Source: CORNELL UNIVERSITY submitted to
NATIONAL ANIMAL GENOME RESEARCH PROGRAM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0177628
Grant No.
(N/A)
Project No.
NYC-473834
Proposal No.
(N/A)
Multistate No.
NRSP-8
Program Code
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2008
Grant Year
(N/A)
Project Director
Antczak, D. F.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
VET JAMES A. BAKER INST. FOR ANIMAL HEALTH
Non Technical Summary
Worldwide demand for high quality animal protein continues to grow at an explosive pace. The fundamental basis for all of animal breeding and the continuing genetic improvement of all agricultural animal species exists within the genome. By developing genome maps for the equine (and other) species, it will ensure increased agricultural efficiency, profitability, and global competitiveness.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30438101080100%
Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1080 - Genetics;
Goals / Objectives
1. Enhance and integrate genetic and physical maps of agriculturally important animals for cross species comparisons and sequence annotation. 2. Facilitate integration of genomic, transcriptional, proteomic and metabolomic approaches toward better understanding of biological mechanisms underlying economically important traits. 3. Facilitate and implement bioinformatic tools to extract, analyze, store and disseminate information.
Project Methods
The approach is to produce and characterize microsatellite markers for linkage mapping of the horse genome (objective #1). Application will include long term studies of genetic spects of reproductive efficiency (objective #2 and #3). This research is being conducted in collaboration with other members of an international workshop group convened to map the genome of the horse.

Progress 10/01/03 to 09/30/08

Outputs
OUTPUTS: During the period 2003 to 2008 inclusive my laboratory worked with the Horse Genome Project workshop on several aspects of genome mapping in the horse. This has culminated in the complete genome sequencing of the horse. This sequence is now available on the World Wide Web on several database, including the following: http://www.uky.edu/Ag/Horsemap/ http://www.ncbi.nlm.nih.gov/genome/guide/horse/ http://www.ncbi.nlm.nih.gov/sites/entrezdb=genomeprj&cmd=Retrieve&do pt=Overview&list_uids=11760 http://genome.ucsc.edu/cgi-bin/hgGateway hgsid=120831969&clade=mammal&org=Horse&db=0 http://www.ensembl.org/Equus_caballus/Info/Index The information in the horse genome has been used to produce two important genome wide resources: 1) a 60,000 member single nucleotide polymorphism (SNP) chip for detecting genetic variation in horses all across the genome, and 2) a 14,000 member gene expression microarray that can be used to estimate gene expression levels in all major genetic pathways. These results have been widely distributed through publications in scientific journals, articles in trade magazines for stakeholders (e.g. horse owners and breeders), and articles in newspapers, radio, and television. PARTICIPANTS: Project Director: D. F. Antczak Technician: Donald Miller Partner organizations: Harry M. Zweig Memorial Fund for Equine Research in New York State Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University National Institutes of Health Dorothy Russell Havemeyer Foundation, Inc. Morris Animal Foundation Collaborators: Dr. Dorothy Ainsworth, Cornell University Dr. Bettina Wagner, Cornell University Many others collaborating scientists in the Horse Genome Project Workshop, from the US and abroad. Training: Several Cornell undergraduates, veterinary students, and graduate students have participated on this project during its lifetime. TARGET AUDIENCES: The target audience is horsemen and women, including horse breeders and owners. As described in a previous section, the Horse Genome Project Workshop members have made numerous presentations to this audience and also reached it through various media channels. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The information in the horse genome has been used by equine geneticists and clinicians to identify mutant genes causing inherited diseases of the horse. Genetic tests developed with this knowledge is available to inform horse breeders on mating strategies that will result in offspring free of particular genetic diseases. Use of the new SNP chip described above will accelerate the identification of additional genes controlling important traits in the horse. In parallel, the new expression microarray is finding wide application in studies of pathology and physiology of the horse

Publications

  • Wagner, B., Hillegas, J. M., Brinker, D., Horohov, D. W., and Antczak, D. F. (2008). Characterization of monoclonal antibodies to equine interleukin-10 and detection of T regulatory 1 cells in horses. Vet. Immunol. Immunopathol. 122:57-64.
  • de Mestre, A.M., Miller, D., Roberson, M.S., Liford, J., Chizmar, L., McLaughlin, K.E. and Antczak, D.F. (2008) Glial cells missing 1 is induced in differentiating equine chorionic girdle trophoblast cells. Biol. Reprod. Oct 29 [Epub ahead of print].


