中国兽医杂志2025，Vol.61Issue(11)：35-40,6.DOI:10.20157/j.cnki.zgsyzz.2025.11.005
基于CRISPR/Cas9技术构建DF-1细胞chGSDME基因敲除株
Construction of chGSDME Gene Knockout In DF-1 Cells Using CRISPR/Cas9 Technology
摘要
Abstract
To clarify the correlation between the chicken embryo fibroblast(DF-1)cell line chicken gasdermin E(chGSDME)gene and virus-induced pyroptosis in avian cells,this study employed the clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9(CRISPR/Cas9)gene-editing system.Single guide RNA(sgRNA)targeting the chGSDME gene were designed,and knockout plasmids were constructed and transfected into DF-1 cells.Successfully transfected cells were subjected to single-cell cloning,and Western blot(WB)and gene sequencing were used to identify cells with successful chGSDME gene knockout,designated DF-1-ΔchGSDME.Cell viability and functional assays were then performed.The results demonstrated that both the knockout plasmids and DF-1-ΔchGSDME cells were successfully constructed.Compared with wild-type DF-1(WT)cells,DF-1-ΔchGSDME cells exhibited no significant difference in viability(P>0.05),indicating that deletion of the chGSDME gene does not affect normal cell growth.Infection of WT cells with avian reovirus(ARV),infectious bursal disease virus(IBDV),or vesicular stomatitis virus(VSV)induced cell pyroptosis.However,compared with WT cells,infection of DF-1-ΔchGSDME cells with ARV,IBDV,or VSV resulted in significantly reduced lactate dehydrogenase(LDH)release in the supernatant(P<0.01 or P<0.05),indicating suppression of virus-induced pyroptosis.In conclusion,we successfully constructed a chGSDME knockout DF-1 cell line,and the chGSDME gene was found to be closely associated with virus-induced pyroptosis in avian cells,providing a useful reference for further studies on chGSDME.关键词
CRISPR/Cas9/基因敲除/禽膜穿孔蛋白E(chGSDME)/鸡胚成纤维细胞(DF-1)Key words
CRISPR/Cas9/gene knockout/chicken gasdermin E(chGSDME)/chicken embryonic fibroblast(DF-1)分类
畜牧业引用本文复制引用
郑好,高丽,曹红,郑世军,王永强,陈志,杨霞,张素,赵一萌,汤君宇,曹静怡,李瑾瑜,赖沁妍..基于CRISPR/Cas9技术构建DF-1细胞chGSDME基因敲除株[J].中国兽医杂志,2025,61(11):35-40,6.基金项目
国家自然科学基金(32072850) （32072850）
国家现代农业产业技术体系-蛋鸡(CARS-40) （CARS-40）
