Core--Gene expression

该类型表达的目的是促进在真核细胞中表达感兴趣的基因，并在大肠杆菌中从这些基因生产蛋白质。该计划中的大多数单独项目都包含这样的目标，即在肺部、II型细胞或细胞系中实现基因在体内的有效表达是必要的或可取的。实现这一目标的最有效方法一直是将该构建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，已经开发了一些方法，使这一过程更快，尽管它仍然相对昂贵和劳动密集型。Core A将使用最近开发的一种重组DNA方法来制备腺病毒DNA，这种方法允许在体外构建和在大肠杆菌中制备DNA。然后，DNA将被包装成病毒，并使用由细胞培养核心(核心D)提供的HEK 293细胞进行扩增。该计划中的一些项目还将需要大规模的蛋白质制剂来进行分析或抗体。核心A将准备表达标签蛋白的结构，并使用用于亲和层析的标签纯化表达的蛋白质。此外，一些项目需要突变特定的氨基酸残基。核心A将进行定点突变，如果需要，还将对所产生的蛋白质进行纯化。最后，核心A将提供其他领域的分子生物学专业知识，例如，为项目中的任何项目提供文库筛选和反义和诱骗寡核苷酸的使用。

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.