shRNA - (Jul/15/2010 )

Hi,
I'm using two systems trying to get knockdown of gene the one is the pSUPER RETRO and the other is the tet on/off
I made stables cell line in both system selecting with antibiotic, somehow when I'm doing real time PCR I'm not getting knock down. the constract was validated before in different cell line.
any IDEA what is the cause?

thanks
Michal

michalcn on Jul 16 2010, 04:14 AM said:

Did you check your constructs before yo did the stable cell lines? You can check 72h after transfection, to check if your constructs are working, or do the transfection and 48-72 h after treat 2-3 days with puro (if you have puro resistance) and do the real time.

I ve seen that sometimes, even using lenti or retrovirus, you knockdown the expression of your gene, but it appears to recover in time. So I infect the cells, treat for 2-3 days with puro, and then do de rt pcr 1,2,3,4,5 days after that. The first and the second day I got a nice silencing, but after that the expression is recover.
Maybe you have the same problem.
Check first that the constructs work, and you can see it by wb or real time pcr, and once you are sure, you can start with the stables

Thanks for your reply,

But I'm still wondering because I'm not doing transfection but infection so when the cells getting the resistance to the antibiotic they actually getting the construct right?
so how can they loose the silencing?

thank you
Michal

michalcn on Jul 16 2010, 06:37 PM said:

No idea! But that was what I saw...and actually I reead that in some papers before...but dont ask me how...yeah, I infect the cells as well.
You should check them right after the puro selection

May be the following problems:
1. PRC primer set on the same exon
2. Genomic DNA contamination or gene homologue
3. Feedback regulation / compensation
4. cell culture condition (better change or split cells before assay)