Nucleic acid-binding properties of human low density lipoprotein

描述（由申请人提供）：本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）的基因递送系统（AraVecTM），用于将广泛的遗传构建体引入各种细胞类型。在这些初步研究中，我们将对LDL可用作基因递送载体的细胞类型进行调查。将从美国典型培养物保藏中心（ATCC）获得许多细胞系，包括HeLa细胞、人成纤维细胞、人MCF 7、人HEP 1、人HepG 2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。含有由人巨细胞病毒立即早期2（HCMV IE 2）启动子驱动的绿色荧光蛋白（GFP）基因的市售质粒将作为与天然人LDL的复合物用于这些研究。将确定每种细胞类型的LDL-DNA掺入细胞和GFP表达的时间过程。我们还将鉴定HCMV和丙型肝炎病毒基因组的片段，其以高亲和力结合LDL。该项目是为成功的基因替代疗法和DNA疫苗开发实验和临床协议的必要步骤，用于各种使人衰弱或致命的人类疾病。这些数据将用于未来的研究，其中遗传构建体被设计为具有增加的对LDL的结合亲和力和适当的细胞特异性表达。这些数据也将有助于阐明LDL在体内的功能，而不是脂质转运。 该项目预计将提高该机构基于研究的培训能力，旨在吸引西班牙裔学生参与尖端生物技术研究。研究生和科学专业的学生将有机会在PI的监督下参与实验研究，在全国会议上展示他们的项目，并共同撰写出版物。

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.