Stable Transfection

In stable transfection, the genes of interest are successfully integrated into the cellular genome and are passed it on to daughter cells. The stable transfected cells can grow continuously over a long period of time without significant change in the expression level of the target gene. This is a key method in reducing the costs of biopharmaceuticals or for long-term protein production. The stable transfected cells are the best choice not only for the protein production but also for research in various fields, including signal transduction, ion channel research, drug target screening, and immunotherapy drug development.

At Biologics International Corp (BIC), we are proud of our ability to offer you the stable cell line service which covers all your needs in the aspects of research assay and protein/antibody production. Please contact us for any questions.

Stable Transfection Cell Line for Protein/Antibody Production

The most widely used mammalian expression system for protein production in commercial production is gene amplification using dihydrofolate reductase-deficient (DHFR-) chinese hamster ovary (CHO) cells by DHFR-mediated gene amplification [1]. The target protein gene is constructed on the same plasmid as the DHFR-mediated gene, followed by transfection into DHFR- CHO cells. After 48 hours of transfection, the transfected cells are plated into 6-well plates for screening the single clones or cell pools.

The next step is enhancement of the protein expression level of stable transfected cells. A common method is MTX pressure selection, which could bind to and inhibit the DHFR enzyme, leading to cell death. The concentration of pressure is gradually increased for multi-round selections, and as a result, only the clones with higher expression will survive. Then, these clones undergo monoclonal screening by limited dilution to select clones with the highest expression.

In addition, the glutamine synthetase (GS) gene is another widely used selectable marker. The pressure selection of the GS amplification system is MSX, which enables suppression of the endogenous glutamine while allowing the use of GS vector for recombinant expression. The GS system has higher amplification efficiency than the DHFR gene amplification system.

After pressure selection, the cell growth and the expression stability of the selected clones are evaluated using ELISA. The stability test is conducted for dozens of generations (such as 50 generations). Upon the results of growth and production profile, the top 3-5 clones with the highest expression level and stability are selected for freezing and delivery.

We can simplify the process of stable cell line construction and save your research and development time using our self-developed CHO cells and by adopting the DHFR/GS expression system.

Stable Transfection Cell Line for Research Assay

The development of a stable cell line for research assay is relatively faster and easier than a stable cell line for protein/antibody production. In stable transfection, the use of selectable markers (usually an antibiotic) that co-expresses with the gene of interest allows researchers to distinguish transient from stable transfections. With continued antibiotic treatment of cells, only the stable transfected cells are able to survive for long periods of time, whereas the transient transfected cells die due to the lack of antibiotic resistance.

Unlike the stable cell line service for protein/antibody production, the construction of stable cell line for research assay does not require pressure selection. Instead, positive clones are selected for the stability (for 10 passages) and mycoplasma tests directly.

The development of stable cell line for research assay is time-consuming and complex. BIC has accumulated comprehensive knowledge about optimal antibiotic concentrations, from which we have successfully cultured dozens of cell lines (CHO, HEK293, HeLa, M14, THP-1, BHK21, HFF-1, HepG2, MCF-7, Vero and more). You can also view our stable cell line generation for research assay and gene knock-out services that are the best match for your application.

The Difference between Transient and Stable Transfection

The generation of stable transfected cells begins with a transient transfection, followed by successfully integration the plasmid DNA into the cellular genome. This method is a much rarer occurrence but an important and serendipitous process. Unlike a transient transfected cell, a stable transfected cell will continuously express the transfected DNA and will be passed on to future generations of the cell.

Cell Transfection	Transient Transfection	Stable Transfection
Timeline of expression	Rapid (3-4 weeks)	Lengthy (12-18 weeks)
Cost	Relatively inexpensive	Expensive
Expression level	Relatively low	High and stable
Foreign gene	Lost in few days	Exists over a long period of time
Secreted or membrane proteins	Good to yield	Good to yield
Intracellular proteins	Hard to yield	Hard to yield
Folding and post-translational modifications	Yes	Yes

References

1. E. V. Voronina, Y. A. Seregin, et al. Design of a stable cell line producing a recombinant monoclonal anti-TNFα antibody based on a CHO cell line. SpringerPlus, 2016, 5:1584.

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