5 replies to this topic

[flashboy]

any advice on how to design antisense oligo's so i can knock out gene expression in my cells? how long should they be? exon and intron? etc etc

[rassen]

Hi flashboy,

Antisense oligo design may be tricky because there are many things to be considered, for example, they do not anneal to an unintended mRNA, they don't contain motifs known to invoke immunostimulatory responses such as four contiguous G residues, palindromes of 6 or more bases and CG motifs.

There are online tools for designing antisense oligo such as
http://biotools.idtdna.com/antisense/

You can also consider using RNAi to knockout genes.

good luck

R

Edited by rassen, 24 March 2004 - 08:29 PM.

[flashboy]

RNAi....? my supervisor mentioned something about that, again how would i go about using that?

[rassen]

I agree. you should go for RNAi. Antisense RNA seems to have been obsolete.

Here are some good resources for you to start.
Ambion:
http://www.ambion.co...RNAi/index.html

Invitrogen:
http://www.invitrogen.com/rnai

[flashboy]

ok, so one thing i don't understand, if i get the RNAi, transfect it in and then wait 48 hours to prove the gene has been silence3d, what can i go with the cells then? how long will the gene be silenced for? can i grow these cells and passage them? or is it basically silence the gene and then run whatever assay i want in the same well they were transfected in?

thanks in advance for any help you can offer guys

[green]

There are several strategies for introducing siRNA to the cell. You can transfect synthesized siRNA (21nt) into cells and the resulting knocking down of target gene won't last for over a week. If you want to establish stable knockdown of your gene, you have to construct a vector containing a siRNA cassete, when transfected into the cell, it will continuously produce siRNA, resulting stable knockdown of your gene.