Laboratory of Applied Reproductive Science, Faculty of Agriculture, Shinshu University, Nagano, Japan.
Department of Neurophysiology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.
PLoS One. 2023 Jun 9;18(6):e0287065. doi: 10.1371/journal.pone.0287065. eCollection 2023.
Generating non-human primate models of human diseases is important for the development of therapeutic strategies especially for neurodegenerative diseases. The common marmoset has attracted attention as a new experimental animal model, and many transgenic marmosets have been produced using lentiviral vector-mediated transgenesis. However, lentiviral vectors have a size limitation of up to 8 kb in length for transgene applications. Therefore, the present study aimed to optimize a piggyBac transposon-mediated gene transfer method in which transgenes longer than 8 kb were injected into the perivitelline space of marmoset embryos, followed by electroporation. We constructed a long piggyBac vector carrying the gene responsible for Alzheimer's disease. The optimal weight ratio of the piggyBac transgene vector to the piggyBac transposase mRNA was examined using mouse embryos. Transgene integration into the genome was confirmed in 70.7% of embryonic stem cells established from embryos injected with 1000 ng of transgene and transposase mRNA. Under these conditions, long transgenes were introduced into marmoset embryos. All embryos survived after transgene introduction treatment, and transgenes were detected in 70% of marmoset embryos. The transposon-mediated gene transfer method developed in this study can be applied to the genetic modification of non-human primates, as well as large animals.
生成人类疾病的非人类灵长类动物模型对于治疗策略的发展很重要,特别是对于神经退行性疾病。食蟹猴作为一种新的实验动物模型引起了人们的关注,许多转基因食蟹猴已经通过慢病毒载体介导的转基因技术产生。然而,慢病毒载体在转基因应用中长度限制为 8kb 以内。因此,本研究旨在优化一种基于 piggyBac 转座子的基因转移方法,该方法将长度超过 8kb 的转基因注入食蟹猴胚胎的卵周隙中,然后进行电穿孔。我们构建了一个携带阿尔茨海默病相关基因的长 piggyBac 载体。使用小鼠胚胎检查了 piggyBac 转基因载体与 piggyBac 转座酶 mRNA 的最佳重量比。用 1000ng 转基因和转座酶 mRNA 注射的胚胎建立的胚胎干细胞中,有 70.7%证实了转基因整合到基因组中。在这些条件下,长转基因被导入食蟹猴胚胎。转基因导入处理后所有胚胎均存活,70%的食蟹猴胚胎中检测到转基因。本研究开发的转座子介导的基因转移方法可应用于非人类灵长类动物和大动物的遗传修饰。
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