simple questions on oligo delivery to embryo

maneesh maneesh at drunkenbastards.com
Fri Apr 2 09:17:38 EST 2004


[please post reply as my email box is filled with spam]

Hi all,
I am working on a qualifying proposal (outside the field of research
which I am participitating in, I will not actually perform the
experiment but it should be all be sound) and I have come up with some
silly idea of studying miRNA:miRNA interactions in drosophila (see Lai
et. al in RNA(2004) 10:171).
I am not going in hoping to blow the minds of my commitee, just come
up with something sensible.

I am particularly interested in studying the potential regulation of 

miR-4 and miR-79 
by 
miR-9a, miR-9b, miR-9c.

miR-4 and miR-79 have complmentary Brd box binding domains, and the
miR9-a and miR-9c have perfectly conserved Brd box on the miRNA*
strand.  I haven't been able to trace through all the predicted
targets at the miRNA registry, but most appear to be invovled in
development and  Lai says that the Brd box consensus sequence
implicates these genes in the Notch signalling pathway.  I believe
that defective Notch pathways have either embryonic lethal phenotypes
or flies that keep folded wings and which die quickly.

So in proposing to understand the potential roles of these 5 miRNAs
and how they relate to each other, I will have to be able to
selectively express and silence them and (verify then look for) Notch
pathway defective phenotypes. A recent issue of PLOS describes 2'-OMe
RNA oligos as selective inhibitors of miRNA function, which appear to
work well in cell culture.

Now one thing I embarassingly wasn't thinking about was, that I am
working with embryos and not cell culture (doh! I have done mammalian
cell culture and transfection before).  I can see a few ways to
deliver miRNA constructs to cells, but I'll have some problems
acheiving silencing in trying to deliver chemically modified oligos to
embryos...won't I?

If anyone can suggest an idea to me, or suggest some other way to shut
off the miRNAs that I am interested in, please let me know or point me
to a reference/tutorial....if you see any gaping holes in my basic
proposal please hit me with the cluestick.

Many thanks in advance
[please remember to post ;)]




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