Hartmut Land

We report the development of an advanced system for transfer and expression of exogenous genes in mammalian cells based on Moloney murine leukemia virus (Mo MuLV). Extensive deletion/mutagenesis analysis to identify cis-acting signals involved in virus transmission has led to the design of a family of novel, highly efficient retroviral vectors and a partner helper-free packaging cell line. The pBabe retroviral vector constructs transmit inserted genes at high titres and express them from the Mo MuLV Long Terminal Repeat (LTR). Each of these vectors has been constructed with one of four different dominantly acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin, respectively. The high titre ecotropic helper free packaging cell line, omega E, was designed in conjunction with the pBabe vectors to reduce the risk of generation of wild type Mo MuLV via homologous recombination events. The omega E cell line was generated with separate gagpol and ecotropic env expression constructs with minimal sequence overlap and decreased sequence homology achieved by 'codon wobbling'. Homologous env coding sequences were deleted from the pBabe vectors without diminishing recombinant vector titre. Together, the pBabe vectors and omega E cell line should prove useful in experiments where highest frequencies of gene transfer, or concomitant expression of several different genes within a single cell are required with minimal risk of helper virus contamination.

Two dozen cellular proto-oncogenes have been discovered to date through the study of retroviruses and the use of gene transfer. They form a structurally and functionally heterogeneous group. At least five distinct mechanisms are responsible for their conversion to active oncogenes. Recent work provides experimental strategies by which many of these oncogenes, as well as oncogenes of DNA tumor viruses, may be placed into functional categories. These procedures may lead to definition of a small number of common pathways through which the various oncogenes act to transform cells.