Core 1: Viral Vector Core

项目摘要--核心1 病毒载体核心(核心1)将为所有PPG项目提供定制的慢病毒或逆转录病毒载体。 慢病毒或逆转录病毒载体颗粒是高效基因工程所必需的基本试剂。 HTLV-1感染细胞。载体颗粒将被用来过度表达或降低宿主、病毒或 新的融合基因。通过递送shRNA或CRISPR/Cas9将实现表达减少。这个 将降低表达的载体与野生型和功能性互补的载体进行匹配 具有沉默突变的突变基因，以阻止shRNA或CRISPR识别。慢病毒载体颗粒将 还提供荧光和/或选择基因。CRISPR/Cas9载体诱导的同源定向修复 将修改HTLV-1基因组序列。整合酶缺陷型慢病毒载体将被用来量化Off-Off 所有CRISPR/Cas9载体的目标效果。Core将与各个PPG实验室合作开发 用于HTLV-1感染细胞修饰的CRISPR/CAS9试剂和方法学。核心还将 开发一种新型载体，将Cas9在空间和时间上的表达限制为转录活性 HTLV-1感染细胞，并减少潜在的非靶标编辑。核心的具体目标是：目标1提供 定制逆转录病毒载体，目标2用于设计、验证和优化CRISPR/Cas9载体，目标3用于开发 可诱导、自限的HTLV-1 CRISPR/Cas9载体。慢病毒或逆转录病毒载体颗粒由 核心将服务于这个PPG的1-3个项目。

PROJECT SUMMARY – CORE 1 The Viral Vector Core (Core 1) will provide customized lentiviral or retroviral vectors to all PPG projects. Lentiviral or retroviral vector particles are fundamental reagents necessary for the efficient genetic engineering of HTLV-1 infected cells. The vector particles will be used to overexpress or reduce expression of host, viral, or novel fusion genes. Reduced expression will be achieved by delivery of either shRNA or CRISPR/Cas9. The vectors for reduced expression will be matched with vectors for complementation with wild type and functional mutant genes with silent mutations to prevent shRNA or CRISPR recognition. Lentiviral vector particles will also provide fluorescent and/or selection genes. Homology directed repair induced by a CRISPR/Cas9 vector will modify the HTLV-1 genome sequence. Integrase defective lentiviral vectors will be used to quantify the off- target effects of all CRISPR/Cas9 vectors. The Core will work with individual PPG labs to develop CRISPR/Cas9 reagents and methodologies for modification of HTLV-1 infected cells. The Core will also develop a novel vector that limits Cas9 expression both spatially and temporally to transcriptionally active HTLV-1 infected cells and reduce potential off-target editing. Specific aims of the core are: Aim 1 to provide custom retroviral vectors, Aim 2 to design, validate, and optimize CRISPR/Cas9 vectors, Aim 3 to develop an inducible, self-limiting HTLV-1 CRISPR/Cas9 vector. The lentiviral or retroviral vector particles produced by the Core will service Projects 1-3 of this PPG.