Construction of klhdc7b Gene Knockout Human Normal Hepatocyte L-02 Stable Strain by CRISPR/Cas9 Technology and its Preliminary Biological Function Analysis

Yuqing Gong ； Xiangyu Ma ； Menglin Feng ； Sanqiang Li ； Lengxin Duan

《International Core Journal of Engineering》 8卷6期 (2022/06) Pp. 47-58

https://doi.org/10.6919/ICJE.202206_8(6).0007

摘要

To knock out the klhdc7b gene in L-02 cells using CRISPR/Cas9 genome engineering technology, and screen klhdc7b knockout L-02 stable strains for analysis of its biological functions. Using the klhdc7b gene sequence retrieved from NCBI, three CRISPR sequences were designed for construction of three eukaryotic recombinant expression plasmids which could simultaneously express sgRNA, Cas9 and puromycin screening markers.After the activity of the transformants were verified by RT-qPCR, using respectively the three recombinant plasmids and transfection reagent infect L-02 cells in an optimal ratio. The stable klhdc7b knockout strains were screened through the stress of puromycin, and the knockout effect was detected by Western blotting.MTT assay and flow cytometry were used to detect the effects of klhdc7b on cell proliferation and apoptosis in normal L-02 cells and L-02 cells under tunicamycin-induced endoplasmic reticulum stress.Verified by RT-qPCR, the most effective guide RNA was sgRNA1, and Western Blot detection showed that the expression of klhdc7b protein in the monoclonal cells was significantly lower than that in the empty group and the normal group (p＜0.001).MTT assay showed that klhdc7b had no significant effect on the proliferation of L-02 cells when they were in a normal state; when L-02 cells were in a state of endoplasmic reticulum stress, klhdc7b played a role in inhibiting cell proliferation. The results of flow cytometry showed that when L-02 cells were in a normal state, klhdc7b had no significant effect on cell apoptosis; when L-02 cells were in a state of endoplasmic reticulum stress, klhdc7b played a role in promoting cell apoptosis. The L-02 klhdc7b knockout stable strain was successfully constructed, and our preliminary experiments demonstrated that klhdc7b gene can inhibit proliferation and promote apoptosis in L-02 cells under ER stress.

關鍵字

L-02 ； Gene Knockout ； klhdc7b ； CRISPR / Cas9 ； Proliferation ； Apoptosis

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