Core D GET iN

核心 D 获取

• 批准号: 10700045
• 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
• 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/10700045
• 关键词: 3-Dimensional; Archives; Biology; CRISPR interference; CRISPR-mediated transcriptional activation; Cell Line; Cell Reprogramming; Cells; Cellular Stress; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complementary DNA; Complex; Consultations; 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; bench-to-bedside translation; biobank; cell bank; constitutive expression; 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; nanolipoprotein particles; novel; nuclease; overexpression; promoter; protein complex; skin barrier; small hairpin RNA; targeted delivery; three dimensional cell culture; transcription factor; viral DNA

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.

基因修饰，如基因敲除、过表达或编辑，是阐明 基因功能，这有可能推进治疗干预。将遗传物质输送到 然而，众所周知，原代皮肤细胞是困难的，并且通过表皮屏障递送是一个巨大的挑战。 基因编辑、转导和纳米技术（GET iN）核心将应用于皮肤研究 最先进和创新的基因操作方法。其中包括CRISPR/Cas9技术 以及用于将DNA、RNA和其它生物分子递送到皮肤中的病毒和非病毒方法。具体地说， 核心将产生用于体外和体内基因过表达和沉默的高滴度病毒原液 应用.核心将提供在普遍存在或皮肤细胞特异性条件下组成型/诱导型表达的病毒。 启动子和病毒表达载体，用于广泛的特定应用。这些措施包括：i）转录 因子荧光素酶报告子; ii）细胞标记/追踪和活细胞成像; iii）原代皮肤细胞的永生化; iv）诱导多能干（iPS）细胞的产生;和核心将提供 在原代皮肤细胞中使用基于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（在组成型或诱导型启动子下），和非- 重组Cas9蛋白+RNA -蛋白复合物（RNPC）形式的合成gRNAS的病毒递送。 我们还将提供CRISPR应用（例如，CRISPRa和CRISPRi）使用核酸酶失活的Cas9（dCas 9） 来调节基因启动子的活性。我们将开发新型非病毒基因递送方法 在原代皮肤细胞、皮肤来源的细胞系和体内皮肤中的过表达/沉默。核心已经调整了 新型纳米峰探针电穿孔（NFPE），一种在西北大学开发的用于靶向 递送至单细胞，用于引入病毒、DNA、RNA、蛋白质、染料和药物。核心还将促进 使用球形取向的纳米载体（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和药物递送至细胞和皮肤。 SBDRC成员还将能够使用基因枪将DNA和RNA传递到皮肤细胞。我们将建立 档案和生物储存库，并提供有关皮肤细胞和皮肤的递送选择的咨询。具有宽 一系列定制的病毒载体和独特的试剂，新型CRISPR/Cas9服务，以及创新的 纳米技术/NFPE交付方法，该核心将为基因工程提供最先进的策略。 在2D和3D皮肤培养以及体内皮肤中的操作。我们的服务以及与其他 SBDRC和西北核心，将进一步加强皮肤生物学和疾病的研究在NU，并最终 促进从实验室到临床的研究转化。