Biology Reference
In-Depth Information
A method of gene targeting involves the insertion of the FLP-FRT system into
the chromosomes of
D. melanogaster
via
P
-mediated transformation (
Golic et al.
1997
).
P
-mediated transformation is essentially a random event and transgenes
end up scattered throughout the genome in multiple copies, which results in
position-effect variation in expression and gene silencing. The ability to target a
transgene to a single site makes comparison of transgenes and their regulatory
elements simpler because the various constructs can all be evaluated within a
single chromosomal environment.
The FLP-FRT site-specific recombination system is based on a target site (FRT)
and the FLP site-specific recombinase enzyme system of yeast (
Kilby et al. 1993
).
This system has two 34-bp target sites for recombination. The recombinase can
mediate site-specific recombination resulting in inversions or deletions between
the recombination sites. Or, it could result in integration of exogenous DNA into
these sites, supplied by injected plasmids containing the gene of interest.
Another approach to gene targeting uses
D. melanogaster
's endogenous
DNA-repair machinery and recombination to substitute one allele for another
at a targeted gene or to integrate DNA at a target site as determined by DNA
sequence homology (
Rong and Golic 2000
). This provides
Drosophila
geneticists
with the ability to do 'reverse genetics' (
Engels 2000
).
Reverse genetics
is the
induction of a mutation in a gene in order to determine its phenotype.
P
-element transformation is limited by the size of the DNA that can be inserted,
so large genes or gene complexes cannot be inserted. Many
P
elements insert into
regulatory elements of genes (possibly disrupting expression), and expression may
be affected by their position in the chromosome. To resolve these issues,
Venken
et al. (2006)
constructed a bacterial artificial chromosome (BAC) that can contain
DNA inserts as large as 133 kb. BACs insert large DNA fragments into specific sites
in the genome, allowing functional analysis of
Drosophila
genes.
Because of the concern that TE vectors could be remobilized by endogenous
transposases or by insects mating with insects containing functional trans-
posases, possibly resulting in horizontal transfer of the transgene to other
species or loss of the transgene in the modified population, methods were
developed by
Nimmo et al. (2006)
,
Schetelig et al. (2011)
, and
Tkachuk et al.
(2011)
to stabilize transgenic lines.
Nimmo et al. (2006)
modified the genome of
Aedes aegypti
,
Schetelig et al. (2009)
modified the Mediterranean fruit fly, and
Tkachuk et al. (2011)
modified
D. melanogaster
.
Tkachuk et al. (2011)
used hom-
ing endonucleases to delete transposon and marker sequences while retaining
the transgene and its regulatory elements in the genome. See Section 9.19.5 for
a description of homing endonucleases.