Progress 10/01/06 to 09/30/07

Outputs
In 2007 our research group continued our long-standing collaboration with other member laboratories of the Horse Genome Project. Of particular significance was the decision by the NIH to undertake whole genome sequencing of the horse. The raw sequence data was obtained at the MIT Broad Institute sequencing center. The horse chosen for the sequencing was a Thoroughbred mare from our research herd at Cornell. She was chosen because of her relatively high level of inbreeding. She is part of a group of horses selected for homozygosity at the Major Histocompatibility Complex, and she is related to the stallion that was the donor horse for the equine Bacterial Artificial Chromosome (BAC) library that was produced several years ago. Our laboratory has been using the raw sequence since the middle of 2006 to produce PCR primers for genes expressed in the early embryo and placenta. The primers are being tested on samples of equine conceptuses obtained by non-surgical uterine lavage of mares between 15 and 30 days of pregnancy. The conceptuses are microdissected into their component tissues before RNA is made from them. We also continued to make progress in translating information from the horse genome into the in vitro production of proteins of immunological interest (Wagner et al., 2007). This technical advance lead to Dr. Bettina Wagner's participation as a leading member of a new USDA consortium grant to develop new monoclonal antibody reagents to horse immune system molecules. The major genetic focus of our laboratory remains the Major Histocompatibility Complex (MHC) of the horse. We have continued to study the polymorphisms and gene structure of MHC class I genes of the horse, using reagents produced in previous years.

Impacts
Our research has added significantly to the understanding of the overall organization of the equine genome, particularly of the equine Major Histocompatibility Complex. This information is important for applications to equine health, and in comparison to similar information about other species, for a better understanding of the mechanisms of evolution. In particular, our work focuses on genes of the immune system. Increased knowledge of the horse immune system is necessary for the development of better vaccines, and for understanding diseases that involve the immune system, such as allergies. Another application of this research is in reproduction. Our laboratory studies how the fetus avoids destruction by the immune system of the mother during pregnancy.

Publications

  • No publications reported this period


Progress 01/01/06 to 12/31/06

Outputs
In 2006 our research group continued our long-standing collaboration with other member laboratories of the Horse Genome Project. Of particular significance was the decision by the NIH to undertake whole genome sequencing of the horse. The raw sequence data was obtained in 2006 at the MIT Broad Institute sequencing center. The horse chosen for the sequencing was a Thoroughbred mare from our research herd at Cornell. She was chosen because of her relatively high level of inbreeding. She is part of a group of horses selected for homozygosity at the Major Histocompatibility Complex, and she is related to the stallion that was the donor horse for the equine Bacterial Artificial Chromosome (BAC) library that was produced several years ago. Our laboratory has been using the raw sequence since the middle of 2006 to produce PCR primers for genes expressed in the early embryo and placenta. The primers are being tested on samples of equine conceptuses obtained by non-surgical uterine lavage of mares between 15 and 30 days of pregnancy. The conceptuses are microdissected into their component tissues before RNA is made from them. We also continued to make progress in translating information from the horse genome into the in vitro production of proteins of immunological interest (Wagner et al., 2006). This technical advance lead to Dr. Bettina Wagner's participation as a leading member of a new USDA consortium grant to develop new monoclonal antibody reagents to horse immune system molecules. The major genetic focus of our laboratory remains the Major Histocompatibility Complex (MHC) of the horse. We have continued to study the polymorphisms and gene structure of MHC class I genes of the horse, using reagents produced in previous years.

