Construction of a Novel Fatty Liver Cell Model with Low Density Lipoprotein Receptor Gene Knockout Based On Cas9/Single Guide RNA Technique
Shandong First Medical University, No.619 Great Wall Road, Taian, Shangdong 271000, China; 1Department of infection control center, Linyi people’s hospital, No. 27 Jiefang Road, Linyi, Shandong 276000, China; 2Department of Cardiovescular Disease, Renmin Hospital of Wuhan University, No. 238 Jiefang Road, Wuhan, Hubei 430060, China
Department of Cardiovescular Disease, Renmin Hospital of Wuhan University, No. 238 Jiefang Road, Wuhan, Hubei 430060, China, E-mail: Lkwang999@163.com
Clustered regularly interspaced short palindromic repeats/Cas9 gene editing technique was used to construct the human liver Lo2 stable cell line with low density lipoprotein receptor gene deletion. According to the design principle of clustered regularly interspaced short palindromic repeats/Cas9 target, the single guide RNA specifically target low density lipoprotein receptor gene was designed and the recombinant plasmid lenticlustered regularly interspaced short palindromic repeats v2 was used to construct the recombinant plasmid which can express this single guide RNA and Cas9 protein. After sequencing identification, the recombinant plasmid was transferred into Human embryonic kidney 293T cells together with retroviral packaging plasmids VSVG and PAX2. 48 h after transfection, the virus supernatant was collected and directly infected with human liver Lo2 cell. Puromycin was used to screen low density lipoprotein receptor-deficient Lo2 cells, which was verified by polymerase chain reaction, western blotting, and immunofluorescence staining. Then polymerase chain reaction and western blotting were used to verify the effects of low density lipoprotein receptor knockout on the function, lipid metabolism, glucose metabolism pathway, and insulin resistance of the Lo2 cell line. In this paper, the clustered regularly interspaced short palindromic repeats/Cas9 plasmid targeting low density lipoprotein receptor was successfully constructed. The results of polymerase chain reaction and western blotting showed that the human liver Lo2 cell with a stable knockout of low density lipoprotein receptor was obtained. The results of immunofluorescence staining showed that compared with the control group, the content of prealbumin in low density lipoprotein receptor knockout cells decreased significantly, while the content of diacylglycerol increased. At the same time, low density lipoprotein receptor gene knockout can increase the expression level of fatty acid synthase, peroxisome proliferator-activated receptor-γ, sterol regulatory element binding protein 1c, and inhibit the expression of carnitine palmitoyltransferase 1a. In addition, compared with the control group, there was no significant difference in mRNA expression and protein level of mammalian target of rapamycin and phosphatidylinositol-3-kinase, and there was no significant change in epsilon isoform of protein kinase C and insulin receptor substrates 1 expression level. The Lo2 cell line with low density lipoprotein receptor gene knockout was constructed by clustered regularly interspaced short palindromic repeats/Cas9 technique, and the role of low density lipoprotein receptor in lipid metabolism and glucose metabolism was proved by Polymerase chain reaction and Western blotting. The low density lipoprotein receptor knockout polyclonal and monoclonal cells constructed in this study can provide a useful tool for future research.