I'd like to transfect HepG2 cells with the glucagon receptor and look at cyclic AMP response (amongst other things). This is of course a receptor usually expressed in hepatocytes, but in with the cells I have, there is very little response to glucagon (as has been observed by others). I'm hoping that overexpressing the receptor in this cell line will allow me to study downstream cellular responses to glucagon in a more meaningful way than using HEK293 etc.
Does anyone have any experience of producing a stable transfection with these cells? Are there any things to watch out for? I have very little transfection experience so far. In the lab people tend to use PEI or lipofectamine 2000.
Hi, in our hands they are pretty hard to transfect with low efficiency , using Lipofectamine LTX which to us gives best transfection rates. Maybe you can try to transduce them with a lentivirus and establish a stable cell line that way. HepG2 are permissive to lentis. Good luck!
We have similar experience that HepG2 are more difficult to transfect as compared to Hek293 cells. However, I could establish several stable transfections in HepG2 just using simple calcium phosphate protocol for transfection, so it should also work with lipofectamine or PEI.
Even if you have a very low transfection efficiency of 5 % (as example) that would give you thousands of transfected cells, which can than be further selected, so just try it.
Hi Ben Jones,
HepG2 cells are hard to transfect and i would suggest to use Huh7 cells, hepatoma cell line. Huh7 cells are very easy to transfect
Sathish
I have optimized a recipe for transfecting HepG2 with LipofectaminLTX. It's proved to work with beta-galactosidase and Period 2. I'm working to find out if the amplitude of overexpression is capable of significantly influencing the downstream signals.
CaCl2 ttransfection works well with HepG2.
If possible I would suggest using lentivirus - this way you get a permeant expression of your receptor. You can also use this receptor to later sort the cells out (using FACS) and than you'll end up with nearly 100% of cells expressing your receptor permanently.
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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