Hi folks
Here's the summary of the survey started a couple of weeks ago. It
has taken a while to get this minimal sample size, hence the delay in
posting the summary. Thanks to all of you who participated.
I have received reports about 31 DsRed constructs from 13
scientists/labs. Of those, 25 were fluorescent and 6 produced no
fluorescence. There is no evident correlation with the expression
system/conditions or the type of fusion partner (soluble, membrane,
etc.). All 6 negative constructs had no linker but there were many
positives that had none either. 3 of them were fluorescent when fused
to GFP instead of DsRed.
Of the fluorescent constructs, 16 were reported to localize properly
while 5 formed abnormal structures (described as aggregates,
clusters, speckles, vacuolar structures). Again, there was no
apparent correlation with the type or natural localization of the
fusion partner. Aggregation was reported to be
concentration-dependent in 2 cases. One source reported naked DsRed
to localize reproducibly to mitochondria.
An interesting comment comes from Thomas Hughes who is "wondering about
the three potential splice sites that were accidentally introduced
during the 'humanization.'". I haven't verified this myself and
Clontech was surprised when I told them about it but they will look
into it. If true, this might explain the lack of
fluorescence/expression in at least some of the cases.
Given the variety of constructs and expression systems and the
relatively small sample, the conclusions that can be drawn are
limited. About half of the constructs seem to work. There is no
apparent pattern visible that leads to success or failure. For most
dysfunctional constructs, there are similar ones that work. There is
weak evidence, that a linker might be helpful. Aggregation (and
possibly cytotoxicity associated with it) seems to be one of the more
frequent issues. Not surprisingly, aggregation seems to depend on
expression levels.
That's the somewhat meager outcome of the survey. I'll try to answer
specific question within the limits of confidentiality the
participants were granted. In any case, I hope the 50% success rate
will encourage some of you who have heard predominantly negative
reports to give DsRed a try anyway. Not because I have any kind of
commercial interest in Clontech, but simply because an improved user
base will provide additional information and greater motivation to
solve the problems associated with this highly useful tool.
Best regards,
Beat
LIFE IMAGING SERVICES
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