Impacts
Our research has added significantly to the understanding of the overall organization of the equine genome, particularly of the equine Major Histocompatibility Complex. This information is important for applications to equine health, and in comparison to similar information about other species, for a better understanding of the mechanisms of evolution. In particular, our work focuses on genes of the immune system. Increased knowledge of the horse immune system is necessary for the development of better vaccines, and for understanding diseases that involve the immune system, such as allergies. Another application of this research is in reproduction. Our laboratory studies how the fetus avoids destruction by the immune system of the mother during pregnancy.

Publications

  • Wagner, B., Hillegas, J.M., Antczak, D.F. 2006. A monoclonal antibody to equine interleukin-4. Vet Immunol Immunopathol. 110:363-367.


Progress 01/01/05 to 12/31/05

Outputs
In 2005 our research group continued our long-standing collaboration with other member laboratories of the Horse Genome Project. This work has resulted in the production of a first generation equine microarray for expression profiling. This array is expected to be available for testing in early 2006. It contains just over 8,000 unique horse ESTs. We also made significant progress in translating information from the horse genome into the in vitro production of proteins of immunological interest (Wagner et al., 2005). This technical advance lead to the participation of Dr. Bettina Wagner as a leading member of a new USDA consortium grant to develop new monoclonal antibody reagents to horse immune system molecules. The major genetic focus of our laboratory remains the Major Histocompatibility Complex (MHC) of the horse. In the past year we achieved a significant breakthrough in characterization of horse MHC class I genes (Tallmadge and Antczak, 2005). We identified 7 MHC class I genes that are expressed as mRNA by lymphocytes from horses carrying the Equine Leukocyte Antigen (ELA) A3 haplotype. Our laboratory also participated in a workshop that defined rules for naming MHC genes and alleles in domestic species (Robinson et al., 2006). Our laboratory will serve as the duty lab responsible for entering new horse MHC sequences into the IPD database (http://www.ebi.ac.uk/ipd).

Impacts
Our research has added significantly to the understanding of the overall organization of the equine genome, particularly of the equine Major Histocompatibility Complex. This information is important for applications to equine health, and in comparison to similar information about other species, for a better understanding of the mechanisms of evolution. In particular, our work focuses on genes of the immune system. Increased knowledge of the horse immune system is necessary for the development of better vaccines, and for understanding diseases that involve the immune system, such as allergies. Another application of this research is in reproduction. Our laboratory studies how the fetus avoids destruction by the immune system of the mother during pregnancy.

Publications

  • Wagner, B., Robeson, J., McCracken, M., Wattrang, E., and Antczak, D. F. 2005. Horse cytokine/IgG fusion proteins, mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines. Vet. Immunol. Immunopathol. 105:1-14.
  • Tallmadge, R.L. and Antczak, D. F. 2005. Genomic characterization of MHC class I genes of the horse. Immunogenetics 57:763-774.
  • Ellis, S.A., Bontrop, R.E., Antczak, D.F., Ballingall, K., Davies, C.J., Kaufman, J., Kennedy, L.J., Robinson, J., Smith, D.M., Stear, M.J., Stet, R.J.M., Waller, M.J., Walter, L., and Marsh, S.G.E. 2006. ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005. Immunogenetics Jan.3; :1-6 [Epub ahead of print]


Progress 01/01/04 to 12/31/04

Outputs
In 2004 our laboratory published two important papers on the genetics of immune response genes in the horse. The first was a collaborative study with Dr. Masanori Kasahara of Japan on the genes encoding the cellular receptors on Natural Killer cells in the horse (Takahashi et al., 2004). The second paper described a Bacterial Artificial Chromosome (BAC) contig map of the horse Immunoglobulin heavy chain constant gene region. In this study we identified a total of 7 IgG isotypes and the gene encoding horse IgD (Wagner et al., 2004). In other research we have continued to investigate the genomic structure and polymorphism of MHC class I and class II genes of the horse, using the recently constructed BAC contig of the equine MHC region. Finally, we have continued to participate in collaborative research with other members of the Horse Genome Project who work under the auspices of the NRSP-8 project.

