This work aims to establish the CRISPR/Cas9 system of knocking out geneAIP1for producing nephocyte cell(293T)of human embryo in which geneAIP1 efficiently, stably and permanently is knocked out. Three 20 bp sgRNAs(sp1, sp2 and sp3)targeting AIP1 exons were designed and inserted in PX458 vector to construct PX458-sgRNA expression vector for knockout. CRISPR/Cas9 efficiency of knockout was assessed using T7E1 assay. sgRNA of the maximum knockout efficiency was inserted into lentiCRISPRv2 vector to construct lentiCRISPRv2-sgRNA expression vector. The correct recombinant plasmid was transfected into 293T cells. The supernatant was collected and filtered, and then infected the 293T cells. The stable 293T cell lines were generated by limiting diluting the cells in which genesAIP1 were knocked out successfully. The expression level ofAIP1 in the stable 293T cells were detected by Western blot. Three sgRNAs of AIP1 were correctly inserted into PX458 vector respectively, T7E1 verified that the knockout rate of AIP1sgRNAsp2 was the maximum. LentiCRISPRv2-sgRNAsp2 expression vector ofAIP1-knockout was successfully constructed, and infected 293T cells. The stable 293T cell lines withAIP1-expression-deficient were obtained by Western blot. In conclusion, the stable 293T cell lines ofAIP1-knockout were successfully generated by CRISPR/Cas9 system, which provides the foundation for further studying the functions ofAIP1gene.%旨在构建AIP1基因CRISPR/Cas9敲除体系,获得高效、稳定、永久性AIP1敲除的人胚肾细胞株(293T)。针对AIP1的外显子设计3个20 bp的sgRNA(sp1、sp2和sp3)。与PX458载体连接,构建PX458-sgRNA敲除表达载体。T7E1实验评估敲除效率。将敲除效率最高的sgRNA与lentiCRISPRv2慢病毒载体连接,构建lentiCRISPRv2-sgRNA敲除表达载体,将阳性重组质粒导入病毒包装细胞293T中,收集病毒上清液转染293T细胞。对敲除成功的293T细胞通过有限稀释法构建敲除AIP1基因的稳定细胞株。Western blot测定转染后293T细胞中AIP1蛋白的表达量。结果显示,测序证实AIP1的3个靶序列分别正确插入PX458表达载体中,T7E1实验证实AIP1sgRNAsp2靶向敲除效率最高;成功构建lentiCRISPRv2-sgRNAsp2 AIP1敲除表达载体,并包装病毒,感染293T细胞;Western blot证实获得稳定的AIP1基因表达缺失的293T细胞株。建立了能稳定敲除AIP1基因的CRISPR/Cas9慢病毒系统,成功获得AIP1敲除的293T稳定细胞株。
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