军事医学2024，Vol.48Issue(6)：421-428,8.DOI:10.7644/j.issn.1674-9960.2024.06.004
基于CRISPR/Cas9系统构建重组人丁酰胆碱酯酶定向整合的山羊胎儿成纤维细胞株
Construction of recombinant human butyrylcholinesterase directed inte-grated goat fetal fibroblast cell lines based on CRISPR/Cas9 system
摘要
Abstract
Objective To construct recombinant human butyrylcholinesterase(rhBChE)knock-in goat fetal fibroblast cell lines(GFFs)by using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated homology-directed repair mechanism for subsequent production of goat expressing rhBChE.Methods The efficient sgRNA sites targeting goat[3-casein(CSN2)gene were designed and screened,and the targeting efficiency of the sgRNA in goat mammary epithelial cells(GMECs)was confirmed by electro-transfection,flow sorting,and sequencing of PCR products.The red fluorescent reporter gene homology repair vector(P2A-mCherry)targeting the sgRNA was constructed,and then the integration and expression efficiency was detected by flow cytometry.The rhBChE homology repair vector(P2A-rhBChE)targeting the sgRNA of CSN2 gene was constructed in GFFs,the rhBChE positive cell clones were obtained via electro-transfection and flow sorting,and the rhBChE knock-in cell lines was identified by sequencing of PCR products.Results The sgRNA4 was identified as an efficient target of goat CSN2 gene,which could be also used for targeted integration of other genes.Three rhBChE knock-in cell lines were successfully constructed.Conclusion The rhBChE knock-in GFFs targeting goat CSN2 gene lays the foundation for the production of mammary bioreactors expressing rhBChE.关键词
丁酰胆碱酯酶/CRISPR/Cas9/CSN2基因/同源定向修复/山羊胎儿成纤维细胞Key words
butyrylcholinesterase/CRISPR/Cas9/CSN2 gene/homology-directed repair/goat fetal fibroblasts分类
生物科学引用本文复制引用
毋云鹏,邱业峰,唐玉玲,孙天奇,秦佟童,张睿,法云智..基于CRISPR/Cas9系统构建重组人丁酰胆碱酯酶定向整合的山羊胎儿成纤维细胞株[J].军事医学,2024,48(6):421-428,8.基金项目
全军实验动物专项课题(SYDW[2020]07号) （SYDW[2020]07号）
