Cloning of retroviral insertion sites possibly involved in growth-factor autonomy

A protocol has been established which allows obtaining growth-factor-independent cell mutants by retroviral insertion mutagenesis in vitro. In order to identify gene alterations possibly leading to growth-factor independency, two techniques were established for the cloning of retroviral insertion sites. One technique makes use of the amplification of retroviral flanking fragments by inverse polymerase chain reaction (IPCR). The other strategy involves complementation of a truncated kanamycin gene present in a bacterial plasmid vector by a neomycin gene fragment originating from the retroviral vector, which allows direct selection for kanamycin resistant bacterial cell clones. Using these techniques flanking fragments with several putative genes have been obtained. One flanking fragment shows high homology to the rat ionotrophic glutamate receptor, i.e. GluR5.