3 replies to this topic

[Thapa]

Hi,
I have generated a stable clone using Lentiviral shRNA-mediated knockdown. The knockdown clones have suppressed expression of my target protein A.

Now, I want to rescue or re-express the same protein to compare its biological functions.

Can I use pcDNA3.0 vectors to overexpress the same target (protein A) in my stable clones?

Since my cells express siRNAs for the same target, dont they suppress pcDNA based overexpression of same gene?

Many thanks,

Edited by Thapa, 04 July 2012 - 01:47 PM.

[bob1]

The expression will depend on the relative expression of the siRNA and the plasmid.  When you generated the siRNA line, you should have a also made a control line with a non-expressing or scrambled siRNA to prove that it isn't just the insertion of the construct that is causing the effect.  You should compare the control line with the siRNA expressing line...

[Thapa]

bob1, on 05 July 2012 - 01:32 AM, said:

The expression will depend on the relative expression of the siRNA and the plasmid.  When you generated the siRNA line, you should have a also made a control line with a non-expressing or scrambled siRNA to prove that it isn't just the insertion of the construct that is causing the effect.  You should compare the control line with the siRNA expressing line...

You are right bob1 but I have done the experiments as you mentioned above. Now what I want to do is "rescue experiments". How to re-express or rescue the same protein in the stably-knockdown cells?

[bob1]

I was meaning that you should compare the expression of the "rescue" plasmid in the control and KD lines.  If you can see a difference in the protein production then the KD line is blocking some, but not all, of the expression from the plasmid.