Low transfection efficiency and lack of protein expresson

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angels
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Low transfection efficiency and lack of protein expresson

Post by angels » Wed Jan 10, 2007 5:17 pm

Can anyone help me with my transfection/protein expression problems?? I have produced several truncated versions of a gene (checked and confirmed to be correct and in frame by sequencing). I used the expression vector pcDNA3.1 and transfected these constructs into CHO's and a colonic adenoma cell line. Transient transfections in CHO's yielded the best reults with the smallest construct (98kDa) but transfection efficiency seemed to decrease with increase in construct size (the largest in its non-glycoslyated form is 278KDa!!..its a whopper!!). Furthermore Western blotting revealed the presence of the 98KDa product but not the larger ones....is this problem solely a matter of construct size or am I missing something more basic???
I have also slot blotted and immunostained the corresponding lysates, immunopercipitates and supernatnats of the aforementioined transfections which didn't work either.......any and all suggestions and comments would be most welcome.

Angels

p33
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Post by p33 » Thu Jan 11, 2007 6:33 pm

It is certainly true that the increase of the size of the construct in a big extent will decrease the final output in terms of transfection effiency. One obvious reason you could think of is that when you transfect equal ng of DNA with larger size, you are actually transfecting less moles of DNA in comparison to the one that is smaller in size. Right? Another reason you could think of is that larger DNA molecule is more easier to be broken when you purified them. And the supercoiled, at least, the circular form of the construct are the only forms that could be transfected. Sure, there are several other realted reasons, and some of them I believe they are not clearly yet. Franky, I am also new to transfection, but here is my suggestion if you think it worth a trial: Try to select for a stable transformants by contransfecting your intrested construct with another construct prossessing a antibiotic resistent gene (eg. neomycin, hygromycin, ...) After 2-3 weeks of selection, you will be able to get a better expression hopefully. Good luck!

angels
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Post by angels » Fri Jan 12, 2007 12:16 pm

Thankyou P33, I will think about your suggestion and see if I can design a transient transfection experiment as a 'quick look-see'.

angels

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