3D Tissue Models

Carla Green

TypeWhite Paper Summary

My lab is interested in gene expression in photoreceptors, with a major emphasis on genes and processes that are controlled by the endogenous retinal circadian clock. This clock controls many rhythmic processes within the retina, but the mechanism of this control is not known. My lab focuses on the study of genes involved in the control of these rhythmic cellular events. We use the frog, Xenopus laevis, as a model system for these studies, because the retinas (and pineal glands) of these frogs contain an intact circadian system that can be maintained in culture. We have identified and cloned several genes that are involved in photoreceptor clock function.

We are currently using Xenopus embryos to study the ontogeny of the circadian clock and light sensitivity. We have been able to show that isolated pineal glands from 2 day old embryos are light-sensitive and are capable of producing rhythmic melatonin release in culture in cyclic light. Circadian control of this process occurs slightly later: by 4 days of development, the isolated pineals can synthesize melatonin rhythmically in constant darkness. Similar experiments have been done with cultured embryonic eyes. We are interested in using this developmental system to study the molecular events that are required to generate the functional internal circadian timing system.

One of the ways that we are approaching these questions is to use a newly described technique for generation of transgenic Xenopus. This will be the basis for the development of a system that could be broadly used for functional studies of molecular mechanisms within photoreceptors. We have been able to target the expression of reporter genes such as GFP (green fluorescent protein) to specific photoreceptor subtypes with the use of promoters such as the rod opsin promoter. We will continue to isolate and test promoters in this system so that we will have a repertoir of cell type-specific promoters that will allow directed expression of various genes of interest. This will be useful for a whole array of photoreceptor studies, such as overexpression of various genes, introduction of dominant negatives, or overexpression of antisense mRNAs to achieve functional knockouts. Furthermore, generation of transgenic lines expressing rhythmic reporters will further extend our studies of clock function by allowing real time analysis of gene expression in living embryos.

Name:Carla Green

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