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High titre stocks of replication-defective retroviral vectors are generally prepared by stable introduction of cloned proviraJ DNA into packaging cell lines that supply in-trans all the proteins necessary for viral assembly (1). However, different vectors are packaged with different efficiencies and the expression of transgenes and activity of heterologous internal promoter elements may vary widely, dependent upon poorly understood and often ill-defined features of recombinant proviral structure (2). Optimization and comparative screening of proviral structures by isolation and selection of stable producer cell clones is thus an extremely time consuming and labour intensive procedure. A more rapid and simpler alternative for the preparation of high titre retroviral stocks is the short-term production of virus via transient transfection of packaging cells. This approach often yields low viral titres due to the inefficiency of standard transfection procedure such as the use of CaPO4-DNA precipitates (3,4). However, methods have been recently described for the transient production of high titre retroviral stocks using CaPO4-DNA precipitates and Cos cells or other SV40 large T antigen expressing cells (5,6). Here we describe a simple method using high efficiency Lipofectamine-mediated gene transfer to transiently and reproducably prepare recombinant retroviral vectors at titres in excess of 10 cfu/ml. Cultures of the ecotropic packaging cell line Ampli-GPE (7) were seeded in 60 mm dishes (1.3 X 10, cells/cm) and 24 hours later cells were transfected by exposure to Lipofectamine-DNA complexes prepared according to the manufacturer (Gibco BRL, Paisley, Scotland).

Production of high titrehelper-freerecombinant retroviral vectors by lipofection