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Construction method of GS gene-knockout CHO-K1 cell strain and suspension cell monocloning

A CHO-K1, gene knockout technology, applied in the biological field, can solve the problems of genome contamination, heavy workload, time-consuming and energy-consuming efficiency, etc., to improve the efficiency of GS gene knockout, reduce the risk of contamination, and avoid genome The effect of restructuring

Inactive Publication Date: 2021-09-07
上海碧博生物医药科技有限公司
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Problems solved by technology

Although conventional gene knockout by homologous recombination has been used in basic research and production of industrial cell lines, the conventional gene knockout by homologous recombination is time-consuming, labor-intensive and inefficient, which is not conducive to the development of CHO cell engineering; At present, the more commonly used method is to knock out the GS gene through CRISPR / Cas gene editing technology. However, this method is generally carried out at the DNA level, and genome reorganization is inevitable, and the genome has a certain risk of contamination. At the same time, GS gene knockout The removal efficiency needs to be improved; the screening of GS gene knockout CHO-K1 cells requires the use of compounds and a large workload

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  • Construction method of GS gene-knockout CHO-K1 cell strain and suspension cell monocloning
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  • Construction method of GS gene-knockout CHO-K1 cell strain and suspension cell monocloning

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Embodiment 1

[0044] figure 1 A method for constructing a GS gene knockout CHO-K1 cell line and a method for constructing a suspension cell monoclonal cell bank are shown.

[0045] A method for constructing a GS gene knockout CHO-K1 cell line, comprising the steps of:

[0046] Step A: Based on the nucleotide sequence of GS exon-5 in CHO-K1 cells, design and select sgRNA-GS57 for knocking out the GS gene. The specific operations are as follows:

[0047]Step A1: Determine the gene sequence of CHO-K1 cells and select the GS knockout gene, including the following steps:

[0048] Step A11 Adhesive culture of the CHO-K1 cell line purchased from the American Type Culture Collection (ATCC), the purchased batch number is 62960170;

[0049] In the above step A11, the culture conditions are as follows: the culture bottle is selected as T75 culture bottle, the culture medium is selected as F12K medium containing 10% fetal bovine serum, and the amount of culture medium is 15ml; the culture time is 2 d...

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Abstract

The invention discloses a construction method of a GS gene-knockout CHO-K1 cell strain. The construction method comprises the following steps: performing adherent culture on CHO-K1 cells, designing and selecting sgRNA, forming a transfection mixture by the selected sgRNA, EGFP mRNA and Cas9mRNA, and performing co-transfection on CHO-K1 to obtain the GS gene-knockout CHO-K1 cell strain. According to the method, the CHO-K1 cells are co-transfected by using the RNA transfection mixture, the method is a GS knockout method based on RNA, meanwhile, a CRISPR / Cas9 gene editing means and GFP screening are combined, thus genome reforming at the DNA level is avoided, the risk that the genome is polluted is reduced, the GS gene knockout efficiency is improved, and the workload of cell screening is reduced; and verification on the GS gene-knockout CHO-K1 cell strain obtained by the invention shows that the GS gene knockout efficiency meets the expectation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a GS gene knockout CHO-K1 cell line and monoclonalization of suspension cells. Background technique [0002] Chinese hamster ovary cells (CHO cells) have become one of the most widely used host cells for the production of therapeutic recombinant proteins. It can quickly identify those rare high-producing cell lines among a large number of low-producing and non-productive cells, and achieve an effective and efficient clinical cell line development process, which is of great significance for accelerating the development of biopharmaceuticals. [0003] The two most common CHO expression systems for recombinant protein production utilize dihydrofolate reductase (DHFR)-based methotrexate (MTX)-based selection methods or glutamine synthetase (GS)-based Methoxine (MSX) selection method. Among them, the GS gene knockout cell line based on the CHO-K1 cell line is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N15/55C12N15/52C12N5/10
CPCC12N15/87C12N9/22C12N9/93C12N5/0682C12Y603/01002C12N2510/00
Inventor 焦鹏徐琦陈少勇林文龙李睿黄启龙
Owner 上海碧博生物医药科技有限公司
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