Marcia,
You do not mention in your email how long you have been selecting these
cells with G418 and I assume your 60/40 split was achieved by viewing a
pool of cells. However, there is a fairly large amount of literature
which shows that even selected, "stable integrants" can lose the
expression of the gene eg GFP in your case, depending on the site of
integration, some clones faster than others (even using good old pIRES
Neo).
I'm not that au fait with macrophage culture but could you as an answer
sort a number of single cell clones, either directly on your cell sorter
if you have one or by dilution cloning and follow the expression of the
clones over three or four months? Those clones still expressing GFP at
that time will probably remain stable.
Hope that's helpful.
Simon
Simon QJ Rice
Gene Expression Sciences/Protein Biochemistry
GlaxoSmithKline R&D, UK.
mlh2207 <mlh2207@ksu.edu>
01-May-2002 18:49
To: "Cytometry Mailing List"
cc:
Subject: sorting GFP macrophages
Hello all. We have created GFP macrophages using the pIRESneo vector with
the
EGFP gene inserted into the MCS. The cells were then selected using G418.
Here's the problem...even with selection we have two distinct populations,
one
negative and one positive. We are now trying to sort the cells, but cannot
eliminate the negative population. The best sort results that we have
accomplished is 60% positive and 40% negative populations. Does anyone
have
any clues or suggestions on why this is happening and what we can do to
reduce/eliminate the negative population?
We had hopes of using these cells for in vivo studies.
Thanks,
Marcia
Marcia Hart
GRA
Division of Biology
Kansas State University
19 Ackert Hall
Manhattan, KS
Office:(785)532-6795
E-mail: mlh2207@ksu.edu
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