A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.

Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic ␤-cell apoptosis and heart failure. Here, we demonstrate in cultured cells that the relative toxicity of two common dietary long chain fatty acids is related to channeling of these lipids to distinct cellular metabolic fates. Oleic acid supplementation leads to triglyceride accumulation and is well tolerated, whereas excess palmitic acid is poorly incorporated into triglyceride and causes apoptosis. Unsaturated fatty acids rescue palmitate-induced apoptosis by channeling palmitate into triglyceride pools and away from pathways leading to apoptosis. Moreover, in the setting of impaired triglyceride synthesis, oleate induces lipotoxicity. Our findings support a model of cellular lipid metabolism in which unsaturated fatty acids serve a protective function against lipotoxicity though promotion of triglyceride accumulation.

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), ...