Core 1: Viral Vector Core

核心1：病毒载体核心

基本信息

批准号：
10251305
负责人：
KRISTINE E YODER
金额：
$ 14.19万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-04-21 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10251305
关键词：
Binding Biological Assay CRISPR/Cas technology Cancer Model Cells Cloning Clustered Regularly Interspaced Short Palindromic Repeats Complement Consult Coupled Custom DNA Elementary Particles Engineering Enzyme-Linked Immunosorbent Assay Feline Immunodeficiency Virus Gene Expression Genes Genetic Engineering Genetic Recombination Genetic Transcription Genome Guide RNA HIV-1 Human T-lymphotropic virus 1 Individual Initiator Codon Integrase Laboratories Leadership Lentivirus Vector Luciferases Methodology Methods Modification Murine leukemia virus Mutation Neomycin Pathogenicity Phosphorylation Point Mutation Proteomics Proviruses Puromycin RNA Binding RNA-Directed DNA Polymerase Reagent Reproducibility Research Personnel Resources Response Elements Retroviral Vector Retroviridae Services Silent Mutation Spumavirus Taxes Time Transgenes Variant Vesicular stomatitis Indiana virus Viral Viral Genome Viral Vector Visual Work base cell type cost design expression vector fusion gene gene function genetic manipulation genome editing homologous recombination hygromycin A interest mutant novel overexpression particle prevent promoter prototype repaired selective expression skills small hairpin RNA success tool vector vector control

项目摘要

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.

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需的基本试剂 HTLV-1感染细胞的载体颗粒将用于过度表达或减少宿主、病毒或病毒的表达。新的融合基因可通过 shRNA 或 CRISPR/Cas9 来实现。用于降低表达的载体将与用于与野生型和功能性互补的载体相匹配具有沉默突变的突变基因会阻止shRNA或CRISPR识别慢病毒载体颗粒。还提供由 CRISPR/Cas9 载体诱导的荧光和/或选择基因。将修改 HTLV-1 基因组序列，整合酶缺陷的慢病毒载体将用于量化关闭。 The Core 将与各个 PPG 实验室合作开发所有 CRISPR/Cas9 载体的靶向效果。 The Core 还将用于修饰 HTLV-1 感染细胞的 CRISPR/Cas9 试剂和方法。开发一种新的载体，限制 Cas9 在空间和时间上的表达，使其具有转录活性 HTLV-1感染细胞并减少潜在的脱靶编辑的核心具体目标是：目标1提供。定制逆转录病毒载体，目标 2 设计、验证和优化 CRISPR/Cas9 载体，目标 3 开发诱导型、自限性 HTLV-1 CRISPR/Cas9 载体由 HTLV-1 CRISPR/Cas9 产生的慢病毒或逆转录病毒载体颗粒。 Core 将为该 PPG 的项目 1-3 提供服务。