Core--Gene expression

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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.