The identification of tumor suppressor genes has been inherently difficult due to the lack of a selectable phenotype. Previous successful attempts to isolate such genes have relied almost exclusively on labor-intensive methods, eg. positional cloning. This proposal describes the application of a functional expression cloning system for the novel purpose of examining rhabdomyosarcoma for the presence of tumor suppressor gene(s). Briefly, RD cells derived from human embryonal rhabryonal rhabdomyosarcoma tumor will be transfected with a normal human fibroblast cDNA library constructed in lambdapCEV29, a lambda-plasmid composite vector (phagemid). This expression cloning system, which was previously developed in this laboratory, has been engineered to allow for: 1) high efficiency transfection of mammalian cells, 2) repressible expression of cDNA, and 3) efficient means of plasmid rescue. Transfected RD cells containing the putative tumor suppressor gene(s) will be identified by the selection of colonies exhibiting a growth-inhibited/differentiated phenotype under conditions of promoter activation/induced gene expression. We have developed a biological selection assay utilizing FACS (Fluorescence Activated Cell Sorting) as a means of isolating cells exhibiting a growth-inhibited phenotype. Using a lipophilic dye, PKH-2, which binds uniformly to cell membrances and divides evenly between cells with each cell division, RD transfected cells will be labeled and then subsequently analyzed for fluorescence intensity using FACS. Relatively growth-inhibited cells will be isolated on the basis of the highest level of retained fluorescence after gene expression is induced. Alternatively, RD transfectants will be selected on the basis of growth inhibition using a morphological inspection/manual selection method. In addition to these selection methods based on a growth-inhibited phenotype, RD tranfectants will also be screened for expression of surface markers consistent with differentiation. This approach will also utilize FACS to isolate RD transfectants which exhibit a normal, differentiated phenotype. Finally, plasmids will be isolated from selected cells to allow for physical and functional characterization of cDNAs encoding a putative tumor suppressor gene(s).
Specific Aims : 1) Construction of a normal human cDNA library using an expression cloning vector with an inducible promoter. 2) Identification of potential tumor suppressor genes in RD cells using an expression cloning method. Selection of RD transfectants based on induction of a growth- inhibited or differentiated phenotype will be attempted. 3) Molecular characterization of putative tumor suppressor gene(s).
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