Abstract
Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs. NHP pluripotent stem cells (PSCs) share key molecular signatures with the early embryo, which is an important target for genomic modification. Therefore, PSCs are a valuable test system for the validation of embryonic genome editing approaches. In the present study, we made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing. These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. These findings facilitate efficient testing of genome editing approaches in NHP-PSC before their in vivo application.
Highlights
Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations
The induced pluripotent stem cells (iPSCs) lines were generated from skin fibroblasts from 2 adult macaques (DPZ_iRhpb#1–3, male) (DPZ_iRhpb#4, female)
Between 100 and 150 colonies were identified per reprogramming experiment (100–150 primary colonies/1 × 106 transfected cells), resulting in a reprogramming efficiency of approximately 0.08–0.12% (~ 16–25 colonies per primary plate; 0.2 × 1 05 cells per primary plate)
Summary
Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. We made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. NHP-PSCs share many features with the pluripotent cells of the early e mbryo[6,26] They are the best available test system to study embryonic genome editing ex vivo and to predict, upon directed differentiation into specific cell types, potential phenotypic alterations that might be observed later on in the animal. Using this work-flow, it is possible to simultaneously evaluate both, the potential phenotypic consequences for the animals as well as the potential value of the in vivo model
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
