建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.032

生物技术通报 ›› 2015, Vol. 31 ›› Issue (8): 219-224.doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.032

建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因

苏甲林¹, 阙彪¹, 张继勤², 李锦辉¹, 王敏², 纪卫东²

1.广州医科大学附属第一医院,广东省泌尿外科重点实验室,广州 510230; 2.中山大学附属第一医院,广州 510080

收稿日期:2015-04-14 出版日期:2015-08-21 发布日期:2015-08-22
作者简介:苏甲林,男,学士,研究方向：泌尿系肿瘤与表观遗传学;E-mail：sujialin125@126.com
基金资助:
国家自然科学基金项目（81272350,81472999）

Establishment of a CRISPR/Cas9 Lentiviral System for Knockout Gene AIP1 of Human

Su Jialin¹, Que Biao¹, Zhang Jiqin², Li Jinhui¹, Wang Min², Ji Weidong²

1. The First Affiliated Hospital of Guangzhou Medical University,Guangdong Key Laboratory of Urology,Guangzhou 510230; 2. The First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080

Received:2015-04-14 Published:2015-08-21 Online:2015-08-22

摘要/Abstract

摘要： 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。

关键词: CRISPR/Cas9, 慢病毒, AIP1, 稳定细胞株

Abstract: This work aims to establish the CRISPR/Cas9 system of knocking out gene AIP1 for producing nephocyte cell（293T）of human embryo in which gene AIP1 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 genes AIP1 were knocked out successfully. The expression level of AIP1 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 of AIP1-knockout was successfully constructed, and infected 293T cells. The stable 293T cell lines with AIP1-expression-deficient were obtained by Western blot. In conclusion, the stable 293T cell lines of AIP1-knockout were successfully generated by CRISPR/Cas9 system, which provides the foundation for further studying the functions of AIP1 gene.

Key words: CRISPR/Cas9, lentiviral, AIP1, stable cell lines

苏甲林, 阙彪, 张继勤, 李锦辉, 王敏, 纪卫东. 建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因[J]. 生物技术通报, 2015, 31(8): 219-224.

Su Jialin, Que Biao, Zhang Jiqin, Li Jinhui, Wang Min, Ji Weidong. Establishment of a CRISPR/Cas9 Lentiviral System for Knockout Gene AIP1 of Human[J]. Biotechnology Bulletin, 2015, 31(8): 219-224.

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建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因

Establishment of a CRISPR/Cas9 Lentiviral System for Knockout Gene AIP1 of Human

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