Bringing more light into blood stem cells
Researchers from the German Cancer Research Center (DKFZ) in Heidelberg were able to devise genetic labelling in the blood stem cells of mice, which could be induced and switched on by the addition of a certain drug. Hence, the researchers were able to observe how blood cells develop out of stem cells, as reported in a recent publication in the journal Nature.
Earlier work of the group of the BIOSS researcher Prof. Dr. Michael Reth has shown that the DNA-recombinase Cre can be regulated remotely and induced by the substance tamoxifen. The researchers attached the tamoxifen-binding Mer domains to Cre’s N- and C-terminal part, thus generating the MerCreMer chimera. Cre is a DNA recombinase that can recognize and delete genes flanked by LoxP sites, the recognition sites for Cre activity. Such “flanked by LoxP site” or “floxed” genes can be altered in the presence of Cre. With a similar strategy one can also activate genes whose expression is inhibited by a floxed stop cassette.
Reth’s group has shown that the MerCreMer enzyme only becomes active in cells in the presence of the inducer Tamoxifen. Their first publication of this method in 1996 ended with the sentence "the MerCreMer system thus may be developed into a useful new genetic tool to mutate and delete genes from the genome of a living organism". At that time, however, this was not well received by one of the reviewers of the manuscript who wrote "this method will for sure never work in vivo".
Now, nearly 20 years later, the MerCreMer technology is used in many laboratories all over the world to delete or induce genes in living organism as described in more than 40 publications. The newest example for the usefulness of the MerCreMer system is the work of Prof. Dr. Hans-Reimer Rodewald and his team at the DKFZ. They inserted the MerCreMer cDNA into a gene locus that is only expressed in haematopoietic stem cells (HSCs), which give rise to all the other blood cells. The research team then crossed the MerCreMer mice with those carrying a conditional yellow fluorescence protein (YFP) gene with a “floxed” stop cassette. Application of tamoxifen to these mice resulted in YFP expression and thus marked haematopoietic stem cells in an inducible and temporally controlled fashion.
By following the YFP marker and its appearance in the different offspring of the haematopoietic lineage, the authors could show for the first time the in vivo kinetic of the production of blood cells that all derive from the haematopoietic stem cell lineage. One surprising finding of this study was that it takes quite a long time - more than 40 days - before mature blood cells have developed from the labelled HSC. Furthermore the researchers showed that the immediate offspring of the HSC also have stem cell like properties and can generate the precursors of the different haematopoietic cell lineages for a long time. The research group can now use this in vivo model to test how mutations are affecting haematopoietic stem cells and the production of the mature blood lineage cells.
At the Cluster of Excellence BIOSS Centre for Biological Signalling Studies, researchers are currently testing a whole range of new strategies which aim at regulating genes as well as signalling and cellular processes. The MerCreMer plasmid is available at the BIOSS Toolbox together with all necessary sequence information.
The press notice released by the DKFZ:
Original publication:
Fundamental properties of unperturbed haematopoiesis from stem cells in vivo.
Busch K, Klapproth K, Barile M, Flossdorf M, Holland-Letz T, Schlenner SM, Reth M, Höfer T, Rodewald HR.
Nature. 2015 Feb 11. doi: 10.1038/nature14242. [Epub ahead of print]
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature14242.html