Drosophila ORFeome. In vitro and in vivo resource for the research community.

The fruitfly (Drosophila melanogaster) is an important model organism for the studies of normal development and different disease states. It has a relatively small but well annotated genome encoding for approximately 15 000 genes, of which 13500 are protein coding genes. Drosophila has been a successful model organism partly because the great majority (close to 80%) of human disease genes have homologous genes in fruitfly making it a very good model to study various diseases. The rapid life cycle and powerful methods to analyse gene function makes fruitfly an attractive system to study gene function even in a large scale.
Understanding the function of each and every gene and protein in an organism is the ultimate goal in biology, and this is likely to be significantly easier to achieve in Drosophila than in human. Currently, depending on the organism, up to half of the genes identified through genome sequencing studies are of unknown function and it is a major challenge in the future to understand the function and interactions of all these genes.
Another challenge in biology is to understand how to exploit the gene information to improve human health. There has been intensive research focus on human disease genes, which when mutated lead to disease. However, a disease mutation does not always lead to disease. Furthermore, very often a drug targeting such a disease gene fails to cure the disease due to unexpected effects. Thus we need to learn more about the other genes that are regulating these diseased genes and deciding factors if a disease gene mutation will cause disease. Some of these genes may not have been identified and that is why we need a new tool to identify such disease regulating genes.
Genetic research has traditionally used two approaches to study gene function which are complementary in nature. These are decreasing the amount of an expressed gene by mutations or gene silencing or by increasing the expression of the gene with additional copies of the gene. Whereas decreasing the amount of gene expression in a large scale has been relatively easy by generation and utilization of mutant collections or gene silencing, increasing the gene expression has been hampered due to lack of suitable gene collections.
We aim to isolate the majority of the Drosophila protein coding genes to form such a large-scale collection of genes, which can be expressed in both cultured cells and in live fruitflies to study the function of these genes in different biological processes such as cell growth and animal development. Such genome-scale collections have been or are being made for other species, including the collections of worm (C. elegans), mouse and human genes, but no collections are available for screens in live animals.
Our aim is that the Drosophila gene collection becomes a resource for the whole scientific community. Having a flexible and thoroughly characterized gene collection covering most of the genome would save significant amount of time and resources and enable researchers to enhance their scientific output. This resource would also be important for the identification of new genes regulating disease progression or any other biological processes.
Following the tradition in Drosophila research where openness and free distribution of research reagents and clones is promoted, this resource of genes, as plasmids and fly strains, will be made available for all researchers.

We aim to generate a fully sequenced Drosophila melanogaster ORFeome collection in a plasmid format and as transgenic flies expressing these genes using site-specific phiC31 integration. Although ORF collections have been done for other species, there are no genome-scale metazoan in vivo collections. The collection would be a community resource to complement the large-scale RNA interference (RNAi) approaches by us and others in the recent years. Our laboratory is interested in using this resource for studying growth in cell culture assays and in vivo in the future.
The genome scale ORF collection would provide tools to systematically study the functions of individual genes in vitro, in cultured cells and in selected tissues using the GAL4/UAS system. In comparison to RNA interference libraries, an ORF collection is more versatile. It can also be used to study localization of the proteins in cells and tissues or to identify binding partners for the proteins using yeast two hybrid and tandem affinity purifications. Importantly, the ORF expression approach allows also identification of redundant functions which is not possible with RNAi and our preliminary in vivo analyses identify phenotypes for 15% of the genes which show no phenotype by RNAi.
To prepare the ORF collection to the highest standards, we are fully sequencing the original template cDNA using RNAseq and use that as a reference sequence to compare the sequences of the ORF clones. This allows us to distinct between true biological sequence variation and PCR generated mutations. This type of careful analysis has not been made for any other collection so far. Finally, we will set up a database with clone, gene and sequence information in a database format for easy access, analysis and distribution to researchers worldwide.

This work is expected to directly and indirectly benefit research and society at multiple levels, not only in UK but also worldwide. Most if not all Drosophila researchers would be interested in being able to use a characterized plasmid collection and ready-made transgenic flies for their own research as this would save their time and funding. These effects would be expected to be immediate and sustained.
This collection is also expected to immediately boost new approaches to study large-scale biology and the datasets generated by using this resource would have a wide impact on the systems biology community in the next years to come and would be expected to last until "next generation" approaches are identified and realized. Within a 5-10 year scope, any findings relevant to human disease from the studies using the resource could boost translational research focusing on the development of new diagnostic or therapeutic means to improve human health. One potential application would be to study which genes decrease the fitness of the fly and extrapolate these findings to find new approached to decrease the spread of diseases like malaria in the developing countries.
It is not unconceivable that some people would utilize the resource for commercial activities. This could happen very rapidly in terms of commercial "research and development" type of activities. For example, the large-scale yeast two-hybrid Drosophila protein interaction map was done in collaboration with CuraGen Corporation and academic groups in US with the aim of identifying novel drug targets. However, it is difficult to predict what these activities might be, but they are likely to concentrate around the health sector.
Another immediate benefit of the project is the gain of knowledge and transferable skills obtained by the post-doctoral research assistant. In addition to immediate research skills learner, he/she would be able to learn project management and dissemination of results to research community and wider public. These skills are likely to be desired not only in research sector, but in any other future employment. Any other people working later on with the collection would learn modern research approaches to enhance the output of their laboratories and host institutions.
Having a standardized gene set which is widely used by the research community would also benefit BBSRC by strengthening the BBSCR brand as a supporter of internationally recognized key systems biology resources and as a supporter of international collaborations.
Finally, by having an impact on multiple levels of basic and translational research, this project would be expected to improve human health and well-being. These effects would be expected to be most indirect and take the longest amount of time to realize, the time scale in this would be likely 10-30 years before seeing any significant impact. However, given the track record of Drosophila genetic research on human health, these are very likely to occur.