Forcing overexpression of a GOI - (Sep/03/2011 )

Hi

I originally posted this in the 'stem cell' section of cell culture, however it seem nobody really reads posts in there. So apologies for the cross posting, but I feel I might have more success in this section of the forum.

I'm a final year PhD student working on overexpressing a few GOIs in human ES cells. Up to now I have good data however I am missing one key thing - overexpression of a gene that I've been trying to do for a year now.

I'm overexpressing with the pCAG constitutive expression vector. I've managed to express GFP using this vector as a control, and one GOI that worked well. Sadly, another GOI is refusing to overexpress and I have no idea why.

I've sequenced the full vector with genscript and the vector sequence is 100% correct. So, it's not the DNA sequence that's the problem.

My cells are surviving in selection and so definitely have the vector in. When I come to do RT-qPCR for my GOI I get absolutely no overexpression over basal levels. I don't think my PCR is a problem. I've tried various Applied Biosystems Assay on Demands, and also designed my own primer probe mixes. Nothing.

Are there any companies out there that help with overexpressing genes for a cost? Or does anybody else have any idea what might be going on here? The GOI I previously managed to overexpress well is overexpressing 1800x over basal levels. That's huge. As such, I have no idea why my other gene isn't working.

Thanks for any ideas you may have. I could do with getting this working as it was supposed to be a whole chapter of my thesis based on the overexpression of this gene :s !!

Cheers!

Have you tried expressing this vector in other cells to see if it works?

That does seem the obvious thing to do. But no, I haven't.

Any cell types yo uwould reccommend? Easy to transfect cells?

Thanks.

the question of bob1 would have been also my first question.

I would recommend HEK293 cells for the transfection.

Yeah, 293 or HeLa would be good, but basically anything that is well known for transfecting easily.

You could also look at the cells to see if you see an increase in apoptosis following transfection.

Did you have a kozak sequence in the plasmid, to enhance the GOI expression?

We worked with CAG promoters with several different type of introns, some as long as 1kb. Did you use the same introns for previous CAG construct too?

Also, does the expression cassette contains WPRE ?

On another thought, is there any reason you have to use CAG promoter? CMV should work well in ES cells too. Maybe you can use CMV vector if CAG doesn't work for any reason.

Yes I do use a kozak sequence to enhance expression.

I'm using cag as cmv seems to be silenced shortly after transfection into ES cells.

I'm not expressing introns. I'm expressing coding regions only (exons from atg start to tag stop codons), and my gene is 1950bp long. The gene I have previously managed to overexpress is 1450bp long.

Not sure about that additional bit you mention in the cassette. Would you like to see the vector sequence?