The generation of stably-transfected cell lines is critical for a wide range of applications. It is applied to the production of recombinant proteins, gene function studies, as well as drug discovery assays. Stable expression of target gene in cell lines overcomes the low transfection efficiency of transient expression and produces more target proteins.
There are two types of stable cell lines. One is achieved by eukaryotic vectors that harbor elements for episomal maintenance in the nucleus of a transfected cell. Another is achieved via direct integration of the transfected plasmid into the target cells genome. Episomal stability is often limited and episomal plasmid elements is often restricted to certain species, therefore integration into the host cell chromosome is in common use. Although integration into the host cell chromosome is a rare event and, for most purposes, clonal events have to be isolated, stability of the intended genetic modification usually is much higher.
Major challenges for generation of stable cell lines are low transfection efficiency and/or integration frequency. Stable expression can be influenced by the transfection method used. The transfection method determines the cell type for stable integration. It is known that liposome reagents can be used to transfer DNA into adherent cell lines. While viral methods or electrotransfection are used to deliver DNA into primary cells or notoriously difficult-to-transfect suspension cell lines. Unfortunately, viral methods suffer from several limitations, such as time consuming production of vectors and safety concerns, while electrotransfection suffer from the low cell survival rate.
In order to select stably-transfected cells, a selection marker must be co-expressed with the target protein. The marker gene could be on either the same plasmid vector or a second, co-transfected vector. There are a variety of systems for selecting transfected cells, including resistance to antibiotics puromycin, neomycin, DHFR, and glutamine synthetase. After gene transfer, cells are developed in medium containing the selective agent. Only those cells which have contained the drug resistant gene survive.
Methods to Generate Stable Cell Lines
Depending on the scope of the experiment, several options are used for the generation of a stable cell line. A mixed population of drug resistant cells can be used directly for experimental analysis with the advantage of generating fast results, but also the disadvantage of dealing with an undefined and genetically mixed cell population. Another option is to generate a monoclonal cell line. In this method, it is necessary to dilute the resistant cells by plating in 96-well plates in such a way that culture as single and isolated cells. Subsequently, the cloning of single cell may be repeated several times to obtain 100% clonal purity. This culture method can be used for screening experiments or conduction studies by using a homogenous and defined cell system.
In conclusion, depending on the type of expression you’re interested in and the construct that you are incorporating, there are many different approaches for generation of stable cell lines. This protocol is specific for the generation of a monoclonal cell line that resistance to antibiotics G418 (neomycin). The end result that you are looking for is a population of cells in which 100% of cells are expressing your fusion protein.
Culture conditions (passage, split rhythm, number, etc.) of your selected cell type are critical for generation of stable cell lines. For optimal results, we recommend following the cell culture recommendations of the supplier (e.g. ATCC) for the respective cell type. In general, for promoting good proliferation and cell physiology, the cell line should be passaged two days before the experiment. Besides, cell passage should not be higher than 30 due to the possibility of interference with integration efficiency.
1. Choose the G418 Concentration
Susceptibility to G418 is different among cell lines, which many even vary with cell passage numbers. The selection condition (e.g. G418 concentration, plating density) for your specific cell type needs to be determined experimentally. Determine the minimum level G418 concentration to guarantee the minimum impact to cell growth. Note that the active concentration of stock G418 can vary considerably from batch to batch. Therefore, we recommend testing G418 sensitivity for every new batch. The final plating density depends on the specific cell type and the G418 concentration. We therefore recommend matrix titration of G418 and titration of cell number for determination of plating density in one 96-well plate.
For transfection, please follow the manufacturer’s instruction of your transfection system. The important thing is to transfect the expression plasmid containing the target gene and the sequence for a drug resistance gene into your cells. We suggest setting a negative control of untransfected cells for selection. Besides, it is much better to check the transfection efficiency and integration frequency of your experiment with a GFP-control plasmid.
3. Cell Selection Post-Transfection
4. Analysis of Stable Cell Lines
Once you have obtained resistant cell lines, you should expand the cells and assay your target gene compared with positive and negative control. You can detect the expression of fusion protein by an appropriate analysis method such as western blot, microscopy, ELISA, as well as flow cytometry.
Creative BioMart provides stable cell line services to meet your specific needs. Not only have we developed cell lines stable at expression level, but also established stable cell lines ready for assay development.