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Gal4-binding sites (upstream activating sequence, or “UAS” sites). These
components are kept in separate stocks, and Gal4 by itself does not usually
induce obvious defects; only by crossing to bring the driver and responder
transgenes into the same animal is the responder transgene expressed for
phenotypic evaluation. There are many hundreds of fly stocks expressing
Gal4 in various spatial and temporal patterns, facilitating diverse
tissue-specific misexpression experiments. The Gal4 collections are comple-
mented by many thousands of fly stocks containing UAS-responsive genes,
either generated by directed cloning of individual genes or frommobilization
of UAS-bearing transposons around the genome (often termed “EP” ele-
ments). These resources permit a mind-boggling number of
in vivo
misex-
pression experiments to be conducted with ease (
R
rth
et al
., 1998
).
3.1. Misexpression of miRNAs from genomic EP insertions
While the Gal4-UAS system was originally developed for the purpose of
conducting gain-of-function analysis of protein-coding genes (
Brand and
Perrimon, 1993
), it has found wide adoption for the misexpression of RNAi
transgenes for knockdowns (
Dietzl
et al
., 2007
;
Kennerdell and Carthew,
2000
). Moreover, as mentioned above, a number of miRNA loci serve as
P
hotspots and are thus represented in EP or other UAS-responsive insertion
collections (
Fig. 8.2
A). A number of these miRNA insertions routinely
score as hits in diverse gain-of-function screens (including
mir-7
,
mir-8
,
mir-14
,
mir-282
,
mir-278
,
mir-310-313
cluster, and
bantam
), implying that
this system is effective for misexpression of miRNAs. This is probably due
to the endogenous transcription of most miRNAs by RNA Polymerase II
(
Lee
et al
., 2004
), as with protein-coding genes.
Indeed, many of these miRNA hotspots emerged from gain-of-function
screens conducted prior to the general recognition of miRNAs in
Drosophila
in 2001 (
Lagos-Quintana
et al
., 2001
) or prior to the initial large-scale
annotations of
Drosophila
miRNAs in 2003 (
Aravin
et al
., 2003
;
Lai
et al
.,
2003
) and thus were not likely recognized as miRNA loci at the time of
their genetic isolation. Examples of miRNA hits in pre-miRNA era screens
include that a gain-of-function screen for loci that could affect adult bristle
sensory organs recovered
mir-7
,
mir-278
, and
bantam
(
Abdelilah-Seyfried
et al
., 2000
); that a misexpression screen for genes affecting motor axons and
synaptogenesis identified
bantam
(
Kraut
et al
., 2001
); and that an ectopic
expression study for modifiers of dorsal thorax formation hit
mir-278
(
Pena-
Rangel
et al
., 2002
). Now that miRNAs are better annotated in the
Drosophila
genome, nearly all genome-wide misexpression studies end up
recovering one or more of the set of EP-miRNA hotspots.
Most of these miRNA misexpression hits have yet to be followed up,
but they provide possible entry points to understanding miRNA biology. In
addition, EP-induced dominant phenotypes provide a genetic entry point
