Molecular and Bioinformatic support for the European Xenopus Resource Centre

Lead Research Organisation: University of Portsmouth
Department Name: School of Biological Sciences

Abstract

The understanding of the basic processes of life and how these can go wrong, for example in disease or during
development, is almost completely dependent on studies using "model" animals that are particularly well understood and
experimentally tractable. Arguably the most versatile of these model animals are the embryos of Xenopus frogs. In order to
make the most effective and efficient use of these models, the genetically altered animals themselves and the special
molecular tools (generally DNA and antibodies) that are used to work with them are shared within the research community.
To do this, resource centres exist for each organism. The centre for Xenopus is the European Xenopus Resource Centre
(EXRC), and this application is to support the unique collection of molecular tools held there. Staff at the centre acquire the
molecular tools from laboratories around the world, grow more of them them and check that they work as expected. The
details of the tools are then entered on the database for this model organism, which is called Xenbase. It has sophisticated
searching and linking functions so that researchers can find the tools easily. When they are requested, the tools are sent
from the EXRC to the user who pays for the cost of their production.

Our aim, using the funding requested here, is first to continue providing the current molecular tools, the curation of which
was originally supported by the BBSRC. These tools allow researchers to determine where in embryos genes are switched
on, to make transgenic frogs and to test the function of genes. We are also requesting funding to develop two new tools for
the community. Antibodies are needed by biologists to "see" proteins in cells and embryos; for some years now the
Xenopus community has been aware of a lack of antibodies for Xenopus research. The School where the EXRC is situated
has been raising antibodies successfully since the mid-1980s and does so at a scale that allows it to supply commercially;
we propose to make this antibody raising pipeline available to Xenopus labs and support them with preliminary
characterisation of the antiserum produced. The other new tool is the physical frog genome (all of the frog DNA) in very
large fragments called BACS. We will pick BACS that span the whole genome and make these available to the community.
BACS let researchers improve the genome sequence of frogs, rescue mutations and identify the regions of DNA that
control specific genes. Since working with BACS is a highly specialised skill, we have recruited an expert BAC scientist to
develop this project part time.

This set of molecular tools, when added to the transgenic, mutant and wild-type lines held at the EXRC and the US
National Xenopus Resource (NXR), together with the training programmes provided at the NXR, give Xenopus scientists
an excellent resource infrastructure that enhances the efficiency and effectiveness of their research programmes.

Technical Summary

Xenopus frogs are one of the most versatile model organisms for studying many aspects of medically relevant basic biology. Here, we request funding for the continuation and further development of an existing Biological resource: the molecular tools used to study cell biology, biochemistry and development in Xenopus laevis and tropicalis.

These tools are collected, quality assured and distributed by the European Xenopus Resource Centre (EXRC) and we also request support to enable these resources to be accessed by the research community using the bioinformatics resource Xenbase, the core funding of which is from the NIH. Current reagents include a collection of plasmids from Xenopus labs for in situ hybridisation and expression, ESTs, fosmids and antibodies. We will continue to supply these and attempt to improve this provision continuously. In the next funding period we will also introduce support for labs wishing to raise further Xenopus-specific antibodies, a need identified by the research community and particularly important now that systems approaches are identifying a great deal of control at the protein, not RNA, level in the embryo. The second expansion is to provide the community with a readily accessible BAC tiling path. Again this is important not only to refine the X. tropicalis genome sequence, but also for rescue experiments in the increasing numbers of frog mutants generated by TILLING and TALEN/ZFN approaches.

The EXRC already collaborates closely with Xenbase and the US National Xenopus Resource (NXR) and is now working with the Japanese X. tropicalis resource to ensure we hold duplicates of frog lines to guard against a catastrophe at any site, but each centre has its own focus and the molecular resources are unique to the EXRC. Taken together with the mutant and transgenic lines held and distributed by the EXRC and its collaborators, these molecular resources will enhance the efficiency and effectiveness of research using this model.

Planned Impact

The impact of the EXRC has both direct and indirect components. The 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 lines, something that many labs cannot do. The impact of the basic research carried out in these labs on human health is made clear in the background section of the case for support

The commercial beneficiaries of the EXRC include pharmaceutical and other biotechnology companies that use the Xenopus model. In some cases these users have been able to stop keeping Xenopus themselves as a result of regular support from the Centre. This not only reduces their costs and the regulatory burden on them, but also 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. Overall the EXRC contributes to the 3Rs in many ways.

By acting as a "hub" for Xenopus researchers and using our established communication networks we are building, the EXRC is able to inform public bodies and charities of the research community's views and lobby on its behalf. An example is joining with the Wellome Trust and RCUK to present a case against a ban on Xenopus movements for DEFRA in response to the spread of chytridiomycosis. Another is providing feedback for the RSPCA on its Xenopus-related publication.

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, 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 on the Centre's staff have been trained in using Xenbase and in computational bioinformatics.

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. 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 birdies 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.

Publications

10 25 50
 
Description We have used resources developed to show that Xenopus carrying Bd are present in all lab colonies in the UK but that feral Xenopus do not transmit this disease to british amphibia living alongside them. This award has contributed to our development of robust sperm cryopreservation techniques in Xenopus which will improve animal welfare.

We have used the resources to develop a new technique that helps to understand how genes are regulated in embryos
Exploitation Route The method will be used widely by biomedical researchers.

The finding about Bd may be used to inform government policy on amphibian health
Sectors Environment,Healthcare,Manufacturing, including Industrial Biotechology

 
Description The European Xenopus Resource Centre (EXRC)
Amount £1,374,932 (GBP)
Funding ID 101480/Z/13/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2013 
End 08/2018
 
Title Antibody purification 
Description The methodology for optimal antibody production and analysis specifically for frogs has been identified and publicised 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact None yet 
 
Title Xenopus resource websearch 
Description Searchable website for all of the libraries, antibody collections, animal strains and GA animal lines made or curated at the EXRC. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Increased use of EXRC 
URL https://xenopusresource.org
 
Description Nanobody techniques 
Organisation Icahn School of Medicine at Mount Sinai
Country United States 
Sector Academic/University 
PI Contribution Person to help make and analyse nanobody libraries
Collaborator Contribution Training for making and analysis of nanobody libraries
Impact technology transfered to UK
Start Year 2014
 
Description UCB nano bodies 
Organisation UCB Pharma
Department UCB Celltech
Country United Kingdom 
Sector Private 
PI Contribution We are developing the antigens and providing the Llamas for this work
Collaborator Contribution Expertise and training
Impact Training of researcher Provision of naive libraries
Start Year 2015
 
Description Hampshire Science Fair 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Demonstrations and discussions of embryo development and of using the frog as a model organism together with introducing the 3Rs and how we contribute to these.
Year(s) Of Engagement Activity 2017
URL http://nearme.thebigbangfair.co.uk/Event/