DsRed is part of Clontech’s Living Colors fluorescent protein line. These proteins are great for use as molecular tags or reporters, especially in live cell assays. They come in six different colors that can be combined for multiple imaging and analysis. DsRed-Mito is part of the subcellular localization vectors, labeling endosomes, endoplasmic reticulum, nucleus, mitochondria, etc. It is a mammalian expression vector with the mitochondrial targeting sequence fused to the 5’ end of the DsRed vector. It contains a CMV promoter, pUC, f1, and SV40 origins of replication, and Neo/Kan resistance.
I have used the DsRed-Mito vector extensively for use in labeling mitochondria in mouse hippocampal cells. It labels the mitochondria along the axons quite well and a little “highway” can be seen. Using Effectene, the cells are transfected at various stages after plating with a typical 10-20% transfection rate. Stably transfected eukaryotic cells can be selected using G418. Antibodies are also available for use in Western blots and protein expression. The vector is not at all toxic to the cells, and time-lapse imaging can be used to track the movement of the mitochondria along the axons and dendrites. The fluorescent labeling persists for at least as long as the cells stay alive, which can be several weeks. A typical Cy3 filter can be used visualize the transfected cells, and there is very little photobleaching or reduction of signal. I have visualized cells for up to an hour, taking pictures every 3-10 seconds with little reduction in signal strength. Having used Mitotracker before, I found DsRed-Mito to be brighter, more persistent and an easier label to use for the mitochondria. The red fluorescence also allows it to be used in conjunction with GFP labeled cells or other double or triple-labeling experiments.
The only drawback would be the ~8-12 hours required before the protein expresses the red fluorescence. It requires extra time to plate cells, perform transfection (usually overnight) and then wait for the labeling and expression of the DsRed by the mitochondria. If time is a great constraint, the Mitotracker is a much faster label. However, once the mitochondria are labeled, they can be used for multiple experiments for several weeks with no apparent toxicity. For studies that involve the movements, localization, fusion/fission of mitochondria, or various other studies, this expression vector can be vital and very informative.
| Comment on this Review |
Comments? Questions? Discuss this review of DsRed-Mito Living Colors Vector From Clontech (Takara Bio) with the author of this review, Elizabeth Hawkes, and the rest of the Biocompare community.
|
Elizabeth Hawkes
Research Associate
Department of Ophthalmology
University of California, San Francisco
United States
more articles...
