The Laboratory of Cardiovascular Science is committed to identifying those gene products which are differentially expressed in embryonic development through exploitation of the cardiac developmental model of embryonic stem and embryonic carcinoma cell differentiation and through interaction with the Laboratory of Genetics and their expertise in making and analyzing specialized libraries and chips. With the recent developments in heart regarding the role of zinc-finger transcription factors (GATA4), homeodomain containing transcription factors (Nkx2.5) and basic helix loop helix proteins (eHAND and dHAND), identification of novel transcription factors should be possible based on sequence data alone; however, the factors responsible for cardiac induction and many aspects of differentiation are entirely unknown. Serial analysis of gene expression (SAGE) yields a quantitative, representative and comprehensive differential gene expression profile. Using several time points in the precise developmental profile of embryonic stem or carcinoma cell differentiation to cardiomyocytes, SAGE analysis can generate a quantitative transcript characterization of tens of thousands of transcripts that is much more rapid and economical than other techniques. During the past four months we have used an RT-PCR based technique to determine the time points where a number of mesodermal and cardiac-restricted gene products are expressed in in vitro differentiated EC derived cardiomyocytes. Results show that the earliest contractions occur on Day 5.5 of differentiation (3+2.5 or 4+1.5). Some contractile protein genes (beta MHC) are expressed before the third day of differentiation , but we show that a clear induction of expression of a alpha MHC and MLC2V starts relatively later (5+0.5 and 3+1 for alpha MHC and MLC2V respectively. GATA-4, a transcription factor involved in the regulation of cardiac contractile protein gene expression is highly present in the earliest stages of differentiation, and form 3+1.5 days of differentiation, its expression is reduced to the level of adult heart tissue. This indicates that this time period corresponds to an early stage of cardiac differentiation. We are using the protocols of SAGE to analyze the expressed sequence profile during this time period . This work is ongoing.
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