Impacts
Our research has added significantly to the understanding of the overall organization of the equine genome. This information is important for applications to equine health, and in comparison to similar information about other species, for a better understanding of the mechanisms of evolution. In particular, our work focuses on genes of the immune system. Increased knowledge of the horse immune system is necessary for the development of better vaccines, and for understanding diseases that involve the immune system, such as allergies. Another application of this research is in reproduction. Our laboratory studies how the fetus avoids destruction by the immune system of the mother during pregnancy.

Publications

  • Takahashi, T., Yawata, M., Raudsepp, T., Lear, T. L., Chowdhary, B. P., Antczak, D. F., and Kasahara, M. 2004. Natural killer cell receptors in the horse: evidence for the existence of multiple transcribed LY49 genes. European J. of Immunology 34: 773-784.
  • Wagner, B., Miller, D. C., Lear, T. L., and Antczak, D. F. 2004. The Complete Map of the Ig Heavy Chain Constant Gene Region Reveals Evidence for Seven IgG Isotypes and for IgD in the Horse. J. Immunol. 173: 3230-3242.


Progress 01/01/03 to 12/31/03

Outputs
In 2003 we published the first full length sequence of the horse beta-2 microglobulin gene, including the upstream regulatory region (Tallmadge et al., 2003). This allowed comparison with the sequence of a horse Major Histocompatibility complex (MHC) class I gene published in the previous year. We have found evidence that these genes are coordinately regulated in the horse placenta during early development. In other work on the MHC, we collaborated with other members of the multi-state project to construct the first Bacterial Artificial Chromosome (BAC) contig map of the horse MHC (Gustafson et al., 2003). This is a major advance that will enable detailed study of the various subregions of the horse MHC. In another study of genes of the equine immune system, we published a characterization of the horse IGHA gene (Wagner et al., 2003). We also participated in two major gene mapping studies that were published in 2003: the construction of the first generation radiation hybrid map of the horse (Chowdhary et al., 2003) and the second iteration of the international equine gene mapping workshop half-sibling linkage map (Guerin et al., 2003).

Impacts
Our research has added significantly to the understanding of the overall organization of the equine genome. This information is important for applications to equine health, and in comparison to similar information about other species, for a better understanding of the mechanisms of evolution. In particular, our work focuses on genes of the immune system. Increased knowledge of the horse immune system is necessary for the development of better vaccines, and for understanding diseases that involve the immune system, such as allergies. Another application of this research is in reproduction. Our laboratory studies how the fetus avoids destruction by the immune system of the mother during pregnancy.

Publications

  • Tallmadge, R.L., Lear, T.L., Johnson, A.K., Guerin, G., Millon, L.V., Carpenter, S.L. and Antczak, D.F. 2003. Characterization of the beta-2-microglobulin gene of the horse. Immunogenetics 54:725-733.
  • Chowdhary, B.P., Raudsepp, T., Kata, S.R., Goh, G., Millon, L.V., Allan, V., Piumi, F., Guerin, G., Swinburne, J., Binns, M., Mickelson, J., Murray, J., Antczak, D.F., Womack, J.E., Skow, L.C. 2003. The first generation whole genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes. Genome Research 13:742-751.
  • Guerin, G., Bailey, E., Bernoco, D., Anderson, I., Antczak, D.F., Bell, K., Biros, I., Bowling, A.T., Brandon, R., Cholewinski, G., Colling, D., Eggleston, M., Flynn, J., Gralak, B., Hasegawa, T., Ketchum, M., Lindgren, G., Lyons, L., Millon, L.V., Mariat, D., Murray, J., Neau, A., Roed, K., Sandberg, K., Skow, L.C., Tammen, I., Van Dyk, E., Weiss, B., and Zeigle, J. 2003. The Second Iteration of the International Equine Gene Mapping Workshop: Half-Sibling Linkage Map. Anim. Genet. 34:161-168.
  • Wagner, B., Greiser-Wilke, I., and Antczak, D.F. 2003. Characterization of the horse (Equus caballus) IGHA gene. Immunogenetics 55:552-560.
  • Gustafson, A. L., Tallmadge, R. L., Ramlachan, N., Miller, D., Bird, H., Antczak, D. F., Raudsepp, T., Chowdhary, B. P., and Skow, L. C. 2003. An ordered BAC contig map of the equine Major Histocompatibility Complex. Cytogenetic and Genome Research 102:189-195.


