Dicer knockdown to show miRNA mediated target mRNA expression? - (Mar/20/2007 )
I would like to determine if my protein of interest is able to regulate the expression of select mRNAs by way of miRNA. To do this I was going to use siRNA directed against dicer1 in human cells along with either a control siRNA or an siRNA targeting my protein of interest. Most studies like this appear to use siRNA against dicer, but I'm concerned that the effects on miRNA levels and subsequently their target mRNA levels, will change too slowly. Does anyone know if there are any cell lines available with either dicer knocked out or stably knocked down? Thanks.
You can transiently knock down Dicer using Morpholino oligos. The following reference shows activity of Morpholinos against Dicer in zebrafish, but they will work as well when delivered into the cytosol of cells in culture.
Wienholds E, Koudijs MJ, Van Eeden FJ, Cuppen E, Plasterk RH. The microRNA-producing enzyme Dicer1 is essential for zebrafish development. Nat Genet. 2003 Nov;35(3):217-8. Epub 2003 Oct 05.
Vogelstein's group has generated Dicer knockout cell lines including Hct116, RKO and DLD1 (http://www.pnas.org/cgi/content/full/103/10/3687?ck=nck). The cells are available upon request.
There are potential problems with dicer kockdown or knockout. Dicer 1 may not be the only enzyme that generates mature microRNAs from pre-miRNAs. In addition, after dicer is knocked out cells may have adapted to the changes and microRNA regulated genes may not be identifiable. I think temporary knockdown of dicer with siRNA is more approporiate.
I agree with pcrman.
Without more info it seems like a circuitous route. I'd first see if your select mRNAs are predicted known miRNA targets. You can then use a premir/antimir system to see if the mRNA's are affected.
Thank you all for your helpful comments. I looked at Vogelsteins paper and I am concerned that only 50% of miRNAs are dysregulated in the KO cells which supports your idea that there are other dicer like enzymes allowing some miRNA biogenesis to occur. With the dicer knockdown, I'm concerned that the miRNA half life is longer than that of dicer itself so I won't know how long after I knockdown dicer that I'll be able to knockdown my protein of interest. Overall though, I do think this is the best strategy. Thanks.
I agree with the others, however, to be miRNA-specific, one should probably knockdown/out DGCR8; a dicer knockdown probably won't be specific to miRNA biogenesis.
Nat Genet. 2007 Mar;39(3):380-5. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal.
how many types of dicer are there in human cells? i guess drosophila has different types of RNAse III including dicer 1 and 2
anyone have idea if we knockdown dicer 1 how will it affect RNAi to dsRNA?