Nucleic acid-binding properties of human low density lipoprotein

描述(申请人提供)：该项目的目标是探索和进一步开发一种基于低密度脂蛋白(LDL)的天然基因传递系统(AraVecTM)，用于将广泛的基因结构引入各种细胞类型。在这些初步研究中，我们将对可将低密度脂蛋白用作基因输送载体的细胞类型进行调查。将从美国典型培养库(ATCC)获得HeLa细胞、人成纤维细胞、人MCF7、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。商业上可获得的含有由人巨细胞病毒即刻早期2(HCMV IE2)启动子驱动的绿色荧光蛋白(GFP)基因的质粒将作为与天然人低密度脂蛋白的复合体用于这些研究。将确定每种细胞类型的低密度脂蛋白-DNA掺入细胞和GFP表达的时间进程。我们还将鉴定与低密度脂蛋白高亲和力结合的巨细胞病毒和丙型肝炎病毒基因组片段。该项目是为各种致衰性或致命性人类疾病开发成功的基因替代疗法和DNA疫苗的实验和临床方案中必要的一步。这些数据将用于未来的研究，在这些研究中，设计的基因结构与低密度脂蛋白具有更高的结合亲和力，并具有适当的细胞特异性表达。这些数据还将有助于阐明低密度脂蛋白在体内的功能，而不是脂类运输。 该项目预计将增加该机构以研究为基础的培训能力，旨在吸引拉美裔学生参与尖端生物技术研究。研究生和理科专业的学生将有机会参加国际和平研究所指导下的实验研究，在全国会议上展示他们的项目，并与人合著出版物。

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.