transfecting large gene in mammalian cells - difficulties in transfecting large gene in mammalian cells (Oct/01/2008 )

Dear all,

i am very new in the field of cell biology and i experience a lot of problems.
Therefore i would appreciate your help.

I want to transfect mammalian cells with a large gene (8 kb) fused with GFP. For this purpose i chose the pcDEST53 vector from invitrogen where i cloned the gene of interest .
The cells i used by now are HEK293 and COS1.
As i understood, because the size of the gene is so big i have to use higher amount of DNA for transfection. So i used 3 µgDNA/well.

I tried a couple of transfection reagents (Lipofectamine, Metafectene, Fugene) and also several different transfection conditions.
However, i do not get any expression at all (as assayed by western blot and fluorescence).

My question is whether the size of the gene is a limiting factor for efficient transfection.
Do people usually transfect genes that are so big?
Can i use 3 µg of DNA /well independent of the size of the culture plate?

In literature i have seen that people usually perform cotransfection experiments where they use 2 plasmids: one with GFP and one with the gene of interest, In molar ration 1:15 and basically count the green cells.
What is the purpose of doing so? How can somebody be sure that the cells which get GFP also get the gene of interest? Does this make sense?

Thank you in advance!
Sarah

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Hi Sarah,

I have a few questions for you (so we can help you a bit better):

Is 8kb the size of the open reading frame, or the size of the whole gene including GFP?



Are you blotting for GFP or your gene of interest? Are you sure that everything is in-frame and that the promoter you're using is active in the destination cell lines?



Co-transfection is normally pretty reliable: bulk transfection methods such as lipofectamine and calcium phosphate precipitation don't deliver single plasmids to cells. Rather, both methods generate large globs of DNA containing up to 20 plasmids, and a cell will take up one of these. If you mix different plasmids, there is a high likelihood that at least one copy of each will end up in each 'blob'. So if you transfect a GFP-expressing plasmid alongside your experimental one, any cells that are green (i.e. have taken up plasmid DNA) are also likely to carry a few copies of your plasmid.

Ginger

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Hi Ginger,

thanks very much for your reply.

The ORF is 8 kb.

i am blotting for both GFP or gene of interest. there is no signal in any of these cases. The transfection efficienty is so low that only 5 cells are green 48h after transfection, therefore i think this explains why there is no signal in western blot.

i have sequenced the plasmid and the frame is correct.

Sarah

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I have done 12 kb plasmid with lipo2000; your 18 kb plasmid is not much larger. I know ppl use adenoviral plasmid (~30 kb) to make recombinant virus. So the size of 18 kb should not be the upper limit.

You mentioned 3 ug/well, what size of the plate are you using? It is low for 6 well plate, for which, you can use up to 8 ug DNA/well if toxicity is OK. When you chose the ratio between DNA and transfection reagent, you can use a ratio that is on the lower side of the recommended ratio, for example, 1:3, DNA to lipo2000.

Good luck.

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First, I recommend you try an empty vector (only GFP) control transfection. This will tell you if the problem is specifically your vector or the cells/transfection. If you can get good transfection with GFP alone but not with your GFP construct, you know the problem is in the DNA. What is your 260/280 ratio? How concentrated is your DNA? Are you sure there is no remaining EtOH from the prep?

How high passage are your cells? The older they are the more difficult it is to transfect (usually) but 293 cells are known for their ability to transfect easily. Given that you have tried multiple transfection reagents, I'm hesitant to think it's a reagent problem. You always split your cells the day before you transfect, correct?

Hmmm.. what else? How long after the transfection do you check for fluorescence? Do you wash the cells before? I'm just wondering if what you are trying to express is highly toxic and kills the cells before you check for expression. Do you see fewer cells after the transfection than before? I had this happen to me once and didn't figure it out until I looked at the cells around 10-12 hours after transfection. Green cells that died within the next 6 hours (what a fun gene to study)!

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The size of the plasmid or gene is not limiting. I guess the transfection could be the problem. Also check the purity of the DNA.

Optimize transfection only with GFP plasmid and with 293 cells, one can get nearly 100% transfection. After this try your 8kb plasmid. By the way, we regularly transfect ~12kb plasmids and works efficiently.

Post your transfection protocol, may be we could help.

Good Luck

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