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In JoVE (1)
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Articles by Cynthia L. Montana in JoVE
Количественная активность СНГ-Регуляторных элементов в мышь Retina от эксплантов Электропорация
Cynthia L. Montana, Connie A. Myers, Joseph C. Corbo
Department of Pathology and Immunology, Washington University School of Medicine
Этот протокол описывает простой и недорогой способ для количественной оценки активности цис-регуляторных элементов (например, усилитель / промоутеров) в живой мыши сетчатки через эксплантов электропорации. Препарата ДНК, сетчатке глаза рассечение, электропорация, сетчатки культуры эксплантов, и после фиксации анализ и количественное описаны.
Other articles by Cynthia L. Montana on PubMed
Pharmacogenomics. Mar, 2008 | Pubmed ID: 18303969
The photoreceptor cells of the retina are subject to a wide range of genetic diseases. This review summarizes current knowledge regarding an important group of retinal diseases caused by mutations in photoreceptor-enriched genes. In addition, progress toward treatment of a variety of these diseases in animal models via adeno-associated virus gene therapy is described. Although no human trials have yet been initiated to treat diseases caused by mutations in photoreceptor-enriched genes, there is a great deal of optimism regarding the prospects of treating these diseases using adeno-associated virus gene therapy.
Transcriptional Regulation of Neural Retina Leucine Zipper (Nrl), a Photoreceptor Cell Fate Determinant
The Journal of Biological Chemistry. Oct, 2011 | Pubmed ID: 21865162
The transcription factor neural retina leucine zipper (Nrl) is a critical determinant of rod photoreceptor cell fate and a key regulator of rod differentiation. Nrl(-/-) rod precursors fail to turn on rod genes and instead differentiate as cones. Furthermore, NRL mutations in humans cause retinitis pigmentosa. Despite the developmental and clinical significance of this gene, little is known about the transcriptional regulation of Nrl itself. In this study, we sought to define the cis- and trans-acting factors responsible for initiation and maintenance of Nrl transcription in the mouse retina. Utilizing a quantitative mouse retinal explant electroporation assay, we discovered a phylogenetically conserved, 30-base pair region immediately upstream of the transcription start site that is required for Nrl promoter activity. This region contains binding sites for the retinal transcription factors CRX, OTX2, and RORβ, and point mutations in these sites completely abolish promoter activity in living retinas. Gel-shift experiments show that CRX, OTX2, and RORβ can bind to the critical region in vitro, whereas ChIP experiments demonstrate binding of CRX and OTX2 to the critical region in vivo. Thus, our results indicate that CRX, OTX2, and RORβ directly regulate Nrl transcription by binding to critical sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by OTX2 and RORβ and later maintained at high levels by CRX and RORβ.