Figure 1: Novel ‘shuttle’ analysis for determination of cancer therapyresistance factors. In this scheme total RNA is extracted from cancer cells that are therapy resistant. This is then converted into cDNA and cloned into the shuttle expression vector, pECV25 (see text). Plasmids are isolated from individual bacterial clones and pooled into groups of 50 and used to transiently transfect the parent therapy-sensitive cancer cell line. Individual therapyresistant transfectants are selected and plasmid DNA extracted. This can then be transformed back into E. coli and DNA from selected clones repeat transfected into parent therapy-sensitive cancer cell lines in order to establish unique transfectants. Alternatively, plasmid DNAs extracted from the first-round of transfection into the parent cancer cells can be restriction digested and nick labelled and dot-blot hydridized against the E. coli colonies enabling unique inserts to be delineated – only after firstly hybridizing against excess unlabelled vector DNA. These clones can be confirmed as carrying therapy-resistance encoding sequences by the second round of transfection into the parent cell line. Suitably isolated plasmid clones can be analyzed to determine the nature of the insert and proteins can be expressed and function assessed.