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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.

基因修饰，如基因敲低、过表达或编辑，是最有效的解释方法