编辑PFF细胞的P53基因及其信号通路中重要基因的表达分析

doi:10.11843/j.issn.0366-6964.2019.11.005

摘要/Abstract

摘要： 旨在编辑PFF细胞的P53基因，并分析其信号通路中重要基因的表达。本研究首先将设计好的两条sgRNAs装载入PX459 V2.0质粒中，订购两条携带241W、242S、266H突变位点的单链寡核苷酸（SSODN）作为供体DNA（一条带有前两个突变，另一条带有266H突变），利用电转法将打靶载体和SSODNs共转染到PFF细胞中，筛选单克隆细胞后检测打靶区域的编辑情况，并检测编辑细胞中信号通路重要基因（MDM2、FAS、WIP1、BAX、P21和DD1α）的表达及检测编辑细胞的增殖能力。结果表明，在筛选到的107个单克隆细胞中，分别有4个为两个位点（R241和R242）纯合子和杂合子编辑型，1个为R266位点纯合子编辑型、63个纯合子缺失型、11个序列倒置型、11个纯合子点插入型、3个杂合子缺失型（或混合克隆）和10个野生型，未获得3个位点同时被编辑的细胞。RT-PCR和Western blotting结果显示，纯合子缺失型细胞中P53几乎不表达；RT-PCR检测显示，在纯合子缺失型细胞和双位点编辑型细胞中MDM2、FAS、BAX、P21基因的表达量极显著下降（P<0.01或P<0.001）；Western blotting结果显示，在纯合子缺失型细胞中，未检测到P21基因的表达蛋白，MDM2的表达量也较明显地下降。CCK-8检测试验表明，编辑型细胞的增殖能力显著（P<0.05）或极显著（P<0.01或P<0.001）高于野生型细胞。综上所述，本试验成功地将PFF细胞中P53基因的两个野生型位点编辑成与人肿瘤发生相关的位点，P53基因被编辑后显著影响了其信号通路中部分重要基因的表达，本试验所获得双位点编辑细胞，为下一步研制P53点编辑模型猪奠定了重要的基础。

Abstract: The aim of this study was to edit the P53 gene, and analyze the expression of vital genes in its signaling pathway. Firstly, two designed sgRNAs were loaded into the PX459 V2.0 plasmid, and two single-stranded oligonucleotides (SSODN) carrying 241W, 242S, 266H mutations were ordered as donor DNA (one carrying 241W and 242S mutations, the other one carrying 266H mutation). The targeting vectors and SSODNs were co-transfected into PFF cells by electroporation, and monoclonal cells were collected. Then editing events in targeting region in isolated cells were detected, and expression of vital genes (MDM2, FAS, WIP1, BAX, P21 and DD1α) in P53 signaling pathway were detected and the proliferative capacity of the gene editing cells were identified. Among the isolated 107 monoclonal cells, each 4 clones were homozygous and heterozygous modification of R241 and R242, 1 clone was homozygous modification of R266, 63 clones contained homozygous deletions, 11 clones carried homozygous inversions, 11 clones contained homozygous insertions, 3 clones carried heterozygous deletions (or mixed clones) and 10 clones were wild-type. No cell with simultaneous modification of 3 sites was found. RT-PCR and Western blotting analysis showed that P53 expression was not detected in the cells with homozygous deletions. RT-PCR result indicated that the expression levels of MDM2, FAS, BAX and P21 were extremely significant decreased (P<0.01 or P<0.001) in cells carrying homozygous deletions and homozygous modification of R241 and R242. Western blotting result showed that P21 protein was undetectable, and MDM2 protein was obviously decreased in cells contained homozygous deletions. The CCK-8 test indicated that the proliferation capacity of the edited cells significantly (P<0.05) or extremely significantly (P<0.01 or P<0.001) increased compared with the wild-type cells. In conclusion, we successfully obtained PFF cells that simultaneously modified two sites in P53 gene related to human tumorigenesis. P53 gene editing obviously affected the expression levels of vital genes in its signaling pathway. The acquirement of cells with modification of two sites laid the solid foundation for further generating P53 point edited model in pigs.

中图分类号:

S828.2

乔传民, 刘为伟, 杨强, 江浩筠, 黄路生, 幸宇云. 编辑PFF细胞的P53基因及其信号通路中重要基因的表达分析[J]. 畜牧兽医学报, 2019, 50(11): 2215-2225.

QIAO Chuanmin, LIU Weiwei, YANG Qiang, JIANG Haoyun, HUANG Lusheng, XING Yuyun. P53 Gene Editing in PFF and Expression Analyses of Vital Genes in Its Signaling Pathway[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(11): 2215-2225.

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