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| Status |
Public on Mar 12, 2008 |
| Title |
Efficient generation of continuous cell lines from Drosophila embryos by expression of oncogenic Ras |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by array
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| Summary |
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
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| 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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| Contributor(s) |
Simcox A, Chen T, Butchar JP, Justiniano S |
| Citation missing |
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| Submission date |
Mar 11, 2008 |
| Last update date |
Jul 06, 2016 |
| Contact name |
Amanda Simcox |
| E-mail(s) |
simcox.1@osu.edu
|
| Organization name |
The Ohio State University
|
| Department |
Molecular Genetics
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| Lab |
Simcox
|
| Street address |
484 W. 12th Avenue
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43210 |
| Country |
USA |
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| Platforms (1) |
| GPL72 |
[DrosGenome1] Affymetrix Drosophila Genome Array |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA107465 |
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