Generation and Breeding of EGFP-Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling.

Charis Drummer,Kerstin Mätz-Rensing,Wilfried A Kues,Edgar-John Vogt,Michael Heistermann,Berit Roshani,Sebastian Kügler,Tamara Becker,Rüdiger Behr

doi:10.3390/cells10030505

Abstract

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

Highlights

Non-human primates (NHP) reflect human anatomy, physiology, and genetic constitution much better than other animal species [1,2]
Lentiviral enhanced green fluorescent protein (EGFP) transgenesis of NHP has been achieved in proof of concept studies using marmosets (Callithrix jacchus) [29] and macaques (Macaca mulatta and Macaca fascicularis) [30,31,32]
We used natural preimplantation embryos instead of in vitro fertilization (IVF) embryos, and injected them with lentiviral vector encoding EGFP under the control of the human ubiquitin C promoter, which was previously shown to mediate stable transgene expression in founders and F1 progeny of mice [28,48] and rats [28]

Summary

Introduction

Non-human primates (NHP) reflect human anatomy, physiology, and genetic constitution much better than other animal species [1,2]. This is of relevance in many research areas including neurobiology [2,3,4,5,6,7,8], reproductive biology [9,10,11], and genetics [11,12,13,14]. Since the spontaneous occurrence of many diseases in NHP has not been observed so far, genetic modification of NHP is a promising approach to generate realistic and meaningful animal models to better understand and eventually treat human diseases and to illuminate primate biology. Tomioka and colleagues reported transmission of a transgene through the germline from the mother to one pup by natural mating in marmosets [33]

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Conclusion