol/L B/B homodimerizer had no significant effect on the growth of WT DF-1 cells.However,after adding B/B homodimerizer to CAG-iCaspase9-mCherry DF-1 cells,the number of cells extremely significantly decreased compared to the blank control group(P<0.01,the same below),and normal cell morphology was lost,which gradually becoming round or even apoptotic.The success-fully established DAZL-iCaspase9-EGFP PGCs still specifically and highly expressed germ cell related genes such as Cvh,Nanog,PouV,DAZL,Cdh,and Ddx4.Proteins such as DAZL,SSEA1,and CVH could also be identified at the protein level,indicating that the insertion of iCaspase9 fragments did not alter the germ cell specificity of PGCs.Under the induction effect of 0.25 nmol/L B/B homodimerizer,the number of DAZL-iCaspae9-EGFP PGCs significantly de-creased and almost all of them were eliminated,indicating that the iCaspase9 system can induce cell apoptosis and effec-tively eliminate endogenous PGCs.[Conclusion]The DAZL-iCaspase9-EGFP PGCs constructed using the iCaspase9 sys-tem can significantly eliminate endogenous PGCs under B/B homodimerizer induction without altering their germ cell characteristics,which provides a receptor chicken embryo without endogenous PGCs for establishing efficient chicken germplasm resource recovery and gene editing chicken preparation research.关键词
鸡/原始生殖细胞(PGCs)/iCaspase9/基因编辑/细胞消除Key words
chicken/primordial germ cells(PGCs)/iCaspase9/gene editing/ablation effect分类
农业科技引用本文复制引用
孙玲玲,黄振文,田奎,张嘉乐,郑基坛,濮黎萍,陆阳清..利用iCaspase9特异性诱导鸡原始生殖细胞消除的研究[J].南方农业学报,2023,54(11):3340-3348,9.基金项目
国家重点研发计划项目(31960157) （31960157）
广西重点研发计划项目(桂科AB21220005) National Key Research and Development Program of China(31960157) （桂科AB21220005）
Guangxi Key Research and Development Project(Guike AB21220005) （Guike AB21220005）
