Platform Degenerative Diseases, German Primate Center-Leibniz Institute for Primate Research, Kellnerweg 4, 37077 Göttingen, Germany.
DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, 37099 Göttingen, Germany.
Cells. 2021 Feb 27;10(3):505. doi: 10.3390/cells10030505.
Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.
非人类灵长类动物(NHP)的基因修饰为现实疾病模型铺平了道路。普通狨猴是一种越来越多地用于生物医学研究的 NHP 物种。尽管发明了 RNA 引导的核酸酶,但在 NHP 中过表达蛋白质的一种策略仍然是慢病毒转导。我们通过慢病毒转导自然着床前胚胎,生成了三只雄性和一只雌性增强型绿色荧光蛋白(EGFP)转基因创始狨猴。所有创始者通过自然交配完成了转基因的种系传递,共产生了 20 只转基因后代(总共 45 只幼崽;44%是转基因)。这表明转基因配子即使与野生型配子竞争也能够进行自然受精。重要的是,90%的转基因后代表现出转基因沉默,这与啮齿动物形成鲜明对比,在啮齿动物中,相同的转基因促进了强大的 EGFP 表达。此外,我们一直在混合野生型/转基因后代中发现体细胞,但迄今为止没有发现生殖细胞嵌合体。体细胞嵌合体导致相应野生型同窝仔的假阳性基因分型。为了通过聚合酶链反应(PCR)从皮肤样本中区分转基因和转基因嵌合体动物,建立了嵌合细胞耗竭方案。总之,可以通过自然交配建立一群转基因狨猴,但需要特定的要求,包括仔细选择启动子。
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