Naturally sterile hybrid mice for the production of embryo transfer recipients
Lead Research Organisation:
University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics
Abstract
Genetically modified (GM) mouse models provide a powerful means of attributing function to DNA sequences. As investigations of how DNA variation and mutation contributes to disease advance, demand for GM mouse models increases. Recently, this demand has been aided by the new CRISPR technology, which permits the production of models at unprecedented efficiencies.
To generate and distribute GM mouse models, embryos are manipulated and/or cultured in vitro and require transfer into recipient female mice who then act as foster mothers for the transferred embryos. These foster females are prepared by breeding with sterile male mice, which renders them in a state known as pseudopregnancy, enabling them to receive embryos and carry them to term.
Traditionally the male mice used for this purpose have been made sterile by vasectomy, which requires that the male mice be anaesthetized and surgery performed. After recovery, the male mice are tested for sterility prior to being used to generate recipient female mice for embryo transfer. As an alternative, certain genetically modified mice strains are known where the gene knock-out or transgene results in male sterility and these can be used instead of vasectomized males. Clearly, these genetically sterile male mice provide a good alternative, avoiding the need for performing surgical vasectomy, however the breeding of these genetically sterile mice lines requires lots of breeding procedures and many mice are wasted in their production. Furthermore, some of the genetic constructs used to achieve genetic sterility are not 100% reliable and random inactivation of these constructs sometimes results in a restored fertility. Again, mice must then be tested for sterility prior to use.
We have been exploring hybrid mice, which are the offspring that are produced when two mice of different strains or species are mated together. Certain hybrids show a natural male sterility that is present in 100% of the males. These phenomenon is well recognized from nature - a common example would be the sterile mule that is generated when a horse and a donkey interbreed.
In preliminary experiments, we have used these naturally sterile hybrids for the production of embryo transfer recipients and find that they are just as good at producing foster mothers as the standard vasectomized males. The use of these sterile hybrids represents an important 3Rs impact, as, similar to genetically sterile males, surgical vasectomies are no longer needed. However, when compared to genetically sterile male, the production of naturally sterile hybrids is much more efficient as all males resulting from the hybrid interbreeding are sterile and can be used. In contrast genetically sterile males are inefficiently produced and there is considerable wastage.
We plan to continue our work with sterile hybrids for this purpose, but would like to disseminate the method to other research centres and have asked collaborators at the University of Manchester and at the Mary Lyons Centre, Harwell, to test the performance of these sterile hybrids against standard vasectomized males and genetically sterile males. With a confirmation of this approach at three independent sites, we then hope to convince the centres world-wide who produce and distribute GM mice to adopt this strategy and hope to cryopreserve many hybrid embryos to facilitate the transfer of this technology.
In addition, we plan to establish whether the female hybrids could serve as embryo transfer recipients, meaning that there would be no wastage of mice from the production of the sterile hybrids.
To generate and distribute GM mouse models, embryos are manipulated and/or cultured in vitro and require transfer into recipient female mice who then act as foster mothers for the transferred embryos. These foster females are prepared by breeding with sterile male mice, which renders them in a state known as pseudopregnancy, enabling them to receive embryos and carry them to term.
Traditionally the male mice used for this purpose have been made sterile by vasectomy, which requires that the male mice be anaesthetized and surgery performed. After recovery, the male mice are tested for sterility prior to being used to generate recipient female mice for embryo transfer. As an alternative, certain genetically modified mice strains are known where the gene knock-out or transgene results in male sterility and these can be used instead of vasectomized males. Clearly, these genetically sterile male mice provide a good alternative, avoiding the need for performing surgical vasectomy, however the breeding of these genetically sterile mice lines requires lots of breeding procedures and many mice are wasted in their production. Furthermore, some of the genetic constructs used to achieve genetic sterility are not 100% reliable and random inactivation of these constructs sometimes results in a restored fertility. Again, mice must then be tested for sterility prior to use.
We have been exploring hybrid mice, which are the offspring that are produced when two mice of different strains or species are mated together. Certain hybrids show a natural male sterility that is present in 100% of the males. These phenomenon is well recognized from nature - a common example would be the sterile mule that is generated when a horse and a donkey interbreed.
In preliminary experiments, we have used these naturally sterile hybrids for the production of embryo transfer recipients and find that they are just as good at producing foster mothers as the standard vasectomized males. The use of these sterile hybrids represents an important 3Rs impact, as, similar to genetically sterile males, surgical vasectomies are no longer needed. However, when compared to genetically sterile male, the production of naturally sterile hybrids is much more efficient as all males resulting from the hybrid interbreeding are sterile and can be used. In contrast genetically sterile males are inefficiently produced and there is considerable wastage.
