Development of induced pluripotent stem (iPS) cells from mutant mouse models in order to reduce animal use
Lead Research Organisation:
University of Sussex
Department Name: Sch of Life Sciences
Abstract
We and others have shown that mice with mutations in the gene coding for dynein (a motor protein involved in many cellular processes including cell division and transport of intracellular material) show defective motor and sensory functions. Moreover, mutations in dynactin - a protein partner of dynein - have been found to cause a form motor neurone disease in humans. Because of their wide range of functions and implications in neuronal survival, these proteins are being studied by research groups around the world using the mouse as a model organism. Such studies require breeding of these mice to provide the necessary amount of neurones needed for research. In addition, neurones isolated from mice are not able to proliferate and die within 2-3 weeks in culture, thus necessitating continuous breeding of large numbers of mice.
Recently, a novel technique has been developed to change the mouse fibroblasts into a type of stem cells called iPS cell. The iPS cells could subsequently be converted into any cell type including neurones. This, therefore, has provided a unique opportunity to generate an easily accessible and abundant resource for the production of neurones and other cell types from the mutant mice, without the need for breeding large numbers of mice. Thus, in the proposed project we are aiming to generate iPS cells from two of the mouse strains with mutations in dynein. We will then be able to produce neuronal cells from these iPS cells and use them for research into dynein function. Successful completion of this project will result in significant reduction in breeding mice with harmful mutations in dynein and in time and costs involved in their breeding and the experimental procedures. In addition, we will be able to share the iPS cells with other labs around the world and signify the use of iPS-cell technology in studying other mouse models.
Recently, a novel technique has been developed to change the mouse fibroblasts into a type of stem cells called iPS cell. The iPS cells could subsequently be converted into any cell type including neurones. This, therefore, has provided a unique opportunity to generate an easily accessible and abundant resource for the production of neurones and other cell types from the mutant mice, without the need for breeding large numbers of mice. Thus, in the proposed project we are aiming to generate iPS cells from two of the mouse strains with mutations in dynein. We will then be able to produce neuronal cells from these iPS cells and use them for research into dynein function. Successful completion of this project will result in significant reduction in breeding mice with harmful mutations in dynein and in time and costs involved in their breeding and the experimental procedures. In addition, we will be able to share the iPS cells with other labs around the world and signify the use of iPS-cell technology in studying other mouse models.
Technical Summary
Cytoplasmic dynein is a large multi-subunit microtubule associated protein involved in a range of cellular processes including transport of cargo in the minus-end direction (retrograde transport). We and others have shown that mutations in the heavy chain subunit of cytoplasmic dynein (Dync1h1) in the Legs at odd angles (Loa), Abnormal rear leg (Arl), Cramping 1 (Cra1), and Sprawling (Swl) mouse models cause neurodegeneration as a result of perturbation of neuron-specific functions of dynein.
We have shown that the F580Y and W1206R substitutions in DYNC1H1 in the Loa and Arl, respectively, result in severe loss of motor and sensory neurones. Analysis of the cells isolate from Loa and Arl suggest that the neuronal loss in these mice is a result of significant defects in the dynein-mediated retrograde axonal transport and endocytic membrane trafficking of trophic factors. Interestingly mutations in dynactin ? a multi-subunit protein partner of dynein, which contributes to retrograde transport and regulates dynein function? have been identified in human families with a late-onset form of motor neurone disease.
These mouse models, therefore, provide invaluable tools for elucidating the mechanisms that control and regulate the motor and cargo transport functions of dynein, and its role in neuronal health and disease. However, large numbers of mice and lengthy breeding and dissection schemes are needed to obtain the required motor and sensory primary neurones for such analyses. Thus, in this application we are proposing to produce ?induced pluripotent stem (iPS) cells? from mouse embryonic fibroblasts (MEFs) isolated from Loa and Arl mice, by following the detailed and well established protocol described by Yamanaka and colleagues. We will then be able to differentiate these iPS cells into neuronal cells using established protocols. All the expertise for carrying out this project is available in our groups. Successful completion of this project will result in significant reduction in breeding mice with harmful mutations in dynein, and in time and costs involved in their breeding and the experimental procedures. In addition, we will be able to share the iPS cells with other labs around the world and signify the use of iPS-cell technology in studying other mouse models.
We have shown that the F580Y and W1206R substitutions in DYNC1H1 in the Loa and Arl, respectively, result in severe loss of motor and sensory neurones. Analysis of the cells isolate from Loa and Arl suggest that the neuronal loss in these mice is a result of significant defects in the dynein-mediated retrograde axonal transport and endocytic membrane trafficking of trophic factors. Interestingly mutations in dynactin ? a multi-subunit protein partner of dynein, which contributes to retrograde transport and regulates dynein function? have been identified in human families with a late-onset form of motor neurone disease.
These mouse models, therefore, provide invaluable tools for elucidating the mechanisms that control and regulate the motor and cargo transport functions of dynein, and its role in neuronal health and disease. However, large numbers of mice and lengthy breeding and dissection schemes are needed to obtain the required motor and sensory primary neurones for such analyses. Thus, in this application we are proposing to produce ?induced pluripotent stem (iPS) cells? from mouse embryonic fibroblasts (MEFs) isolated from Loa and Arl mice, by following the detailed and well established protocol described by Yamanaka and colleagues. We will then be able to differentiate these iPS cells into neuronal cells using established protocols. All the expertise for carrying out this project is available in our groups. Successful completion of this project will result in significant reduction in breeding mice with harmful mutations in dynein, and in time and costs involved in their breeding and the experimental procedures. In addition, we will be able to share the iPS cells with other labs around the world and signify the use of iPS-cell technology in studying other mouse models.