transfection of chick cell - (May/03/2007 )
hi guys!
i recently started my grad career and i have been trying, in vain, for the past few months to look at the best reagent for transfecting chicken cell lines with a gfp plasmid, just to standardize. i have used the SL-29's from ATCC, two primary cell lines, one of them embryonic, and have preety much tried every reagent i could find, Lipofectamine 2000, lipo-with the PLUS, superfect, effectin... calcium phosphate, you name it... i dont have access to an electroporator..
the eventual aim is to look at RNAi. papers talk about reagents used, but none i have found talk about transfection efficiency, the best i've gotten is 30-35% if that..... and its different for every cell line and reagent combination...
has anyone tried, successfully, to transfect ANY chicken cell line... or has any idea what has been used to good effect by someone!
i came across this forum just today and registerd instantly.... any help would be appreciated..
thanks,
vik.
I dont know what % is the minimal goal you want to achieve, but it sounds like you have tried real hard. How about adenovirus? Is that an option for you? You probably can make one for yourself fast than trying to get these methods working for you.
Longmuir KJ et al (2001) Optimization of a peptide/non-cationic lipid gene delivery system for effective microinjection into chicken embryo in vivo. Mol Ther. 4:66-74. This is not a simple system though. Maybe you can call them up and see if collaboration is a possibility.
Thanks for the reply.
I guess 70-80% transfection would be ideal for looking at knockdown. Unfortunately I am not looking at anything that might have an obvious phenotype. So, transfecting embryos and then the subsequent RNA isolations might be tougher, especially considering I need a high throughput system for screening different/numerous shRNA constructs to see which ones work best. But I agree, looking at the results that I have been getting, adeno or lenti-virus might be the better bet in the longer run.
Thanks,
Vik.