Glut4 gene knockout sgRNA, A549 cell line and its construction method

A gene knockout and construction method technology, applied in the field of genetic engineering, can solve the problems of uptake and cell function impact, and achieve the effect of complete knockout and reduced glucose uptake

Active Publication Date: 2021-06-15
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, whether the loss of GLUT4 gene expression will affect the glucose uptake and cell function of A549 cells has not been reported yet.

Method used

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  • Glut4 gene knockout sgRNA, A549 cell line and its construction method
  • Glut4 gene knockout sgRNA, A549 cell line and its construction method
  • Glut4 gene knockout sgRNA, A549 cell line and its construction method

Examples

Experimental program
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Embodiment 1

[0030] Example 1 A549 cell line knocked out of GLUT4 gene and its construction method

[0031] 1. Sequence design of GLUT4 gene sgRNA

[0032] Search the human GLUT4 gene sequence on the NCBI website to find the exon sequence and send the exon sequence to the website http: / / crispr.mit.edu / , and then obtain the exon information of the GLUT4 gene from the website, from EXON 1 -EXON 6 selects EXON 3--GAGCTACAATGAGACGTGGC (as shown in SEQ ID NO: 1) as the target sequence of the sgRNA, designs the restriction endonuclease site at both ends of the sgRNA according to the bBSI restriction endonuclease, and adds CACCG constitutes the sequence SEQ ID NO: 2; AAAC is added to the 5' end of the reverse complementary sequence. Nucleotide C is added to the 3' end to form the sequence SEQ ID NO: 3, and finally the designed plasmid is sent to a biological company for synthesis.

[0033] Table 1 - sgRNA primer sequences

[0034]

[0035] 2. Construction of pX458 plasmid expression vector:...

Embodiment 2

[0071] Example 2 Effect of wild-type A549 cell line and GLUT4 knockout A549 cell line on glucose uptake

[0072] In this embodiment, the glucose uptake is detected by Confocal technology after the cell patch is cultured, which specifically includes the following steps:

[0073] 1. Grouping: experimental group and control group; the experimental group is the A549 cell line with GLUT4 gene knockout; the control group is the wild-type A549 cell line. Culture the cells of the experimental group and the control group separately, and when the cells cover the bottom of the dish, digest the cells, enrich the digested cells with 1mL of culture medium, then discard 850mL of the cell suspension, and reserve 150mL of the cell suspension for future use. Add 3 mL of culture medium to the remaining 150 mL of solution for dilution.

[0074] 2. Use tweezers to take out the standby glass slide soaked in alcohol, dry it on an alcohol lamp, place it in a glass petri dish, take 1mL of diluted cul...

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Abstract

The invention provides a GLUT4 gene knockout sgRNA, an A549 cell line and a construction method thereof. The sgRNA for knocking out the GLUT4 gene provided by the present invention has a target sequence on exon 3 of the GLUT4 gene, and the target sequence is shown in SEQ ID NO: 1; the present invention also provides the A549 cell line for knocking out the GLUT4 gene As well as the construction method, the expression vector pX458‑GLUT4 is used as the targeting carrier of the GLUT4 gene to transfect A549 cells to obtain a monoclonal cell line with knockout of the GLUT4 gene; the present invention utilizes the CRISPR / Cas9 system to knock out the GLUT4 gene from the A549 cells to obtain Both gene and protein levels have confirmed that GLUT4 has been successfully knocked out, providing an ideal cell model for studying the effects of GLUT4 gene expression loss on glucose uptake and cell function in A549 cells.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to sgRNA for GLUT4 gene knockout, A549 cell line, construction method and application thereof. Background technique [0002] A549 cells are a human lung adenocarcinoma epithelial cell line. It is very similar to type II alveolar cells in shape, structure and composition of metabolites. The latter is a type of spherical alveolar cells with a diameter of about 9 microns. Its main function is to synthesize and secrete some lipoprotein active substances, reduce alveolar air The surface tension of the interface increases alveolar stability and prevents edema. Therefore, A549 cells have been used as a model to study type II alveolar cell responses. On the other hand, as a kind of non-small cell lung cancer, it has certain significance in lung cancer research. As we all know, cancer cells have the ability of uncontrolled unlimited proliferation and continuous invasion and mi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N9/22C12N5/10C12Q1/02C12Q1/6869
CPCC12N5/0693C12N9/22C12N15/113C12N15/85C12N15/907C12N2310/10C12N2510/00C12Q1/6869G01N33/5011C12Q2531/113
Inventor 赵平刘佳张颖
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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