Progress 01/01/02 to 12/31/02

Outputs
Aim 1: In 2001 we published the first full genomic sequence of a horse MHC class I gene, including approx. 1,000 bp upstream of the start codon, in a region containing several regulatory elements. This was followed by a parallel characterization of the horse beta-2 microglobulin gene. Mining a new (BAC) Library for Immune System Genes: In previous years we had worked with BAC clones provided by colleagues in France & Texas from equine BAC libraries produced at those sites. The libraries suffered from lack of coverage, & the origin & MHC types of the horses used to construct the libraries were unknown. These deficiencies were remedied in 2002, with the completion of a new BAC library by our colleague, Dr. de Jong. The library was made using DNA from one of Cornell's MHC homozygous research stallions.. During the past 6 months the library was successfully screened for MHC class I genes. It is important to understand which and how many horse MHC class I genes are expressed in the placenta, and how their expression is regulated. The new sequence data and analysis of other MHC class I containing clones will help define the MHC class I region of the horse and provide locus specific gene probes for functional studies of these genes in the developing horse placenta. Aim 2: In 2002 we published a study of the expression of MHC class I and beta-2 microglobulin genes in the horse placenta using RT-PCR and Northern blotting assays (Bacon et al., 2002). Northern blotting suffers from a relative lack of sensitivity compared to quantitative real time PCR, and it also requires more tissue, a particular disadvantage in studies of early embryonic tissue. Over the past two years we have developed Q-RT-PCR primers for MHC class I and beta-2 microglobulin genes, and additionally for several control genes, that are being applied to placental tissues from early horse conceptuses to confirm and extend the studies cited above.

Impacts
Genetic studies of the horse in the past decade have already resulted in the molecular identification of three of the most important inherited diseases of horses and the development of commercially available tests for the defective genes. The widespread use of these tests would result in the elimination of these three genetic diseases from the horse population. This would have a positive outcome on horse breeders and owners, and reduce animal suffering. Additional applications of genomics in equine husbandry and medicine hold promise for developing new approaches to treatment and prevention of many important diseases and blocks to production, such as infections, lameness, and reduced breeding efficiency. The molecular tools of the horse genome project, including whole genome scanning using the markers of the linkage map, and expression profiling using individual genes and gene arrays, are already being applied to many problems such as these.

Publications

  • Carpenter, S., Baker, J.M., Bacon, S.J., Hopman, T., Maher, J., Ellis, S.A., and Antczak, D.F. 2001. Molecular and functional characterization of genes encoding horse MHC class I antigens. Immunogenetics 53:802-809
  • Bacon, S.J., Ellis, S.A., and Antczak, D.F. 2002. Control of expression of Major Histocompatibility Complex genes in horse trophoblast. Biol. Repro. 66:1612-1620.


