Construction of Porcine KLF4 Gene Knockout Cell Using CRISPR/Cas9 Technology and Its Effect on Cell Viability
[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 KLF4 gene knockout on cell viability and cell cycle.[Method]Three pairs of sgRNAs(sgRNA1,sgRNA2 and sgRNA3)were designed in exon 1 of porcine KLF4 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 CruiserTM 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 KLF4 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 CruiserTM Enzyme digestion.TA clone sequencing results indicated that 116 and 137 bp sequences were deleted in the two alleles of KLF4 gene,respectively.Western blotting results showed that KLF4 protein was not expressed in KLF4 gene knockout cells.Analysis of cell viability and cell cycle showed that KLF4 gene knockout extremely significantly inhibited the cell viability(P＜0.01),and induced cell cycle arrest at G0/S transition.[Conclusion]KLF4 gene knockout IPEC-J2 cells were constructed in this study using CRISPR/Cas9 technique.KLF4 gene knockout could inhibit cell viability and result in cell cycle arrest at G0/S transition.KLF4 gene knockout cells could provide the materials for further exploring functions and molecular mechanisms of KLF4 gene.
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