The objectives of this proposal are to examine the biological role of the retinoblastoma (RB) gene in cellular growth and differentiation. The hypothesis to be tested is that the RB gene functions in tissue development to regulate the growth rate from that of rapidly dividing stem cells to the more slowly dividing mature differentiated cells. To study this question we will produce transgenic mice that have either a generalized or tissue- specific abnormal level of RB gene expression. Transgenic mice will be produced that have incorporated an RB minigene under the control of its won promotor, or under the control of tissue-specific promotors from the insulin gene, growth hormone gene, or the T cell receptor beta-chain gene. Transgenic mice that have incorporated many copies of these chimeric genes will be analyzed for expression at the level of mRNA and protein. Those mouse lines that show high levels of expression will be analyzed for the development of beta-islet cells, the anterior pituitary gland, and the thymus, respectively. These experiments may indicate whether the RB gene product, pp110RB, is specifically involved in the regulation of tissue development. In a second set of experiments, we will product transgenic mice using chimeric RB minigenes that are transcribed in the anti-sense orientation. In mice that have incorporated many copies of the gene, expression of the transgenic anti-sense and endogenous RNAs molecules will be examined. Those mice that show a tissue-specific diminution of RB gene expression will be studied for normal tissue growth and development. In addition, the mice will be monitored for an increased spontaneous or induced incidence of malignancy. In a third set of experiments, we will isolate pluripotent embryonic stem (ES) cells that have a heterozygous defective RB gene. These ES cells will be used to generate chimeric mice by injection of the cells into mouse blastocysts. Mosaic mice will be bred for presence of a homozygous or heterozygous defective RB gene. Lines of mice containing the heterozygous defective RB gene will constitute a direct model for inherited retinoblastoma. We will study a number of parameters involved in the onset of retinoblastoma in these mice. These experiments will provide information concerning the biological function of at least one """"""""tumor suppressor"""""""" gene and provide a model for studying the onset of malignancy in patients with a heterozygous defect in the RB gene.
