DESCRIPTION (provided by applicant): The purpose of this proposal is to derive a photo-activatable gene expression system compatible with two-photon microscopy that will allow the induction of a single gene within a single cell in the tissue of a living animal. This will provide a valuable resource enabling two novel lines of research in gene expression: generating a high temporal and spatial resolution probe for assessing the mechanism of transcription and affording the means to evaluate the downstream effects of a particular gene product in a cell within tissue where all the surrounding cells serve as controls. Hence the microenvironment of the affected cell and its interactions with neighboring cells can be rigorously addressed as a function of the activated gene. We have provided a proof-of- principle for this approach in cultured cells using this funding mechanism. In this previous work, we have constructed a reporter gene and cell line that can be activated through the ecdysteroid receptor and is thereby orthogonal to mammalian transcriptional activation. In collaboration with our co-PI, David Lawrence, a synthetic organic chemist, we caged the ecdysteroid analog ponasterone and established that it could be activated by a laser at 340nm. We built an uncaging microscope that was capable of focusing the laser beam into a micron spot on a cell and demonstrated the activation of a reporter gene. The gene activation was detected through nascent RNAs containing a stem-loop repeat that binds a fluorescent capsid protein from the phage MS2 and this RNA, is translated into a blue fluorescent protein containing a sequence that concentrates in peroxisomes. This proposal describes how we intend to adapt this system so that it is compatible with investigations in living tissues. This involves the design and use of a caging group that is sensitive to two-photon excitation, the construction of animal models using xenograft cells, the development of methods to image into tissues (the expertise of the Co-PI John Condeelis) with high resolution and single molecule sensitivity and the design and operation of customized approaches to collect the images (the expertise of the Co-PI Ben Ovryn) and powerful computational analysis tools (the expertise of the Co-PI Lin Ji). Finally, we intend to construct a transgenic mouse into which we have inserted a photo-activatable gene for a physiologically relevant protein, to determine its effects on a single cell in a tissue. Public Health Relevance: We have discovered a way to activate a gene using only a focused laser beam. The procedure uses an insect hormone that we can block with a compound that can be cleaved off by light. We have built a microscope that allows us to do this in cells and tissues in a living animal so we can investigate exactly how genes turn on. This enables us to induce a single cell to synthesize any protein and then determine the actions of that protein in cells. that controls how and where other proteins are made in the cell. We are particularly interested in proteins that cause cancer, oncogenes, and in determining how these proteins can induce cells to produce tumors.
|Effective start/end date||9/30/08 → 7/31/14|
- National Institute of General Medical Sciences: $711,684.00
- National Institute of General Medical Sciences: $718,099.00
- National Institute of General Medical Sciences: $734,237.00
- National Institute of General Medical Sciences: $704,186.00
- Molecular Biology
- Cell Biology
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