Abstract

Genetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases. The common marmoset (Callithrix jacchus) is a non-human primate species adequate for the production of genetically modified animals because of their small body size and high reproductive capacity. Autologous embryo transfer (AET) is routinely utilized in assisted reproductive technologies for humans but not for experimental animals. This study has developed a novel method for efficiently producing mutant marmosets using AET and CRISPR/Cas9 systems. The embryos were recovered from oviducts of naturally mated females, injected with Cas9/guide RNA, and transferred into the oviducts of the donors. This AET method can reduce the time for in vitro culture of embryos to less than 30 min. This method uses an embryo donor as the recipient, thus reducing the number of animals and allowing for “Reduction” in the 3R principles of humane experimental technique. Furthermore, this method can utilize nulliparous females as well as parous females. We applied our novel method and generated the 6 marmosets carrying mutations in the fragile X mental retardation 1 (FMR1) gene using only 18 females including 14 nulliparous females.

Highlights

  • Genetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases

  • This study has developed a novel method for efficiently producing mutant marmosets using Autologous embryo transfer (AET) combined with the CRISPR/Cas[9] system (Fig. 1)

  • As far as we know, this is the first report of the production of newborns by AET in non-human primates

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Summary

Introduction

Genetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases. The embryos were recovered from oviducts of naturally mated females, injected with Cas9/guide RNA, and transferred into the oviducts of the donors This AET method can reduce the time for in vitro culture of embryos to less than 30 min. This method uses an embryo donor as the recipient, reducing the number of animals and allowing for “Reduction” in the 3R principles of humane experimental technique. (3) microinjection of DNA, RNA, or proteins of genome editing methods such as TALENs and CRISPR/Cas[9] systems into embryos and culture in vitro for several days, and (4) transfer of injected embryos into the uteri of another set of females, recipient females.

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