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Puromycin - shRNA - stable cell line for knock down


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#1 rrn

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Posted 05 October 2012 - 05:50 AM

Hi,

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 ?

Thanks,

RRN

#2 bob1

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Posted 05 October 2012 - 01:36 PM

Perhaps your shRNA is not very efficient, perhaps you didn;t use the right amount of puromycin - this should be titrated on non-transfected cells before using for selection.

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?

#3 rrn

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Posted 05 October 2012 - 07:32 PM

I did do a puromycin kill curve .. and selected the puromycin amount to be used. Non transfected at same cell density of seeding dies completely..

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 !

#4 bob1

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Posted 05 October 2012 - 11:56 PM

Did you do quantitative PCR or just look for a band on a gel? If you did qPCR, then you should have also done a control where you looked at non-transfected cells RNA levels as well, so you will know if your shRNA construct is working or not. If you just did a gel - you realise that this is an end-point measure - the presence of ANY transcript could easily amplify over 30-40 cycles and give you a band... you need to do qPCR with the appropriate controls. Even the most efficient KD will only result in something like 90% KD - so there will still be transcript there. The protein is the real measure of the KD in the end - that's the functional bit after all.

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.

#5 rrn

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Posted 06 October 2012 - 04:57 AM

No i did qRT PCR only !! With control cells.. also for gene of interest as well as B-actin for internal control ! p21 was surprisingly highly expressed in the knock down set compared to control cells...

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 !




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