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A modified cho cell and its use

A technology for cells and uses, applied in the field of cell engineering and genetic engineering, can solve problems such as limited production capacity and inability of cells to grow.

Active Publication Date: 2020-07-24
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the domesticated CHO cell lines generally have the following problems: the cells cannot achieve stable and high-density growth; the production capacity of the cells is relatively limited (usually less than 10 pg / cell / day) when integrating foreign genes to stably express foreign proteins )

Method used

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  • A modified cho cell and its use
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  • A modified cho cell and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Example 1: Screening and Characterization of CHO-9618s Cell Line

[0118] 1.1. Screening of CHO-K1 cell line (CHO-9618s) capable of high-density growth under serum-free and suspension conditions

[0119] The CHO-K1 cell line (ATCC CRL-9618) purchased from ATCC was cultured adherently in DMEM / F12 medium (abbreviated as DF12 medium) supplemented with 10% FBS, and the CHO in the logarithmic growth phase was selected. - K1 cells. The cultured cells were observed through a microscope. The results showed that the CHO-K1 cells were in the shape of willow leaves and grew well on the wall ( figure 1 A).

[0120] The medium was replaced with a 1:1 mixture of DF12 medium and SFM IV medium (the final concentration of FBS was 5%), and the cells were continued to be cultured statically for at least 1 week. Then, replace the medium with a 1:4 mixture of DF12 medium and SFM IV medium (the final concentration of FBS is 2%), and continue to culture the cells statically until the cell...

Embodiment 2

[0131] Construction and characterization of embodiment 2.CHO-NIDVD

[0132] In this example, the cell line CHO-9618s was modified to construct a new cell line CHO-NIDVD, in which the GS gene and the FUT8 gene had been knocked out.

[0133] 2.1. Construction of CHO-NIDVD cell line

[0134] Briefly, the cell line CHO-9618s was modified according to the gene editing method described in Ran, F.A. et al. Genome engineering using the CRISPR-Cas9system. Nature protocols 8(143):2281-2308 (2013); The gene Zeocin (Zeo) replaces the fifth exon of the GS gene to achieve knockout of the GS gene; and, introduces a frameshift mutation (ie, inserts a base) in the seventh exon of the FUT8 gene , to achieve knockout of the FUT8 gene. The nucleotide sequence of the fifth exon of the GS gene (NCBI accession number: NW_003613921.1; positions 1430036-1435423) is shown in SEQ ID NO:1. The nucleotide sequence of the seventh exon of the FUT8 gene (NCBI accession number: NW_003613860.1; positions 60...

Embodiment 3

[0149] Example 3. Transient expression of foreign proteins in CHO-NIDVD

[0150] The expression plasmid PTT5-GFP encoding green fluorescent protein was transfected into CHO-NIDVD cells in logarithmic growth phase. After 48 hours, observe and take pictures under a fluorescent microscope. Observation results such as Figure 13 shown. The results showed that CHO-NIDVD can be used to express foreign proteins (such as green fluorescent protein).

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Abstract

The invention relates to modified CHO cells and application thereof. The modified CHO cells can realize stable and high-density growth under condition of serum-free suspension culture, and preferably,the modified CHO cells do not express functional glutamine synthetase (GS) and fucosyltransferase 8 (FUT8). The modified CHO cells are particularly applicable to high-level eukaryotic expression of recombinant proteins (especially antibodies), and thus have good application prospects in the fields of genetic engineering and protein engineering.

Description

technical field [0001] This application relates to the fields of genetic engineering, protein engineering and cell engineering. Specifically, the present application relates to a modified Chinese hamster ovary cell (CHO) and its application. The modified CHO cells can grow stably and at high density under serum-free, suspension culture conditions, and preferably, do not express functional glutamine synthetase (glutamine synthetase, GS) and fucosyl transfer Enzyme 8 (Fucosyltransferase 8, FUT8). Such modified CHO cells are particularly suitable for high-level eukaryotic expression of recombinant proteins (especially antibodies), thus having broad application prospects in the fields of genetic engineering and protein engineering. Background technique [0002] Since the U.S. FDA approved Genetech's recombinant insulin in 1982, recombinant proteins have been successfully used in scientific research, diagnosis, treatment and prevention of various diseases. Currently, recombina...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C07K16/00C12R1/91
CPCC07K16/00C12N9/1051C12N9/93C12Y204/01068C12Y603/01002
Inventor 罗文新游敏陈奋天夏宁邵
Owner XIAMEN UNIV
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