There is currently a significant need for populations of animals with specified genotypes which cannot be satisfied by the importation of animals from the wild or by the identification and propagation of valuable founder animals by selective breeding. Indian-origin, rhesus macaques carrying the MHC class 1 allele, A*01, are particularly needed for vaccine development research. The objective of this application is to demonstrate that ARTs can be applied to the rapid, efficient propagation of a valuable founder animal and to the production of identical twins using existing technology, thereby establishing a new approach to satisfying animal requirements of the biomedical research community. The rationale for focusing on a homozygous, founder male is simple. All offspring produced by this animal will be Mamu-A*01 positive. Moreover, if heterozygous carriers of the A*01 allele are used as oocyte donors, 50% of the offspring will be homozygous for the allele, creating additional founder animals. The applicants have access to a male at the NERPRC that is presumed homozygous for the Mamu-A*01 allele based on the fact that 100% (15 of 15; K. Mansfield, personal communication) of his offspring are Mamu-A*01 positive. The number of sperm in an average ejaculate, when used in conjunction with ICSI, can result in the production of hundreds, if not thousands, of embryos. Sperm collection, preservation during transportation or storage and use of ICSI is established technology in the applicants' laboratory, allowing a high probability of success. The investigators propose to establish a new paradigm for the efficient, cost effective propagation of founder monkeys and populations of valuable, genetically defined macaques, including identical twins, through conduction of the following specific aims: 1) Produce 25 Mamu-A*01 positive animals in the first year and 50 animals per year for the duration of this study using sperm from a homozygous Mamu-A*01 positive donor.
This aim will establish the paradigm and create new populations of heterozygous and homozygous animals without impacting the natural reproduction of either the sperm or the oocyte donors. The applicants will confirm homozygosity in candidate animals by creating embryos with their gametes and monitoring the presence or absence of the Mamu-A*01 allele in these embryos; and 2) Employ blastomere separation and culture or embryo splitting technology to increase embryo numbers and to produce genetically identical animals. The applicants will select the optimal approach to twinning in year 1 and produce 5 sets of identical twins per year during years 2-5 of the study.
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