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Help with forcing overexpression in hES cells

overexpression ES cells transfection

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#1 andyg2886

andyg2886

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Posted 02 September 2011 - 03:39 PM

Hi

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!





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