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M. Thiersch, P. Blank, M. Samardzija, C. Grimm; Conditional Knock Out of HIF-La and the Consequences for Hypoxia Induced Retinal Neuroprotection in a Model of Light Induced Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2007;48(13):631.
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Hypoxic preconditioning protects the retina from light induced photoreceptor apoptosis. The hypoxic pretreatment stabilizes the transcription factor hypoxia inducible factor-1a (HIF-1a) in the retina. This leads to the upregulation of several HIF-1 target genes including erythropoietin (Epo) and to neuroprotection.To elucidate the role of HIF-1a in the observed neuroprotection, we generate and test different conditional HIF-1a knockouts using the cre-lox system.
Tamoxifen (TAM) inducible Prp-Cre, GFAP-cre and Nestin-Cre mice were bred on a ROSA26 background to analyze the expression pattern of cre. To generate tissue specific HIF-1a knockouts, mice were intercrossed with HIF-1aflox/ko mice. TAM was injected twice a day, 5 days in a row with a concentration of 1mg TAM per 40g starting around PND 21. Knock out efficiency was estimated by real time RT-PCR and Western Blot. Mice will be exposed to low 6% oxygen for 6 hours for hypoxic preconditioning and exposed to white fluorescent light to test for the efficacy of neuroprotection in the partial absence of HIF-1a.
Cre expression controlled by the Prp promoter was mainly detected in the photoreceptor layer. GFAP- and Nestin-promoters directed cre expression predominantly to the inner retina and to the ganglion cell layer. RT-PCR data revealed, that TAM-induced, cre-mediated excision of HIF-1a in the retina occurred with an efficiency of almost 80%. Nestin-Cre mediated excision was only 25% effective. The effect on partial HIF-1a ablation on hypoxic preconditioning and neuroprotection will be presented.
TAM inducible Prp-Cre;HIF-1aflox/ko mice are a very good tool to analyze the impact of lacking HIF-1a in the photoreceptor layer, especially since the retina is allowed to develop normally before the excision of the target gene is initiated. Nestin-Cre might be a useful tool to investigate the lack of HIF-1a in the inner retina. These mice will be tested in our model of light induced retinal degeneration to investigate whether the lack of HIF-1a in different cells of the retina has an impact on hypoxia induced neuroprotection.
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