Method for knocking out KDM2A gene of HEK293T cells with CRISPR-CAS9 technology

A CAS9, gene technology, applied in the field of genetic engineering, can solve the problem of unclear cell apoptosis, and achieve the effect of complete knockout, complete and thorough effect, and easy operation.

Inactive Publication Date: 2018-09-07
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have shown that KDM2A can promote cell growth and migration, but how it affects apoptosis is still unclear

Method used

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  • Method for knocking out KDM2A gene of HEK293T cells with CRISPR-CAS9 technology
  • Method for knocking out KDM2A gene of HEK293T cells with CRISPR-CAS9 technology
  • Method for knocking out KDM2A gene of HEK293T cells with CRISPR-CAS9 technology

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Embodiment 1

[0019] Example 1: A method for knocking out the KDM2A gene in HEK293T cells using CRISPR-CAS9 technology.

[0020] 1. Experimental materials

[0021] Eukaryotic expression vector pSpCas9(BB)-2A-GFP(pX458), human HEK293T cells.

[0022] 2. Construction of CRISPR / Cas9 targeting vector

[0023] (1) Determination of target sequence

[0024] The exon information of the human KDM2A gene (NM_012308.2) was obtained according to the website http: / / asia.ensembl.org / index.html, and exon 14 and exon 18 were selected as target sequences. Then design the sgRNA sequence, add the BbsI restriction site sequence (as shown in SEQ ID NO: 1, 2) to its 5' end, and synthesize its complementary sequence (as shown in SEQ ID NO: 3, 4).

[0025] (2) The target sequence is connected to the pX458 plasmid

[0026] ① Digest the pX458 empty vector, see Table 1 for the 20 μL reaction system, and digest overnight at 37°C.

[0027] Table 1 pX458 empty plasmid digestion system

[0028] Element

...

Embodiment 2

[0061] Example 2: The effects of KDM2A on the proliferation and apoptosis of wild-type cell lines and knockout lines were detected by flow cytometry

[0062] 1. In order to detect how the knockout of KDM2A gene will affect the cells specifically, we carried out EdU cell proliferation detection and Annexin V-FITC / PI cell apoptosis detection on the wild-type HEK293T cells and the knockout line respectively. The operation process is as follows:

[0063] (1) Inoculate 1×10 per well in a six-well plate 5 ~3×10 6 cells, cultured to the normal stage;

[0064] (2) EdU medium was prepared according to the instructions, and a blank control without EdU medium was set up, so as to perform dye background analysis of flow cytometry data. After replacing the cell culture medium with EdU medium and control medium, add 1 mL to each well and incubate for 2 hours;

[0065] (3) Transfer the cells to a 1.5mL EP tube, collect the cells and discard the supernatant, resuspend the cells in PBS, an...

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Abstract

The invention provides a method for knocking out a KDM2A gene of HEK293T cells with a CRISPR-CAS9 technology. The method comprises the steps as follows: (1), cell transfection; (2), cell genome extraction; (3), PCR identification; (4), gene level verification; (5), protein level verification: whether K3, K7-1 and K7-2 are KDM2A gene-deficient is further verified by Western blotting after a mutantcell line is verified at the gene level. The invention further provides a KDM2A knockout HEK293T cell line prepared with method and an application of the KDM2A knockout HEK293T cell line as a cell model in study of cell proliferation and apoptosis signaling pathways involved in KDM2A methylase.

Description

technical field [0001] The invention provides a method for knocking out the KDM2A gene of HEK293T cells by using CRISPR-CAS9 technology, which belongs to the technical field of genetic engineering. Background technique [0002] HEK cells are derived from human embryonic kidney cells. HEK293T cells are a variant of HEK cells. They are transfected with SV40 large T antigen and can proliferate in vitro. This cell line proliferates quickly and has high transfection efficiency. It is commonly used in protein expression. The lysine-dependent demethylase family has extremely important effects on cell proliferation and apoptosis, which determines its potential application as a tumor therapy. One of the notable features of cell carcinogenesis is the dysfunction of epigenetic regulation caused by the dysregulation of the expression of methylase and demethylase. In this demethylase family, KDM2A is a member that has not been well understood so far. The main function of KDM2A is to r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N5/10
CPCC12N5/0603C12N5/0686C12N9/0071C12N9/22C12N15/113C12N15/85C12N15/907C12N2310/10C12N2510/00C12Y114/11027
Inventor 彭勇波许文豪梁大焱
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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