Generation of Transgenic Sperm Expressing GFP by Lentivirus Transduction of Spermatogonial Stem Cells In Vivo in Cynomolgus Monkeys.
Generation of Transgenic Sperm Expressing GFP by Lentivirus Transduction of Spermatogonial Stem Cells In Vivo in Cynomolgus Monkeys.
复制标题
DOI：
10.3390/vetsci10020104
复制
发表时间：
2023-02-01
影响因子：
2.4
通讯作者：
中科院分区：
文献类型：
--
作者：
基因编辑技术有望成为建立人类疾病动物模型的有效途径。使用精原干细胞和精子作为基因编辑对象操作简单。然而，目前尚无对精原干细胞进行基因编辑以获得转基因精子的研究，低温保存是否会对转基因精子产生不良影响也尚不清楚。此外，很少有明确的细胞表面标记物用于鉴定灵长类动物精原干细胞。在本实验中，我们利用超声引导注射针进入睾丸网间隙，将增强的绿色荧光蛋白慢病毒从睾丸网注入精小管。最后，我们成功获得了转基因精子，其冷冻和恢复速度与野生动物相似。该方法有望成为建立动物疾病编辑模型的另一种有效途径，并将在人类疾病的研究以及新药和治疗方法的开发中发挥重要作用。由于非人类灵长类动物在基因组学、代谢组学、生理和病理特征上与人类高度相似，它们被认为是生物医学研究的最佳模型。然而，由于靶向效率和作为卵母细胞/受精卵供体所需的NHPs数量的限制，通过传统方法，如在单细胞胚胎原核中微量注射产生转基因NHPs是不可能的。以精原干细胞（SSCs）为基因编辑靶点，产生经过基因编辑的精子进行受精，被证明是建立基因编辑动物疾病模型的有效途径。在本实验中，我们利用超声引导回声密集注射针进入睾丸网间隙，使EGFP慢病毒在正压下从睾丸网缓慢注入精小管。我们发现Thy1可以作为食蟹猴SSCs的表面标记物，证实SSCs携带GFP基因。最后，我们成功获得了转基因精子，其冷冻率和恢复率与WT动物相似。
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. 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.
登录
查看更多内容
影响因子：
3.7
作者：
Sinha N;Pilder S;Vijayaraghavan S
通讯作者：
Vijayaraghavan S
DOI：
10.1073/pnas.0508780102
发表时间：
2005-11-29
影响因子：
11.1
作者：
Hamra, FK;Chapman, KM;Garbers, DL
通讯作者：
Garbers, DL
DOI：
10.1152/ajpheart.00060.2007
发表时间：
2007-07-01
影响因子：
4.8
作者：
Michalkiewicz, Mieczyslaw;Michalkiewicz, Teresa;Cowley, Allen W., Jr.
通讯作者：
Cowley, Allen W., Jr.
影响因子：
64.5
作者：
Niu, Yuyu;Shen, Bin;Sha, Jiahao
通讯作者：
Sha, Jiahao
过度表达 MeCP2 的转基因猴中的自闭症样行为和种系传播。
影响因子：
64.8
作者：
Liu, Zhen;Li, Xiao;Qiu, Zilong
通讯作者：
Qiu, Zilong