The overall goal of our studies is to generate and characterize synthetic smooth muscle cell-selective promoters that are active in different tissues of the genitourinary tract. These promoters will be useful for targeting expression of genes to these tissues and they will be utilized in our studies to determine the molecular mechanisms mediating smooth muscle cell-selective expression of proteins.
In Aim 1 the telokin promoter, will be used to generate transgenic mice that selectively express EGFP in smooth muscle cells of the genitourinary and digestive tracts. These transgenic mice will be used to isolate pure populations of smooth muscle cells from these tissues by fluorescence activated cell sorting of freshly dissociated cells. These mice will be made available to other investigators that require pure populations of GU-tract smooth muscle cells for their investigations, they will also be in our studies to determine the molecular mechanisms mediating smooth muscle cell-selective expression of proteins in different tissues of the genitourinary tract.
In aim 2, synthetic chimeric promoters comprised of fragments of the telokin and SM22 (z promoters will be generated and analyzed for their ability to direct 13-galactosidase expression to smooth muscle cells within selected tissues of the genitourinary tract in transgenic mice. Preliminary data show that the telokin AT/CArG element can selectively increase transgene expression in bladder smooth muscle cells. This suggests that smooth muscle cells in each of the tissues of the genitourinary tract must express either distinct transcription factors or differentially regulate the activity of common factors that bind to this region of the telokin promoter. To identify these factors experiments described in Aim 3, we will compare the expression of proteins known to be able to bind this sequence and also use the AT/CArG element as a affinity probe to isolate proteins present in nuclear extracts of pure populations of smooth muscle cells obtained from bladder, uterus and gut of EGFP transgenic mice.
In aim 4 it is proposed to use a global proteomic approach to identify additional nuclear proteins that are differentially expressed or modified in distinct smooth muscle tissues of the genitourinary tract. Together these studies will provide a comprehensive analysis of the mechanisms regulating smooth muscle cell-selective gene expression in the genitourinary tract. ? ? ?
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