Autocrine stimulation after transfer of the granulocyte/macrophage colony-stimulating factor gene and autonomous growth are distinct but interdependent steps in the oncogenic pathway ( ABSTRACTAutocrine stimulation of cells by aberrant synthesis of growth factor may lead to malignant transformation, either as a direct consequence of endogenous factor production or as a first step of a series of successive events.

The use of retroviral vectors for gene transfer into animals has been severely hampered by the lack of provirus transcription in the early embryo and embryonic stem (ES) cells. This primary block in provirus expression is maintained in differentiated cells by acis-acting mechanism that is not well characterized. Retroviral vectors based on the murine embryonal stem cell virus (MESV), which overcome the transcriptional block in ES cells, were constructed to investigate this secondary mechanism. These vectors transferred G418 resistance to ES cells with the same efficiency as to fibroblasts, but overall transcript levels were greatly reduced. A mosaic but stable expression pattern was observed when single cells from G418-resistant clones were replated in G418 or assayed for expression of LacZ or interleukin-3. The expression levels in independent clones were variable and correlated inversely with methylation. However, a second, more pronounced, block to transcription was found upon differentiation induction. Differentiation of the infected ES cells to cells permissive for retroviral expression resulted in repression and complete extinction of provirus expression. Extinction was not accompanied by increased levels of methylation. Provirus expression is thus regulated by two independentcis-acting mechanisms: (i) partial repression in the undifferentiated state, accompanied by increased methylation but compatible with long-term, low expression of retroviral genes, and (ii) total repression and extinction during early stages of differentiation, apparently independent of changes in methylation. These results indicate a time window early during the transition from an undifferentiated to a differentiated stage in which provirus expression is silenced. The mechanisms are presently unknown, but elucidation of these events will have an important impact on vector development for targeting stem cells and for gene therapy.

A coculture system of a murine erythroblastic leukemia cell line mia which meets many of these requirements. 10,11 The leu-(ELM-D) with its supportive stromal cell line (MS-5) was estabkemic cells require contact with primary cultured stromal lay- unless indicated. Routinely, GM-CSF (5 U/ml), rGM-CSF