Award details

transgenic expression of UPRT as a novel tool for tagging RNA in specific tissues of the mouse

Reference	BB/K013424/1
Principal Investigator / Supervisor	Dr Martin Turner
Co-Investigators / Co-Supervisors	Dr Sebastian Lukasiak
Institution	Babraham Institute
Department	Immunology
Funding type	Research
Value (£)	109,685
Status	Completed
Type	Research Grant
Start date	01/07/2013
End date	30/06/2014
Duration	12 months

Abstract

Summary (technical) Transgenic expression of the enzyme uracil phosphoribosyltransferase (UPRT), which is lacking in mammals, allows conversion of uracil into uridine monophosphate. When 4-thiouracil is instead used it is converted to 4-thiouridine which is known to be incorporated efficiently into the RNA of mammalian cells. This makes possible the use of specific thio-reactive biotinylation reagents to label thiolated RNA which can be purified by means of its high affinity for streptavidin. We have generated a mouse that expresses UPRT conditionally under the control of Cre recombinase so as to allow cell-type specific labelling of RNA. We have verified that the transgene is functional and now want to demonstrate the power of the tool. Thus we will isolate RNA from T cells within the liver (these are a minority population) after administration of thiouracil to mice in which the transgene is activated by CD4Cre. Additionally, we will use a photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR CLIP) to identify RNA protein associations in thymic lymphocytes. Finally we will test the ability of transgene to mediate incorporation of 5-bromouracil or 5-ethynyl uracil into RNA. The approach will allow investigations into RNA in any field of biology for which a suitable Cre transgenic mouse is available.

Summary

Summary (Lay) RNA is synthesised using information encoded by DNA and it mediates many of the functions of the genome as well as producing proteins. Understanding the regulation of RNA is a priority for the understanding of cell function. Like DNA sequences, RNA sequences are made up for four bases, but, unlike DNA, RNA contains the nucleoside uridine which is composed of the pyrimidine uracil. Uracil is not incorporated into the RNA of mouse or human cells as the required enzymes for this pathway are not encoded in the mammalian genome. Consequently, controlled expression of the enzyme uracil phosphoribosyltransferase (UPRT) which converts uracil into uridine monophosphate allows the incorporation of uracil in cell-type specific or temporally controlled manner labelling of RNA. We have generated a transgenic mouse that expresses UPRT in a controllable way and we want to demonstrate the broad utility of this as a tool for purification of RNA or the identification of RNA protein interactions in rare cell subsets in complex mixtures of cells. This is an improvement because it will open access to the RNA makeup of some cell types that cannot be easily purified. Furthermore, the act of purification is known to change the quantity and quality of the RNA within the cell. Thus by reducing the numbers of manipulations a more accurate readout of RNA content or RNA-protein interactions will be obtained.

Impact Summary

The tool that we propose to validate will be practically used by academic and commercial sector scientists who want to study RNA. It will lead, in the longer-term, to datasets that will be exploited by bioinformatics experts. The tool has been designed for maximal utility; it will be applicable to any cell type in which cre-dependent gene expression can be controlled. We can imagine further uses for the transgenic system such as the conversion of pro-drugs to more toxic forms which may provide a new system for tissue specific cell ablation. We anticipate that further uses will be devised for the system once it becomes available to other scientists. The successful take-up of the tool will also represent a contribution to the refinement of the use of animals in research which will benefit the scientific community and the research councils reputation for adherence to the three Rs of animal research. Understanding how RNA is regulated in key rare cell populations is necessary if we are to understand the process of differentiation or the response of cells to environmental changes or stress. This understanding will be needed to exploit the regenerative potential of stem cells. Because of this we think charities interested in regenerative medicine may benefit from this; these include the Wellcome Trust or other charities focussed on research into the diseases of ageing such as cancer, arthritis and diabetes. Certainly, the LLR which has invested in the work of the Turner group will be a beneficiary. The postdoctoral scientist who is the recognised researcher on this grant will have the opportunity to conclude this project and will present the data in various ways. He will be the first author on the resulting publication and develop writing skills during the project. He will also develop project management skills. This will have significant positive impact on this individual's career.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	Tools and Resources Development Fund (TRDF) [2006-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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