Each of the projects in the program will utilize the Cell Biology Core Unit, which is designed to provide purified cultures of neonatal rat myocardial cells for the various projects. In many cases, the cultured cells will be used to identify candidate signaling molecules and pathways that can activate hypertrophy in the in vitro cultured myocardial cell system. The cell system has been extremely well- characterized, and numerous approaches for documenting cause-effect relationships between specific signaling molecules and the activation of a hypertrophic response have been developed within the context of our previous program. As a result, a number of other laboratories now have adopted similar approaches to map signaling pathways in this in vitro cardiac muscle cell system. This Core Unit will assist in providing the cultured cells, and any other assistance related to the use of the in vitro assay system. In addition to providing purified cultures of neonatal rat ventricular myocardial cells, this Core Unit will also provide adult mouse cardiac myocytes. Our laboratory has extensive experience in the culturing of embryonic, neonatal, and adult muscle cells from various cardiac chamber compartments. Another important function of the core is to generate recombinant adenoviral vectors for gene transfer in neonatal rat myocardial cells. In certain cases, adenoviral vectors will primarily be utilized in cultured myocardial cells to direct the expression of dominantly active signaling molecules, although we have also generated adenoviral vectors which will bring in the CRE recombinase, to serve as an alternative approach for conditional gene targeting in animals which harbor """"""""floxed"""""""" alleles of interest, in a manner analogous to studies utilizing transgenic approaches to introduce CRE recombinase. Accordingly, there are three specific functions for this core unit: a) To provide purified cultures of neonatal rat ventricular myocardial cells and related techniques for gene transfer in these cells. b) To provide purified adult ventricular muscle cells from various transgenic and gene-targeted mouse lines for analysis. c) to generate and utilize recombinant adenoviral vectors for gene transfer in neonatal rat myocardial cells for various projects in the Program.
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