Transgenic Analysis of Neural Differentiation

Carson, John

Abstract

The process of cellular differentiation is regulated by the interaction of gene-specific cis acting sequences with cell- specific transacting factors. In homogeneous tissues, cis-trans regulation of differentiation has been studied to advantage using transgenic techniques whereby exogenous gene sequences are introduced into homogeneous populations of differentiated cells by bulk methods such as transfection or viral mediated infection and the pattern of expression of the exogenous gene sequences in the recipient cells is analyzed. However in the nervous system the specific differentiated phenotype of a particular cell is often dependent on the interaction of that cell with a variety of different cell types. Furthermore, many neural cell types exhibit polarized or dendritic morphologies and the pattern of gene expression may be regionally differentiated within a single cell. This means that cis-trans regulation of differentiation in neural cells cannot be studied effectively using homogeneous cell populations and bulk methods for introducing exogenous gene sequences and analyzing gene expression. We propose to solve this problem by developing a system for cell-directed transgenic studies of neural differentiation. There are three components to the system.. The first component is a sophisticated microinjection system. This will enable us to identify specific neural cells using morphological, topographical and immunological criteria, and to microinject the identified cells with specific exogenous gene sequences (DNA or mRNA). The second component is a tissue culture and containment facility where the transgenic neural cells will be grown and maintained under precisely controlled environmental and containment conditions. The third component is a cell-resolved imaging system to analyze the intracellular pattern of gene expression in individual transgenic neural cells. It is essential that the three components of this system be established in a centralized location within close physical proximity of each other. We have requested part of the cost of establishing this system for cell-directed transgenic studies of neural cell differentiation through the High Technology Grants program of the State of Connecticut Board of Higher Education. In this application we are requesting the remaining cost of establishing the system. The major users of the system are working on glial and neuronal cell differentiation in rodent and avian nervous system. The system will enhance the research programs of each of the major users as well as of many other workers in the field of neuroscience at UCHC. It will bridge the technological barrier which has until now separated the fields of cellular neurobiology and molecular biology, providing neuroscientists access to the powerful techniques of molecular biology, and enabling molecular biologists to address important questions of neural differentiation.