摘要
【目的】试验旨在利用CRISPR/Cas9技术构建Krüppel样因子4(Krüppel-like factor 4,KLF4)基因敲除的猪小肠上皮细胞,并探究KLF4基因敲除对于细胞活性和细胞周期的影响。【方法】在猪KLF4基因转录本第1外显子区域设计3条sgRNAs(sgRNA1、sgRNA2和sgRNA3),经退火形成的双链DNA与线性化pGK1.1载体连接,产物转化大肠杆菌Top10感受态细胞进行鉴定,并将重组载体转染至猪小肠上皮细胞(IPEC-J2)。PCR扩增敲除位点附近序列,并通过测序判断sgRNA敲除效率;利用CruiserTMEnzyme酶切鉴定阳性细胞克隆,通过TA克隆测序鉴定敲除序列;利用Western blotting检测基因敲除细胞中KLF4蛋白表达情况。利用CCK-8和流式细胞术检测KLF4基因敲除后细胞活性和细胞周期的变化。【结果】重组载体测序结果显示,sgRNAs与pGK1.1成功连接。分析敲除效率发现,3个sgRNAs均可对靶序列进行敲除,其中sgRNA3有较高的敲除效率。PCR产物经CruiserTMEnzyme酶切筛选出2个阳性单克隆细胞。TA克隆测序分析发现,KLF4基因2个等位基因序列分别缺失116和137 bp。Western blotting结果表明,KLF4基因敲除细胞中未见KLF4蛋白表达。细胞活性及细胞周期分析显示,敲除KLF4基因极显著抑制了细胞活性(P<0.01),并导致G0/S细胞周期阻滞。【结论】本研究利用CRISPR/Cas9技术构建了KLF4基因敲除的IPEC-J2细胞,且KLF4基因敲除可抑制细胞活性,并引起G0/S细胞周期阻滞。KLF4基因敲除细胞可为进一步探究KLF4基因功能及分子机制提供材料。
【Objective】The aim of this study was to knockout Krüppel-like factor 4(KLF4)gene in porcine small intestinal epithelial cells using CRISPR/Cas9 technique,and investigate the effects of KLF 4 gene knockout on cell viability and cell cycle.【Method】Three pairs of sgRNAs(sgRNA1,sgRNA2 and sgRNA3)were designed in exon 1 of porcine KLF 4 gene.The double stranded DNA after annealing was ligated with the linearized pGK1.1 vector,and the products was transferred into Escherichia coli Top10 competent cells for identification.The recombinant vectors were transfected into porcine intestinal epithelial cells(IPEC-J2).The sequences adjacent to the target sites were amplified by PCR,and the knockout efficiency of sgRNA was verified by sequencing.The positive cell clones were selected by Cruiser TM Enzyme digestion,and the knockout sequences were checked by TA clone sequencing.The expression of KLF4 protein in gene knockout cells was detected by Western blotting.The changes of cell viability and cell cycle of KLF 4 gene knockout cells were analyzed by CCK-8 and flow cytometry.【Result】Sequencing of the recombinant vectors showed that sgRNAs were successfully ligated with the pGK1.1 vector.Analysis of knockout efficiency demonstrated that all three sgRNAs could lead to gene knockout,of which sgRNA3 achieved higher knockout efficiency.Two positive cell clones were selected by Cruiser TM Enzyme digestion.TA clone sequencing results indicated that 116 and 137 bp sequences were deleted in the two alleles of KLF 4 gene,respectively.Western blotting results showed that KLF4 protein was not expressed in KLF 4 gene knockout cells.Analysis of cell viability and cell cycle showed that KLF 4 gene knockout extremely significantly inhibited the cell viability(P<0.01),and induced cell cycle arrest at G0/S transition.【Conclusion】KLF 4 gene knockout IPEC-J2 cells were constructed in this study using CRISPR/Cas9 technique.KLF 4 gene knockout could inhibit cell viability and result in cell cycle arrest at G0/S transition.KLF 4 gene knockout cells could provide the materials for further exploring functions and molecular mechanisms of KLF 4 gene.
作者
董娇
陆繁
方晓敏
陈瑜哲
包文斌
王海飞
DONG Jiao;LU Fan;FANG Xiaomin;CHEN Yuzhe;BAO Wenbin;WANG Haifei(College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China;Institute of Food Safety and Nutrition,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Livestock and Poultry Slaughtering and Quarantine Center of Kunshan,Kunshan 215300,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2024年第3期893-902,共10页
China Animal Husbandry & Veterinary Medicine
基金
江苏省高等学校基础科学研究重大项目(22KJA230002)
扬州大学大学生科技创新基金项目(X20220634)。
