shRNAmir and CRISPR sgRNA Library Construction Core

shRNAmir 和 CRISPR sgRNA 文库构建核心

基本信息

批准号：
10488582
负责人：
Matthew Eugene Pipkin
金额：
$ 14.01万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10488582
关键词：
B-Lymphocytes Biological CD8-Positive T-Lymphocytes CRISPR/Cas technology Cell Maintenance Cell Surface Receptors Cell physiology Cells Chromatin Cloning Clustered Regularly Interspaced Short Palindromic Repeats Complement Core Facility Custom DNA DNA Library Data Development Doctor of Philosophy Evolution Follow-Up Studies Gene Targeting Gene Transfer Generations Genes Goals Guide RNA Human Immune response Immune system In Vitro Individual Infection Laboratories Lentivirus Vector Libraries Mediating Memory Methods MicroRNAs Mus Phenotype Publishing RNA Interference Reagent Regulation Research Institute Retroviral Vector System T memory cell T-Lymphocyte Technology Time Tissues acute infection analysis pipeline base differential expression effector T cell experience flexibility gene function genome-wide in vivo innovation loss of function novel novel strategies plasmid DNA small hairpin RNA tool transcription factor tumor vector virtual

项目摘要

PROJECT SUMMARY / ABSTRACT Core B (Pipkin) Short hairpin RNAs in microRNA contexts (shRNAmirs) and CRISPR-Cas9 guide RNA (sgRNA) are powerful tools for experimental RNA interference (RNAi) and gene-disruption in the immune system, respectively. However, virtually all current approaches require pre-activation of naive cells to render them susceptible for gene transfer, typically with retroviral or lentiviral vectors, and cause constitutive RNAi or immediate gene-disruption. These issues complicate studying genes that function prior to retroviral delivery, and clarifying the temporal requirements of genes at different stages of immune responses. In Core B, we describe development of multiple innovative tools and methods that substantially expands the utility of shRNAmirs and sgRNAs to study gene function. We demonstrate generation of unmanipulated naive CD4 and CD8 T cells carrying conditionally regulated retroviral constructs that enable conducting inducible and reversible RNAi in vivo, and extend this system to a novel approach that facilitates large in vivo pooled screens using inducible shRNAmirs in untouched naive cells. We also demonstrate CRISPR-Cas9 guide RNA (sgRNA) gene disruption in primary B and T cells, and its use during in vivo immune responses to analyze B and T cell function. The combination of these shRNAmir and sgRNA approaches are synergistic and constitute an innovative and rigorous experimental platform for rapidly dissecting gene function in T and B cells during in vivo immune responses. Core B builds on multiple existing libraries of high-fidelity shRNAmir clones, which include those that target all genes encoding mammalian chromatin regulator factors (312 genes, ~1,700 shRNAmirs), and all mammalian transcription factors (2,083 genes, ~8,000 shRNAmirs). The overarching goal of Core B is to provide essential tools for executing the proposed in vivo loss-of-function studies proposed in Projects 1-3 of this P01. Our specific objectives are to sub- clone pooled shRNAmir libraries using an inducible vector for conditional pooled screens in naive T cells that are proposed in Projects 2 and 3 (Aim 1); to produce high-quality, ready-to-transfect retroviral plasmid DNA from cloned libraries of shRNAmirs and sgRNAs for Projects 1-3 (Aim 2); and to re-apply existing arrayed shRNAmir libraries and build custom shRNAmir and gRNA clone sets in appropriate vectors based on gene targets prioritized during the evolution of Projects 1-3 to facilitate detailed follow-up studies (Aim 3).

项目摘要 /摘要核B（pipkin） MicroRNA上下文（Shrnamirs）和CRISPR-CAS9指南RNA（SGRNA）中的短发夹RNA功能强大免疫系统中的实验RNA干扰（RNAi）和基因 - 局部的工具。但是，几乎所有当前的方法都需要对幼稚细胞进行预激活，以使其容易受到基因的影响转移，通常带有逆转录病毒或慢病毒载体，并引起本构的RNAi或立即基因 - 脱离。这些问题使研究基因在逆转录病毒之前起作用并澄清时间复杂免疫反应不同阶段基因的要求。在核心B中，我们描述了多个的发展创新的工具和方法基本扩展了Shrnamirs和SgrNA的实用性研究基因功能。我们证明了未经操纵的幼稚CD4和CD8 T细胞的产生调节的逆转录病毒构建体能够在体内进行可诱导和可逆的RNAi，并扩展采用新型方法的系统，该方法促进了使用诱导的shrnamirs在未触及的情况下促进大型体内合并屏幕天真的细胞。我们还展示了在原代B和T细胞中的CRISPR-CAS9引导RNA（SGRNA）基因破坏，及其在体内免疫反应中的使用以分析B和T细胞功能。这些的结合 Shrnamir和Sgrna方法是协同作用的，构成了创新和严格的实验在体内免疫反应过程中，用于快速剖析T和B细胞基因功能的平台。核心B建立在高保真shrnamir克隆的多个现有库，其中包括针对所有基因编码的库哺乳动物染色质调节剂因子（312个基因，〜1,700 Shrnamirs）和所有哺乳动物转录因子（2,083个基因，约8,000个Shrnamirs）。核心B的总体目标是提供执行的基本工具拟议的在本P01项目1-3中提出的体内功能丧失研究。我们的具体目标是克隆的shrnamir库使用可诱导载体在幼稚的T细胞中有条件合并的屏幕在项目2和3（目标1）中提出；为了产生高质量的，现成的逆转录病毒质粒DNA 来自Shrnamirs和SGRNA的克隆图书馆，用于1-3项目（AIM 2）；并重新应用现有的数组 Shrnamir图书馆并建立基于基因的适当向量中的自定义Shrnamir和Grna克隆集合在项目1-3的演变过程中，目标优先考虑，以促进详细的后续研究（AIM 3）。