Progress 01/01/00 to 12/31/00

Outputs
The role of the Cornell laboratory in the Regional Project has been to characterize genomic clones containing specific genes from equine Bacterial Artificial Chromosome (BAC) libraries produced by other Regional Project participants. They have concentrated on genes of the immune system, with emphasis on the Major Histocompatibility Complex (MHC). BAC clones containing the horse beta-2 microglobulin gene: Beta-2 microglobulin (beta-2 m) is the invariant light chain which forms a complex with MHC class I heavy chains and is responsible for stable expression of the MHC class I molecule at the cell surface. It is under study as part of an investigation of the control of expression of MHC genes in the equine placenta (Carpenter et al., 2000, abst.). In mid-1999 two BAC clones were received containing the horse beta-2 m gene from Dr. Gerard Guerin of INRA in Jouay-en-Josas in France. Dr. Guerin is a regular member of the Horse Genome Project Workshop group, and he has produced an equine BAC library for use by equine geneticists (Godard et al., 1998). Plans are underway for characterization of the horse beta-2 m gene. Southern blotting using a probe generated from a beta-2 m CDNA (Ellis and Martin, 1993) confirmed that both of these BACs contained the gene, but one BAC contained a larger positive fragment at approximately 8 kb. This was selected for subcloning in order to obtain as much genomic sequence as possible, including the promoter region. The horse beta-2 m gene was localized to band q23-q25 of horse chromosome I (ECAL) by fluorescence in situ hybridization (FISH) performed by our Horse Genome Project colleagues at the University of Kentucky using one of the BAC clones. ECAL contains parts of human chromosome 15 (HSA 1 5) [Raudsepp et al., 1996), and the human beta2 m gene is located on HSA15. Therefore, it is likely that the assignment of horse beta-2 m to ECAL is correct. Subsequent data obtained indicates there may also be a pseudogene contained in the same region. This is probably close enough to the active gene not to be resolved by FISH mapping. The 8 kb band identified from the BAC digest to contain beta-2 m was in fact a doublet of two bands very similar in size. Both of these bands are positive for beta-2 m by PCR. Sequencing of PCR products generated from the central region of this gene in several equine species revealed the presence of a frameshift mutation which is further evidence for the presence of a pseudogene coainplifying with the active copy. Long range PCR has also identified products across putative intron sites which correspond to both the sizes predicted by genomic DNA and CDNA. This implies that the pseudogene may be processed. Genomic sequence has been obtained from intron 2 of beta-2 m from the BAC clone. Further work would seek to subclone the active copy of this gene and elucidate the promoter sequence and also investigate the presence of the pseudogene.

Impacts
By developing genome maps for the equine (and other) species, it will ensure increased agricultural efficiency, profitability, and global competitiveness.

Publications

  • Bailey, E., Marti, E., Fraser, D.G., Antczak, D, and Lazary, S. (2000) Immunogenetics of the horse: In: The Genetics of the Horse, chapt. 7 (A.T. Bowling and A. Ruvinsky, eds. )CAB Internatinal, Oxon, U.K., pp. 123-155.


Progress 01/01/99 to 12/31/99

Outputs
Two Bacterial Artificial Chromosome (BAC) clones have been obtained from a horse genomic library constructed in the laboratory of Dr. Gerard Guerin in France. Dr. Guerin is the lead scientist from his laboratory, and he is a full collaborating member of the International Horse Genome Project and the USDA's NRSP-8 National Council Genome Project (Equine Section). The clones were identified by screening the library with PCR primers for horse beta-2 microglobulin sent by our laboratory to France. At Cornell we are investigating the structure and genetics of the horse beta-2 microglobulin gene which lies within these clones. Current work focuses on subcloning smaller fragments for sequencing. The characteristics of the horse beta-2 microglobulin gene will be compared to those of a horse Major Histocompatibility Complex (MHC) class I gene that has already been characterized in our laboratory. We are studying the mechanism by which MHC antigen expression is suppressed by the trophoblast cells which form the outer layer of the placenta.

Impacts
This research is important for understanding pregnancy loss and for developing new methods of contraception. It also has relevance to clinical organ transplantation in humans, and in cancer biology.

Publications

  • No publications reported this period