Construction of recombinant human butyrylcholinesterase directed inte-grated goat fetal fibroblast cell lines based on CRISPR/Cas9 system
10.7644/j.issn.1674-9960.2024.06.004
- VernacularTitle: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
- Author:
Yunpeng WU
1
;
Yefeng QIU
;
Yuling TANG
;
Tianqi SUN
;
Tongtong QIN
;
Rui ZHANG
;
Yunzhi FA
Author Information
1. 军事科学院军事医学研究院,北京 100850
- Keywords:
butyrylcholinesterase;
CRISPR/Cas9;
CSN2 gene;
homology-directed repair;
goat fetal fibroblasts
- From:
Military Medical Sciences
2024;48(6):421-428
- CountryChina
- Language:Chinese
-
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.
