Core--Gene expression

类型表达核心，核心A的目的是促进真核细胞中感兴趣的基因的表达和这些基因在大肠杆菌中的蛋白质生产。该计划中的大多数单独项目都包含有必要或希望在肺、II型细胞或细胞系中实现基因在体内的有效表达的目标。实现这一目标的最有效方法是将构建体置于复制缺陷腺病毒中，直到最近，这是一项非常艰巨和昂贵的任务。最近，已经开发的方法使这一过程更加迅速，尽管它仍然相对昂贵和劳动密集。Core A将使用最近开发的重组DNA方法制备腺病毒DNA，该方法可以在大肠杆菌中进行体外构建和DNA制备。然后将DNA包装成病毒，并使用细胞培养核心（Core D）提供的HEK 293细胞进行扩增。本项目部分项目还需要大规模制备用于分析的蛋白质或抗体。Core A将制备表达标记蛋白的结构，并使用标签纯化表达蛋白，用于亲和层析。此外，一些项目需要突变特定的氨基酸残基。核心A将执行定点诱变，并在需要时纯化产生的蛋白质。最后，Core 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和诱饵寡核苷酸的使用。

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.