Figure 2: Screening mechanism for isolation of genes involved in determining senescence bypass via ‘shuttle’ expression vector. In this scheme total RNA is extracted from cancer cells. 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 stably transfect selected terminally differentiated cells with limited in vitro replicative capacity. Individual transfectants are selected that are hygromycin resistant and display ankorage–dependent growth and multiple PDL (see text). DNA is isolated and restriction digested and labelled and hydridized against excess unlabelled vector DNA. Resultant labelled DNA is then used to dot-blot hydridize against E.coli clones from step 3 enabling unique inserts to be delineated. Control hydridization against vector alone should be negative. These clones can be confirmed as carrying immortalising genes by a second round of transfection into senescing cell line. Suitably isolated plasmid clones can be analyzed to determine the nature of the insert and proteins can be expressed and function assessed.