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Transgenic models of mammalian meiotic exit

Periodic Report Summary 2 - PERRY/MII/TG (Transgenic models of mammalian meiotic exit)

Summary of the project
This award has achieved its main goals of facilitating the relocation of a mouse molecular embryology laboratory from Japan to Europe and generating novel transgenic mouse lines. Transitions of this nature are inevitably time-consuming and non-trivial: in our case, they involved compliance with new national and international laws and guidelines, ensuring effective husbandry and establishing micromanipulation. My laboratory generates gene-modified mouse strains as a central facet of its work, so we produce transgenic lines de novo via micromanipulation. It was with a view to achieving this end in particular that IRG grant support was sought and it has been a success in two principal aspects:
 It has allowed us to establish piezo-actuated micromanipulation ('piezo') in the UK. Piezo is at the heart of the technologies we have developed to produce transgenic mice. (Injecting solid material much larger than 1 μm into mouse eggs is difficult because they are exquisitely sensitive, but piezo overcomes this problem.) To confirm success, we undertook one of the most exacting piezo protocols, nuclear transfer cloning, and produced England's first cloned mouse (Fig. 1).
 Using piezo, we have produced multiple transgenic lines, which have been used in work that is being prepared for publication. In particular, we have produced proteins fused to proteins that fluoresce in the green or red range. The proteins are expressed in eggs and are selected because they give us information about intracellular events immediately following fertilization. This method is especially powerful because once the transgenic lines have been produced, they can be used to provide information about the earliest moments of development. Some of these successes have surpassed expectations and will be published in due course. Examples (see Fig. 2) include transgenes encoding fusions to structural elements of the cell, regulators of the cell cycle, reporters for genes active in stem cells and proteins that are associated with DNA. This last category allows us non-invasively to observe chromatin exchange immediately after fertilization; this is a fundamental step in the initiation of the developmental programme and is at the core of our work. In some cases, we have introduced transgenes onto background strains with special applications, such as the strain 129Sv.
REA comments on the Interim Report
The project (€100,000) has indeed contributed to publishable data from transgenic lines generated in this work, and although they have not yet been published, we expect the first submissions to begin in the first half of 2014. This will set in train a series of allied dissemination activities, including presentations at meetings. The small independent research group comprises three people: Dr Perry and two post-doctoral researchers. Dr Perry has tenure at the University of Bath and has a reasonable teaching load, including a lecture block, seminar series, tutorials and post- and under-graduate project supervision. We are seeking to expand the group, a process that begins with good science that is published. Until the group has reached a sufficient critical mass to be internationally competitive we do not have the resources to provide "work stations for visitors, etc". Indeed, without major funding, securing even one single work-station is difficult in the UK.
Context and potential impacts of the work
This work will influence academic communities via publication in international journals. Papers will be published soon; they form part of a multifaceted approach involving European collaborations, and take time. However, we expect that the first manuscripts reporting IRG-backed work will be submitted in the coming weeks. In addition, it will be presented in an ongoing tour of presentations at European institutions and meetings. Publication informs international policy when it achieves a high profile, such as a recent News & Views by Dr Perry on human nuclear transfer cloning and published in Nature Biotechnology. More news of our work can be found on our regularly-updated lab website: http://www.egg2embryo.com/
To develop and further raise the profile of our work, we have collected striking preliminary nanotechnology data that forms the basis of a trans-disciplinary, international collaboration with physicists in Spain. Our exchanges will enable essential physics-biology knowledge transfer and preliminary data collection that is expected to produce a collaborative grant proposal to the EU Future and Emergent Technologies call in 2015.
We anticipate that IRG-supported work will lead to clinical impacts in infertility, cancer and regenerative medicine. It has allowed us to pursue and develop clinical collaborations in Germany in the area of cancer aetiology. This relationship facilitates knowledge transfer to top oncologists with access to a large archive of diverse human cancer samples, aided by frequent meetings. We also have a major collaboration in the US to exploit our findings in the arena of human Assisted Reproductive Technology (ART). Where phenotypes in our work resemble clinical phenotypes in impaired fertility, our transgenic models will reveal underlying cellular behaviour. In the longer term (around 5 years) it is anticipated that this will information will be commercialized , for example, as it pertains to infertility markers or for treatment.
Finally, we recognize several pathways to maximizing the impact of this work in regenerative medicine. First, through companies involved in translational stem cell research, we will ensure that our transgenic lines are used to improve the efficacy of nuclear transfer cloning in mice so that they can be translated to human nuclear transfer ES cells. Secondly, our work has clear implications for porcine genome manipulation. In a long-standing collaboration with a preeminent group in Japan, we develop technologies in the mouse and extend them to pigs. One corollary is the establishment xenotransplantation in the UK with the South West UK Regional Pre-clinical Biomedical Research Centre. Thirdly, we will use transgenic mice to evaluate new approaches to stem cell induction. For example, we have produced transgenic stem cell reporter mouse lines from which we have derived somatic cells with which to evaluate the reprogramming potential of putative stem cell factors. Success will be extended to human cells that will ultimately be characterized in systems that include engineered pigs.