Method for screening CHO cell line high-expression site

A high-expression site and cell line technology, applied in the field of screening high-expression sites, can solve problems such as high cost and difficulty

Inactive Publication Date: 2018-01-09
JIANGNAN UNIV
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Problems solved by technology

On the one hand, this method objectively requires researchers to already have high-expression cell lines, and to master the difficult and expensive TLA technology to find integration sites;

Method used

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  • Method for screening CHO cell line high-expression site

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Embodiment 1

[0028] A method for screening high-expression sites in CHO cell lines, the method integrates a lentivirus with a green fluorescent gene into the CHO cell genome, collects and expands monoclonal cells with high fluorescent expression by flow sorting After culturing, the corresponding high-expression integration sites were screened out by chromosome walking technology.

[0029] The method comprises the following specific steps:

[0030] (1) Construct lentiviruses with fluorescent labels;

[0031] Transform the three plasmids pLVX-CMV-MCS-T2A-Zsgreen, pSPAX2 and pMD2G into E. coli strain DH5α (CB101-03) from Tiangen Company. In the LB medium, shake culture at 37°C and 250rpm for 24 hours, and then use the plasmid mass extraction kit DP117 from Tiangen Company to extract the three kinds of plasmids used for the preparation of lentivirus;

[0032] Inoculate the resuscitated HEK-293T cells into a T75 culture flask (the culture medium is complete medium containing 10% FBSDMEM), and...

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Abstract

The invention discloses a method for screening a CHO cell line high-expression site. The method comprises the following steps: integrating slow virus with a green fluorescence gene into a CHO cell genome, collecting monoclonal cells with high fluorescence expression quantity by a flow sorting method, performing expanding culture, and then screening out a corresponding high-expression integration site by using a chromosome walking technology. According to the method, a CRISPR / Cas9-mediated site-specific integration technology is combined, a to be expressed foreign gene can be quickly and accurately into a found site, and a foreign protein high-expression cell line can be quickly and efficiently obtained.

Description

technical field [0001] The invention relates to the technical field of biological genes, in particular to a method for infecting CHO cells with a lentivirus with a green fluorescent gene and screening out high expression sites. Background technique [0002] Chinese Hamster Ovary cell (CHO), as the main cell line in the field of biopharmaceuticals, has been developed into many different types of CHO cell lines, even those that can be used to expand gene copy number; then, transgenic An increase in copy number does not necessarily mean a significant increase in the yield of the target protein; and even when protein expression increases, such expression is often unstable. In addition, the currently commonly used method for constructing stable cells is time-consuming and labor-intensive, mainly because a large number of monoclonal screening processes need to be repeated, so it is generally expected in the field of pathway engineering to develop a method that can obtain Highly e...

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N5/10
CPCC12N5/10C12N15/65C12N15/867
Inventor 周松涛金坚
Owner JIANGNAN UNIV
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