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alternatively elicit nonspecific toxic effects caused by simultaneous misre-
gulation of hundreds of transcripts. As further examples, misexpression of
many miRNAs elicits specific developmental phenotypes reflecting gain- or
loss-of-function of the core cell signaling pathways (
Hagen and Lai, 2008
),
such as Notch (
Lai and Posakony, 1997
;
Lai
et al
., 1998, 2005
;
Stark
et al
.,
2003
), Hippo (
Brennecke
et al
., 2003
), Hedgehog (
Friggi-Grelin
et al
.,
2008
), and Wnt (
Silver
et al
., 2007
). Efforts are underway to extend
plasmid-based UAS-miRNA libraries used to screen tissue culture cells
(
Silver
et al
., 2007
) into transgenes, for systematic
in vivo
phenotypic screen-
ing in the animal (Y. Chou, F. Bejarano, D. Bortolamiol-Becet, and E. C.
Lai, Unpublished). We anticipate that a similar set of resources in the mouse
would have a high probability of revealing a great diversity of disease-
relevant miRNA activities, most of which would probably not be easily
anticipated from extensive lists of miRNA target predictions.
Conversely, the generation and analysis of more miRNA deletion alleles
are heavily anticipated. In contrast to the
C. elegans
system, where very few
miRNA mutants have overt phenotypes (
Miska
et al
., 2007
), even when
examining multiple mutants of entire families (
Alvarez-Saavedra and
Horvitz, 2010
), the extant literature indicates an impressive set of develop-
mental and physiological defects associated with many different
Drosophila
miRNA mutants, under nonperturbed conditions. Perhaps this reflects the
rich phenotypic assays available in
Drosophila
, and we suspect that mamma-
lian systems (e.g., mice and humans) are similarly phenotypically rich. It is
worth considering that many seemingly inert
C. elegans
miRNA deletions
generate phenotypes in genetically sensitized backgrounds (
Brenner
et al
.,
2010
). Similarly, environmental stress can strongly enhance the phenotypes
of the
Drosophila mir-7
mutant (
Li
et al
., 2009
). Therefore, future miRNA
deletion studies in flies should take advantage of both normal and sensitized
backgrounds.
The flexibility of manipulating endogenous miRNA loci will be greatly
increased by recent upgrades to HR technologies. These include the devel-
opment of “ends-out” targeting for direct allele replacement (
Gong and
Golic, 2003
) and various modifications that improve the ease and efficiency
of detecting candidate targeting events (
Huang
et al
., 2009
). Even more
valuable are the development of “genomic engineering” strategies to permit
rapid construction of allelic variants using a founder line modified to contain
phage phiC31 integrase recognition sites (
Fig. 8.1
F;
Choi
et al
., 2009
;
Gao
et al
., 2008
;
Huang
et al
., 2009
). This is probably the way forward for
generation of reverse-engineered alleles in
Drosophila
, including miRNAs
(
Weng
et al
., 2009
). For example, one can imagine generating one founder
mutant allele that deletes a miRNA locus, then quickly using that as a
platform to reintroduce the miRNA to demonstrate rescue of the targeted
allele, to insert a nuclear enhancer trap marker, a membrane-localized
marker (e.g., useful with neural genes to delineate their projections), a
