Creation of Drosophila cell lines by overexpression of Ras
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE9366 | GEO | 2007/10/19
SECONDARY ACCESSION(S): PRJNA103069
REPOSITORIES: GEO
Similar Datasets
Project description: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.
2008-06-16 | E-GEOD-9366 | biostudies-arrayexpress
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
2008-03-12 | GSE10781 | GEO
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.
2008-04-05 | E-GEOD-10781 | biostudies-arrayexpress
Project description:We have used microarrays to identify genes expressed and required for the second mitotic wave (SMW) during eye development. Eye discs expressing Spitz under the control of GMR Gal4 have no SMW as Spitz promotes G1 arrest, ectopic differentiation also occures. To control for the ectopic differentiation, Spi expressing eye antennal discs were compared to eye antennal discs expressing activated RasV12. In discs expresseding RasV12 under the control of GMRGal4 the SMW takes place normally prior to any ectopic differentiation. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. Experiment Overall Design: Drosophila eye antennal imaginal discs expressing either UAS RasV12 or UAS Spi under the control of GMRGal4 were dissected from 3rd instar larvae for RNA extraction and hybridization on Affymetrix microarrays.
2008-06-14 | E-GEOD-6300 | biostudies-arrayexpress
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.
2023-05-01 | GSE219105 | GEO
Project description:The goals of this study were to identify gene transcription changes in cells expressing activated H-RasV12 or activated versions of the ras effector domain mutants RasV12G37, RasV12S35, and RasV12C40 for the purpose of identifying common or unique transcription alterations in cells transformed by different ras signal transduction pathways. Keywords: genetic modification design HME16C mammary epithelial cells infected with pLRT control vector, or H-RasV12-, V12G37-, V12S35-, or V12C40-expressing vectors were each treated with 250ng/mL doxycycline and total RNA prepared at two separate times to give two biological replicates. Then dye swap experiments were performed with each biological replicate.
2008-08-01 | E-GEOD-8916 | biostudies-arrayexpress
Project description:Epigenetically silenced Ink4a-Arf locus is activated by loss of H3K27me3 in cellular senescence, where secreted factor expression is also involved. Here we analyzed epigenome and transcriptome alteration during Ras-induced senescence using mouse embryonic fibroblast (MEF). Seventeen genes with H3K27me3 loss and H3K4me3 gain showed marked upregulation, including p16Ink4a and Bmp2, a secreted factor for BMP/SMAD signal. Smad6, specific BMP/SMAD pathway inhibitor, was identified as the only one gene showing de novo H3K27 trimethylation with H3K4me3, resulting in strong repression. Ras-activated cells senesced with SMAD1/5/8 phosphorylation, and they escaped from senescence with decreased SMAD1/5/8 phosphorylation when introducing Smad6 or knocking-down Bmp2. Mouse embryonic fibroblasts were established from 13.5 embryonic day embryos of C57/B6. After cells were passaged twice (MEFp2), cells were infected with retroviruses for 48 hours. Then cells were exposed to 4 M-NM-<g/mL peuromycin for selection during days 0-3, and were passed on days 3, 7, and 10. Retroviral vectors for Ras was constructed by cloning cDNAs for wild type HRAS (RasG12) and mutated HRAS (RasV12) by reverse-transcription PCR products from HMEC and SK-BR3 cell RNA, respectively, with N-terminal FLAG tag into pMX vector that contains puromycin resistance gene. Mock pMX vector (mock), and vectors containing RasG12 and oncogenic RasV12 were transfected into plat-E packaging cells using FuGENE 6 Transfection Reagent (Roche, Germany) to prepare retroviruses. Smad6 cDNA with N-termainal 6x Myc tag was also cloned into pMX vector. To knock down Bmp2, double strand oligonucleotide DNA to express small hairpin RNA against Bmp2 (shBmp2) was cloned into RNAi-Ready pSIREN-RetroQ Vector (Clontech, CA). Viral packaging for Smad6 and shBmp2 retrovirus vectors was also done using plat-E cells. For genome-wide transcription analysis, GeneChip Mouse Genome 430 2.0 Array (Affimetrix) was used. For global normalization, the average signal in an array was made equal to 100. Chromatin immunoprecipitation (ChIP)-sequencing was performed. MEFp2 cells and cells with mock, RasG12 or RasV12 infection at day 10 were cross-linked with 1% formaldehyde for 10 min at room temperature and were prepared for ChIP. ChIP using anti-H3K4me3 (ab8580, abcam, rabbit polyclonal) or H3K27me3 (07-142, Upstate, rabbit polyclonal) antibody was performed as described previously. Sample preparation for ChIP-sequencing was performed according to the manufacturer's instructions (Ilumina), and sequencing was performed using Solexa Giga sequencer.
2011-12-22 | E-GEOD-18125 | biostudies-arrayexpress
Project description:The goals of this study were to identify gene transcription changes in cells expressing activated H-RasV12 or activated versions of the ras effector domain mutants RasV12G37, RasV12S35, and RasV12C40 for the purpose of identifying common or unique transcription alterations in cells transformed by different ras signal transduction pathways. Keywords: genetic modification design
2008-08-01 | GSE8916 | GEO
Project description:Transcriptional profiling of normal human diploid fibroblasts(TIG-3) comparing empty vector transducted cells with RasV12 transducted cells.
2014-04-10 | GSE49862 | GEO
Project description:Transcriptional profiling of normal human diploid fibroblasts(TIG-3) comparing empty vector transducted cells with RasV12 transducted cells. TIG-3 cells were subjected to infection with retrovirus encoding RasV12 or empty vector. Total RNA was isolated using TRIzol reagent and were analyzed using the human 3D-Gene DNA chip (Toray) that contains 25000 genes. The genome wide transcriptional resp
2014-04-10 | E-GEOD-49862 | biostudies-arrayexpress