We plan to continue our work with sterile hybrids for this purpose, but would like to disseminate the method to other research centres and have asked collaborators at the University of Manchester and at the Mary Lyons Centre, Harwell, to test the performance of these sterile hybrids against standard vasectomized males and genetically sterile males. With a confirmation of this approach at three independent sites, we then hope to convince the centres world-wide who produce and distribute GM mice to adopt this strategy and hope to cryopreserve many hybrid embryos to facilitate the transfer of this technology.
In addition, we plan to establish whether the female hybrids could serve as embryo transfer recipients, meaning that there would be no wastage of mice from the production of the sterile hybrids.
Technical Summary
To produce and distribute genetically modified (GM) mouse models, pseudopregnant embryo transfer recipients are required. For their preparation, vasectomized mice are commonly used, generated by surgical procedures associated with pain and discomfort. Sterile GM strains provide a non-surgical replacement. However, maintenance of GM strains requires extensive breeding and genotyping, which are regulated procedures and their breeding leads to a large wastage of animals, as mice of incorrect genotype are discarded.
In contrast, certain wild-type hybrid males, generated by the simple intercrossing of two different wild-type strains, are sterile. We have been exploring the use of these sterile hybrids as alternatives for producing pseudopregnant embryo transfer recipients and find their behaviour to be indistinguishable from those generated by vasectomized males.
The method provides two substantial 3R impacts
- refinement, when comparing the use of these naturally sterile hybrids with vasectomized males, as clearly no surgical procedures are necessary
- reduction, when comparing the use of these naturally sterile hybrids with sterile GM mice, as the breeding is substantially more efficient, with all males born from production crosses being suitable for use.
The value of a 3Rs initiative is only as good as the uptake in the community. We thus plan to select a naturally sterile hybrid, which can be generated from strains which are common and easily sourced: the F1 hybrid between Mus spretus and C57BL/6 is a good candidate and we have confirmed the sterility of this strain and its good husbandry characteristics.
We seek funding from the NC3Rs to validate the use of this hybrid as a replacement for surgical vasectomized or sterile GM males. Importantly, we plan to achieve this comparison within three different research environments and thus validate its dissemination to achieve a maximal 3Rs impact.
In contrast, certain wild-type hybrid males, generated by the simple intercrossing of two different wild-type strains, are sterile. We have been exploring the use of these sterile hybrids as alternatives for producing pseudopregnant embryo transfer recipients and find their behaviour to be indistinguishable from those generated by vasectomized males.
The method provides two substantial 3R impacts
- refinement, when comparing the use of these naturally sterile hybrids with vasectomized males, as clearly no surgical procedures are necessary
- reduction, when comparing the use of these naturally sterile hybrids with sterile GM mice, as the breeding is substantially more efficient, with all males born from production crosses being suitable for use.
The value of a 3Rs initiative is only as good as the uptake in the community. We thus plan to select a naturally sterile hybrid, which can be generated from strains which are common and easily sourced: the F1 hybrid between Mus spretus and C57BL/6 is a good candidate and we have confirmed the sterility of this strain and its good husbandry characteristics.
We seek funding from the NC3Rs to validate the use of this hybrid as a replacement for surgical vasectomized or sterile GM males. Importantly, we plan to achieve this comparison within three different research environments and thus validate its dissemination to achieve a maximal 3Rs impact.
Planned Impact
This project aims at using naturally sterile hybrid male mice as a replacement for either surgically vasectomized or genetically sterile male mice
Generating sterile mice by the simple mating of two wild-type strains, can be considered a Refinement over the preparation of sterile males by surgical vasectomy. The breeding of two wild-type strains to generate males which are naturally sterile obviates the need for any surgical procedures.
As an alternative to using vasectomized mice, some genetically modified (GM) mouse strains have a phenotype of male sterility and these models are being used for the preparation of pseudopregnant embryo transfer recipients. Compared to this approach, the idea of using naturally sterile hybrid mice represents a Reduction, since their production is more efficient and fewer mice are wasted in their production. Sterile hybrids are made by mating two strains of mice together and 100% of the male offspring are sterile and can be used to generate pseudopregnant embryo transfer recipients. In contrast GM sterile male mice have to be generated by mating heterozygous mice (in the case of Knock-outs) or hemizygous transgenic females (in the case of transgenes). Not only does the breeding GM mice constitute a regulated procedure and necessitates genotyping, but only a subset of the male mice produced are of the correct genotype and subsequently sterile. A large wastage of mice results from the maintenance and production of these genetically sterile strains.
I have estimated that 12,000 sterile male mice are required each year in the world-wide production facilities responsible for the generation and distribution of GM mice.
