Award details

Exploiting cis-limited antigens in livestock

Reference	BB/J00877X/1
Principal Investigator / Supervisor	Professor Nabeel Affara
Co-Investigators / Co-Supervisors	Dr Peter Ellis
Institution	University of Cambridge
Department	Pathology
Funding type	Research
Value (£)	314,220
Status	Completed
Type	Research Grant
Start date	14/04/2012
End date	13/04/2015
Duration	36 months

Abstract

This project applies two complementary approaches to investigate the molecular control of offspring gender balance: (1) a search for endogenous sex specific antigens in a mouse model system using transcriptomic technology; (2) development of a sperm "tagging" system allowing for antigenic separation of X- and Y-bearing sperm. (1) Male mice with deletions on Yq show a non-Mendelian sex ratio among their offspring caused by a functional difference in the fertilising ability of X- and Y-bearing sperm. We reason that this functional difference must be rooted in a difference in the transcript content of the developing X- and Y- cells. Alternative explanations, such as a difference in translation efficiency or post-translation modification, pose a bootstrapping problem: how does that difference itself arise? We will carry out a systematic search for the predicted "responder" gene via microarray analysis of purified, flow sorted spermatid populations in Yq deleted mice, in matched controls, and in mice transgenic for a candidate distorter gene. (2) We will generate "marker" transgenes that selectively label transgene-bearing sperm. IVF protocols for sorting these sperm will be developed, and finally the tag system will be deployed in a mouse model to allow for sperm selection.

Summary

The ability to pre-select the sex of offspring would be hugely important in animal husbandry. In many farm species such as pigs or dairy cattle, female offspring are much more economically important than males, while in other species such as racehorses or beef cattle, the opposite situation holds. In particular, meat from adult male boars can have an "off" flavour known as boar taint, meaning it is uneconomical to raise intact male pigs to high weights. Currently, boar taint is avoided by early slaughter or castration of male pigs, which is ethically undesirable as well as economically wasteful. We are trying to discover ways to sort sperm before fertilisation occurs, i.e. to distinguish between sperm bearing an X chromosome (which will give female offspring) and those bearing a Y chromosome (which will give males). Currently the only way to do this is by using a dye to stain the DNA in the sperm head, and separating the sperm that have more DNA from those that have less - this works because the X chromosome is slightly larger than the Y. This "flow-sorting" process is slow and cumbersome and not commercially viable for some species such as pig. Also, the flow-sorting process involves staining the DNA and shining an ultraviolet laser on it, both of which can potentially damage the DNA. There is thus a strong need for more efficient and safer ways of separating X and Y sperm. We are using two approaches to tackle the problem. Firstly, we are looking to see whether X and Y sperm naturally carry markers on their surface allowing them to be distinguished. We will do this by using flow-sorting to separate developing X- and Y-bearing sperm and looking to see which genes are active in each cell type. If we find genes which are only switched on in one of the two cell types, these are likely to be be useful in developing ways to separate the two. We will carry out this search in normal mice, in a strain of mice which naturally produce 60% female offspring, and ina transgenic strain which has altered levels of a gene we believe to be involved. From previous work, we know that in these strains with a skewed sex ratio, equal numbers of X- and Y-bearing sperm are produced, but that the Y sperm are less effective at fertilising the egg. This means that these strains will help us to home in on genes of interest - i.e. the ones which make X and Y sperm different from each other. Secondly, we want to test whether we can separate sperm using an artificial surface marker. To do this, we will breed transgenic mice which express a marker protein in their sperm cells. Ideally, sperm cells containg the transgene will show this "tag" on the cell surface, while those which do not contain the transgene will remain untagged. This will then allow us to separate the two types of sperm and only use untagged sperm to breed the next generation. In livestock, this would enable sperm selection while preventing genetically modified organisms entering the food chain.

Impact Summary

This project aims to discover specific cell surface antigens allowing the separation of X and Y-bearing sperm and the pre-selection of offspring sex. We also aim to develop synthetic "tags" selectively identifying transgenic sperm. These goals, if realised, would have very substantial commercial impacts. Beneficiaries include, but are not limited to: 1) Farming industry Pre-selection of offspring sex would reduce the number of "undesirable" offspring generated. This will mean less culling of unwanted males/females, more efficient meat production requiring smaller breeding populations, and reduced waste generation / environmental impact. 2) Rare species conservation Captive breeding programmes for endangered species rely critically on the generation of breeding females to maintain the population. Offspring sex selection would thus help ensure the stability of such schemes. 3) Research and biotechnology companies producing transgenic animals In some species, current methods of targeted transgenesis require the creation of chimeric founders, which transmit the transgene to subsequent generations at high or low levels depending on the degree of germ line chimerism. The ability to specifically sort transgenic sperm from non-transgenic sperm would make this latter step more efficient. In terms of timescale, if we are able to identify suitable cell surface markers on X and Y sperm, then commercial development and testing could begin rapidly, within the duration of this project, subject of course to regulatory approval. The sperm tagging project is currently pre-competitive and will require further rounds of optimisation before a commercial sex selection technology can be developed (5-7 years). However, if the search for endogenous cell surface markers is unsuccessful, then the transgenic tag technology is a plausible route to selection of X and Y sperm, given that flow-sorting is slow, cumbersome and not commercially viable in most farm species.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Animal Health, Animal Welfare, The 3 Rs (Replacement, Reduction and Refinement of animals in research)
Research Priority	Animal Health
Research Initiative	LINK: Responsive Mode [2010-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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