94591
Targeted Mutagenesis of the Genes flin-7 by Homologous Recombination in Drosophila melanogaster
Presenters: Sheila P. Nguyen and Hao Nguyen (not presenting)
Mentors: Dr. Peter J. Bryant and Dr. Cecilia De Lorenzo
In the last 35 years, the fruit fly Drosophila has been intensively used as a model system to study the genetic changes that occur in cancer cells. One of the best studied tumor suppressor genes in Drosophila is lethal(1)discs large-1, encoding the Discs large (Dlg) protein, which functions in larval brain and imaginal discs. Similar to other tumor suppressor genes in Drosophila, Dlg has human homologs implicated in various cancer types. Preliminary evidence indicated that Dlg may exert its tumor suppressive function by regulating the localization and/or function of DER (the Drosophila Epidermal Growth Factor Receptor) either directly or through its interaction with the putative cytoskeletal proteins Flin-7. To test these ideas, we are producing genetic mutations of flin-7 using homologous recombination, a new technique in Drosophila that uses DNA repair and recombination mechanisms to replace normal DNA with exogenous mutant DNA at a target locus. To create the necessary DNA construct, we used pGEM-T as an intermediate cloning vector, which increased the efficiency of subcloning into the final vector pW25. This construct contained the necessary elements for its own insertion into fly genome and facilitated recombination. Thus far, we have created the flin-7 construct, injected it into fly embryos, screened and identified eight lines of transformants, and obtained nine mutant candidates. We are in the process of carrying out phenotypic and genotypic analysis. The study of the phenotypic defects caused by the flin-7 mutation will show whether the gene product have the suggested functions in controlling cell proliferation.