EHE zebrafish model progressing

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31 December 2018

In the third quarter the EHE Rare Cancer Charity was delighted to sign a one-year research contract with the Bateson Centre at the University of Sheffield, the first dedicated EHE research project in the UK. Working in collaboration with Dr Rubin, the project will explore the feasibility of creating a valid EHE model using zebrafish, attempting to genetically modify the fish to express the TAZ-CAMTA1 protein in endothelial cells, and thereafter describe the phenotype associated with this model, focusing on the vascular morphology. TAZ-CAMTA1 is used as this fusion protein is believed to drive EHE.

September saw the start of the project and Dr Fredericus Van Eeden, one of the Principle Investigators, was pleased to be able to report that a good start had been achieved! “We have just started working on the EHE project” reported Dr Van Eeden. “We received the EHE clones, and because it is an essential 'building block' on which most of our work will be based, we decided to recheck the sequence of the inserts and found them to be perfect; thanks to Dr Brian Rubin for that”. The team then successfully  cloned the gene into a plasmid that can be used to make mRNA for embryo injection, allowing them to create global overexpression of the fusion gene in the embryo, and see its effect. They were also delighted when a tricky PCR (polymerase chain reaction) that was needed to get it into the Gateway cloning system (used to make transgenesis construct) worked."

At year end good progress continued to be made, creating the first construct (fli1::WWTR-CAMTA1) which will lead to expression of the oncogene in endothelial cells that make up the blood vessels. This construct has been injected into zebrafish. As the WWTR-CAMTA1 oncogene does not have any visual markers itself, the team have added an extra marker gene (clmc2::EGFP) which colours the heart of the embryo green in the DNA construct to create transgenic fish. “We have injected this construct into zebrafish embryos. When such an injection is performed, we expect the injected DNA in be taken up into the genome in a subset of the cells of the embryo. Therefore if the injection is successful a subset of the cells in the embryo will get it, and if these cells are part of the heart, they will express the green transgenesis marker. As can be seen from the picture here (arrows point at the heart)  this is indeed the case. Embryos have varying amounts of green cells in the heart. We are raising these embryos now, and hope to be able to start identifying adult animals that transmit  the transgene in their germ cells in early March.

Dr Van Eeden and his team are continuing the project and we look forward to posting further positive reports in the future.

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