Sigma-Aldrich Product Name Ampli-GPE, 95050229 biological source mouse embryo growth mode Adherent

Mouse NIH Swiss embryo, transfected with retroviral DNA

The retrovirus packaging Ampli-GPE (clone A57) cells were obtained from NIH3T3 fibroblasts (Sigma Catalogue number. 93061524) being co-transfected with two kinds of recombinant chimeric proviral genes in the BMG Neo vector. To reduce the homology between proviral DNA in the packaging cell and the retroviral vector, the gag-pol and env genes of Moloney murine leukaemia virus were separated onto two different plasmids in which the 5′ long terminal repeat (LTR) and the 3′ LTR had been replaced by the mouse metallothionein I promoter or the rabbit ß-globin gene. This expression and amplification system produces a viral titre high enough for gene transfer to somatic cells (up to 5x1,000,000 CFU/ml) and avoids the generation of replication-competent viruses.

Retrovirus packaging, gene transfer assays

DMEM + 2mM Glutamine + 250μg/ml G418 + 10% Foetal Bovine Serum (FBS).

Split sub-confluent cultures (70-80%) 1:2 to 1:6 i.e. seeding at 2-5x10,000 cells/cm² using 0.25% trypsin or trypsin/EDTA; 5% CO₂; 37°C.

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