Project description: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 cells have undergone more than 90 population doublings and therefore constitute continuous cell lines. 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 silenced. We successfully created several cell lines and found 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 mechanism to promote proliferation in Drosophila primary culture and serves as an efficient means to generate continuous cell lines. This offers an opportunity to create cell lines of specific genotypes and potentially of a given cell type. Keywords: Cell type comparison
Project description:Expression of activated Ras, RasV12, provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here we used restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type specific genes and the expected alignment with single cell sequencing data in several cases. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center.
Project description:ChIP against Opbp followed by next generation sequencing of Drosophila melanogaster embryos. Samples were sequenced using Illumina HiSeq and include four biological replicates, with both mock and input controls.
