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Detecting and visualising proteins in Xenopus embryos

Student thesis: Doctoral Thesis

There are records of animals used for experimental research more than 2000 years ago (Hajar, 2011) and nowadays such use is controlled. UK animal research regulations date to 1876, and were most recently revised in 2013. The appropriate use of animals in biomedical research allows the elucidation of crucial information that can be used to enhance human and animal life while being humane.
Two Xenopus species (African Clawed Frogs), are major model organisms used to study Cell and Developmental Biology. As in most animal models, mRNA levels during development are used as a proxy for protein abundance. However, recent proteomic analysis has revealed that, for at least half the genes, this assumption is wrong in vertebrate models. To visualize and assay proteins and to test their function, antibodies are needed. There have been many attempts to generate Xenopus-specific antibodies by traditional methods. These, however, had limited success and the lack of these antibodies imposes a severe research bottleneck. This project tested different approaches to raising antibodies and compared their suitability. Libraries of VHH (camelid single chain antibodies) raised against Xenopus protein mixtures were acquired from a collaborator and screened for Xenopus-specific VHHs; only VHHs against yolk proteins were isolated. To improve on this, antigen generation strategies were used both in vitro and in human cell cultures. Two proteins were successfully expressed in vitro, but yields were lower than expected. HEK (Human Embryonic Kidney) cell expression was also successful. However these DNA-binding transcription factors proved challenging to solubilise and purify. Finally, commercially synthetized peptides were used to immunise llamas with a single antigen and their plasma was screened for an immune response with positive results. Experiments to test whether VHH would have advantages in Xenopus compared with conventional antibodies were carried out with a donated VHH against human β-catenin. To understand the differences between raising VHH and conventional antibodies, a sheep was immunized with kash5 and the resulting antibody characterized. There are proteins, usually closely related members of the same family, which are difficult to tell apart using standard antibody detection. For these, a different approach to visualising the endogenous protein was undertaken. As proof of principle, the endogenous locus of Xenopus Gata2 was tagged with HA using CRISPR cas9 to target the site, followed by homologous DNA repair to add the tag. This approach showed low incidence but the positive insertion of the HA tag into the C-terminal of Gata2. Overall, this work has shown that Xenopus transcription factor proteins can be expressed in vitro and in vivo, however, either the quantity obtained or their solubility makes these products not suitable to raise antibodies against them. The alternative of using long peptides or MAPs (Multiple Antigenic Peptides) became a suitable option. The successful tagging of an endogenous protein was shown to also be possible approach. This approach showed low effectivity but a few cases of HA tag insertion at the C-terminal of Gata2 were nevertheless observed.
Original languageEnglish
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Award dateSep 2018

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