Question:
I have recently stably-transfected a melanoma cell line with a ß-gal
reporter plasmid (pCR3-ß-gal). However, following 2 weeks of
G418-selection only one out of approximately 100 G418-resistant
colonies expressed ß-gal!
Since: (1) the G418 concentration was the minimal dose which killed
all untransfected cells in a 2 week assay and (2) transient
transfections performed at the same time (and same conditions) as the
stable transfections indicated that 10-20% of the cells expressed
ß-gal, I cannot understand why only 1% of the stably-transfected
colonies expressed ß-gal.
I'd be grateful if anyone has any comments or ideas?
Answer:
I do not know the exact composition of your vector but my guess is
that it has two promoters - one for G418-R, the second for b-gal. When
you select for G418 the other promoter gradually shuts down.
You may want to check papers by Proudfoot (early eighties, I think)
who did some elegant studies on this subject.
