Model Organisms in Drug Discovery

104 DROSOPHILA AS A TOOL FOR DRUG DISCOVERY
genome and linked genes can be rapidly determined. However, transposon
insertion is sensitive to DNA sequence context, thus is less ‘random’ compared
with chemical mutagens. The combination of the PiggyBac transposon and
the FLP/FRT mitotic recombination system is likely to make a significant
contribution to genetic screens in the near future (Nystedt et al., 2002).
Targeted mutagenesis
The complete sequencing of the Drosophila genome heightens the need for
targeted mutagenesis methods based on gene sequences. This is especially true
for systematic functional analysis of gene families, such as kinases,
phosphatases and proteases, which are highly relevant gene families for
drug discovery. Only recently has targeted mutagenesis been achieved in
Drosophila. These methods include homologous recombination, synthetic
sequence-specific zinc finger nuclease (ZFN) and in vivo dsRNAi.
The lack of embryonic stem cell lines of Drosophila has hampered, for many
years, the use of homologous recombination to knock-out genes. However, a
method has been developed recently to induce homologous recombination in
vivo (Gloor, 2001; Rong et al., 2002). A transgene is made to carry a donor
element with a DNA fragment from the target gene and a marker gene. The
DNA fragment has engineered mutations and an I-SceI endonuclease
recognition site in the middle; the entire donor element is flanked by flipase
(FLP) recognition sites. A circular extrachromosomal donor DNA element is
induced by expression of the FLP-site-specific recombinase in vivo. The I-SceI
endonuclease, also expressed from a transgene, converts the circular DNA
into a linear recombinogenic molecule. A successful homologous recombination
event will insert the marker and mutations in the target gene. The
frequency of targeting events in the germline depends on the target genes. In a
study of five targeted genes, the homologous recombination frequency varies
from 1 in 1500 gametes for one gene, to 1 in 34 000 gametes for another gene
(Rong et al., 2002). Owing to the need for a donor transgene with engineered
mutation for transfer and a low homologous recombination frequency, the
gene targeting is still too inefficient to meet the demands of drug discovery.
Incorporating positive and negative selection schemes should relieve the
screening burden and thus increase throughput (Gloor, 2001).
Another method for gene targeting is based on ZFN. A C 2H 2-type zinc finger
can specifically bind to a DNA site with three nucleotides. Thus a collection of
64 zinc fingers are needed to bind any one of the 64 triplets. Because C 2H 2 zinc
fingers act in a modular manner, by stringing several zinc fingers together it is
possible to create a protein with multiple zinc fingers that can bind any sequence
of interest (Beerli and Barbas, 2002). A ZFN is a chimeric protein with a nonspecific
DNA cleavage domain and zinc fingers for sequence-specific DNA