Molecular and bioinformatic resources for research using Xenopus

Much of our understanding of how the human body functions and of how diseases arise comes from studying "model" organisms in which, unlike humans, we can do experiments. Clawed frogs (Xenopus) are model organisms with an amazing track record, contributing to discoveries such as how cell division is controlled and how nuclei from adult cells retain the potential to programme stem cells. These Nobel-winning discoveries underpin cancer biology and regenerative medicine, respectively.
The clawed frog is a versatile model serving developmental biologists, cell biologists, biochemists and ecotoxicologists. Gene editing has recently made it an excellent organism in which to perform genetic studies. It shares much of its genome structure with humans. These studies require normal or genetically altered frogs and the molecular toolkit used for experiments, e.g. the physical strands of DNA corresponding to specific genes. Whilst 3 centres collaborate worldwide to provide frogs, the European Xenopus Resource Centre (EXRC) is the sole provider of the molecular toolkit. This application is to support that specific activity. The EXRC supplies researchers with between 2500 and 3500 quality assured resources each year at cost, thus providing savings both for researchers and the bodies that fund them.
We apply for support to continue our work, including supplying DNA in forms that allow RNA products of specific genes to be detected or that overexpress proteins to test their function. Renewed support will let us continue to collect and curate the antibodies raised by Xenopus researchers that allow scientists to detect specific proteins. The need for antibodies has been rated as the greatest requirement of the Xenopus research community for the last 5 years. This is because several studies using physical methods to analyse the proteins in embryonic cells show that the levels of protein and the mRNA that encodes it are only very poorly related. Until now, mRNA (which is relatively easy to quantify and visualize) has mainly been used as a "proxy" for protein. We now know that this is not valid and antibodies are needed to visualise proteins directly. Visualising Xenopus proteins is the major new work proposed in this application.
Many companies raise antibodies; however their quality is widely questioned and they are very rarely targeted against Xenopus. To discover which antibodies recognise important Xenopus proteins we will take suggestions for target proteins from the Centre's users. We will then identify which of the proteins against which antibodies were raised (most often human) is most similar to the target frog protein. A small amount of this antibody will then be obtained and we will test whether it will recognise the target protein in human cells (to check that the antibody works) and frog cells. If it does work in frog cells then it will be tested on Xenopus embryos. Thus, new antibodies that work in frogs will be identified.
For some of the proteins multiple attempts have failed to raise antibodies recognizing them or they are so similar to other proteins that it is impossible to make an antibody that recognises them specifically. The current approach to visualizing them is to add a small piece of protein onto their end by manipulating the DNA sequence encoding them (called epitope tagging - the tag is then visualised by a standard antibody). This tagged protein is then made in organisms either by injection of the RNA it encodes or by inserting the DNA randomly into the target animal's genome. Either method may make the protein in inappropriate amounts or in the wrong cells, thus giving inaccurate results in experiments. Gene editing allows us to overcome this problem. Recent experiments show that we can add the epitope tag DNA to the endogenous gene of an animal so the tagged protein will be produced at the same level as the normal one and in the right cells. This will allow researchers to visualize these difficult proteins.

Xenopus are key model organisms for research in cell and developmental biology and biochemistry. Gene editing allows site-directed mutations and knock outs to be performed in them so frogs are also becoming a widely used model to study human genetic diseases. Three resource centres provide genetically altered Xenopus to the community. The EXRC also collects, curates and distributes the molecular tools for Xenopus research. This application is to support this unique activity.
The EXRC distributes 2500-3500 resources annually at cost. This renewal will allow us to maintain our current activity: collecting, verifying and distributing molecular resources including expression plasmids, in situ probes, an ORFeome, a fosmid library, two BAC libraries and antibodies. We will also develop the molecular resources further as part of this application: obtaining and distributing 2 new ORFeomes, and improving the detection of specific proteins in Xenopus. Protein detection is the community's highest priority since "omic" studies have shown that, in the embryo, mRNA and protein levels correlate poorly. Analysing mRNA therefore often does not reflect the presence of the protein.
We will identify monoclonal antibodies raised against proteins from other organisms that recognise the Xenopus orthologue. Target choice will be community-driven. Antibodies will be tested by staining human (positive control) and Xenopus cells, and by immunoblotting; those that stain Xenopus will be tested on embryos and the resulting data placed online (whether positive or negative). This approach will enhance the number of antibodies available to Xenopus researchers and provide useful data for models. To detect key proteins for which there are no antibodies, we will epitope tag the endogenous locus of the cognate gene using gene editing, avoiding the miss-expression seen with expression from exogenous constructs. Together these will significantly increase the number of proteins detectable in Xenopus.

The impact of the EXRC has both direct and indirect components. Indirect impact results from the very nature of a resource centre and comes from enhancing the outputs of all the Xenopus users' laboratories by making their research more efficient. The centre allows them to do experiments that they lack the material or facilities to do in their own institutions. This can include providing unique reagents or by establishing transgenic or gene edited lines, something that many labs cannot do due to the technical demands and need for growing frogs from embryos. The impact of the research from these laboratories is extensive: basic studies on gene regulatory networks, intracellular trafficking, cell polarity, DNA replication and repair, embryo patterning and cell signaling make Xenopus famous. Alongside this now are several close, well-organised collaborations with groups of clinical geneticists carrying out more applied research using Xenopus to understand rare genetic diseases.
The commercial beneficiaries of the EXRC include a small number of pharmaceutical/ biotechnology companies that use the Xenopus model. Some of these users have been able to stop keeping Xenopus themselves as a result of regular support from the centre. This reduces their costs and the regulatory burden on them and directly impacts the 3Rs by reducing the number of Xenopus used. Further reduction in the number of frogs used by industry has been achieved by training their staff to freeze sperm, so fewer live animals need keeping for access to GA lines: this too reduces costs. A major potential impact on animal welfare in the next funding period is the roll out of much wider use of frozen sperm, since it requires fewer male frogs to be used than currently. Overall the EXRC contributes to the 3Rs in many ways.
By acting as a "hub" for Xenopus researchers and using our established communication networks, the EXRC is able to inform public bodies and charities of the research community's views and lobby on its behalf. An example was joining with the Wellome Trust and RCUK to present a case against a ban on Xenopus movements to DEFRA in response to the spread of chytridiomycosis. Another is providing feedback for the RSPCA on its Xenopus-related publication and attending a wide variety of animals in science meetings (for example the MRC's animals' forum) to represent the Xenopus community.
Whilst running training courses for working with Xenopus is not part of the EXRC remit (this is carried out by the NXR, since Wood's Hole is particularly well resourced for such courses), several scientists each year visit the EXRC for training in specific techniques, for example transgenesis, gene editing, sperm freezing or for training in frog welfare. In addition, those coming to perform their experiments at the EXRC are trained as a "by-product" of the research hotel function. Scientists and an administrator on the Centre's staff have also been trained in using Xenbase, the Xenopus community's bioinformatics resource.
Xenopus embryos were historically used for teaching in Schools and to stimulate interest in Biology, but this is very seldom possible now. EXRC staff therefore take embryos, microscopes and their knowledge and enthusiasm out to schools and to science displays in the local area. Two members of the staff have been trained at presenting animal research to school children by Understanding Animal Research. For school students studying A-level, more advanced classes are held at the University.
Overall the EXRC contributes to "impact" directly by: enhancing the effectiveness of commercial organisations and hence the UK's competitiveness, providing feedback or advice from the community to public bodies and charities, providing advanced training for academic scientists and those based in the commercial sector, again enhancing the UK's competitiveness, and by stimulating interest in Biology in young people whilst educating them thus improving STEM subject uptake.