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shRNAmir and CRISPR sgRNA Library Construction Core

shRNAmir 和 CRISPR sgRNA 文库构建核心

基本信息

批准号：
10024585
负责人：
Matthew Eugene Pipkin
金额：
$ 11.29万
依托单位：
LA JOLLA INSTITUTE FOR IMMUNOLOGY
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 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10024585
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