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Can I target non-coding sequence by siRNA - (May/16/2005 )

Hi,

I am trying to examine the function of mutated protein in our cell lines. However, they have Western detectable level of endogenous wild-type protein. I am now looking for a method which can abolish the expression of endogenous protein while the exogenous mutant protein is unaffected. Since homologous recombinant is too difficult, I prefer RNAi. My plan is to design vector based siRNA that targets the intron sequence and select stable transfectant. Is that reasonable and applicable? (Can RISC present or RNAi occur in nucleus?) What about antisense? Please give me some advice.

Thank you very much for your help!

Wei

-w_emu-

Hi Wei,

Yes, RNAi is operable in the nucleus. No one has tried silencing gene by targeting intron, but you can target the promoter. There are reports published last year in Nature and Science showing gene silencing by targeting promoter by siRNA, called siRNA directed transcriptional silencing. To ensure success, select target sites that contain some CpG sites and are located in a CpG island if there is any. Please be aware that this strategy may not work in every cell, for every gene, by every siRNA. All depend on your luck because little is known about this phenomenon.

Good luck.

-pcrman-

The best way to do this is - no siRNA, just over-express your mutant protein in your target cells. The expressed mutant proteins will compete with endogenous wild-type one and you will see the effect. Incorporation of siRNA will increase another layer of complexity......Think it through.

-joy_maf-

hi
i have same problem that endogenous protein will effect the activity of introduced gene. In my case, I tried to knockdown human RBM6 in hela cells but keep the mouse RBM6 introduced. these two are very similar, so i targeted human rbm6 5UTR and 3UTR. I am doing it at the moment. will get results in 2 weeks. I ll let you know whether the siRNAs targeting these two areas work. Also as pcrman said, different genes act differently to differentsiRNA, but you can always have a go!!

-hsm142-

QUOTE (hsm142 @ May 26 2005, 05:49 AM)
hi
i have same problem that endogenous protein will effect the activity of introduced gene. In my case, I tried to knockdown human RBM6 in hela cells but keep the mouse RBM6 introduced. these two are very similar, so i targeted human rbm6 5UTR and 3UTR. I am doing it at the moment. will get results in 2 weeks. I ll let you know whether the siRNAs targeting these two areas work. Also as pcrman said, different genes act differently to differentsiRNA, but you can always have a go!!


Your situation is different. You're trying to introduce wild-type mouse RBM6 into a human line (which has a wild-type human RBM6). It is reasonable to reduce noise (the human RBM6) while increase signal (mouse RBM6) by siRNA targeting endogenous human RBM6.
But w_emu's problem is regarding mutant and wild-type proteins!!!

-joy_maf-

hi
recently i've read that morpholino targetting an intronic sequence would have as a result an alternative splicing that exclude the targeted intron sequence... Assuming that morpholinos are actually blocking translation and not same mechanism as siRNA, may i introduce the question of siRNA mediated alternative splicing???

What do you think about it?

-fred_33-

This may be the most logical approach. You may co-transfect siRNA vector to silence the endogenous protein together with an expression vector for your mutant protein. Your mutant protein plasmid must contains nucleotide mismatches (around 3-4) without changing your protein sequence (because of codon degeneracy) in order to avoid silencing. This is akin to "rescue experiments" in siRNA validation

-Custer-