Hi
1- I did transient transfection for hek293 cells by 6 kb plasmid after 48 h the cells expressed my target protein at approximately 60%, I used lipofectamine 2000 in a DNA: LIPO ratio 1:4
then in a separate experiment I repeated the transfection to generate stable clone, I split the cells, 24 h after the transfection and the day after add G418 500 ug/ml
after two weeks cells clusters were formed, after a month I screen the transfection efficiency and the result was negative
I repeated the transfection but adding the G418 in the splitting day and the results came negative again
what could be the reason for this? why it work fine with transient only not stable? how can I make sure the cells will express my target earlier than 1 month? any suggestions?
HEK293 should be transfected with a suitable reagent like this one (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/). A 6kb plasmid isn't that large, but you should still use a complex condenser to make sure that it has an easier time entering into cells.
Is 1:4 the optimal DNA:lipo 2000 ratio for the transfection of your plasmid in HEK293T cells? If that was your starting point, I would attempt to optimise the ratio to obtain the best possible transfection efficiency. Once optimised, I would upscale the transfection as generating stable clones via transient transfection has a very low efficiency as Weber Lin already mentioned.
I would also add G418 48hrs post transfection to allow your positively transfected cells sufficient time to begin expressing the resistance gene off the plasmid. You could also consider harvesting conditioned medium from a culture of HEK293T cells to use post-transfection as it will contain all the nutrients and factors required to promote HEK293T cell growth under antibiotic selection.
Did you linearize your plasmid to insure that the recombination would not disrupt your gene expression?
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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