Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Drosophila cell lines overexpressing Ras

ABSTRACT: RasV12 was used to drive the generation of cell lines from primary cultures. Lines have been passaged for over a year, indicating that they are true lines. Experiment Overall Design: RasV12 drove the creation of cell lines from primary culture. The array datasets were created from passage 11 cells and compared to datasets of established cell lines, as well as embryonic, imaginal disc and whole-fly sets.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Amanda Simcox

PROVIDER: E-GEOD-9366 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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. Experiment Overall Design: Cells from a RasV12 line at passage 12, CL8 cell line and L2 cell line were grown to 70% confluence and RNA was extracted (Quiagen RNeasy). Three samples of RasV12, four samples of CL8 and one sample of L2 cell line, derived from independent T-flasks were processed. Targets were generated and hybridized to DrosGenome 1 Affymetrix gene chips using standard procedures (Affymetrix.com). All analyses were done using the Bioconductor suite of packages (www.bioconductor.org) in R (www.r-project.org). Expression values were calculated using the GC Robust Multiarray Average (GCRMA) method and statistical tests for differential expression were done using the 'limma' package. Clustering was performed on the top 20% of genes ranked by standard deviation, using 1-correlation as the distance measure and an average linkage. For class discrimination analysis, the 'pamr' package was used.

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Transcription profiling of Drosophila cell lines overexpressing Ras

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