Live Bgal Detection: SUMMARY

Richard S Sucgang rss19 at cunixa.cc.columbia.edu
Thu Dec 17 21:08:44 EST 1992


A while back, I posted a request for a method for detecting a reporter
gene, like beta galatosidase, in situ, without killing the cells, or,
alternatively, a way to preferentially kill the cells that carry the
reporter. I received quite a few responses, which I have summarized here:

A. Fluorescent detection of Beta Galactosidase

This was recommended the most often, by quite a few people. A substrate
called Imagene is available from Molecular Probes (503-344-3007), which
reportedly is cleaved to form a fluorescent product. It is loaded into
cells by hypotonic shock or scrape loading. In intended to use this in
Dictyostelium cells, but David Knecht reports low rates of success
getting the substrate into the cells.

credits to:  
Jing Huang  (pnj at stein.u.washington.edu) (he called it FDG)
Jim Garbern (provided the ff references: 
             PNAS 85:2603 (1988) 
             Science 215:815 (1991)
             Genes and Development 6:591 (1992)).
David R. Micklem  (drm21 at mbuk.bio.cam.ac.uk, provided the ff references:
             Fiering et al, Cytometry 12:291-301,1991 
             Nolan et al PNAS USA 85:2603-2607,1988)
Frank Kolakowski  (lfk at eastman1.mit.edu)
Thomas A. Pressley (pressley at oac.hsc.uth.tmc.edu)

B. Other answers

Rich Buckholz (RGB12955%USA.decnet at usav01.glaxo.com) suggests the use of
secreted placental alkaline phosphatase as a reporter, since it can be
assayed outside the cell. Problem: most cells have a kind of endogenous
alkaline phosphatase, most membrane bound or secreted. No dice here. 

Shuomo (sbhattac at mrc-crc.ac.uk) recommends using the human growth hormone
expression system, where the HgH gets secreted, and is assayable by
antibody. Not very in situ, though. Still, thanks. 

Martin Kennedy (CYTOGEN at chmeds.ac.nz) addressed the issue of the suicide
selection: 

"The Diptheria toxin subunit A gene has been used to selectively kill
tissues in transgenic mice by hooking it up to tissue specific promoters. 
It has also been used in positive - negative selection protocols for ES
cells...The Herpes virus thymidine kinase gene is also very useful. 
Cells carrying it are sensitive to the antiviral agent gangcyclovir, and
so you can selectively kill any cells expressing the HSV tk gene."

Borrelli, E. et al. (1988).  Targeting of an inducible toxic phenotype
in animal cells.  PNAS 85, 7572-7576  

Bernstein, A., and Breitman, M. (1989).  Genetic ablation in transgenic
mice. Mol. Biol Med. 6, 523-530  (a good review of the area).

Palmiter, R.D., et al.  (1987)  Cell lineage ablation in transgenic mice
by cell specific expressio of a toxin gene.  Cell 50, 435-443

Maxwell, F., et al. (1987)  Cloning....and expression of Diptheria toxin
A gene  Mol. Cell. Biol. 7, 1576-1579.

Breitman, M. et al. (1990).  Genetic ablation in transgenic mice with an 
attenuated diptheria toxin A gene.  Mol. Cell. Biol. 10, 474-479

Caillet-Fauquet et al. (1990)  EMBO J. 9, 2989-2995

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To everyone who responded, many many thanks. For a limited time, reprints
of this summary are available by e-mail.

-rich
rss19 at cunixa.cc.columbia.edu
"I *LIKE* peanut butter! "



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