Molecular and bioinformatic resources for research using Xenopus

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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.