CRISPR/Cas9介导的CD71基因编辑IPI-2I细胞系的建立

doi:10.16431/j.cnki.1671-7236.2025.10.001

摘要/Abstract

摘要： 【目的】通过CRISPR/Cas9技术获得分化抗原71(cluster of differentiation 71，CD71)基因编辑的猪回肠上皮(immortalized porcine intestinal-2I，IPI-2I)细胞系，为进一步在细胞水平上研究CD71基因功能及其在猪传染性胃肠炎病毒(Transmissible gastroenteritis virus，TGEV)感染中的作用提供材料。【方法】针对CD71基因第3外显子及其相邻内含子区域设计5条sgRNAs，并将其连接至pX458-GFP载体；在猪胎儿成纤维(porcine embryonic fibroblast，PEF)细胞中通过T7E1酶切试验对不同sgRNA载体进行活性检测；选择切割效率较高的2个sgRNAs载体质粒分别电转染至IPI-2I细胞中，48 h后通过流式细胞仪收集带有绿色荧光蛋白(green fluorescent protein，GFP)的单个细胞至96孔细胞培养板中扩大培养，筛选单克隆细胞；通过PCR扩增和TA克隆技术对单克隆细胞进行基因型鉴定，获得CD71基因编辑的IPI-2I细胞系。【结果】质粒测序结果表明，5条sgRNAs均成功连接至pX458-GFP载体；T7E1酶切结果显示，转染的5个sgRNAs质粒组均产生了切割，其中，pX458-sgRNA1和pX458-sgRNA4切割效率较高，分别为35.8%和44.1%。流式细胞术分选结果显示，转染pX458-sgRNA1和pX458-sgRNA4的阳性细胞比例分别为29.8%和25.7%；PCR扩增结果显示，获得的65株单克隆细胞中有14株细胞发生了基因编辑，编辑效率为22%；TA克隆结果显示，14株基因编辑细胞中有5株为单等位基因编辑，9株为双等位基因编辑。【结论】本研究利用CRISPR/Cas9技术成功构建了CD71基因编辑的IPI-2I细胞系，获得了CD71基因单等位基因编辑和双等位基因编辑细胞。试验结果为明确CD71基因功能及其介导TGEV感染的作用机制提供了良好的细胞模型。

关键词: 猪; CRISPR/Cas9; CD71基因; IPI-2I细胞

Abstract: 【Objective】 This experiment aimed to obtain cluster of differentiation 71 (CD71) gene-edited immortalized porcine intestinal-2I (IPI-2I) cell lines through CRISPR/Cas9 technology，which provided materials for further study of CD71 gene function and its role in Transmissible gastroenteritis virus (TGEV) infection at the cellular level.【Method】 Five sgRNAs were designed for the third exon and its adjacent intron region of CD71 gene,which were ligated into the pX458-GFP vector.The activity of different sgRNA vectors was detected by T7E1 enzymatic cleavage in porcine embryonic fibroblast (PEF) cells.Two sgRNA vector plasmids with higher cleavage efficiency were selected to be electrotransfected into IPI-2I cells,and after 48 h,single cells with green fluorescent protein (GFP) were collected by flow cytometry,and then cultured in 96-well cell culture plates to screen the monoclonal cells.The monoclonal cells were genotyped by PCR amplification and TA cloning to obtain the CD71 gene-edited IPI-2I cell line.【Result】 The results of plasmid sequencing showed that all five sgRNAs were successfully ligated to the pX458-GFP vector.T7E1 enzyme digestion results showed that cleavage occurred in all five transfected sgRNA plasmids.Among them,the cleavage efficiencies of pX458-sgRNA1 and pX458-sgRNA4 were relatively high,which were 35.8% and 44.1%,respectively.The results of flow cytometry sorting showed that the proportion of positive cells transfected with pX458-sgRNA1 and pX458-sgRNA4 were 29.8% and 25.7%,respectively.PCR amplification results showed that among the 65 monoclonal cells obtained,14 cells occurred gene editing,with an editing efficiency of 22%. TA cloning results showed that among the 14 gene-edited cells,5 were monoallelic edited and 9 were biallelic edited.【Conclusion】 In this study,the CD71 gene-edited IPI-2I cell line was successfully constructed using CRISPR/Cas9 technology,and CD71 gene monoallelic and biallelic gene-edited cells were obtained.The results provided a good cell model for clarifying the function of CD71 gene and its mechanism of mediating TGEV infection.

Key words: pig; CRISPR/Cas9; CD71 gene; IPI-2I cells

中图分类号:

S828.9

聂雨欣, 袁茂莎, 王婉洁, 王楠, 孙亚茹, 刘志国, 牟玉莲. CRISPR/Cas9介导的CD71基因编辑IPI-2I细胞系的建立[J]. 中国畜牧兽医, 2025, 52(10): 4527-4537.

NIE Yuxin, YUAN Maosha, WANG Wanjie, WANG Nan, SUN Yaru, LIU Zhiguo, MU Yulian. Establishment of CRISPR/Cas9-mediated CD71 Gene Editing IPI-2I Cell Line[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4527-4537.

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