Selection for expression of the gene requires transcription from a cellular promoter, and consequently a mutation in a cellular gene, and the activity of the tagged gene can be followed by staining for b galactosidase activity. It is therefore designed to function when it is inserted in an intron.
Retroviruses insert as a single copy per locus, with no rearrangement of flanking sequences.
They have a preference for insertions at the 5' end of genes, often upstream of the initiator ATG, and the splice acceptor sequence we use does not appear to be bypassed. As a result, the majority of the mutations generated using our gene trap vectors are predicted to lead to null alleles. This has in fact been verified in all of the insertions we have analyzed to date at a molecular level. To identify and mutate growth factor or retinoid regulated genes, we have performed induction trapping by monitoring differential expression of the reporter gene, in ES cells or their differentiated derivatives. In some cases, such as ROSA49, we have replica plated ES cells infected with ROSA b gal and tested for differential lacZ activity upon exposure to exogenous factors.