Screening method of GS (glutamine synthetase) expression system cell strains

Abstract

The invention discloses a screening method of GS (glutamine synthetase) expression system cell strains. The method comprises the screening steps as follows: after an expression vector containing a target gene is transfected into host cells, the host cells are cultured in a 5% CO2 incubator at 37 DEG C for 3-4 weeks under the MSX (methionine sulfoximine) pressurization condition, the fermentation liquid expression is detected, positive clone cells are screened out and subjected to scale-up culture until the cell viability reaches 85% or above, monoclonal screening is performed with a limited dilution method, host cells with the density being 1*10<4>-1*10<5> / well are added to a 96-well plate during screening and cultured in the 5% CO2 incubator at 37 DEG C for 3-5 weeks, positive monoclonalcell strains are screened out, and the GS expression system cell strains are obtained. The host feeder cells with the density being 1*10<4>-1*10<5> / well are added during cloning, the average cloning efficiency can reach 22.1%, and the monoclonal efficiency is substantially improved.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a screening method for GS expression system cell lines. Background technique [0002] Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia, which is the only way glutamine is formed in mammals. GS is essential for the survival of mammalian cells in glutamine-free media. Some mammalian cell lines, such as mouse myeloma cells, do not express enough GS to survive without the additional addition of glutamine. While other cell lines, such as Chinese hamster ovary cells (CHO), can express enough GS to survive without the need for additional glutamine. In this case, the GS inhibitor methioninesulfoximine (MSX) can be used to inhibit the endogenous GS activity, and only the transfected cells containing the exogenous GS gene can survive. [0003] The glutamine synthetase (GS) expression system is one of the mammalian expression systems widely used in...

AI Technical Summary

Problems solved by technology

However, this method has high requirements on the growth state of cells, and the cloning efficiency is extremely low.

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