The use of naturally sterile hybrids instead of surgically prepared vasectomized mice would therefore result in a Refinement impact of 12,000 / year. Considering the naturally sterile hybrids instead of genetically sterile mice, the Reduction impact would be 24,000 (for a transgenic sterile line, where hemizygous transgenic males are sterile) or 72,000 (for a knock-out line, where homozygous males are sterile).
A further 3Rs impact could result through the use of female hybrid mice produced during the generation of the hybrid males as embryo transfer recipients. This would mean that there would be no wastage of animals from the breeding required to generate the males. Assuming 12,000 mice sterile hybrid males are required per year, then 12,000 female hybrids would be generated, providing a further Reduction of this amount.
The project aids the dissemination of this 3Rs initiative by coordinating the validation of the naturally sterile hybrids for the purpose of generating pseudopregnant embryo transfer females within three research environments - University of Oxford, Harwell, University of Manchester. Furthermore, the dissemination and impact of the approach will be maximized by preparing a cryoarchive of the sterile hybrid strain for distribution.
After presenting the preliminary data from our studies with naturally sterile hybrids, we were contacted by many groups who are interested in adopting this approach within their own core facilities. Subsequently, we anticipate that the 3Rs impact both locally and within the wider research community, both nationally and internationally will be considerable.
The economic consequences of the research results could reduce the cost of studies that require GM mouse models substantially, reducing the cost of basic biomedical research and drug discovery programmes. Positive societal consequences of more economic biomedical research are clear, in that research and development tackling genetic disease and exploring novel therapeutics of all sorts is facilitated.
The hybrid strain will be made freely available to the academic community and extensive dissemination at both academic and technical meetings is planned. Public awareness of this 3R research will be encouraged by interactions with press and participation at Science Festivals.
Generating sterile mice by the simple mating of two wild-type strains, can be considered a Refinement over the preparation of sterile males by surgical vasectomy. The breeding of two wild-type strains to generate males which are naturally sterile obviates the need for any surgical procedures.
As an alternative to using vasectomized mice, some genetically modified (GM) mouse strains have a phenotype of male sterility and these models are being used for the preparation of pseudopregnant embryo transfer recipients. Compared to this approach, the idea of using naturally sterile hybrid mice represents a Reduction, since their production is more efficient and fewer mice are wasted in their production. Sterile hybrids are made by mating two strains of mice together and 100% of the male offspring are sterile and can be used to generate pseudopregnant embryo transfer recipients. In contrast GM sterile male mice have to be generated by mating heterozygous mice (in the case of Knock-outs) or hemizygous transgenic females (in the case of transgenes). Not only does the breeding GM mice constitute a regulated procedure and necessitates genotyping, but only a subset of the male mice produced are of the correct genotype and subsequently sterile. A large wastage of mice results from the maintenance and production of these genetically sterile strains.
I have estimated that 12,000 sterile male mice are required each year in the world-wide production facilities responsible for the generation and distribution of GM mice.
The use of naturally sterile hybrids instead of surgically prepared vasectomized mice would therefore result in a Refinement impact of 12,000 / year. Considering the naturally sterile hybrids instead of genetically sterile mice, the Reduction impact would be 24,000 (for a transgenic sterile line, where hemizygous transgenic males are sterile) or 72,000 (for a knock-out line, where homozygous males are sterile).
A further 3Rs impact could result through the use of female hybrid mice produced during the generation of the hybrid males as embryo transfer recipients. This would mean that there would be no wastage of animals from the breeding required to generate the males. Assuming 12,000 mice sterile hybrid males are required per year, then 12,000 female hybrids would be generated, providing a further Reduction of this amount.
The project aids the dissemination of this 3Rs initiative by coordinating the validation of the naturally sterile hybrids for the purpose of generating pseudopregnant embryo transfer females within three research environments - University of Oxford, Harwell, University of Manchester. Furthermore, the dissemination and impact of the approach will be maximized by preparing a cryoarchive of the sterile hybrid strain for distribution.
After presenting the preliminary data from our studies with naturally sterile hybrids, we were contacted by many groups who are interested in adopting this approach within their own core facilities. Subsequently, we anticipate that the 3Rs impact both locally and within the wider research community, both nationally and internationally will be considerable.
The economic consequences of the research results could reduce the cost of studies that require GM mouse models substantially, reducing the cost of basic biomedical research and drug discovery programmes. Positive societal consequences of more economic biomedical research are clear, in that research and development tackling genetic disease and exploring novel therapeutics of all sorts is facilitated.
The hybrid strain will be made freely available to the academic community and extensive dissemination at both academic and technical meetings is planned. Public awareness of this 3R research will be encouraged by interactions with press and participation at Science Festivals.