Author: Lin, Zhaoru; Gilbert, Robert J. C.; Brierley, Ian
Title: Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U(6)A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting Document date: 2012_6_28
ID: kjet3e50_13
Snippet: Frameshifting assays in tissue culture COS 7 cells were maintained in Dulbecco's modification of Eagle's medium supplemented with 10% (vol/vol) fetal calf serum, 1% (vol/vol) penicillin/streptomycin and 1% (wt/vol) 25 mM glutamine. Plasmids were transfected using a commercial liposome method (Lipofectamine 2000, Invitrogen). 4 Â 10 4 cells were seeded per well in 24-well plates and grown for 18-24 h until 80% confluency was reached. Transfection.....
Document: Frameshifting assays in tissue culture COS 7 cells were maintained in Dulbecco's modification of Eagle's medium supplemented with 10% (vol/vol) fetal calf serum, 1% (vol/vol) penicillin/streptomycin and 1% (wt/vol) 25 mM glutamine. Plasmids were transfected using a commercial liposome method (Lipofectamine 2000, Invitrogen). 4 Â 10 4 cells were seeded per well in 24-well plates and grown for 18-24 h until 80% confluency was reached. Transfection mixtures (containing plasmid DNA, serum-free medium [Optimem; Gibco-BRL] and Lipofectamine 2000) were set up as recommended by the manufacturer and added directly (dropwise) to the tissue culture cell growth medium. The cells were harvested 24 h post-transfection and reporter gene expression determined using a dual luciferase assay system kit (Promega). Each data point represents the mean value (± SEM) from six separate transfections.
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