CORE--VECTOR PRODUCTION

Vector Core将通过提供肿瘤逆转录病毒和慢病毒（基于HIV-1和SIV）载体的开发、制备和表征方面的专业知识来支持所有项目。基于鼠白血病病毒（MuLV）的肿瘤逆转录病毒载体将通过产生特异性载体生产细胞来生产。可获得基于不同包膜假型的多种包装细胞，包括嗜亲性、亲嗜性、巨猿白血病病毒（GALV）、水泡性口炎病毒G（VSV-G）蛋白和猫内源性病毒（RD 114）。在一些情况下，还将通过用载体和包装质粒瞬时转染293 T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术分析在适当的靶细胞中的GFP表达来确定。将通过Southern印迹分析对所有载体确认没有重排的准确载体基因组传递。 包装序列普遍存在于所有载体中，将使用标准测定法检测载体生产细胞中是否存在可复制逆转录病毒。核心还将开发和制备各种基于HIV-1和SIV的载体，这些载体采用293 T细胞/四质粒转染方法进行自失活（SIN）设计。具有几种不同内部启动子元件的载体是可用的。将使用真实的时间PCR对基于HIV-1和SIV的载体制备物进行滴定，以定量基因组传递至适当靶细胞中。可通过超滤和/或超离心浓缩载体制剂。使用1）ELISA评估传代靶细胞中gag抗原的表达，和2） 一种检测是否存在传递和表达的达特的测定法。在正在进行的和拟议的工作中，核心将表征和评估是否使用我们的慢病毒载体系统将非复制性gag序列传递到靶细胞中。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表达质粒。此外，开发基于HIV-1和SIV的预包装、包装和载体生产细胞将是核心的主要目标，以提供更高滴度和更安全的载体。

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.