Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse Egr-1 knock-outs during development (p30 and p42)

ABSTRACT: In chicks, the avian homologue of the early growth response protein-1 (ZENK) has been shown to be increased in a special cell type of the retina, the glucagonergic amacrine cells, under conditions that lead to a reduction in eye growth (myopic defocus, recovery of myopia) and decreased under conditions that enhance ocular growth (hyperopic defocus, form-deprivation). The investigation of Egr-1 knock-out mice showed that homozygous knock-out mice with no functional Egr-1 protein developed relative axial myopia at the age of 42 and 56 days, compared to heterozygous- and wildtype Egr-1 knock-out mice. To clarify the role of Egr-1 in the retinal regulation of eye growth, and to get an idea about the biochemical pathways underlying this mechanism, we studied the role of Egr-1 in more detail using Affymetrix microarrays. Experiment Overall Design: Retinal samples of young homozygous Egr-1 knock-out and wildtype mice at the age of 30 days (hm30 and wt30; no difference in axial eye length yet) and 42 days (hm42 and wt42; already a difference in axial eye length of 59 Âµm) were taken to compare the mRNA expression changes over time between these two genotypes and within the same genotype between the two age groups.

ORGANISM(S): Mus musculus

SUBMITTER: Marita Pauline Feldkaemper

PROVIDER: E-GEOD-16974 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Juvenile primates develop myopia when their visual experience is degraded by lid fusion. In response to the abnormal visual input, retinal neural networks cause an excessive growth of the postequatorial segment of the eye, but the mechanism underlying this axial elongation is unknown. By combining analysis of gene expression, injection of the thymidine analog 5-bromo-2'-deoxyuridine and immunocytochemistry, we show that the retinal periphery in both juvenile rhesus macaques and green monkeys harbors a population of mitotically active neuroprogenitor cells that proliferate when the visual experience is altered by lid fusion. Furthermore, the number of dividing cells is highly correlated with the axial elongation of the eye and the resulting myopic refractive error. Thus, the retina undergoes active growth during the postnatal development of the primate eye. This growth is modulated by the visual input and accelerates considerably when the eye develops axial myopia. cDNA subtractions, construction of cDNA libraries and differential screening were performed as previously described (Tkatchenko et al, 2000). Two subtractions were carried out resulting in two cDNA libraries. One library was enriched in clones potentially up-regulated in the retina of the closed eye and the other was enriched in cDNAs potentially down-regulated. One thousand clones from each library were analyzed, and 535 differentially-expressed cDNAs were amplified by PCR and spotted onto glass slides coated with poly-L-lysine (five duplicates for each cDNA). The slides were then processed, hybridized with Cy3- and Cy5-labeled complex cDNA probes and washed in 0.2xSSC as described at http://cmgm.stanford.edu/pbrown/protocols/. Two hybridizations with color swap were carried for each monkey. After hybridization, slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA). The images were acquired and processed using the GenePix Pro 5.1 software package (Axon Instruments). The data obtained were normalized against GAPDH expression level and mean values (the reported values) and P-values (probability associated with a Student's t-test) were calculated for duplicate experiments (n = 10) with color swap using Microsoft Excel. Genes were defined as differentially regulated if P< 0.05. Cluster analysis was performed using the Genesis software package (provided by Alexander Sturn, Graz University of Technology, Austria).

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Transcription profiling of mouse Egr-1 knock-outs during development (p30 and p42)

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