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problem with transient transfection

transfection

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#1 dandan

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Posted 24 August 2011 - 08:47 AM

Hi folks:

I have encountered a very annoying problem with a commercial plasmid we bought from origene. this plasmid is pcvm-flag-entry vector, with a CMV promoter, and my gene of interest is quite big around 100 kda.
i did a transient transfection of this plasmid for 24-72 hrs in hela cells but i could not able to get an overexpression of the inserted gene, i stained with tag antibody to check the transfection efficiency under microscope , it turns out there is only 5-10% of cells are transfected and stained.. however, i used another bcl-2 plasmid as a positive control to transfect at the exact condition, the overexpression of bcl-2 is really good, which means my transfection system is good, but the pcmv plasmid is not express well, what would be the problem, promoter is too weak or my protein is too big? do i need to clone my gene in another vector to have a try ?

can anybody give me some idea regarding to this issue. i highly appreciate.

dan

#2 GNANA

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Posted 24 August 2011 - 08:59 AM

Try to express the same in another system like 293, if the you get the same result then think of subcloning....because i too encountered some plasmids doesnt express well in some models...,i am not sure is it due to the issue with the plamid (though i could express another gene in the same plasmid), or is it with the protein we trying to overexpress!!!....
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 dandan

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Posted 24 August 2011 - 08:58 PM

Hi Gnana, thank you for your reply. I will try out with 293 cells again, although this plasmid doesnt work for 2 cell lines i tried so far.
Do you think there would be the protein itself's problem?

#4 GNANA

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Posted 25 August 2011 - 12:01 AM

wouldnt say no, because for some proteins the basal degradation rate is higher in that case you dont see enough protein...may be you may have to check the mrna...thats why i always prefer phoenix cells (293) to check the expression, it just express anything i put in, if i fail in that then i would think of subcloning in a known good expressing vector or balming protein itself...Another useful tip would be if you are subcloning in another vector add the Kozak sequence in front of ATG so that we totally given all things for the construct to express in any eukaryotic system...good luck...
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#5 dandan

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Posted 25 August 2011 - 01:26 AM

yeh, the mRNA level also came into my mind as well, and coincidently, this afternoon i just did the reverse transcript PCR.
very weird thing is that i cant detect the mrna level of my gene, given a high expression of endogenous protein form. GAPDH works perfect,
as well as a positive control that i used this plasmid as a template, which indicates my pcr condition and primers are working well. cant figure out
what is the problem, why the gene cant be picked up by mrna, but if degradation is the case, why protein level is quite okay.

#6 GNANA

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Posted 25 August 2011 - 02:02 AM

Are you sure you designed the RT primer that is common to all isoform (if there is) of ur gene...??then regarding expression try to find a system that has no endogenous expression of ur gene, this i would do only after giving a try in 293....
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#7 dandan

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Posted 29 August 2011 - 01:31 AM

thanks a lot man.
i have already got a 293 cell line, at the meantime will overexpress the plasmid in some other cell line with a low endogenous expression of the gene, and see how the expression looks like, although i still believe that if the plasmid works, should works in
most of the cell lines regardless the endogeneous expression of the interested gene.





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