steffen@merlin.bcm.tmc.edu (David Steffen) (08/30/90)
Approximately 2 weeks ago, I posted two requests for help with the
promise that I would summarize the responses.
=== Transfecting/Cloning HL60 cells ===
The first request was for suggestions as to how we could
successfully isolate stable transfectants of HL60 cells ( a cell line
established from Human promyelocytic leukemia cells). The difficulty
we were having was an inability to clone these cells. I received no
responses to this request.
However, we have made some progress on this problem in the meantime.
We have experimented with different lots of fetal calf serum, and have
some evidence that their ability to support the growth of HL60 cells
varies tremendously. Preliminary data suggests that with optimal
serum it may be possible to clone HL60 cells. In the past I have
never seen differences between lots of sera. (Most of my work has
been with standard fibroblast cell lines; 3T3 cells, NRK cells, etc.)
Thus, this is new to me. Incidentally, when we tested these same lots
of sera on fibroblast lines we saw no difference between them, but
when we tested them on two T-cell lines, we saw even greater
differences than with the HL60 cells. Further, the two T-cell lines
behaved different one from another.
I would be most interested to hear from any of you about experience
you have had with differences between different lots of sera. Again,
please reply by email (steffen@mbir.bcm.tmc.edu) and I will post a
summary of responses.
=== Repetitive Sequences/Artifacts in Southern Blots ===
My second request was for insight into difficulties we were having
with our Southern blots. A rat probe we had isolated behaved as if it
were single copy on rat (and some other) DNAs but acted as if it
contained repeats in some other DNAs.
I had two (similar) responses to this request; from Hans Vos of the
Netherlands Cancer Institute Amsterdam (hvos@vaxh.nki.nl) and from
John Hancock of the Dept of Genetics, University of Cambridge, U.K.
(@pucc.princeton.edu:JMH14@PHOENIX.CAMBRIDGE.AC.UK). Both of these
responders pointed out to me that there was precidence for the notion
that a sequence can be single copy in some species, but multiple copy
in others. I thank them for this response. We have experiments in
progress to determine if such is the case for the sequences in our
probe.
-David Steffen-