s.d.huen at bham.ac.uk
Tue Apr 26 10:46:19 EST 1994
> In article <6321.lyc at ibms.sinica.edu.tw_POPMail.PC_3.2.2>,
> lyc at IBMS.SINICA.EDU.TW ("L.Y.Chau") wrote:
> > Hi,
> > I am doing transfection exp. on undifferentiated and DMSO-induced
> > differentiated HL-60 cells. The B-gal activity driven by SV-40 promoter is
> > always 3-fold lower in differentiated HL-60. Here comes the question! Is
> > SV-40 promoter down-regulated in differentiated cells? If I need to
> > normalize my transfection data, is there any other promoter I can use which
> > is not regulated during differentiation?
> > Jonghwei
Try either a beta-actin promoter-driven or HCMV IE promoter-driven beta-Gal
promoter. Both are around and commonly used for normalisation. Dunno about
the beta-actin one but the CMV one may not be as constitutive as it is made
out to be (in literature. see Medline). Transfecting NF-kB p50/65 into
cells with this reporter drives it bananas but this is non-physiological to
say the least. OK most other times though it's OK in my hands.
Sounds as if you're using GL2-control in your case. I'd prefer something
stronger with the 50C heat-inactivation step to eliminate endogenous
beta-gal activity (cf. Anal Biochem last year. Works wonders in my
In article <leach-280394235604 at ppp-7-15.bu.edu>, leach at mbcrr.harvard.edu
(Martin Leach) wrote:
> BTW how can a promoter be down-regulated, do you mean the factors to drive
> it are not present in some cell-types!!! Maybe so.
There are repressors of expression operative as well so perhaps
'down-regulated' might well be appropriate. I think the early-to-late
switch in the SV40 promoter (SV40 promoter can theoretically be
bidirectional!!!) is governed by T antigen derepression at sites where
cellular factors bind within the promoter. The MMLV promoter is repressed
in EC cells by cellular factor(s) that go away on differentiation.
David Huen, Institute of Cancer Studies, Univ. of Birmingham, U.K.
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