monitoring promotor activity in individual live cells?

Garry P. Nolan gnolan at cmgm.stanford.edu
Sat Sep 16 19:18:48 EST 1995


In article <1995Sep13.141522.6558 at wisipc.weizmann.ac.il>, David de Graaf
<bmgraaf at dapsas1.weizmann.ac.il> wrote:

> Dear netters, This is a more generalized version of a previous post. I am
> interested in one of the holy grails of molecular biology: a way that will
> allow me to closely follow promotor activity in intact live cells.
>

Check out an old PNAS paper on use of LacZ for single cell assay of gene
expression in living cells (Nolan et al, around 1987 I think, I can't
remember offhand).  It is fully quantitative and there is a kit available
from Molecular Probes, Eugene, Oregon that makes the reagent FDG.  DO NOT
BUY their lipophilic reagent.  It concentrates in lysozymes and you get
background activity.  I've used it very successfully for years.

 
> Green Fluorescent Protein seemed to be the answer until we - and many
> others
> that I have talked to - found that its exprssion was irregualr and not
> quantitative. Autofluorescence also posed a problem in FACS analysis (why
> has
> noone published FACS results since Chalfie mentioned it as a detection
> method
> in the original paper?). I hope this post will give a good discussion of
> available options.

We are actually using it for FACS regularly with retroviruses as the
expression vector.  Works fine.  Autofluorescence is easy to remove.  The
reference is in the PNAS article above (Nolan et al).  Basically it
involves an electronic subtraction protocol where yuou measure the
autofluorescence at another wavelength.  Since autofluorescence is pretty
proportional across wavelengths and comparable from cell to cell, you can
use another wavelength to "subtract" the autofluorescence from the
wavelength you are measuring on (530 or so for GFP or low level
fluorescein).  The original reference is Alberti et al, Cytometry.  Makes
apparently "negative" cell on FACS truly stand out from the background.

good luck

garry nolan

-- 
Garry P. Nolan, Ph.D.
Assistant Professor
Dept. of Molecular Pharmacology
Dept. of Microbiology and Immunology
Stanford Universoty School of Medicine
Stanford, CA 94305
gnolan at cmgm.stanford.edu



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