CRISPR/Cas9载体优化及CHD1L条件性敲除肝癌细胞系的构建

doi:10.13418/j.issn.1001-165x.2020.01.009

Abstract

Abstract: Objective　To verify the effect of CHD1L QGY-7703 hepatoma cell line knockout and the effect on the cell biological functions and then to provide important cells models for studying the role of CHD1L in the development of mechanism of malignant phenotype in cancer cell by using the optimized pLV-Tet3G-CRISPR/Cas9 technology to construct a QGY-7703 hepatoma cell line.　Methods　pLV-Tet3G-Cas9 vector was optimized by point mutation technique to reduce its off-target effect and increase specificity. Cas9 was transformed into eSpCas9. Then, QGY-7703 cell line stably expressing eSpCas9 was obtained by screening. pLVX-mCherry-hU6-SgRNA vector carrying mCherry fluorescent gene was obtained by inserting mCherry gene into pLVX-hU6-SgRNA vector. The sgRNA sequence targeting the CHD1L gene was designed and screened, and the targeting sequence of Hu6-CHD1L-sgRNA was obtained by overlapping PCR method. Cloning the site with cleavage activity into the pLVX-mCherry-hU6-SgRNA vector. The vector was packaged as lentivirus by lentiviral packaging method and transfected into 7703 eSpCas9 cell line. The CHD1L knockout induced by Dox was verified by Western blot. The effects of Dox-induced CHD1L knockout on the biological function of hepatocellular carcinoma cells were detected by Wound Healing and Transwell assay. Results　In this experiment, vector Cas9 of pLV-Tet3G-Cas9 was successfully optimized as sequence eSpCas9. pLVX-mCherry-hU6-CHD1L-SgRNA vector was constructed. The QGY-7703 liver cancer cell line that conditional induction CHD1L gene knocking out was successfully constructed. Cell function experiments showed that the expression of eSpCas9 can be induced by Dox, the migration and invasion ability of these hepatoma cells were significantly decreased after knockout of CHD1L.　Conclusions　This study successfully constructed a conditional CHD1L knockout QGY-7703 liver cancer cell line. This model can be used as an effective tool to study the function and mechanism of specific genes targeting tumors.

Key words: CRISPR/eSpCas9; , , CHD1L; , , SgRNA; , , Hepatoma carcinoma cell

CLC Number:

R730.2

HUANG Li, LIU Shan-shan, ZHANG Xiao-feng, BAI Yin-shan, LIU Ming. The CRISPR/Cas9 system optimization and construction of CHD1L conditional knockout liver cancer cell lines[J]. Chinese Journal of Clinical Anatomy, 2020, 38(1): 39-44.

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The CRISPR/Cas9 system optimization and construction of CHD1L conditional knockout liver cancer cell lines

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