The molecular cloning, cellular expression, and functional study of human caveolin-1.

Abstract

This thesis will discuss the molecular cloning, cellular expression, and functional study of human caveolin-1. This 21 kDa inter-membrane protein is found in cell surface invaginations termed caveolae (Gk: little caves). The expression of caveolin-1 has been shown to be sufficient to form caveolae in cell culture. Caveolin-1 was cloned from human adipose tissue by RT-PCR, sequenced and subcloned into vectors for its eukaryotic over-expression. The eukaryotic expression vectors contained either myc or myc and poly-histidine epitopes to assist in purification. Vectors containing antisense caveolin-1 and a ribozyme targeting caveolin-1 were also created for the targeted disruption of caveolin-1. Caveolin-1 cDNA was transfected into McA-RH7777 rat hepatoma cells, human fibroblasts, human umbilical vein endothelial cells (HUVEC), and Cos-7 cells to evaluate candidate cell lines for stable expression models. Stable cell lines over expressing caveolin-1 were generated in McA-RH7777 and HUVEC models. Confluent HUVEC cells that display cobblestone monolayer morphology were used for the study of transcellular trafficking on Falcon 3-D cell culture inserts. We studied the effect of caveolin-1 expression on cellular cholesterol transport and efflux. (Abstract shortened by UMI.)