stable cell line works, but transient transfections wont - why?

Imre Hunyor via methods%40net.bio.net (by imre.hunyor from gmail.com)
Tue Jul 24 05:20:04 EST 2007


I'm having considerable trouble getting my gene to be transiently expressed
even though the stable cell lines with the same construct have worked
successfully. I'm concerned that this may mean that there is something
inherently wrong with the plasmid before i go ahead with mouse blastocyst
microinjection, but i'm not sure. Is there a reason why stables work, but
transients don't seem to? Even if there is a reason, could it have
implications for expressing the construct in a transgenic mouse line?
I'm transfecting my plasmid into an existing stable line of NIH-3T3 tet-off
cells (G418 resistant). The construct is basically my gene of interest
cloned into pTRE-Tight (Clontech). There is no Kozak sequence present for
this cardiac-specific gene and I have about 100bp of the 5' UTR in the
construct (Q- would introducing a Kozak make a difference considering the
stables worked anyway?). I have verified that my sequence is correct and
managed to set up stable lines with G418/puro selection and selected clones
with good expression (confirmed by Western and also Immunoflourescence of a
HA tagged version of the construct). The problem is, when I try transient
transfections, i get no expression - despite trying various transfection
conditions (Fugene, Genejuice, Lipofectamine with varying DNA/reagent
ratios, +/- Optimem, varying confluence (50-90%) and harvesting times (24h,
48h)). I have not yet verified if there transcription is working with
RT-PCR.
I'd appreciate some advice on this.


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