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Construction method for hepatoma cell line for conditionally-induced knockout overexpressed Chd1l genes

A technology of liver cancer cell line and construction method, which can be applied to tumors/cancer cells, genetically modified cells, other methods of inserting foreign genetic materials, etc. Reference effect, good contrast effect

Inactive Publication Date: 2018-07-31
GUANGZHOU MEDICAL UNIV
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Problems solved by technology

In the embryonic stage of animals, when the expression of the target gene is not required, the addition of Dox will have a negative impact on the growth and development of the fetus

Method used

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  • Construction method for hepatoma cell line for conditionally-induced knockout overexpressed Chd1l genes
  • Construction method for hepatoma cell line for conditionally-induced knockout overexpressed Chd1l genes
  • Construction method for hepatoma cell line for conditionally-induced knockout overexpressed Chd1l genes

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Embodiment 1

[0033] Example 1 Method for Establishing Chd1l-EGFP Liver Cancer Cell Line with Conditional Induction and Knockout of Chd1l Gene

[0034] see figure 1 , a method for establishing a Chd1l-EGFP liver cancer cell line that is conditionally induced to knock out the Chd1l gene in this embodiment, comprising the following steps:

[0035] 1), establishing the 7703 liver cancer cell line of Chd1l-EGFP overexpressing the Chd1l gene

[0036] According to the Chd11 gene sequence (NM_004284), the CMV was constructed to start the expression vector carrying EGFP, and the 7703 liver cancer cell line expressing EGFP and the Chd11-EGFP overexpressing Chd11 were finally established through transgene and drug screening (such as figure 2 ), after detection and analysis, the 7703 liver cancer cell line has the following characteristics: expression of EGFP fluorescence, increased ability of tumorigenicity (such as image 3 ).

[0037] 2), Construction of Chd1l-7703 liver cancer cell line that c...

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Abstract

The invention discloses a hepatoma cell line for conditionally-induced knockout overexpressed Chd1l genes and a construction method thereof. The method comprises the steps that firstly, a hepatoma cell line for overexpressed Chd1l and green fluorescent proteins is constructed; secondly, a carrier for conditionally-induced expressed Cas9 is transferred into the hepatoma cell line; finally, a pLVX-mCherry-hU6-Chd1l-SgRNA carrier with red fluorescence and targeted Chd1l cutting activity is constructed and transferred to the cell line, and the hepatoma cell line for conditionally-induced knockoutoverexpressed Chd1l is obtained at last. The cell line has the advantages of being the hepatoma cell line for overexpressed Chd1l before the induction of doxycycline (Dox) and being the hepatoma cellline for expressed knockout Chd1l after induction. The cell line has good contrast, the simultaneous culture of multiple cell lines can be reduced, the scientific research work is simplified, and thecell line is an ideal tool for gene function research.

Description

technical field [0001] The invention belongs to the field of tumor research, and relates to a Chd11-EGFP liver cancer cell line with conditional induction and knockout of the Chd11 gene and a construction method thereof. Background technique [0002] Chd1l (chromodomain helicase / ATPase DNA binding protein 1-like gene) gene is located in the chromosome 1q21 region, contains the conserved SNF2-N functional domain, helicase functional domain and Macro functional domain, belongs to the SWI2 / SNF2 related ATPase superfamily, and It is related to processes such as chromatin unwinding and DNA repair. In addition to being specifically highly expressed in liver cancer, this gene has abnormal amplification and high expression in various solid tumors, and is closely related to the occurrence, development and prognosis of various tumors. In hepatocellular carcinoma (HCC), the reduction of Chd1l expression by siRNA interference technology has shown great therapeutic potential in its trea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/90C12N5/10
CPCC12N5/0693C12N9/14C12N9/22C12N15/86C12N15/907C12N2510/00C12N2740/15043C12N2800/107C12N2810/10
Inventor 白银山刘珊珊黄丽张晓锋马宁芳
Owner GUANGZHOU MEDICAL UNIV
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