CORE--VECTOR PRODUCTION

核心——矢量制作

• 批准号: 6967760
• 负责人: DEREK A PERSONS
• 金额: $ 21万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967760
• 关键词: Lentivirus; Retroviridae; biomedical facility; biotechnology; enzyme linked immunosorbent assay; flow cytometry; gene expression; gene therapy; green fluorescent proteins; sickle cell anemia; southern blotting; tissue /cell culture; transfection /expression vector; virus genetics

The Vector Core will support all projects by providing expertise in the development, preparation, and characterization of oncoretroviral and lentiviral (HIV-1- and SIV-based) vectors. Murine leukemia virus (MuLV)-based oncoretroviral vectors will be produced through the generation of specific vector producer cells. A wide variety of packaging cells based on different envelope pseudotypes, including amphotropic, ecotropic, gibbon ape leukemia virus (GALV), vesicular stomatitic virus G (VSV-G) protein, and feline endogenous virus (RD114) are available, In some instances, vectors will also be generated through transient transfection of 293T cells with vector and packaging plasmids. Titers of vectors encoding the GFP marker will be determined by flow cytometric analysis for GFP expression in appropriate target cells, Accurate vector genome transmission without rearrangement will be confirmed by Southern blot analysis for all vectors, Vectors lacking GFP will be titered by Southem blot analysis using a probe derived from the packaging sequences which are universally present in all vectors, Vector producer cells will be tested for the presence of replication competent retrovirus using standard assays. The Core will also develop and prepare a variety of both HIV-1- and SIV-based vectors of self-inactivating (SIN) design using a 293T cell/four plasmid transfection method. Vectors with several different internal promoter elements are available. HIV-1- and SIV-based vector preparations will be titered using real time PCR for quantitiation of genome transmission into appropriate target cells. Concentration of vector preparations by both ultrafiltration and/or ultracentrifugation is available. Vector preparations are tested for the presence of replication competent lentivirus (RCL) using 1) an ELISA to assess the expression of gag antigen in passaged target cells and 2) an assay to test for the presence of transmitted and expressed tat. In ongoing and proposed work, the Core will characterize and evaluate whether non-replicative gag sequences are transmitted into target cells using our lentiviral vector systems. If needed, we will develop and test strategies to diminish this process, including using a codon-optimized gagpol expression plasmid. Additionally, the development of both HIV-1- and SIV-based pre-packaging, packaging, and vector producer cells will be a major goal of the Core in order to provide both higher titer and safer vectors.

病媒核心将通过提供开发、制备和表征肿瘤逆转录病毒和慢病毒(基于HIV-1和SIV)病媒的专业知识来支持所有项目。基于小鼠白血病病毒(MuLV)的肿瘤逆转录病毒载体将通过产生特异的载体产生细胞来产生。基于不同包膜伪型的包装细胞有很多，包括两性、生态、长臂猿白血病病毒(GALV)、水泡性口炎病毒G(VSV-G)蛋白和猫内源性病毒(RD114)，在某些情况下，也可以通过用载体和包装质粒瞬时转染293T细胞来产生载体。编码GFP标记的载体的滴度将通过流式细胞仪分析GFP在适当的靶细胞中的表达来确定，所有载体的准确的无重排的载体基因组传输将通过Southern印迹分析来确定，缺乏GFP的载体将通过Souome杂交分析来滴定 对于所有载体中普遍存在的包装序列，将使用标准检测方法检测载体生产细胞中是否存在具有复制能力的逆转录病毒。Core还将开发和准备各种基于HIV-1和SIV的载体，采用293T细胞/四质粒转染法进行自灭活(SIN)设计。具有几个不同内部启动子元件的载体是可用的。基于HIV-1和SIV的载体制剂将使用实时聚合酶链式反应进行滴定，以定量基因组向适当目标细胞的传播。通过超滤和/或超速离心法浓缩载体制剂是可行的。载体制剂检测复制能力慢病毒(RCL)的存在，使用1)ELISA来评估Gag抗原在传代的靶细胞中的表达；2) 一种检测是否存在传递和表达的TAT的化验方法。在正在进行的和拟议的工作中，核心将表征和评估非复制性GAG序列是否使用我们的慢病毒载体系统传输到靶细胞。如果需要，我们将开发和测试减少这一过程的策略，包括使用密码子优化的gagpol表达载体。此外，开发基于HIV-1和SIV的预包装、包装和媒介产生细胞将是Core的主要目标，以便提供更高滴度和更安全的媒介。