DETECTING AND VISUALISING PROTEINS IN XENOPUS EMBRYOS Maya Zita Piccinni This thesis is submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy of the University of Portsmouth. September 2018 Declaration Whilst registered as a candidate for the above degree; I have not been registered for any other research award. The results and conclusions embodied in this thesis are the work of the named candidate and have not been submitted for any other academic award. WORD COUNT 62974 Acknowledgements Is my pleasure to give my special thanks to Peter Coxhead, Darren Gowers and Matt Guille for their support through my degree at Portsmouth, and for believing in me all these years. To Matt Guille and Colin Sharpe, for supporting me through the doctorate years; Colyn Crane-Robinson and Jerome Swinny for their valuable input and patience during our six annual meetings; to Sergei Sokol for allowing me to work in his lab and Keiji Itoh, Jonathan Chamberlain, Martin Devonshire, Simon Streeter, Samantha Griffiths and Liliya Nazlamova for their help in the practical aspects of research, to Anna Philpott for her intellectual input and Victoria Allan for helping us define the kash5 sequence. Very special thanks to my family, Hari, Bali, Mamá, Papá, Alex and children Eirwen, Skye and Edryd, who have been incredibly supportive and patient with me all this time. III Confidentiality and ethical consideration Ethics Statement All experiments and procedures in Xenopus are conducted under the British Home Office PPL 70/6450 held by Professor Matthew Guille in the “European Xenopus Resource Centre” following the Animal (Scientific Procedures) Act of 1986. The animals were regularly overseen by a Named Veterinary Surgeon. All llama vaccination experiments are executed according to UK and EU animal welfare legislation and after approval of the local ethics committee (AWERB). The sequence of the α-β-catenin VHH fragment kindly donated by UCB is confidential. IV Abstract 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 V 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. VI Abbreviations 3’ Three prime 5’ Five prime 3C 3C cleavage tag AAs Amino acids AMP Ampicillin approx Approximately APS Ammonium persulphate BCA assay Bicinchoninic acid assay BLAST Basic local alignment search tool bp Base pair BSA Bovine serum albumin cDNA Coding DNA C-terminal Carboxyl-terminal CV Column volumes dH2O Distilled water DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic acid DNaseI Deoxyribonuclese I dNTP Deoxynucleoside triphosphate DTT Dithiothreitol E. coli Escherichia coli ECL Enhanced chemi- luminescence EDTA Ethylene-diamine-tetraacetic acid EXRC European Xenopus Resource Centre FCS Foetal calf serum Flag3 Polypeptide protein tag g Grams gRNA Guide RNA GST Glutathione S-transferase HA Human influenza hemagglutinin HcAb Heavy- chain only camelid antibody HDR Homology-directed repair VII HEK cells Human Embryonic Kidney cells HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid His histidine HRP Horse- radish peroxide IPTG Isopropyl β-D- thiogalactopyranoside Kb Base x 10-3 kDa Daltons x10-3 Khis6 Lys-his-his-his-his-his-his LB Luria Broth M Molar MAP Multiple antigenic peptide MBP Maltose-binding protein MBS Modified Barth’s saline MCS Multiple coding site mg Grams x10-3 MgCl2 Magnesium chloride ml Litres x10-3 mM Molar x10-3 mmol Moles x10-3 mRFP Monomeric red fluorescent protein mRNA Messenger RNA NaAc Sodium acetate NaCl Sodium chloride Nb Nanobody NEAAs Nonessential amino acids ng Grams x10-9 nmol Moles x10-9 N-terminal Amino-terminal O/N Overnight ODC Ornithine decarboxylase OPPF Oxford Protein Production Facility ORF Open reading frame PAGE Polyacrylamide gel electrophoresis PBMC Peripheral blood mononuclear cell PBS Phosphate buffer saline VIII PCR Polymerase chain reaction PEG Polyethyleneglycol PEI Polyethylenimine pg Grams x 10-12 POI Point of insertion Primer Fw Forward primer Primer Rv Reverse primer RNA Ribonucleic acid RPTPmu Receptor-like protein-tyrosine phosphatase SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis ssDNA Single stranded DNA SS Signal sequence strepII Specifically engineered streptavidin tag TBE Tris/Borate/EDTA Buffer TBS Tris-Buffered saline TBSTw Tris-Buffered saline tween TEMED N,N,N’,N’-Tetramethylethylenediamine TF Transcription factor tRNA Transfer RNA µg Gram x10-6 µL Litres x10-6 µM Molar x10-6 µmol Moles x10-6 UTR Untranslated region VHH Nanobody V Volts v/v Volume/volume v/w Volume/weight wt Wild type xg Relative centrifuge force Xla Xenopus laevis Xtr Xenopus tropicalis IX Contents Declaration ................................................................................................................... II Acknowledgements ...................................................................................................... III Confidentiality and ethical consideration ...................................................................... IV Abstract ........................................................................................................................ V Abbreviations .............................................................................................................. VII Statement of Aims .................................................................................................... XIX List of Tables ............................................................................................................. XX List of Figures ......................................................................................................... XXIV 1 Introduction ............................................................................................................ 1 1.1 General introduction ...................................................................................... 1 1.2 Xenopus as a model organism ...................................................................... 4 1.3 Embryonic development of Xenopus ............................................................. 8 1.4 Xenopus transcriptome, proteome and metabolome ................................... 11 1.5 Antibodies and Research ............................................................................ 19 1.6 Heavy chain-only antibodies ........................................................................ 21 1.7 Importance and