I am having some problems trying to clone a mRNA, maybe due to it's amount compared to other mRNA. I have tried PCR using cDNA from oligo DT, specific or random primers, but with no success. I was wondering if I could clone the full length DNA, transfect it, and get the mature mRNA inside mammalian cells. The gene is 6000 bp long, with several introns on it, and mRNA is 2400 bp long. Any clue?
I am not sure to really understand your point, could you be a bit more specific?
Me too, not sure to really understand your issue.
Why not producing your mRNA by IVT and then transfecting it directly?
Hi Danillo, I think I get your point, you can amplify the genomic sequence, but you are having trouble amplifying the mRNA in the tissue where your gene is endogenously expressed. You are pretty lucky that your gene is only 2.5 times longer than the mature mRNA! Pretty small introns...
So, I have to admit it is not a very standard procedure, but in principle it should work. If you place the gene after a strong promoter, you should get quite an overexpression... hopefully it won't be toxic... Of course in mammalian cells the pre-mRNA will be spliced if you have the genomic sequence there. In fact the presence of one intron normally increases expression of transfected cDNAs, so you might have noticed that in some mammalian expression plasmids there is an intron in the 3' UTR... In this case you'll have the endogenous introns.
Good luck.
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