DsRed Survey - Summary (as far possible)

Beat Ludin bludin at lis.ch
Thu Oct 12 06:50:12 EST 2000

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,


               - visit our web site at http://www.lis.ch
| Dr. B. Ludin
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| CH-4600 Olten             beat.ludin at lis.ch
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