Our projects are driven by our scientific questions, and we use whatever tools and techniques are needed to answer them.
If needed, we find new collaborations to introduce new technologies into the lab. However, the following techniques are routinely used.
Much of our work is based on the use of mouse models. We routinely use conditional knockout mice for Wt1 and different conditionally activated mutants for β-catenin. In addition we use different fluorescent reporter lines, developmental stage-specific Cre drivers and Cre reporters.
In vitro models
Whenever possible we combine our in vivo work with in vitro models carrying the same alleles. These are experiments where the whole kidney or organism context is not an essential aspect, and for experiments that require large number of cells that we cannot isolate from in vivo samples via FACS-sorting. It also greatly reduces our animal numbers. We mainly use mouse embryonic stem cells and primary NPC cultures.
Organ culture and imaging
Our method of choice for analysing kidney developmental phenotypes is the organ culture system. Its pseudo-3D nature gives a much better idea of the spatial organization of developing kidneys, and the imaging of antibody whole-mount stained samples can in the right hands be aesthetically magnificent. In addition, we use a dedicated time-lapse imaging set-up for analysing kidney organ cultures over time. This allows us to study the dynamics of kidney development and, importantly, the dynamics of phenotypes. It also means we get more data per kidney, again reducing our animal numbers.
From Lindstrom et al (2015) Stem Cell Rep. 4, 551-560.
Transcriptomics and epigenetics
Many of the genes in involved in Wilms’ tumours are, maybe not surprisingly, transcriptional regulators that are also controlling normal kidney development. Therefore, an important part of our research is focusing on the transcriptional programs that control NPCs, how these get deregulated in Wilms’ tumours, and how this explains the phenotype of Wilms’ tumours. We are interested in the transcriptional effects of Wilms’ tumour mutations, to explain the phenotypes they cause, and how the normal and mutant gene regulatory networks are organized at the epigenetic level. All of these we try to study as much as possible in freshly isolated FACS sorted material, but if needed we use cultured NPCs.
Gene targeting and genome editing
Mutant models, either mutant mice or cultured NPCs are central to all our work. A lot of the gene targeting required for this we do ourselves, and we have extended expertise in mouse ES technologies, gene targeting, construct design and building. Over the years we have developed and published several high-efficiency vector systems for this, which are available via Addgene. These days, most of our targeting is done with the help of CRISPR/Cas9-based genome editors, and we are involved in many in vitro and in vivo editing project in The Roslin Institute.