4 replies to this topic

[catjo]

Hi,
I'm new to this and am ultimately wanting to have an inducible vector in transgenic mice - to switch gene on and off during development (not sure if this is aiming too high?!). Initially, to check for knockdown I was going to just transfect some siRNAs into cells - is this a good first step, or should I make the shRNA and clone into vector and test function at this point? Is there a way to transfect the hairpin into the cells without vector perhaps...??
Am grateful for any comments, sorry to be so wordy...
C

[pcrman]

To find out a functional siRNA target site before using vector based siRNA, you can order chemically synthesized siRNA which is cheaper, faster, and time saving. In addition, synthesized siRNA has stronger RNAi effect than other siRNAs and less likely causes interferon response. I usually use siRNA from Invitrogen. They charge about $200 for a double-stranded siRNA (21nt, 20 nM) which is enough for lots of transfections.  

Once you find a reliable target with synthesized siRNA, then you can construction your vector to achieve long-term silencing.

Transfecting hsRNA longer than 30 bp may cause interferon response. So that is not a choice.

Edited by pcrman, 16 November 2004 - 10:40 PM.

[ahpaul]

is better to transfect your shRNA with a vector ... the vector that i know is pSM2 from Open Biosystems ... i think they had also just formulated a special transfection reagent called Arrest-In for the shRNA ... from what i know, Open Biosystems has around 60% of the human shRNA library and 50% of the mouse shRNA library ...
so catjo, u r working with shRNA as well ? may i know where are u from?

[catjo]

Hi ahpaul - thanks for the info. I'm in Brisbane, Australia

[mas]

catjo, on Nov 23 2004, 06:22 PM, said:

Hi ahpaul - thanks for the info. I'm in Brisbane, Australia

I have a bad experience with pSM2 shRNA from open biosystems, so be cautious!