Genetic analysis of the fruit fly, Drosophila, is at the forefront of discovery in understanding normal biology and is also important as a model for human disease. The analysis of Drosophila in vivo is the major thrust of the field, but as with any system, in vitro analysis is also important. Recently a new method, using expression of oncogenic Ras, has proven to be a simple genetic way to rapidly produce fly cell lines. This opens up the possibility of making 'custom'lines of certain genotypes and potentially different cell types. The development is expected to result in an increase in the number of cell lines, and puts a new impetus on developing ways to genetically modify Drosophila cultured cells. In this regard, Drosophila lags seriously behind mammalian systems for which multiple methods have been developed for site-specific integration of exogenous DNA. The phage phiC31 integrase allows insertion of donor DNA at target genomic 'landing sites'and has a number of advantages. These include insertion of large pieces of DNA and comparisons between genes expressed from the same genomic locus. The system has been used successfully for site-specific integration in vivo in Drosophila. Here it is proposed to adapt this system for use in vitro. In the first aim, a series of donor vectors will be developed that allow the simple addition of genes of interest and will include features for tagging and expression. A set of 'landing sites'will also be engineered that facilitate easy selection for the desired integration of the donor vector. In the second aim, a series of cell lines will be generated that allow expression of inserted genes at reproducible 'high', 'medium'and 'low'levels. It is expected that this technology-the vectors and the cells will be widely used by the Drosophila community for in vitro analysis in both gene-specific and high-throughput studies.
Genetic analysis of the fruit fly, Drosophila, is invaluable for discovering genes and their functions. High conservation of these genes, and the mechanisms by which they operate, allows for many direct comparisons between flies and mammals. For this reason, Drosophila is an important model for human disease. The proposed studies will significantly advance the technological capability of cell-based genetic studies in Drosophila. The subsequent use of this system by individual investigators to study biological problems in vitro, in gene-specific and high throughput analyses, is expected to impact our understanding of human disease.
| Manivannan, Sathiya N; Jacobsen, Thomas L; Lyon, Peter et al. (2015) Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange. Genetics 201:1319-28 |
| Justiniano, Steven E; Mathew, Anne; Mitra, Sayan et al. (2012) Loss of the tumor suppressor Pten promotes proliferation of Drosophila melanogaster cells in vitro and gives rise to continuous cell lines. PLoS One 7:e31417 |
