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| Status |
Public on Mar 01, 2006 |
| Title |
p300-mediated gene expression during muscle cell survival |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
Type of experiment:
The primary focus of these studies is to define the transcriptional network regulated by ectopic expression of the transcriptional co-activator protein, p300, in order to define its mechanism(s) of action in promoting muscle cell survival.
Experimental factors:
Our laboratory has generated a murine C2-derived myoblast cell line stably expressing an IGF-II cDNA in antisense orientation (C2AS12 cells). These cells proliferate normally in serum-rich growth medium but progressively die in low-serum differentiation medium. Ectopic expression of p300 (a transcriptional co-coactivator with acetyltransferase activity) prevents the progressive cell death induced by serum withdrawal, however, the mechanism of this action is not understood. Further, over-expression of a mutated form of p300, lacking at protein interaction domain (deltaTAZ2), failed to maintain cell viability although it retains catalytic activity. Wild-type and mutant p300 are delivered using recombinant adenoviruses (Ad-p300 and Ad-p300deltaTAZ2) and their expression is regulated by a second recombinant adenovirus encoding the tetracycline transactivator protein (Ad-tTA), affording regulated expression of p300 forms (Tet-off system) and providing a control for viral load.
Using these reagents the overall experiment was as follows: Three parallel series of C2AS12 cells were infected with (1) wt p300 + tTA, (2) p300deltaTAZ2 +tTA, (3) wt p300 +tTA +doxycycline. Infected cells were grown to confluence followed by transfer to low-serum differentiation medium (T0). Cell were isolated at this point and following 24 (T24) hours incubation for RNA isolation. Companion dishes of cells were included for analysis by immunocytochemistry to ensure regulated transgene expression and cell viabilities.
Keywords: time course
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| Overall design |
6 biological replicates, representing 3 treatment groups, two timepoints and 2 biological outcomes (cell survival with p300wt and apoptotic cell death with p300wt+Dox and p300mut)
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| Contributor(s) |
Kuninger D, Rotwein P |
| Citation(s) |
16672693 |
| |
| Submission date |
Feb 09, 2006 |
| Last update date |
Feb 18, 2018 |
| Contact name |
David Kuninger |
| Phone |
503 494 0539
|
| Fax |
503 494 8393
|
| Organization name |
Oregon Health & Science University
|
| Department |
Biochemistry and Molecular Biology
|
| Lab |
Rotwein
|
| Street address |
3181 SW Sam Jackson Park Rd
|
| City |
Portland |
| State/province |
OR |
| ZIP/Postal code |
97239-3098 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL81 |
[MG_U74Av2] Affymetrix Murine Genome U74A Version 2 Array |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA94869 |
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