Characterisation of a Retroviral Insertion Mutagenesis Protocol to Obtain Mutants with Activated Apoptosis Inhibitory Genes

Apoptosis or programmed cell death is a cell autonomous suicide pathway characterised by a reduction in cell volume, chromatin condensation and the activation of an endogenous endonuclease that digest DNA into oligonucleosome length fragments. The cellular machinery necessary for the execution of the death program is present in most cell type in an inactivated form (Raff, 1993) This state of inhibition is dependent on survival signals delivered by exogenous molecules like growth factors or adhesion molecules (Bates, 1994; Harrington, 1994). An increasing number of genes involved in the regulation of apoptosis have been described in the past few years, however the mechanisms responsible for the activation or inactivation of the death program remain poorly understood. The Bcl-2 gene family plays a key role in the regulation of the death program. Over-expression of the Bcl-2 family members such as Bcl-2 or Bcl-XL can inhibit apoptosis induced by removal of survival signals or delivery of a death signal (Oltwai, 1994). To understand the regulation of apoptosis, we would like to identify genes which are involved in the inhibition of apoptosis. These genes could be identified on the basis of homologies with known apoptosis inhibitors or because their product binds to apoptosis regulatory proteins. Alternatively, genes coding for proteins which do not share these properties could be identified by mutagenesis or “random” cDNA expression.