Gene targeting by homologous recombination in the rat

Embryonic stem (ES) cells are remarkable biological entities. They are derived from the pluripotent founder cells of embryos and exhibit the two essential stem cell properties, 1) unlimited proliferation, and 2) the potential to differentiate, in this case into any cell type in the foetus, including the gametes. The ability to propagate mouse ES cells stably in culture through many rounds of cell division, combined with state-of-the-art methods for genetic engineering, has provided a suite of powerful technologies to design genetically modified mice for use as research tools in academia, medicine and biotechnology. Although progress in genetic engineering has largely centered on the mouse, its close relative the rat is in fact the most important and commonly used animal in biomedical and biotechnology research. To further improve the utility of the rat as an experimental model, there has been an international effort over several decades to apply the technologies for genetic engineering developed in the mouse, to the rat. Whilst cell lines have been available for mouse for over 20 years, similar rat cells were only recently isolated (by applicant AGS) using a novel culture protocol that relies on specific chemical inhibitors to suppress the ERK MAPK signaling pathway, a trigger of ES cell differentiation. Rat cell lines maintained in this medium can be transmitted through the germ line and therefore provide a novel route for genetic modification in the rat. Indeed, in a preliminary study we have taken a first step in this process and generated ES cells that carry a targeted mutation in a specific rat gene. Notwithstanding these successes, significant challenges remain in applying genetic engineering efficiently in the rat. The initial reports and our subsequent studies indicate that a better understanding of rat ES cell biology is required to stably maintain their full developmental potential, particularly during the extended periods of culture required to introduce genetic modifications through gene targeting. We also need to assess how well the techniques for genetic modification will be applied to these novel cell lines. The aims of this proposal are 1) to improve the new methods for propagating rat ES cells, 2) apply the tools for genetic engineering to the rat ES cells, and 3) to generate a knock-out rat. To develop and improve the culture conditions in which Rat ES cells are propagated we will examine their physiological and differentiation status in culture. We will examine the molecular profile of rat ES cells grown under different growth conditions to determine whether the cells are subject to particular stresses, and make appropriate adjustments in culture protocols to alleviate these responses. We will also genetically engineer cell lines to allow us to monitor and improve their differentiation status in living cultures. We have noted that rat ES cells tend to spontaneously differentiate into a particular endoderm cell type: we will attempt to neutralise this tendancy through the conditional expression of stem cell factors that have previously been shown to stablilise ES cell states in mouse ES cells. We will also explore the potential of cell lines derived from rat germ cells as vectors for delivering targeted genetic mutations through the germ line of rats. Finally, a major objective in this grant is to generate a targeted knock-out rat. The rat is particularly appropriate laboratory animal for studying brain and behaviour, and is the mainstay of these kinds of studies in academia and industry. We will therefore generate rats carrying an inactivating mutation in the neurotrophin receptor p75NTR a key regulator of neuronal growth, brain development and behaviour. Studies of the knock-out rats will provide insights into the role of p75NTR receptor in regulating complex phenomena such as anxiety, depression and neurological deficits associated with trauma and ageing.

The rat is one of the most important and commonly used animals in biomedical research. Its utility as an experimental model will be further improved by applying genetic engineering technologies developed in the mouse, to the rat. These methods have relied on the availability of authentic mouse ES cells. Equivalent rat cells were only recently isolated (by applicant AGS) using a medium that specifically blocks the ERK MAPK signaling pathway, a trigger of differentiation. These ES cells are germ line competent and therefore provide a novel route for genetic modification in the rat. Indeed, in a preliminary study we have successfully targeted the HPRT gene in rat ES cells. However, the initial reports and our subsequent work indicate that further study is required stabilise the developmental potency of the cells in culture and robustly implement genetic engineering in the rat. The aims of this proposal are 1) to improve the new methodology for propagating rat ES cells, 2) apply contemporary tools for genetic engineering, and 3) to generate a knock-out rat. To investigate how to stabilise the rat cells we will perform molecular profiling to characterize the physiological status of the rat cells in the inhibitor medium. To asses the phenotypic status of cultures we will generate targeted stem cell (Rex1-EGFP) reporter cell lines that will provide feedback in living cultures and facilitate efficient methods to refine culture protocols. Rat ES cells are particularly susceptible to hypoblast differentiation: we will attempt to neutralize this tendancy using transponson mediated delivery of the transcription factors Nanog and/or Klf2. Cells stabilization by these factors may provide a window in which to genetically manipulate the cells. Finally, we will generate a rat knock-out the neurotrophin receptor p75NTR, a molecule that plays a critical role in regulating brain and behaviour, an area of research in which the rat is particularly suitable as an experimental model.

WHO WILL BENEFIT FROM THIS RESEARCH? In the short to medium term the primary beneficiaries of outputs from this study will be the research community (academic and biomedical). In the longer term potential beneficiaries will include the biotechnology and pharmaceutical industry. Products developed by these sectors informed or enabled by the knowledge, technologies and resources developed in this research project could ultimately deliver healthcare benefits to the wider public. These long-term beneficiaries could include those who require treatment for diseases that can be modelled in a physiologically relevant and widely used experimental animal like the rat. HOW WILL THEY BENEFIT FROM THIS RESEARCH? The first step in delivering benefits from this research will be the exploitation of the technologies and cells developed in the project. For example, a major goal of this research is to generate gene targeted knock-outs in the rat. This will require establishing robust methodology for the propagation of fully developmentally competent rat ES cells, the application of currently available gene targeting technology in rat ES cells and the transmission of these cells through the germ line. Genetically engineered rat ES cells will be used to generate transgenic rats that will serve as new genetic models for studying heath and disease in an animal that is already widely used in biomedical research and by the pharmaceutical industry. The larger size and behavioural complexity of the rat makes it particularly amenable to studies where surgery in the mouse is a limiting factor, or when investigating complex aspects of behaviour (see letter of support from Professor Yves Barde). The rat cells can also, through their in vitro differentiation, provide unlimited supplies of genetically modified cells that can be used in in vitro experiments (such as high through put screens) and will complement the in vivo studies. Finally, rat ES cells will be used as a basic research tool to understand more about the basic mechanisms that regulate pluripotency and self renewal in mammals. Importantly, advances in the understanding of rat ES cells, and any technological improvements developed through the course of this programme can be expected to be relevant to efforts to derive pluripotent stem cells from commercially important animals such as pigs, sheep etc. Traditional protocols for ES cell derivation in strain 129 mice have not previously translated to success with larger animals. However, technical developments that allow robust expansion of undifferentiated rat ES cells, which also do not survive in traditional mouse ES cell conditions, may have broader applications with other non-permissive species. WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH? In order to ensure that the benefits of this research can be realised we will communicate our results (knowledge and technologies) in a timely manner at scientific meetings and through the peer-reviewed scientific literature. We will make our Rat ES cells available to other research groups under appropriate material transfer agreements and license arrangements. Subject to appropriate funding we will provide training for other researchers in methods developed during the project. We will seek follow-on funding to consolidate progress in generating transgenic rats in collaboration with other research groups (see letter of support from Professor Yves Barde). We will use the expertise of our technology transfer office and our extensive industrial contacts to seek collaborative demonstration project opportunities with industry as well as with academia.