Core D GET iN

核心 D 获取

基本信息

批准号：
10259800
负责人：
Irina Budunova
金额：
$ 12.13万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-20 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10259800
关键词：
3-Dimensional Archives Biology CRISPR interference Cell Line Cells Cellular Stress Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complementary DNA Complex Consultations Custom Cutaneous DNA DNA delivery Disease Drug Delivery Systems Dyes Education Electroporation Epidermis Epigenetic Process Gene Expression Gene Silencing Generations Genes Genetic Genetic Materials Genome Goals Guide RNA High Density Lipoproteins Human In Vitro Infectious Skin Diseases Injections Label Libraries Luciferases Maps Mediating Methods MicroRNAs Microfluidic Microchips Modification Mus Nanotechnology Organ Organism Pharmaceutical Preparations Proteins RNA RNA delivery Reagent Recombinants Reporter Research Resources Ribonucleoproteins Scientist Service provision Services Skin Small Interfering RNA Spherical Nucleic Acids System Technology Therapeutic Intervention Time Translational Research Universities Viral Viral Vector Virus base bench to bedside biobank cell bank expression vector gene function gene gun genetic approach genetic manipulation genetically modified cells in vivo in vivo imaging induced pluripotent stem cell inducible gene expression innovation knock-down live cell imaging member nanocarrier nanoparticle novel nuclease overexpression promoter protein complex skin barrier small hairpin RNA targeted delivery three dimensional cell culture transcription factor viral DNA

项目摘要

Genetic modification, such as gene knockdown, overexpression or editing, is the most effective way to elucidate gene function, which has the potential to advance therapeutic intervention. The delivery of genetic material into primary skin cells, however, is notoriously difficult and delivery through the epidermal barrier is a great challenge. The Gene Editing, Transduction, and Nanotechnology (GET iN) Core will apply to cutaneous research the most advanced and innovative approaches for genetic manipulation. These include CRISPR/Cas9 technology and both viral and non-viral methods for the delivery into skin of DNA, RNA, and other biomolecules. Specifically, the Core will generate high titer viral stocks for gene overexpression and silencing for in vitro and in vivo applications. The Core will offer viruses for constitutive/inducible expression under ubiquitous or skin cell-specific promoters and viral expression vectors for a broad range of specific applications. These include: i) transcription factor luciferase reporters; ii) cell labeling/tracking and live cell imaging; iii) immortalization of primary skin cells; iv) generation of induced pluripotent stem (iPS) cells; and v) iPS cell reprogramming. The Core will provide reagents for DNA editing and epigenetic modifications using CRISPR-based technologies in primary skin cells and cell lines, using both viral delivery of Cas9 and gRNAs (under constitutive or inducible promoters), and non- viral delivery of recombinant Cas9 protein + synthetic gRNAS in the form of RNA – protein complexes (RNPC). We will also offer CRISPR applications (e.g., CRISPRa and CRISPRi) using nuclease-deactivated Cas9 (dCas9) to regulate gene promoter activity. We will develop novel non-viral delivery methods for gene overexpression/silencing in primary skin cells, skin-derived cell lines and skin in vivo. The Core has adapted the novel Nanofountain Probe Electroporation (NFPE), a micro-fluidic device developed at Northwestern for targeted delivery to single cells, for introducing viruses, DNA, RNA, proteins, dyes, and drugs. The Core will also facilitate the use of spherically oriented nanocarriers (e.g., spherical nucleic acids and high density lipoprotein nanoparticles) for intracellular and transcutaneous DNA, RNA and drug delivery to cells and skin, respectively. SBDRC members will also be able to deliver DNA and RNA to skin cells using the Gene Gun. We will establish archives and biorepositories, and offer consultation on delivery options into skin cells and skin. Having a wide range of customized viral vectors and unique reagents, novel CRISPR/Cas9 services, and innovative nanotechnology/NFPE delivery approaches, this Core will provide the most advanced tactics for genetic manipulation in 2D and 3D skin cultures, as well as skin in vivo. Our services, and collaboration with other SBDRC and Northwestern Cores, will further enhance skin biology and diseases research at NU, and ultimately facilitate the bench to bedside translation of research.

遗传修饰，例如基因敲低，过表达或编辑，是阐明的最有效方法基因功能，具有推进治疗干预的潜力。将遗传物质传递到然而，众所周知，原发性皮肤细胞很难，并且通过表皮屏障传递是一个巨大的挑战。基因编辑，转导和纳米技术（进入）将适用于皮肤研究遗传操作的最先进和创新方法。这些包括CRISPR/CAS9技术以及将递送到DNA，RNA和其他生物分子的皮肤的病毒和非病毒方法。具体来说，核心将产生高滴度病毒库存，用于基因过表达和沉默的体外和体内沉默申请。该核心将在无处不在或皮肤细胞特异性下提供构成性/可诱导表达的病毒启动子和病毒表达向量，用于广泛的特定应用。这些包括：i）转录因素荧光素酶报告者； ii）细胞标记/跟踪和活细胞成像； iii）原代皮细胞的免疫化； iv）产生诱导多能茎（IPS）细胞；和v）IPS细胞重编程。核心将提供使用基于CRISPR的技术在原代皮细胞中使用基于CRISPR的技术进行DNA编辑和表观遗传修饰的试剂和细胞系，使用CAS9和GRNA的病毒递送（在构成或诱导型启动子下）以及非 - 以RNA - 蛋白质复合物（RNPC）形式的重组Cas9蛋白 +合成GRNA的病毒递送。我们还将使用核酸酶量化的CAS9（DCAS9）提供CRISPR应用（例如CRISPRA和CRISPRI）调节基因启动子活性。我们将开发新的非病毒递送方法的基因原代皮肤细胞，皮肤衍生细胞系和体内皮肤的过表达/沉默。核心已经适应了新型纳米探测器电穿孔（NFPE），这是一种在西北开发的微荧光装置传递到单个细胞，用于引入病毒，DNA，RNA，蛋白质，染料和药物。核心也将有助于使用面向球体的纳米载体（例如球形核酸和高密度脂蛋白纳米颗粒）分别用于细胞内和经皮DNA，RNA和药物分别递送至细胞和皮肤。 SBDRC成员还将能够使用基因枪向皮肤细胞传递DNA和RNA。我们将建立档案和生物库，并就皮肤细胞和皮肤的递送选择进行咨询。有宽阔的地方定制的病毒矢量和独特的试剂，新颖的CRISPR/CAS9服务以及创新的范围纳米技术/NFPE交付方法，该核心将为通用提供最先进的策略 2D和3D皮肤培养物以及体内皮肤的操作。我们的服务，并与其他 SBDRC和西北核心将进一步增强NU的皮肤生物学和疾病研究，最终将促进长凳到研究的床边翻译。