From: Derek Gatherer (firstname.lastname@example.org)
Date: Thu 08 Dec 2005 - 09:05:48 GMT
At 21:18 07/12/2005, Dace wrote:
>Here's Dr. Hill's latest response:
>Dr. Gatherer confused the preliminary with decisive experiments. In a
>preliminary experiment (named serial assay), the tested cells were seeded on
>the top of depleted monolayer while in all subsequent, decisive experiments
>(redesigned serial assays) they were seeded into clean flasks. This has been
>said on the bottom of page 213 "....and seeded into separate flasks
>(instead of a depleted monolayer) containing the selection medium."
But that "decisive experiment" is precisely the same setup that was
used as "the control" (not a particularly good one) for the previous
"serial assay". What Hill appears to be saying is that, in the serial assay, mutations appear faster than in passage. Now, I would say, as would most conventional biologists, that this must simply be due to the mutagenic activity of the dying cells in the serial assay
(free radicals released on cell death etc). No problem there.
Then he says that subsequently, in passage, he _also_ gets elevated
mutation rates after several rounds of selection. But what is the
control for that? Since "the control" for the first experiment has
now become the experiment itself, it requires a control of its own.
CH1 cells are a new cell line freshly derived from an explant (p212
top). It's not uncommon for new cells lines in the process of
establishment to undergo genome rearrangement (the karyotypes are
frequently very mashed up compared to the original sources etc). It
would not be surprising if mutation rates increased due to damage to
genes involved in mutation repair.
To say that "the more one selects for resistance the more mutants one
finds" (p.214 top) is meaningless, for the above reason, unless one
can show that in cells never exposed to the mutagen, there are fewer
resistant colonies. Since Hill maintains that in cells never exposed
to the mutagen, there are actually _more_ resistant colonies, this
experiment is by definition control-less.
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