Puromycin - shRNA - stable cell line for knock down
Posted 05 October 2012 - 05:50 AM
I have this sh RNA construct against p21 which has puro resistance. After transfection and pressure.. very few cells.. less than 100 only would have survived.. and they started dividing rapidly and now with the same amount of puro.. there is no cell death. But when I isolated mRNA and made cDNA and did a real time for p21.. the transcript is still there. Should I design different sets of real time primers and check ?
If the stable selection did not work then why the cells are not dying now ? Can cell lines develop puro resistance by any chance ?
Posted 05 October 2012 - 01:36 PM
Did you check whether you can find the shRNA sequence or some other sequence on the plasmid in the colonies?
Is the construct inducible?
Did you check for p21 protein?
Posted 05 October 2012 - 07:32 PM
I did not check at protein level coz at RNA level itself i got p21 PCR product..
If not transfected / construct is bad.. how are these cells surviving with puromycin pressure?
shRNA is in pLKO.1 vector !
Posted 05 October 2012 - 11:56 PM
Occasionally inserted plasmids get methylated as the inserts are different in seqence structure to normal DNA patterns, with just the resistance gene being unmethylated. I don't know how this will work for shRNA constructs thouh.
Posted 06 October 2012 - 04:57 AM
Thats why i was wondering how then these cells survived if they dont have the plasmid inside !
Thanks anyways.. will check up more about the shRNA transfection/functionality !