I am going to make a stable transfected hek293 cell line. My plasmid contains neomycin as selection marker. What concentration of neomycin should be used for selection?
Hello Marianne,
Literature suggests a range between 200 to 500 ug/mL. However, I ll suggest you to do a kill curve to determine optimal concentration for your HEK293 cells. Please make sure these are HEk293 cells and not 293T cells that are resistant to G418.
yes, you need to determine the sensitivity of your HEK293 cells to neomycin first with a kill curve. Start with a high concentration e.g. 2 mg/ml and dilute your way down. Sometimes HEK293 can tolerate a bit more than 500 ug/ml. When you transfect your cells, you should also treat empty HEK293 with the same concentration of neomycin you choose - as a control, to make sure empty HEK293 die and when.
Marianne,
You must do a kill curve as suggested by Ali & Nadra, and select a proper selection concentration. But make sure that you calculate the amount of active neomycin required to prepare the stock solutions and subsequent dilutions. This simple point is often missed and may lead to false selection concentrations.
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
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