Core--Gene expression

核心--基因表达

• 批准号: 6569894
• 负责人: SHELDON I FEINSTEIN
• 金额: $ 24.07万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-10 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6569894
• 关键词: affinity chromatography; biomedical facility; gene expression; lung; molecular biology; protein purification; site directed mutagenesis; tissue /cell culture; transfection; transfection /expression vector

The purpose of the Genre Expression Core, Core A is to facilitate the expression of genes of interest in eukaryotic cells and the production of proteins from these genes in E. coli. Most of the individual projects in this Program contain aims in which it is necessary or desirable to achieve efficient expression of a gene in vivo in lungs, in type II cells or in cell lines. The most efficient method of achieving this has been to place the construct in a replication-defective adenovirus, until recently, a very arduous and expensive task. Recently, methods have been developed which make this process more rapid although it is still relatively expensive and labor intensive. Core A will prepare adenoviral DNA using a recently developed recombinant DNA method which allows in vitro construction and DNA preparation in E. coli. The DNA will then be packaged into virus and amplified using HEK 293 cells supplied by the Cell Culture Core (Core D). Some of the projects in this Program will also need large-scale preparations of protein for analysis or antibody. Core A will prepare constructions for expressing tagged proteins and purify the expressed proteins using tags for affinity chromatography. In addition, some projects need to mutate particular amino acid residues. Core A will perform site directed mutagenesis and, if needed purification of the resulting proteins. Finally, Core A will supply molecular biology expertise in other area, for example library screening and use of antisense and decoy oligonucleotides to any project in the Project.

Genre Expression Core（Core A）的目的是促进目的基因在真核细胞中的表达，并在大肠杆菌中从这些基因产生蛋白质。杆菌该计划中的大多数单个项目都包含这样的目标，即必须或希望在肺、II型细胞或细胞系中实现基因的体内有效表达。实现这一点的最有效的方法是将构建体置于复制缺陷型腺病毒中，直到最近，这是一项非常艰巨和昂贵的任务。最近，已经开发了使该过程更快速的方法，尽管其仍然相对昂贵且劳动密集。核心A将使用最近开发的重组DNA方法制备腺病毒DNA，该方法允许在E.杆菌然后将DNA包装到病毒中，并使用细胞培养核心（核心D）提供的HEK 293细胞进行扩增。本计划中的一些项目还需要大规模制备分析用蛋白质或抗体。核心A将制备用于表达标记蛋白质的构建体，并使用亲和色谱标记纯化表达的蛋白质。此外，一些项目需要突变特定的氨基酸残基。核心A将进行定点诱变，并且如果需要，纯化所得蛋白质。最后，核心A将为项目中的任何项目提供其他领域的分子生物学专业知识，例如文库筛选和反义寡核苷酸和诱饵寡核苷酸的使用。