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How to transfect siRNA into the nucleus for transcriptional silencing?


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#1 green

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Posted 01 February 2005 - 10:39 PM

Hi All,

I am working on siRNA transcriptional gene silencing (through DNA methylation or histone methylation) by targeting non-coding sequences. The major problem for this to work is to get the siRNA to the nucleus. We already know that in RNAi experiment, some siRNA can find it way into the nucleus in some cells possibly through passive difussion. I have used Lipofectamine 2000, and found most cells don't have much siRNA in their nucleus after transfection. I wonder if there are any good transfection reagents, methods of transfection, types of siRNA modification that can lead to better siRNA delievery into the nucleus.

Thank you in advance for your thoughts.

Green

#2 fred_33

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Posted 02 February 2005 - 02:53 AM

hi

why don't you try systems for stable transcription systems as pSuper or pSuper retro?

#3 expresson_help

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Posted 02 February 2005 - 06:52 AM

I don't know if any transfection reagents have been studied for transfection into the nucleus - most are obviously designed for transfection across the cell membrane. A stable expression vector might be the answer as has been previously suggested, such as pSUPER or pEXPRESS.

#4 green

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Posted 02 February 2005 - 09:22 AM

Thank you for your feedback. But, a siRNA expression vector may not be what I wanted, Because once the hairpin siRNA is transcripted, it has to be exported to the cytoplasm where it can mature to siRNA and then the siRNA goes back to the nucleus to function. So even an expression vector is used, the siRNA face the same problem of overcoming the nuclear membrane barrier. On the other hand, vector based siRNA technology may be not a good choice for therapy. I hope to find an efficient way of delivering siRNA to the nucleus.

Edited by green, 02 February 2005 - 10:38 AM.


#5 fred_33

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Posted 02 February 2005 - 09:28 AM

hi
as i know siRNA have not necessary to get in the cytoplasm in order to be processed. In fact, DICER can be in the nucleus and process siRNA in the nucleus. But i must admit that there's no direct evidence that majority of hairpin processing occurs in the nucleus.
What do you think of that?

#6 green

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Posted 02 February 2005 - 10:48 AM

Hi Fred,

Thanks for reply. A recent paper does (nature structure mol bio) show that siRNA directed RNAi and RISC exist in the nuclear compartments of human cells. They used synthetic siRNA for transfection. I am not sure whether hairpin siRNA can be processed in the nucleus into matured siRNA.

#7 expresson_help

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Posted 03 February 2005 - 03:11 AM

Given that miRNAs are processed in the nucleus (I believe) by RISC, it seems likely shRNAs may be too.

#8 fred_33

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Posted 03 February 2005 - 04:20 AM

hi in a recent paper of yoontae lee et al. (EMBO 2004), the autour explains that miRNAs are transcribed as long primary transcripts (pri-miRNAs), which are cropped into the hairpin-shaped pre-miRNAs by nuclear RNase III Drosha (Lee et al, 2003; Kim, 2004). This cleavage event is important because it predetermines mature miRNA sequence and generates optimal substrate for the subsequent events (Lee et al, 2003; Lund et al, 2004). The processing intermediate, pre-miRNA, is exported out of the nucleus by exportin-5 (Exp5), member of the Ran-dependent nuclear transport receptor family (Yi et al, 2003; Bohnsack et al, 2004; Lund et al, 2004). Pre-miRNA is subsequently cleaved by cytoplasmic RNase III Dicer.

But DICER can also been found in the nucleus. In this regard we can postulaute that the nuclear form of dicer may process only the siRNA but not the miRNA...

Two recent paper about miRNA processing should been read :

The nuclear RNase III Drosha initiates microRNA processing, nature vol 425, 415-9 from yoontae lee et al.

Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha EMBO J 2005 vol 24 138-48 from Yan Zeng et al.




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