summary of luciferase in mammalian cell replies to date.

David Adelson davead at prospect.anprod.csiro.au
Thu Jun 16 17:11:51 EST 1994


I was very gratified to receive the prompt replies I got.  In the spirit
of the newsgroup I am posting the direct replies I received.  Hope this
helps someone else as well.=20
  ________________________________________________________________________
  |  Dave Adelson                E-mail: davead at prospect.anprod.csiro.au |
  |  CSIRO Division of Animal Production  * "All opinions are my own"    |
  |  Locked Bag 1, Delivery Centre        * If you don't like my logic,  |
  |  Blacktown, 2148, AUSTRALIA           * use your own.                |
  |----------------------------------------------------------------------|


>From T.Vink at med.ruu.nl  Ukn Jun 10 18:12:23 1994
Return-Path: <T.Vink at med.ruu.nl>
Subject: in vivo luciferase detection
To: davead at prospect.anprod.csiro.au
Organization: Medical Faculty, Utrecht University, The Netherlands

Hello Dave,

I was just looking for this subject a few days ago and could only find=20
this reference Craig,F.F. et al. Biochem. J. 1991 , 276, 637-641.
This article descibes the synthesis of membrane permeable luciferin
esters which supported detection of expressed luciferse in cos cells in viv=
o.
The pictures look nice!! The authors were from Amersham, so I looked in the=
ir
catalogue but could not find anything about these compounds, so they are
probably not available yet. I am very interested in this subject for the
same purposes as you, so could you please keep me posted about any other
reactions on your request.

Tom Vink
Dep. of haematology
University Hospital Utrecht
Utrecht
The Netherlands
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From: "M.E. Couchman" <cou at leicester.ac.uk>
Subject: Re: luciferase in vivo in mammals?
=E0Date: Fri, 10 Jun 1994 09:32:18 +0100 (BST)
>=20
> Can anybody out there tell me if luciferase can be detected in vivo in
> mammalian cells or tissues? In cells this would be as a screen for
> expression and so would have to be non lethal as we want to pick the
> expressing cells and grow them up.

I don't know about mammals, but it has been done in adult fruit flies. I se=
e no
reason why it shouldn't work well in tissue cultures

M. Couchman
Leicester University
UK
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Date: Fri, 10 Jun 94 10:25:06 -0400
From: coady at ere.umontreal.ca (Michael Coady)
Subject: Re: luciferase in vivo in mammals?
Organization: Universite de Montreal

Dear Dave,
=09I looked into this a while back and found that there are
methods for introducing the substrates, but they're not that easy
and the substrate is a bit too pricey for me.  Sorry that I don't
have any references or prices to quote.
=09What I do have is a reference that you should read about
green fluorescent protein (Science, Vol 263, pp 802-805, 1994).
=09If this message looks screwy, it's because I just received a
control message about the system being about to go down.  Anyways,
check out green fluorescent protein, I use it for injections into
Xenopus oocyte nuclei and it works fairly well.  Write if you need
more details.

Mike


--=20
Michael J. Coady
COADY at ERE.UMONTREAL.CA
The opinions expressed above are solely those of the author and do not,
in any way, shape or form, represent the Universite de Montreal.

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Date: 10 Jun 1994 12:42:40 -0800
From: "bates" <dbates at hph.ucdavis.edu>
Subject: Luciferase
To: davead at prospect.anprod.csiro.au


Yes it can. For cells in culture, grow on coverslips (I use small round one=
s),
& add Promega Assay Reagent. Count in Liquid Scintillation Counter. For in
vivo, collagenase treat, homogenise gently to avoid breaking up the cells, =
and
add assay reagent. Works fine in Frogs, and I did it once on a rat.

Good luck, let me know if you get it to work. How are you transfecting in v=
ivo?

Cheers

Dave

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Date: Fri, 10 Jun 1994 15:07:56 -0500 (CDT)
From: Michael Benedik <BCHS1B at jetson.uh.edu>
Subject: Re: luciferase in vivo in mammals?


Yes.

firefly luciferase is what most people think of in the euk work because
it is a single gene product. However you need to break the cells open
to get in  the substrate.

Bacterial luciferase has 2 subunits and therefore is tricky to express
in eucaryotes. However a number of groups have made gene fusios
fusions of the two subunits, and these work in both proks and euks.
Decanol will diffuse in without requireing cell lysis and hence this
can be used in vivo. But for use in mammalian cells see
Pazzagli et al 1992 Analytical Biochemistry 204:315-323.


----------------------------------------------------------------------
 Michael Benedik=09=09=09=09INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences=09
 University of Houston=09=09=09=09BITNET: Benedik at uhou
-----------------------------------------------------------------------
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