PGRN和Rev-erbβ双基因敲除HEK 293细胞系的构建及应用

Construction and application of PGRN and Rev-erbβ double genes knockout HEK293 cell lines

摘要:

为研究PGRN与Rev-erbβ相互作用可能存在的分子机理及其生理意义，在前期构建的Rev-erbβ基因敲除HEK293 C3-6细胞系的基础上，利用CRISPR/Cas9技术构建PGRN和Rev-erbβ双基因敲除的HEK293细胞系。首先，针对PGRN基因设计了4个不同的sgRNA，经筛选将活性较高的PGRN sgRNA 2和sgRNA 3串联，构建携带双PGRN sgRNA和Cas9的慢病毒打靶载体pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro。再将包装的携带Cas9和双PGRN sgRNA的慢病毒感染HEK293 (Rev-erbβ-/-) 细胞，通过筛选、克隆化及测序分析获得双基因敲除的HEK293 C3-6/23 (Rev-erbβ-/-; PGRN-/-) 单克隆细胞系，并用qRT-PCR和Western blotting检测HEK293 (C3-6/23) 细胞中PGRN mRNA和蛋白质的表达。最后，在双基因敲除HEK293（C3-6/ 23）细胞系中，通过回补基因方式研究PGRN介导Rev-erbβ对靶基因启动子转录活性调控研究。在PGRN和Rev-erbβ双基因敲除的HEK293 (C3-6/23) 细胞系中，PGRN基因的两条DNA链均为缺失突变型，PGRN mRNA和蛋白质的表达均未达到检测水平。同时在HEK293 (C3-6/23) 细胞系中研究发现PGRN与Rev-erbβ相互作用，可以增强Rev-erbβ对靶基因启动子转录的调控。利用CRISPR/Cas9系统，成功构建了双基因敲除的HEK293 (Rev-erbβ-/-；PGRN-/-) C3-6/23单克隆细胞系。利用该敲除细胞系研究发现PGRN可以影响Rev-erbβ对靶基因启动子转录的调控，但PGRN参与介导Rev-erbβ转录调控的机制还有待进一步研究。

Abstract:

In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbβ, the PGRN gene in HEK293 (Rev-erbβ-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbβ and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbβ-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbβ on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbβ enhanced the regulation of Rev-erbβ on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbβ-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbβ on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbβ in transcriptional regulation remains to be further studied.