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adding other functionalities to the vector backbone. For example, one can
place recombinogenic sequences such as FRT onto the transposon. When
placed
in trans
with another FRT-bearing insertion, and in the presence of
FLP recombinase, one can induce deletions of genomic DNA between the
FRT sites (
Fig. 8.1
D). The possibility to generate molecularly defined
deletions has motivated the creation of extensive FRT-piggyBac collections
for custom deletions (
Parks
et al
., 2004
;
Ryder
et al
., 2007
;
Thibault
et al
.,
2004
). Several
Drosophila
miRNAs, including
mir-1
(
Kwon
et al
., 2005
),
mir-184
(
Iovino
et al
., 2009
), and
mir-284
(
Karr
et al
., 2009
), were deleted in
this fashion. In the future, “finishing” of the
Drosophila
GDP to 95% of all
genes will be facilitated by yet other transposons. In particular, Minos
combines a broad capacity for random insertion throughout the genome
with the possibility for imprecise excisions (
Fig. 8.1
B;
Bellen
et al
., 2011
).
2.4. miRNA alleles induced by homologous recombination
Since the vast majority of miRNA loci lack useful transposon insertions, or
reside in introns where deletions would affect both miRNA- and protein-
coding genes, targeted methods must be used to generate alleles (
Fig. 8.1
E).
Of course, HR has been widely used in unicellular organisms and mice for
quite some time, but a strategy to perform this in
Drosophila
was only
developed a decade ago (
Rong and Golic, 2000, 2001
;
Rong
et al
., 2002
).
The main limitation in fly, compared to mouse, is the inability to regenerate
an intact organism from an ES-like cell that can be propagated and manipu-
lated in culture. HR requires the introduction of linear molecules, which
unlike circular or supercoiled forms are recombinogenic. This was finally
solved by a two-step procedure for generating the HR substrate
in vivo
,in
the germline, by using an integrated vector with flanking FRT sites and an
internal rare restriction endonuclease site (I-SceI). Expression of FLP
recombinase in the germline excises the targeting vector, converting it
into a circular form that is then linearized by I-SceI cleavage. HR is still a
relatively rare event, requiring extensive screening to identify targeted
alleles. Nevertheless, it has proven to be a reasonably reliable technique
that has been adapted to generate many designer alleles, including a number
of miRNA deletions.
A growing number of
Drosophila
miRNA mutants have been made by
HR, including
mir-1
(
Sokol and Ambros, 2005
),
mir-309/3/286/4/5/6-
1,2,3
cluster (
Bushati
et al
., 2008
),
mir-278
(
Teleman
et al
., 2006
),
let-7/
mir-125/mir-100
cluster (
Caygill and Johnston, 2008
;
Sokol
et al
., 2008
),
mir-9a
(
Li
et al
., 2006
),
mir-12/283/304
cluster (
Friggi-Grelin
et al
., 2008
),
mir-31a
(
Weng
et al
., 2009
),
mir-263a
, and
mir-263b
(
Hilgers
et al
., 2010
).
Their study has uncovered diverse aspects of development and physiology
regulated by miRNAs, many of which will be discussed in subsequent
sections. We highlight here one example from targeted knockout of
mir-9a
.
