Gene targeting by homologous recombination in the rat

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

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.

Technical Summary

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.

Planned Impact

WHO WILL BENEFIT FROM THIS RESEARCH? In the short to medium term the primary beneficiaries of outputs from this research will be the scientific community (academic and industrial). 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 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 requires 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. Interestingly, success in the derivation of rat ES cells, and any technological improvements developed through the course of this programme may also have importance in the derivation of similar types of embryonic cell types from commercially important animals such as pigs, sheep etc. Given that protocols for ES cell derivation in rodents have not simply translated to success with larger animals, technical developments that allow robust expansion of undifferentiated rat ES cells 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 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 these collaborative demonstration project opportunities with industry as well as with academia.

Publications

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Description This grant had three specific objectives:
1. To achieve robust clonal propagation of germ line competent rat ES cells.
2. To introduce locus-specific mutations into Rat ES cells by gene targeting
3. To reliably transmit targeted mutations in Rat ES cells through the germ line to generate
a knock-out rat.
During the course of the grant we have met all three objectives.
1. Robust clonal propagation of germ line competent rat ES cells.
We have derived rat ES cells in a simplified serum-free medium on feeder cells using chemical inhibition of the MEK signalling pathway alone, and by additionally modulating the activity of GSK3 signalling. We reported the successful derivation of germ line competent rat ES cells generated in this manner in our manuscript "Tuning of b-catenin is Required to Stabilise Self-renewal of Rat Embryonic Stem Cells Stem Cells: 31 (2013) 2104-15". In this publication we demonstrated that unlike mouse ES cells, rat cells are vulnerable to high levels of b-catenin signalling which over-rides the self-renewal signals and drives differentiation. Using siRNA and TALEN mediated gene editing our subsequent experiments have demonstrated that high levels of the transcription factor LEF1 in rat ES cells are responsible for this. We also generated rat ES cell lines carrying a stem cell specific knock-in fluorescent reporter (Rex1-EGFP) which allowed us to identify self-renewing stem cells in situ in culture and investigate how to refine the cell culture conditions further, in particular with respect to the effects of modulating signalling and epigenetic status of the cells.
2. Introduction of locus specific mutations into rat ES cells by gene targeting
We have shown that standard homologous dependent gene targeting is efficient in rat ES cells and equivalent to that obtained with mouse ES cells. ('Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells', PLoS One, 5 (2010), e14225.) We have also shown that gene knock-in (Rex-1EGFP, manuscript in preparation) and negative selection (CSF1R, manuscript in preparation) is highly effective (100 and 80% of clones, respectively). We have also applied recently developed gene editing approaches to increase the efficiency of homology dependent gene targeting. We used TALEN gene editors to promote homologous recombination and targeting of the rat genes for IAPP, CFTR, and neurotrophin receptor p75NTR. The efficiencies for these experiments ranged between 20-90%, thus greatly simplifying gene targeting protocols in rat ESC. We have also used CRE recombinase excision to modify targeted loci to eliminate selection cassettes and engineer point mutations (CFTR) and floxed exons (p75NTR).
3. Transmission of targeted mutations through the germ line.
Using good quality cell lines and with careful selection of clones it is possible to ensure germline transmission. Indeed, the majority of the targeted loci have been successfully transmitted through the germ line to generate transgenic lines of rats.
We generated an HPRT deficient rat as a new model of the human disorder, Lesch Nyhan Disease. These rats display altered metabolism in the brain and loss of dopamine and serotonin. Further analysis will be required to determine whether loss of HPRT is associated with behavioural defects. We also made the novel finding that HPRT deficiency in female rats is associated with reduced fertility, suggesting that the purine salvage pathway plays a critical role in oocyte maturation or early preimplantation embryonic development in vivo.
We have also used rat ESCs to generate a number of mutant rats. As outlined in this programme we generated transgenic rats carrying either constitutive or conditional (Floxed) mutations of the neurotrophin receptor p75NTR. We have demonstrated that rats with deletion of exon 2 in the rat p75NTR gene lack any expression of the p75NTR protein, but appear healthy and fertile. Rats carrying a p75NTR allele with a floxed exon 2 express normal levels of the p75NTR protein. We generated a transgenic rat carrying a fluorescent marker knocked into the stem cell reporter Rex1. This rat allows tracking of pluripotency and investigation of the contribution of Rex1 transcription factor in X-chromosome regulation and fertility. We have not detected any deficit in fertility in the Rex1 knock-out rats and are apparently healthy (Manuscript in preparation).
We have generated a CSF1R (Colony stimulating factor 1 receptor) deficient rat that displays novel features compared with the equivalent mouse mutant (manuscript in preparation). and also generated a Cystic fibrosis transmembrane conductance regulator (CFTR) knock-in rat that carries the G551D mutation, that is now being studied by our colleague and collaborator Dr Gerry McLachlan.
Exploitation Route The work on targeting and refinement of cell culture conditions for rat Embryonic stem cells (ESC) will be of use to scientists using rat ESC and stem cells from other species. It has been cited in relation to modifying ESC growth conditions for human naïve ESCs ( Takashima et al., Cell 014: Theunissen et al., Cell Stem Cells 2014)
Sectors Pharmaceuticals and Medical Biotechnology

 
Description The work on targeting and refinement of cell culture conditions for rat Embryonic stem cells (ESC) has been cited in relation to development of modified growth conditions for human naïve ESCs ( Takashima et al., Cell 014: Theunissen et al., Cell Stem Cells 2014)
First Year Of Impact 2014
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title HPRT knock-out rat 
Description Transgenic rat that carries a mutation in the hprt gene. Similar mutations in humans are responsible for Lesch Nyhan Disease, an X-chromosome linked severe neurological disorder. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact The rat is the preferred rodent model for brain and behaviour studies. This transgenic rat will be useful in assessing behavioural effects of hprt deficiency in a laboratory animal - and may provide useful insights into the molecular basis of Lesch Nyhan Disease. 
 
