5 replies to this topic

[hollypop]

Hi,

I have been using the pSILENCER vector to knock down expression of my OVEREXPRESSED target gene with great success >99% protein knockdown on westerns but I am having real problems trying to knockdown the ENDOGENOUS targets it just won't work. I am experiencing the same thing with 3 different target genes has anyone else come across this/ can anyone help?

Cheers.

[fred_33]

hi
did you try to see the effect of psilencer on your gene in te same cell line that do not overexpress the gene?
if so what' the result?

[hollypop]

Hi,

Thanks for your speedy reply. Initially to test my constructs I was co-transfecting my silencers with an expression vector for my target gene into 293 cells which do not express the gene of interest and this was very successful. To test the effects on the endogenous gene the silencers alone were transfected into different cell lines which express the gene of interest and this has been very unsuccessful.

Holly

[fred_33]

hi
have you run a northern blot in order to detect your siRNA products?
did you check the efficiency of transfection?
do you have a selective marker to ensure of it?

maybe can we mail each other in order to discuss more of your problem?

fred

Edited by fred_33, 25 February 2005 - 05:47 AM.

[hollypop]

Hi,

Thanks again for your interest in my problem! In answer to your questions
(1) No I haven't run a northern to check for my siRNAs
(2) I have made constructs that contain both the shRNA under the control of the H1 Pol III promoter and eGFP under the control of the CMV promoter. I have compared the pSILENCER alongside the pSIL-GFP constructs and at western level the appear to be as efficient at knocking down protein levels. My transfection efficiency varies greatly between different cell types and methods of transfection from terrible to acceptable, however GFP expression has allowed me to either sort the cells and assess RNA levels, or stain for my protein of interest using a label such as PE and gate this against GFP expression on the flow.
(3) The pSILENCER plasmid does not have a selectable marker (as far as I know) but my pSILGFP constructs have the neomycin resistance gene but as yet I haven't attempted to make stables.
Hopefully this may shed some light on why I am not seeing endogenous knock down but in an attempt to overcome this problem I have ordered alexa fluor labelled siRNA from qiagen to see if  this helps so fingers crossed!
Thanks
Holly

[fred_33]

hi

for my experiments,  was reading the article from carl sledz in nature cell bio 2003 and a question show up...
is there an expression stimulation of your interest gene by the siRNA?

it could be possible that you see an effect when you have an over quota of protein in your cell, but maybe can't you knock down the endogen protein...

For the possibility of stable cell line, according to the fact you select cells that express a good level pf gfp, i think you should see an effect on protein level.

i cross my fingers for you.
Fred.