Biology Reference
In-Depth Information
2. Transgenic misexpression
One of the most powerful and versatile tools available in Drosophila is the ability to
generate transgenic constructs that can be used to drive the expression of a chosen
gene in a tissue-specific manner by exploiting a yeast transcriptional activation
mechanism. The GAL4/UAS system uses the yeast transcriptional activator GAL4
expressed under the temporal and spatial control of endogenous enhancer/promoter
elements, to drive the expression of a specific transgene (Brand and Perrimon,
1993). The transgene is cloned in a vector containing a minimal promoter coupled
with upstream activator sequences (UASs) that are specifically recognized by the
GAL4 transcription factor. Upon binding of GAL4 to the UAS sites, expression of
the transgene is induced in a tissue-specific manner dependent on the endogenous
enhancer/promoter elements controlling GAL4 expression. There are now many
different GAL4 lines available that drive the expression in a wide variety of tissues
(e.g., nervous system, muscle tissue, or ubiquitously). This technique has been used
with great effect to determine the
consequence of misexpression or over-
expression of fly genes and has been adapted to study the ectopic expression of
human genes and their disease causing aberrant forms. An additional advantage to
modeling late-onset, age-dependent processes, relevant for most neurodegenerative
disorders, is the facility to bypass confounding developmental defects and study the
effects of transgenes only in the adult stage by the use of steroid-induced transgene
expression (Roman
in vivo
et al.
,2001).
3. Genetic screening
Probably, the single greatest advantage of using Drosophila for any area of
biological interest is the ability to conduct relatively unbiased genetic screens
for mutations in other genes that suppress or enhance the phenotypes associated
with the disease model. This approach has the potential to identify cellular
factors that act in the same or parallel pathways to the disease gene and does
not require any
knowledge of the function of the disease gene. The power
of such screening approaches cannot be overstated; human counterparts
corresponding to suppressors identified from screens using Drosophila define
potential targets for therapeutic intervention. It is primarily the feasibility of
conducting such high-throughput screens that sets Drosophila apart from verte-
brate models of disease.
There are a large number of approaches available for conducting genetic
modifier screens in Drosophila (St. Johnston, 2002). One of the most commonly
used approaches involves crossing a collection of enhancer
a priori
) element
insertions (Rorth, 1996) into a disease model background and investigating the
effects of these insertions on the disease model phenotypes. The
P
(
EP
transposons
have been engineered to drive overexpression of sequences flanking the
EP
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