Title Mutant rat carrying a floxed Neurotrophin p75NTR allele 
Description Neurotrophin p75NTR is involved in regulating neuronal survival, stem cells and nociception. Using a TALEN gene editor and homologous recombination we have generated rat embryonic stem cells(ESC) in which we have flanked exon 2 of the rat p75NTR gene with LoxP recombination sites to generate a " floxed" p75NTR allele . We used a correctly targeted rat ESC line to generated a mutant rat that carries the floxed p75NTR allele. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This is the first description of a floxed p75NTR allele in the rat. When mutant rats carrying the floxed p75NTR allele are bred with rats that express CRE recombinase in a tissue restricted pattern this allows conditional deletion of the p75NTR gene in a tissue specific manner. 
 
Title Neurotrophin p75NTR knock-out rat 
Description Neurotrophin p75NTR is involved in regulating neuronal survival, stem cells and nociception. Using a TALEN gene editor and homologous recombination we have generated rat embryonic stem cells(ESC) in which we have deleted exon 2 of the rat p75NTR gene. Using these targeted rat ESCs we have generated a mutant rat that lacks the p75NTR protein. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This is the first p75NTR knock-out rat generated using rat ESCs, and deletion of exon 2 appears to generate a complete null allele that should allow researchers to accurately assess the role of p75NTR in neurogenesis, the brain and nocioception. 
 
Title Rex1-EGFP knock-in transgenic rat 
Description We have generated a transgenic rat in which we have knocked-in a EGFP-IRESneo reporter gene into the first exon of the rat rex1 gene. The rex1 gene is highly expressed in undifferentiated embryo stem cells and in some cells within the germ lineage. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact The rex1-EGFP knock-in gene allows the characterisation of the rex1 expression pattern in vivo in the rat. It also allows the identification and purification of rex1 expressing cells from rat tissues, and the induction or maintenance of the undifferentiated stem cell state in reprogrammed rat cells. 
 
Title Rex1-egfp knock-in rat embryonic stem cells 
Description The rex1-EGFP rat embryonic stem cell (ESC) line carries an egfp reporter knocked into the rat rex1 gene. Since rex1 expression is tightly associated with the undifferentiated embryonic stem cell state, the egfp reporter provides a way to monitor the differentiation status in live stem cell cultures, and purify subpopulations of cells from rat ESC cultures. 
Type Of Material Cell line 
Year Produced 2015 
Provided To Others? Yes  
Impact We have used the rex1-egfp reporter to characterise and refine the culture conditions for propagating undifferentiated rat embryonic stem cells. 
 
Description Generation of ICE-HIP model of diabetes in rat 
Organisation Aarhus University
Country Denmark 
Sector Academic/University 
PI Contribution Assistance with culture, transfection, selection and screening of targeted rat embryonic stem cells
Collaborator Contribution Gene targeting constructs and assistance with culture, transfection, selection and screening of targeted rat ESC clones
Impact Not yet
Start Year 2013
 
Description Generation of a new cystic fibrosis model in the rat 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Assistance in the transfection, selection and screening of CFTR targeted rat ESC clones
Collaborator Contribution Provision of the CFTR targeting vector and screening of CFTR clones
Impact No outputs yet
Start Year 2013
 
Description Generation of motor neuron disease model in rat 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Culture, transfection, isolation and selection of VAPB targeted clones. Construction and validation of TALEN gene editing enzymes
Collaborator Contribution Construction of VAPB gene targeting vector
Impact Not yet
Start Year 2013
 
Description Brainstorming workshop on somatic cell derived bovine gametes/embryos 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Brainstorming Workshop on Somatic Cell derived Bovine Gametes/Embryos
Attended by heads of Indian organisations NDDB, DBT, the company TAG, and international research scientists (New Delhi, 25-26th June 2018)
Presented talk entitled " Pluripotent stem cells: Achievements and Challenges"
Year(s) Of Engagement Activity 2018
 
Description Edinburgh-Tsukuba Symposium: Stem cells and regenerative medicine 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented talk at Symposium entitled " Tuning Self-renewal in rat ESCs" on April 16th 2018 at Tsukuba Medical Research Institute, Japan.
Promoted collaborative discussions.
Year(s) Of Engagement Activity 2018
 
Description Hosting College students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation on stem cells and lab visit by students from Forth Valley College

To quote "Tom

I wanted to write and thank you for the visit that you arranged for the students and myself last week. It was an extremely good experience for the students, and I really enjoyed seeing the labs and hearing about your research. As I said last week I thought the presentations were excellent and perfectly pitched for us an audience.

If I can ever return the favour please feel free to ask. I know you mentioned that outreach is important for putting on your grant applications, and we do lots of outreach work with the local schools. We have developed lots of small experiments that we take out as a roadshow, so if you are ever in a position to need such resources I'd be happy to help.

Thanks and best wishes
Monica


Monica MacLeod

Room 316
Forth Valley College
Grangemouth Road
Falkirk

FK2 9AD

Tel: 01324 403172
Year(s) Of Engagement Activity 2012
 
Description Roslin Institute Annual Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Interactions triggered questions and discussions on the application and ethics of stem cells in biomedicine and animal science

Increased appreciation of the importance of stem cell research in biomedicine and animal science
Year(s) Of Engagement Activity 2011,2012,2013