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Method for easily screening and obtaining target gene knock-out cell line by using CRISPR/Cas9 technology, and product of method

A technology for gene knockout and screening of marker genes, applied in the field of genetic engineering, can solve problems such as difficulties, huge screening workload, and inconvenient research

Inactive Publication Date: 2017-05-10
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PX330 does not have a screening marker gene, which results in a huge screening workload and a relatively low probability of obtaining a positive cell line for gene knockout, which will cause great inconvenience and difficulty to follow-up research

Method used

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  • Method for easily screening and obtaining target gene knock-out cell line by using CRISPR/Cas9 technology, and product of method
  • Method for easily screening and obtaining target gene knock-out cell line by using CRISPR/Cas9 technology, and product of method
  • Method for easily screening and obtaining target gene knock-out cell line by using CRISPR/Cas9 technology, and product of method

Examples

Experimental program
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Effect test

Embodiment

[0049] Example, using CRISPR / Cas9 technology to knock out the Nedd4 gene in BMDM cells and quickly obtain cell lines

[0050] The embodiment uses CRISPR / Cas9 technology to knock out the Nedd4 gene in BMDM cells (the embodiment uses the Nedd4 gene as the target gene, and other target genes are similar to the method in this example) and quickly screen to obtain cell lines, including the following processes:

[0051] 1) Construct the Tia11 recombinant vector carrying the screening marker and the 2A self-cleaving peptide coding sequence. In the embodiment, puromycin (puromycin) or blasticidin (blasticidin) is used as an example of a selection marker, and other selection markers are used similarly to the method in this example.

[0052] Such as figure 1 (A: Tia1l vector structure; B: BamH1-2A-Puro(Bsd)-BGHpA-HindⅢ fragment amplified by Overlapping PCR; C: Tial vector multiple cloning site structure: the multiple cloning site includes BamH Ⅰ, EcoR Ⅴ, Hind Ⅲ enzymes Cutting site, i...

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Abstract

The invention discloses a method for easily screening and obtaining a target gene knock-out cell line by using a CRISPR / Cas9 technology, and a product of the method. According to the method, small guide ribonucleic acid (sgRNA) of a target exon is designed aiming at an exon of a target gene-Nedd 4 gene, a stable knock-out Nedd 4 gene can be obtained by using the CRISPR / Cas9 technology, puromycin or blasticidin screening and Western blot identification, and the cell line which can be screened by using puromycin or blasticidin is obtained. The method combines a PX330 carrier (a CRISPR / Cas9 carrier) and a Tiall carrier for use so as to obtain the target gene knock-out cell line; therefore, an effective tool is provided for researching the functions and action mechanisms of genes.

Description

technical field [0001] The invention belongs to the genome editing technology in the technical field of genetic engineering, and relates to a method and application of using CRISPR / Cas9 technology to knock out a target gene in a cell for directional gene editing and easy screening to obtain a cell line. Background technique [0002] CRISPR / Cas9 genome editing technology, known as "gene magic scissors", is an important tool for targeted gene editing that has emerged in recent years and has been applied to many model organisms, including humans, mice, and rats , zebrafish, Caenorhabditis elegans, plants, and bacteria. This technology uses artificially designed special guide RNA (single-guideRNA, sgRNA, also known as targeting RNA) to mediate the exogenously expressed endonuclease cas9 protein to specifically cut the target on the genomic DNA, and then, The cell's non-homologous end joining repair mechanism (NHEJ) reconnects the genomic DNA at the break, thereby introducing ba...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12R1/91
CPCC12N15/85C07K14/47C12N5/0645C12N2800/107C12N2800/80C12N2810/10
Inventor 刘庆军张市会周虹
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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