DNA/RNA Delivery Core

DNA/RNA 递送核心

• 批准号: 7677673
• 负责人: Irina Budunova
• 金额: $ 13.45万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7677673
• 关键词: Adopted; Area; Arts; Biology; Cell Culture Techniques; Cell Line; Cells; Complementary DNA; Consult; Consultations; DNA; Data; Databases; Educational workshop; Epithelial; Epithelial Cells; Gene Expression; Generations; Genes; Genome; In Vitro; Infection; Information Dissemination; Instruction; Methods; MicroRNAs; Modification; Newsletter; Oligonucleotides; Polymerase; Production; Proteins; RNA; RNA Interference; RNA Sequences; Research; Research Personnel; Resources; Retroviridae; Role; Series; Services; Small Interfering RNA; Subfamily lentivirinae; Techniques; Technology; Training; Universities; Viral; Virus; base; cost; design; gene discovery; gene function; innovation; interest; keratinocyte; member; overexpression; programs; promoter; research study; skin disorder; small hairpin RNA; technique development; tool; vector; web site

The ability to specifically express or delete single gene products within cells is a means of determining gene function. In contrast to cell lines, however, introduction of DNA or RNA into normal cultured keratinocytes is difficult. Viral delivery methods are currently the most effective means of long-term gene overexpression or silencing in keratinocytes in vitro. Recently, new methods have been developed to generate viruses for delivery of various types of RNA sequences such as small hairpin (sh-RNA) and microRNA (miRNA). Since retroviruses and lentiviruses have the ability to integrate into the cell genome, they have become the preferable tool for delivery of exogenous cDNAs and RNAs into keratinocytes. We have adopted, modified and developed a wide range of techniques to use lenti- and retro-viruses to infect different types of epithelial cells in vitro, including primary and transformed keratinocytes. We generated viral expression cassettes that incorporate polymerase type II or polymerase type III promoters necessary for expression of gene cDNAs and RNA sequences. The DNA/RNA Delivery Core will support NU SDRC researchers and attract new investigators in the area of keratinocyte biology by providing service and/or instruction about basic techniques for the development and use of siRNA/shRNA/miRNA and cDNA in experiments involving keratinocytes. This core will also provide NU SDRC researchers with assistance in the use of viral methods for infecting keratinocytes, primarily focusing on retro- and lentiviruses. The Core will offer the expertise and instruction needed for the successful gene overexpression/silencing proposed in the Pilot and Feasibility projects. Assistance in the selection of target sequences and design of primers as well as viral constructs were indicated to be of greatest general interest for the SDRC investigators, and thus is the major service of this Core. Helping SDRC members troubleshoot the steps in the generation of stably infected cell cultures is an important role for the Core. Dissemination of the information about changes in technology through the SDRC Core-directed Website and Newsletter, and the Enrichment Program's Workshop series will enable SDRC investigators to perform their experiments at the most advanced "state-of-the-art" level.

在细胞内特异性表达或删除单个基因产物的能力是决定基因功能的一种手段。 然而，与细胞系相反，将DNA或RNA导入正常培养的角质形成细胞是困难的。病毒 递送方法是目前在哺乳动物中长期基因过表达或沉默的最有效手段， 体外培养角质形成细胞。最近，已经开发了新的方法来产生用于递送各种类型的病毒。 RNA序列，如小发夹（sh-RNA）和microRNA（miRNA）。由于逆转录病毒和慢病毒具有 由于能够整合到细胞基因组中，它们已成为递送外源cDNA的优选工具， RNA进入角质形成细胞。我们已经采用，修改和开发了广泛的技术来使用lenti-和 逆转录病毒在体外感染不同类型的上皮细胞，包括原代和转化的角质形成细胞。我们 产生了病毒表达盒，其掺入了必要的聚合酶II型或聚合酶III型启动子， 用于表达基因cDNA和RNA序列。DNA/RNA交付核心将支持NU SDRC研究人员 并通过提供关于角质形成细胞生物学的基本知识的服务和/或指导 在涉及角质形成细胞的实验中开发和使用siRNA/shRNA/miRNA和cDNA的技术。 该核心还将为NU SDRC研究人员提供使用病毒方法感染角质形成细胞的帮助， 主要关注逆转录病毒和慢病毒。核心将提供所需的专业知识和指导， 在试点和可行性项目中提出的成功的基因过表达/沉默。协助挑选 靶序列和引物设计以及病毒构建体被认为是最普遍感兴趣的， SDRC调查员，因此是该核心的主要服务。帮助SDRC成员解决 产生稳定感染的细胞培养物是Core的重要作用。传播该散页 通过SDRC核心指导网站和新闻通讯，以及丰富计划的 研讨会系列将使SDRC研究人员能够在最先进的“最先进的”环境中进行实验 水平