Biology Reference
In-Depth Information
9.8.4 Targeted Gene Transfer
The ability to replace or modify genes in their normal chromosomal locations,
targeted gene transfer
, is a very valuable genetic tool (
Ballinger and Benzer
1989, Kaiser and Goodwin 1990, Gloor et al. 1991, Sentry and Kaiser 1992,
Lankenau 1995, Siegal and Hartl 1996, Golic et al. 1997, Rong and Golic 2000
).
Several methods have been evaluated to achieve this goal.
The cut-and-paste mechanism of
P
transposition provided a model for insert-
ing a gene into the gap left behind by a
P
(
Figure 9.5
). As noted above,
P
trans-
position leaves a ds gap in the original insertion site, and this gap may be
repaired using a template provided by a sister chromatid, or by a homologous
chromosome containing a homologous DNA sequence, or by an extrachromo-
somal element. If the sister chromatid or homologous chromosome has a second
copy of the
P
, the
P
sequences will be restored in the gap, giving the impression
that transposition has been replicative.
Engels et al. (1990)
proposed a method for
site-directed mutagenesis
(
=
tar-
geted mutagenesis or targeted-gene replacement). The first step is to insert a
P
into the gene of interest, preferably close to the site to be modified. This is fea-
sible because many different colonies of
Drosophila
have been developed that
contain
P
elements in known locations. The next step is to transfer the desired
replacement
gene into a second colony with a
P
vector. Then, individuals from
the first colony are crossed with the second. A source of transposase is added
to promote transposition and targeted gene replacement. In some cases, the
replacement gene serves as the template to fill in the gap left when the
P
trans-
poses. The result is that the original site is converted to the desired introduced
sequence.
The X-linked
white
locus was modified by targeted-gene replacement (gene
conversion) with a success rate of
≈
1% (
Gloor et al. 1991
). Changes ranged from
a few base pairs to alterations of at least 2790 bp. A 1% gene conversion rate is
sufficiently frequent to make targeted gene transfer a practical method for sys-
tematically altering genes in their normal locations to see how their function is
modified. An advantage to targeted gene transfer is that it is possible to insert
genes longer than 40kb by this method.
P
-mediated transposition is limited to
inserting DNA segments less than 40 kb in length.
Nassif and Engels (1993)
investigated the length and stringency of homol-
ogy required for repair of ds-DNA breaks in
Drosophila
germ cells using the
targeted-gene transfer system. They found that a relatively short match (of a
few hundred base pairs) of homologous sequence on either side of the target
