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Most cell biology experiments utilize transient transfection protocols that afford peak gene expression between 24-96 hours post transfection. However, if sustained gene expression is required, generation of stable cell lines is a viable option. Furthermore, stable cell lines selected through limiting dilution/colony-picking provide a genetically homogenous and clonal population.
A. | B. |
Stable Cell Line Generation and Characterization. HEK 293 cells stably expressing EGFP were generated through transient transfection of EGFP and neomycin plasmid DNA vectors. Monoclonal populations were selected by limiting serial dilution and gene stability was verified for at least ten passages. Cells were assessed by fluorescence and phase microscopy (A) and flow cytometry (B).
Stable cell line generation is made possible by the use of positive selection markers such as G418, hygromycin B, puromycin resistance, etc.. Selection markers can be delivered using the same plasmid that contains the gene of interest (in cis), or on a separate plasmid (in trans) that needs to be co-transfected with the plasmid containing the gene of interest. The cis approach is generally easier and has a higher likelihood of producing drug-resistant stable transfectants that express the gene of interest. The trans method of co-transfection is a good alternative in instances where the target construct does not have the antibiotic-resistance gene in the vector backbone. In such cases, a plasmid mixture containing 5 to 10 parts gene expression plasmid and 1 part antibiotic selection marker plasmid can be introduced into cells. This plasmid ratio helps increase the likelihood that the selected cells will express both the gene of interest and the selection marker.
The protocol for generating stable cell lines requires several steps as shown below:
Timeline for Stable Cell Line Generation. Stable cell line generation can take a total of 9-12 weeks to establish the cell line. It is ideal to first determine the optimal antibiotic concentration for selection for each cell type; this can take up to 1 week. Following this, the target plasmids can be transfected for two days and antibiotic selection can be applied. Once the cells are under adequate selection, clone picking and expansion is the final and most time consuming part of stable cell line generation. After the stable cell line is generated, it is preferable to verify stable expression from a few cell passages before freezing stocks.
The first critical step for stable cell line generation is determining the optimal antibiotic concentration for selecting stable cell colonies; the optimal concentration is cell type dependent. A kill curve is a dose-response experiment where the cells are subjected to increasing amounts of antibiotic to determine the minimum antibiotic concentration needed to kill all the cells over the course of one week. Performing a kill curve is recommended with each new cell type or when a new selection antibiotic or different lot of selection antibiotic is used.
Selection Antibiotic | Working concentration range |
---|---|
G418 | 0.1 -2.0 mg/ml |
Hygromycin B | 100 – 500 µg/ml |
Puromycin | 0.25 – 10 µg ml |
G418 Titration for Determining Working Selection Concentration. Increasing amounts of the antibiotic such as G418 (0-1000 µg/ml) is added to duplicate wells of cells plated in complete media in a 24 well plate. Visual toxicity is assessed for upto a week to determine low, optimal and high doses for antibiotic selection.
While performing the kill curve (1 week), optimize transfection conditions in a T75 flask by transfecting a reporter plasmid (such as a GFP encoding plasmid) into cells at high confluence, e.g. 80%. Determine the appropriate dose of plasmid (5-15 µg) and transfection reagent (15-45 µl) in a T75 flask. Observe reporter gene expression (GFP) and toxicity at regular time points over at least a 48 hour period, optimally for 72 hours. Specific tips on optimizing DNA transfection can be found here.
Optional: Linearize your target plasmids before transfection. When generating a stable cell line, the transfected plasmid undergoes recombination during chromosomal integration. The recombination event can occur within any region of the plasmid, including the gene expression or selectable marker cassettes that might disrupt their function. To increase the likelihood that recombination will occur in non-essential plasmid regions, such as the bacterial replicon or bacterial marker gene, linearize the plasmid with restriction enzyme(s) that cut within these non-essential regions. Prior to transfection, purify the linearized DNA by ethanol precipitation, size exclusion or column purification.
The above procedure works well for routinely transfected cell types. For hard-to-transfect cells, another method to generate stable cell transfectants is via lentivirus or retrovirus transduction. In this case, antibiotic resistance harboring virus particles generated after transfection of producer cell types such as HEK293T are used to transduce cells that can then be selected for virus integration. Details on virus production can be found here.
TransIT® Transfection Reagents are Ideal for Virus Production. All TransIT® Transfection Reagents are low toxicity and do not require a media change. Save time and money by adding formed complexes directly to cells in media containing serum and avoiding unnecessary media changes.
Well No. | Antibiotic dose |
---|---|
1 | No antibiotic |
2 | Low dose of antibiotic |
3,4 | Optimal dose of antibiotic in each well |
5,6 | High dose of antibiotic in each well |
The polyclonal cell culture can be further processed to isolate monoclones using different techniques such as:
Of all the above-mentioned methods, limiting dilution is the most cost-effective and frequently adopted technique; a detailed protocol using limiting dilution to generate monoclonal cell lines is as follows:
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