Biology Reference
In-Depth Information
In this review, we concentrate on what has been learned about the
biological usage of miRNAs from fly genetics. The
Drosophila
system is
privileged to have had many researchers devoted to technology develop-
ment over the decades, resulting in a battery of powerful methods for
genetic analysis. We do not intend to present an exhaustive compilation
of fly techniques; rather, we summarize the genetic toolbox that has been
most utilized for miRNA research. We hope that these detailed illustrations
of how specific techniques have illuminated
Drosophila
miRNA biology
may provide a useful reference and comparison for those studying other
animal models (e.g.,
C. elegans
, zebrafish, mouse, and cultured mammalian
cells). As well, in the course of discussing landmark and recent
Drosophila
miRNA literature, we specifically point out general implications from fly
genetics that have particular bearing on the collective endeavor of studying
miRNA functions.
2. Generation of miRNA Mutants
The gold standard for studying gene function in model organisms is
by mutant/deletion alleles within the intact organism. In
Drosophila
and
C. elegans
, large-scale genetic screens using chemical mutagens or transpo-
sons were instrumental in elucidating genes and pathways that control
development. The small size of miRNAs makes them poor mutagenesis
targets. Only through extremely deep genetic screens, and heroic and
stubborn positional cloning efforts, have rare point mutations in miRNA
genes been recognized in nematodes (
Fig. 8.1
A;
Chalfie
et al
., 1981
;
Johnston and Hobert, 2003
;
Lee
et al
., 1993
;
Reinhart
et al
., 2000
). Simi-
larly, a handful of
Drosophila
miRNA loci were associated with notable loss-
of-function phenotypes prior to realization of their encoded small RNAs
(
Brennecke
et al
., 2003
;
Cayirlioglu
et al
., 2008
;
Hardiman
et al
., 2002
;
Hipfner
et al
., 2002
;
Raisin
et al
., 2003
;
Xu
et al
., 2003
). However, in flies as
in all other species, by far the vast majority of miRNA mutants have been
studied by reverse genetics. We begin by reviewing the methods by which
Drosophila
miRNA mutant alleles have been generated.
2.1. Transposon-induced miRNA mutants from forward
genetic screening
The “taming” of transposable elements for experimental use has been
invaluable for the creation of mutant alleles, provide entry points to clone
genes, probe expression patterns, and of course facilitate a majority of
Drosophila
genetic experiments. The general utility of transposon insertion
collections for forward phenotypic screening, as well as serving as an allele
