Research articleGermline transmission in transgenic Huntington's disease monkeys
Introduction
The rationale for developing the Huntington's disease (HD) monkey was to create a large preclinical animal model of HD that could pass the mutant huntingtin (mHTT) transgene down through generations, such that offspring with predictable phenotypes could facilitate basic and preclinical research in HD, which remains a terminal disease today [1], [3]. Thus, with such a large animal model, we could develop and test novel treatments using clinical assessment tools and methods similar to those used in humans [1], [3]. A cohort of three male HD monkeys (rHD6, rHD7 and rHD8; rHDs6–8) carrying the mHTT transgene containing exons 1 to 10 of the human HTT gene with an expanded polyglutamine (polyQ) tract (69–72Q) in exon 1 driven by the human HTT promoter were used as founders for the production of F1 HD monkeys [4], [5]. We have been conducting an ongoing longitudinal study to monitor disease onset and progression in the HD monkeys by using a variety of clinical assessment tools, including noninvasive imaging, cognitive behavioral assessments, and molecular profiling studies [4], [5]. Although our model shows great promise as a preclinical large animal model for HD, the practicality of using the HD monkeys in a clinical research setting depends largely on whether the mHTT transgene can be transmitted through the germline, thereby making the cohort of HD monkeys readily expandable and available to researchers. Unlike rodents and small primate species, such as the marmoset, rhesus macaques reach sexual maturity at approximately 4 to 5 years of age, with a subsequent seasonal reproductive cycle peaking between fall and late spring [1], [6]. Because of this, the inheritability of the mHTT transgene has been assessed only recently with F0 HD monkeys reaching pubertal age.
Section snippets
Animal models
Huntington's disease monkeys were generated by lentiviral-mediated transgenesis as previously described [7]. rHD6, rHD7, and rHD8 are male rhesus macaques that carry exons 1 to 10 of the HTT gene with expanded polyQ repeats (66–74Q, 66–72Q, and 71–74Q), under the control of the human HTT promoter [5], [7]. All monkeys received the same treatments and procedures designed for the longitudinal study, including magnetic resonance imaging scans, cognitive behavioral assessments, and scheduled blood
Derivation and genotyping of transgenic HD monkeys' germline and embryonic stem cells
Germline transmission of the mHTT transgene in HD monkeys was assessed primarily by performing PCR genotyping using primers that specifically amplified a region (including the polyQ expansion in exon 1) of the mHTT transgene (Fig. 1A). To confirm the presence of transgene in germ cells, semen was collected from each of the three F0 founders (rHDs6–8). Genotype results confirmed the presence of the mHTT transgene in HD monkey sperm from each of the F0 HD monkeys (Fig. 1B). Polymerase chain
Discussion
Development of a transgenic nonhuman primate model has proved a challenging task that requires the efficient and innovative use of genetic techniques; not only this, the innate physiological parameters of the primate itself pose a major obstacle because the rhesus macaque does not reach reproductive maturity until 4 to 5 years of age with seasonal breeding cycles [1], [4], [8]. Although genetically modified primate models such as the marmoset have certain physiological advantages, such as their
Acknowledgments
All animal procedures and experiments were performed in a BSL-2 facility at the Yerkes National Primate Research Center (YNPRC) and were approved by the Emory University's Institutional Animal Care and Use Committee. The Yerkes National Primate Research Center is a fully Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility. All animals in the colony are managed in accordance with the applicable United States Department of Agriculture (USDA's)
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