We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and tet°minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 107 colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of _106 colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (>1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replicationcompetent virus. the VSV-G protein, since the constitutive expression of significant levels of VSV-G in most cells is toxic. However, this method of virus production significantly limits the evaluation of the potential applications of the viral pseudotypes, since only small amounts of virus can be easily produced. To overcome these difficulties, we have generated a stable humanderived cell line which constitutively expresses the necessary retroviral proteins for packaging and provides for large amounts of the VSV-G protein by inducible expression. We describe here the manner in which the cell line was constructed and some of the characteristics of the virus that is generated from the cells. MATERIALS AND METHODSCell Lines and Drug Selections. Adenovirus 5-transformed human embryonic kidney 293 cells (10) were obtained from B. Panning (Whitehead Institute). The 293 cells were grown in 293 growth medium containing Dulbecco's modified eagle medium (DMEM) (GIBCO/BRL), 10% (vol/vol) inactivated fetal bovine serum (IFS) (Sigma), 2 mM L-glutamine (GIBCO/BRL), and 50 units/ml penicillin and streptomycin (GIBCO/BRL). Drug selections in transfected 293 cells were performed at 2 ,ug/ml puromycin (Sigma), 0.3 mg/ml G418 (GIBCO/BRL) and 100 jig/ml Zeocin (Invitrogen). All growth media, except where noted, was supplemented with 1 ,ug/ml tetracycline. NIH 3T3 cells (ATCC CRL 1658) were grown in DMEM containing 10% (vol/vol) calf serum (Sigma), ...

Mutualistic interactions benefit both partners, promoting coexistence and genetic diversity. Spatial structure can promote cooperation, but spatial expansions may also make it hard for mutualistic partners to stay together, because genetic drift at the expansion front creates regions of low genetic and species diversity. To explore the antagonism between mutualism and genetic drift, we grew cross-feeding strains of the budding yeast Saccharomyces cerevisiae on agar surfaces as a model for mutualists undergoing spatial expansions. By supplying varying amounts of the exchanged nutrients, we tuned strength and symmetry of the mutualistic interaction. Strong mutualism suppresses genetic demixing during spatial expansions and thereby maintains diversity, but weak or asymmetric mutualism is overwhelmed by genetic drift even when mutualism is still beneficial, slowing growth and reducing diversity. Theoretical modeling using experimentally measured parameters predicts the size of demixed regions and how strong mutualism must be to survive a spatial expansion.evolution | invasion | snow-drift game | microbes | auxotroph

Mutualistic interactions benefit both partners, promoting coexistence and genetic diversity. Spatial structure can promote cooperation, but spatial expansions may also make it hard for mutualistic partners to stay together, because genetic drift at the expansion front creates regions of low genetic and species diversity. To explore the antagonism between mutualism and genetic drift, we grew cross-feeding strains of the budding yeast Saccharomyces cerevisiae on agar surfaces as a model for mutualists undergoing spatial expansions. By supplying varying amounts of the exchanged nutrients, we tuned strength and symmetry of the mutualistic interaction. Strong mutualism suppresses genetic demixing during spatial expansions and thereby maintains diversity, but weak or asymmetric mutualism is overwhelmed by genetic drift even when mutualism is still beneficial, slowing growth and reducing diversity. Theoretical modeling using experimentally measured parameters predicts the size of demixed regions and how strong mutualism must be to survive a spatial expansion.evolution | invasion | snow-drift game | microbes | auxotroph