Beta-gal expression
Graham Smith (prsgs@bath.ac.uk)
Wed, 3 May 1995 15:36:18 +0100 (BST)
We have set up a system using FACS-gal in B16 murine melanoma witha long
term aim of setting up a drug delivery system. By FACS gal we
consistently get a 10-15% transfection but we also see a shift in the
negative peak. We cannot tell whether these really express the gene as
the cells are pigmented and X-gal staining would be masked by the brown
colour of the cells.
The negative control for this assay is a sample not treated with
exogenous DNA, but otherwise permeabilised and treated exactly the same
as the test. We see a two fold increase in fluorescence in the negatives
compared to a 10 fold increase in the definite positives.
We suspect that the dim cells are not positive because we think we would
see a continuum in the level of fluorescence, whereas we really see two
distinct peaks.
We have two questions:
1. Does anyone have experience of this assay to be able to comment on the
system used with regard to methods etc?
2. Can anyone comment on how much difference it would make if the
negative control was carried out with empty vector? In the context of
our department, cutting the insert out is impractical. We could maybe
use calf thymus DNA as a control.
Thanks in advance
Graham
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