In the electroporation (nucleofection) experiments, 1 - 2
Ü? 106 cells were resuspended in 100 μl of transfection
buffer (Table 1). The pmaxGFP plasmid (AMAXA Biosystems,
Gaithersburg, MD) that contains an enhanced
green fluorescent protein (EGFP) gene under regulation
of a cytomegalovirus (CMV) enhancer/promoter element
and is kanamycin resistant, was then added (2 μg/
transfection sample) to the cell suspension. The cell/
DNA mixtures, in 1 cm transfection cuvettes, were
nucleoporated according to a specific predefined program.
Following the electroporation, the cells were incubated
in their respective culture medium pre-heated to
37°C for 10 min, and then seeded into cell type-specific
growth medium. Unless otherwise indicated all nucleofection
experiments were carried out in triplicate using
3 separate dishes for each point.
The MESCs were separated from the SNL feeder cells by short-term (30 min) plating of the trypsinized mixed cell population in Petri dishes not coated with gelatin. The SNL fibroblasts preferentially adhere and the MESCs are readily harvested for nucleofection.
See Table 1 for lots of different cell types used in paper.