Nucleic acid-binding properties of human low density lipoprotein

描述（由申请人提供）：该项目的目标是探索和进一步开发一种基于天然低密度脂蛋白（LDL）的基因传递系统（AraVecTM），用于将广泛的遗传结构引入各种细胞类型。在这些试点研究中，我们将对LDL可用作基因传递载体的细胞类型进行调查。将从美国类型培养收集（ATCC）获得HeLa细胞、人成纤维细胞、人MCF7s、人HEP1、人HepG2细胞、人神经母细胞瘤、人肺和内皮细胞、人前列腺上皮细胞（LnCap）和中国仓鼠卵巢CHO细胞等多个细胞系。市售的含有绿色荧光蛋白（GFP）基因的质粒由人巨细胞病毒早期2 （HCMV IE2）启动子驱动，将用于这些研究中作为与天然人LDL的复合物。测定每种细胞类型LDL-DNA并入细胞的时间进程和GFP的表达。我们还将鉴定HCMV和丙型肝炎病毒基因组中与低密度脂蛋白高亲和力结合的片段。这个项目是为各种使人衰弱或致命的人类疾病制定成功的基因替代疗法和DNA疫苗的实验和临床方案的必要步骤。这些数据将用于未来的研究，在这些研究中，基因结构将被设计成与低密度脂蛋白的结合亲和力增加，并具有适当的细胞特异性表达。这些数据也将有助于阐明LDL除脂质转运外的体内功能。

DESCRIPTION (provided by applicant): 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.