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Neuronal specificity of HSV/Sleeping Beauty amplicon transduction in utero is driven primarily by tropism and cell type composition during embryogenesis

机译:子宫内HSV /睡眠美容扩增子转导的神经元特异性主要受胚胎发生过程中的向性和细胞类型组成的驱动

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摘要

A novel bipartite vector system consisting of the Herpes Simplex Virus (HSV) amplicon and the Sleeping Beauty (SB) transposon was previously shown to efficiently deliver a “transgenon” (integrating transgene) in utero. This vector platform facilitated long-term transgenon expression specifically within neurons and neuronal precursor cells of the rodent brain. However, the mechanism underlying the neurospecificity of the HSV/SB amplicon in the setting of mouse embryogenesis is unknown. We find that embryonic cells expressing the Sox1 “neurocompetence” transcription factor represent the primary targets for HSV amplicon transduction in utero. These cells, which comprise the ependymal and subventricular zones, express significant levels of HMGB1, a co-factor shown to facilitate SB-mediated transposition. Using a conventional, non-integrating amplicon expressing Cre recombinase to embryonically “tag” transduced cells in ROSA26 Cre indicator mice in utero, we found transduced cells were exclusively of the neuronal lineage, but in comparison to HSV/SB-mediated in utero delivery, staining patterns were less widespread and “tagged” neuroprogenitor cells were absent. Our findings demonstrate in utero HSV/SB amplicon gene transfer is primarily neurospecific due to viral tropism and target cell populations present embryonically, where multi-potent cells of the developing embryo are supportive of SB-driven transposition.
机译:先前已证明,由单纯疱疹病毒(HSV)扩增子和“睡美人”(SB)转座子组成的新型两方载体系统可在子宫内高效递送“转基因子”(整合转基因)。该载体平台特别在啮齿动物脑的神经元和神经元前体细胞内促进了长期转基因的表达。但是,在小鼠胚胎发生过程中,HSV / SB扩增子的神经特异性的潜在机制尚不清楚。我们发现表达Sox1“神经竞争能力”转录因子的胚胎细胞代表子宫内HSV扩增子转导的主要靶标。这些包含室管膜区和脑室下区的细胞表达显着水平的HMGB1,HMGB1是一种辅助因子,可促进SB介导的转座。使用常规的非整合型表达Cre重组酶的扩增子在子宫内的ROSA26 Cre指标小鼠的胚胎中“标记”转导的细胞时,我们发现转导的细胞仅属于神经元谱系,但是与HSV / SB介导的子宫内传递相比,染色模式不那么普遍,并且没有“标记”的神经祖细胞。我们的发现表明,子宫内HSV / SB扩增子的基因转移主要是神经特异性的,这归因于病毒的向性和胚胎中存在的靶细胞群,其中发育中的胚胎的多能细胞支持SB驱动的转座。

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