efficient genetic method for establishing drosophila cell lines unlocks the potential to create lines of specific genotypes高效的遗传方法建立果蝇细胞系解锁特定基因型的潜在创造行.pdfVIP

Efficient Genetic Method for Establishing Drosophila Cell Lines Unlocks the Potential to Create Lines of Specific Genotypes Amanda Simcox.*, Sayan Mitra., Sharon Truesdell, Litty Paul, Ting Chen, Jonathan P. Butchar, Steven Justiniano Department of Molecular Genetics, Ohio State University, Columbus, Ohio, United States of America Abstract Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene RasV12 (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle- shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate