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| Status |
Public on Feb 28, 2006 |
| Title |
Targeting of GFP to new-born rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
Purpose: To investigate the gene regulatory networks during photoreceptor differentiation.
Special aims: To generate gene expression profiles of purified photoreceptors at distinct developmental stages and from different genetic backgrounds.
Background: Rod photoreceptor genesis spans a broad temporal window during retinal development. It starts as early as E12.5 and peaks at P0-P2. At E16.5, there are some early born rods but the peak of rod genesis does not occur. At P2, the majority of rod photoreceptors are born. At P6, rod specific structural/functional genes begin to express. At P10, Outer segments morphogenesis is taking place. At 4 weeks, retinal development is complete and retina is functional. Nrl is a rod specific transcription factor and one of the earliest markers of rod photoreceptors. Nrl promoter drives the expression of GFP exclusively to rod photoreceptors shortly after they exit cell cycle. In the Nrl-knockout background, the expression of GFP is detected in S-opsin positive cells, which suggested a cell fate transformation from rods to cones in the absence of Nrl.
Design: GFP positive photoreceptors from the WT-Gfp or Nrl-knockout-Gfp retina were enriched (purified) by FACS at five distinct developmental stages (E16, P2, P6, P10, and 4 weeks). Total RNA was extracted by Trizol reagent. Around 50 ng of total RNA was used for linear amplification and biotin labeling followed Nugen kit protocol. Fragmented cDNA was hybridized on Affymetrix mouse genomic expression array 430 2.0 and then scanned with the standard protocol. Four replicates were performed for each time point.
Conclusion: By comparing the gene expression profiles from different developmental stages, we can obtain novel insights into molecular events underlying photoreceptor differentiation.
Keywords: Transcription factor, development, photoreceptor, retina, neuron, differentiation, gene regulation, microarray, gene profiling, cell type comparison
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| Overall design |
Postmitotic rod precursors and mature rod photoreceptors are tagged by GFP under the control of an Nrl promoter in the wild type background (Wt-Gfp mice). When cross-bred into the Nrl-knockout background (Nrl-ko-Gfp mice), the transformed “S-cones” are tagged by GFP. GFP positive photoreceptors from the Wt-Gfp or Nrl-ko-Gfp retina were enriched (purified) by FACS at five distinct developmental stages (E16, P2, P6, P10, and 4 weeks). Total RNA was extracted by Trizol regent and around 50 ng of total RNA was used for linear amplification and biotin labeling following Nugene kit protocol. Fragmented cDNA was hybridized on Affymetrix mouse genomic expression array 430 2.0 and then scanned with the standard protocol. Four replicates were performed for each time point.
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| Contributor(s) |
Akimoto M, Cheng H, Zhu D, Brzezinski JA, Khanna R, Filippova E, CT Oh E, Jing Y, Linares J, Brooks M, Zareparsi S, Mears AJ, Hero A, Glaser T, Swaroop A |
| Citation(s) |
16505381 |
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| Submission date |
Jan 17, 2006 |
| Last update date |
Feb 11, 2019 |
| Contact name |
Swaroop Anand |
| E-mail(s) |
swaroop@umich.edu
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| Phone |
734-615 2246
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| URL |
http://www.umich.edu/~retina
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| Organization name |
University of Michigan
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| Department |
Ophthalmology & Visual Sciences
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| Street address |
1000 Wall St.
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48105 |
| Country |
USA |
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| Platforms (1) |
| GPL1261 |
[Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array |
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| Samples (39)
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| GSM92610 |
Nrl-ko-Gfp 4 weeks retina replicate 1 |
| GSM92611 |
Nrl-ko-Gfp 4 weeks replicate 2 |
| GSM92612 |
Nrl-ko-Gfp 4 weeks replicate 3 |
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| Relations |
| BioProject |
PRJNA95193 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE4051_Nrl-ko-Gfp-PAdetectioncalls.txt |
1.5 Mb |
(ftp)(http) |
TXT |
| GSE4051_RAW.tar |
253.3 Mb |
(http)(custom) |
TAR (of CEL) |
| GSE4051_wt-Gfp-PAdetectioncalls.txt |
1.5 Mb |
(ftp)(http) |
TXT |
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