CRISPR/Cas9系统敲除山羊CDK2基因载体构建及转染效率检测

Construction and Transfection Efficiency Evaluation of Goat CDK2 Gene Plasmid by CRISPR/Cas9 System

细胞周期蛋白依赖性蛋白激酶-2(CDK2)是细胞通过和进入S期所必须的细胞周期调节激酶,与许多癌细胞的生长有关。然而,目前在山羊上关于CDK2基因敲除及相关功能的研究尚未见报道。研究采用CRISPR/cas9系统,首先设计CDK2基因的sgRNA片段,在合成CDK2 sgRNA核苷酸序列后,构建能够同时表达sgRNA和Cas9 D10A的质粒PYSY-CDK2 sgRNA,连接并转化至大肠杆菌DH5α感受态细胞,对转化子进行验证,最后转染HEK293T细胞,检测细胞荧光和细胞增殖。酶切和测序结果证明,CDK2基因敲除载体构建正确;荧光显微镜检测显示,细胞转染效率在75%以上;细胞增殖检测表明,山羊CDK2敲除质粒不会对人源细胞增殖产生影响。说明采用CRISPR/Cas9构建的载体,可为后续获得山羊CDK2基因缺失型细胞系,研究支原体肺炎感染引发的细胞凋亡分子机制提供理论依据。

CDK2 is a necessary cell cycle regulatory kinase for cells to pass through and enter the S phase.It was reported that CDK2 gene is associated with the growth of many cancer cells.However,there were currently no reports on CDK2 gene knockout and related functions in goats.To address this issue,this present study aimed to construct and transfect the CDK2 plasmid by CRISPR/cas9 system.Firstly,the sgRNA fragment of the CDK2 gene was designed.After synthesizing the CDK2 sgRNA nucleotide sequence,a plasmid PYSY-CDK2 sgRNA was constructed that can simultaneously express sgRNA and Cas9 D10A.The plasmid was connected and transformed into E.coli DH5αcells to validate the transformants.Finally,it was transfected to HEK293T cells to detect cell fluorescence and proliferation.The results of enzyme digestion and sequencing confirmed that the construction of the CDK2 gene knockout vector was correct.Fluorescence microscopy detection showed that the transfection efficiency of cells was above 75%.Cell proliferation detection showed that the goat CDK2 knockout plasmid did not affect the proliferation of human cells.It was indicated that the vector constructed using CRISPR/Cas9 could provide a theoretical basis for obtaining goat CDK2 gene deficient cell lines and studying the molecular mechanism of cell apoptosis caused by Mycoplasma pneumonia infection.