Generation of Transgenic Sperm Expressing GFP by Lentivirus Transduction of Spermatogonial Stem Cells In Vivo in Cynomolgus Monkeys

SIMPLE SUMMARY: Gene editing technology is expected to be an effective way to establish animal models of human diseases. Using spermatogonial stem cells and sperm as gene editing objects is simple to operate. However, there has been no research on gene editing of spermatogonial stem cells to obtain genetically modified sperm, and it is not known whether cryopreservation has adverse effects on genetically modified sperm. Furthermore, there are few clear cell surface markers to identify primate spermatogonial stem cells. In this experiment, we used ultrasound to guide the injection needle into the testis reticulum space to inject the enhanced green fluorescent protein lentivirus from the testis reticulum into seminiferous tubules. Finally, we successfully obtained transgenic sperm, which have a similar freezing and recovery rate to that of wild animals. This approach is expected to be another effective way to establish edited animal disease models, and will play an important role in the research of human diseases and the development of new drugs and therapeutic methods. ABSTRACT: Nonhuman primates (NHPs) have been considered as the best models for biomedical research due to their high similarities in genomic, metabolomic, physiological and pathological features to humans. However, generation of genetically modified NHPs through traditional methods, such as microinjection into the pronuclei of one-cell embryos, is prohibitive due to the targeting efficiency and the number of NHPs needed as oocyte/zygote donors. Using spermatogonial stem cells (SSCs) as the target of gene editing, producing gene-edited sperm for fertilization, is proven to be an effective way to establish gene editing animal disease models. In this experiment, we used ultrasound to guide the echo dense injection needle into the rete testis space, allowing the EGFP lentivirus to be slowly injected at positive pressure from the rete testis into seminiferous tubules. We found Thy1 can be used as a surface marker of cynomolgus monkey SSCs, confirming that SSCs carry the GFP gene. Finally, we successfully obtained transgenic sperm, with a similar freezing and recovery rate to that of WT animals.