Retrotransposon gene engineering

We have used a mobile mouse VL30 genetic element together with retroviral helper cells to efficiently transmit and express chimeric foreign gene sequences in murine and human cells. The construct comprised a cDNA copy of retrotransposon NVL3, an internal promoter [rat cytosolic phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32)] and an expressed bacterial neomycin resistance gene. Thirty to sixty thousand colony forming units/ml (CFU/ml) were recovered from the supernatant of mass cultured psi2 helper cells transfected with the recombinant retrotransposon plasmid DNA. RNA was expressed from both the VL30 long terminal repeat and from the internal PEPCK promoter, resulting in a G418 drug resistance phenotype in recipient cells. Integrated VL30 DNA sequences transduced from psi2 or PA317 retroviral helper cells failed to regenerate detectable replication competent virus. Human and rodent recipient cells transduced by the retrotransposons appeared to bear intact vector sequences after two rounds of transmission by helper cells.