Nucleic acid-binding properties of human low density lipoprotein

Guevara, Natalia

Abstract

The goal of this project is to explore and further develop a native low density lipoprotein (LDL)-based gene delivery system (AraVecTM) for the introduction of a broad range genetic constructs into various cell types. In these pilot studies we will perform a survey of cell types for which LDL can be used as a gene delivery vehicle. A number of cell lines, including HeLa cells, human fibroblasts, human MCF7s, human HEP1, human HepG2 cells, human neuroblastomas, human lung and endothelial cells, human prostate epithelial cells (LnCap), and Chinese hamster ovary CHO cell, will be obtained from American Type Culture Collection (ATCC). Commercially available plasmids containing green fluorescent protein (GFP) gene driven by human cytomegalovirus immediate early 2 (HCMV IE2) promoter will be used in these studies as a complex with native human LDL. Time course of LDL-DNA incorporation into cells and GFP expression will be determined for each cell type. We will also identify fragments of HCMV and Hepatitis C viral genomes that bind to LDL with high affinity. This project is a necessary step in developing experimental and clinical protocols for successful gene replacement therapies and DNA vaccines for a variety of debilitating or lethal human diseases. The data will be used in future studies where genetic constructs are designed with increased binding affinity to LDL and appropriate cell-specific expression. The data will also help to clarify in vivo functions of LDL other than lipid transport. ? ? The project is expected to increase research-based training capacities of the institution, and is aimed at engaging Hispanic students in cutting-edge biotechnology research. Graduate students and science majors will have an opportunity to participate in experimental studies under the PI's supervision, present their projects at national conferences, and co-author publications. ? ? ?