Homologous Recombination in a Secretory Mouse Cell Line

Saint Louis University, Saint Louis MO

Investigators

Linked publications & trials

Abstract

DESCRIPTION (Provided By Applicant): Disruptions of the molecular events that regulate exocytosis/synaptic transmission give rise to a variety of neuropathologies. Model systems that allow exploration of the underlying molecular machinery that control these processes are problematic. This exploratory/developmental grant (R21) application seeks to develop a mouse cell line that can be genetically modified to study vesicle release at the single-cell level, and to provide pilot data for subsequent grant applications. In the nervous system, synaptic transmission is initiated by the entry of Ca2+ into the presynaptic terminal, which activates the fusion of transmitter-filled vesicles with the presynaptic membrane. Synaptotagmin I (syt I), an integral vesicular protein, has been postulated to act as the Ca2+ sensor for exocytosis. One genetic approach to study the function of syt I as a Ca2+ sensor has been the generation of knockout mice. The syt I homozygous knockout mice are viable until 48 hours after birth, and the heterozygous animals are phenotypically indistinguishable from wild-type. These problems, not uncommon in knockout animals, make it difficult to study the functional role of syt I. 1. The first aim is to introduce a targeting vector to disrupt the syt I gene by homologous recombination in a mouse cell line of non-embryonic origin. We have identified a mouse pheochromocytoma cell line that exhibits Ca2+-dependent exocytosis of membrane-bound vesicles. We have designed specific targeting vectors to the syt I gene of the cells. We will attempt to introduce the specific targetig vectors into the cells, to screen cells with positive and negative selection, and to obtain and test the genomic DNA from the screened cells for targeted interruption of the syt I gene. We believe that this unique cell line will serve as an excellent model system to probe the function of syt I with biophysical techniques. 2. The second aim is to characterize Ca 2+-regulated exocytosis in the knockout cell line. We will use capacitance and amperometric measurements, in conjunction with [Ca2+]i measurements, to measure exocytosis from patch-clamped single cells. Cells will be stimulated to elicit Ca2+-dependent secretion. From the analysis of Ca2+-influx and vesicle release, we will determine the Ca2+ dependency of secretion for the knockout cell line and compare it to secretion in wild-type cells. Our short-term goal is to establish a syt I homozygous knockout cell line that can be used to determine whether syt I is the Ca2+ sensor for rapid Ca2+-dependent secretion in these secretory cells. Our long-term goal is to knock out each of the syt isoforms in the mouse cell line, using different selection vectors, and then add each one back to the cell by stable transfection to study the role that different syt isoforms play in secretion.