Tracing synaptic connectivity onto embryonic stem cell-derived neurons.
Tracing synaptic connectivity onto embryonic stem cell-derived neurons.
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DOI：
10.1002/stem.1185
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发表时间：
2012-10
期刊：
影响因子：
5.2
通讯作者：
Arenkiel, Benjamin R.
中科院分区：
文献类型：
--
作者：
Garcia, Isabella;Huang, Longwen;Ung, Kevin;Arenkiel, Benjamin R.
使用基因修饰的狂犬病病毒（RV）的跨突触电路追踪是用于识别神经元之间的突触连接的新兴技术。补充这种方法，它已成为可能的测定基本的分子和细胞特性的神经元谱系来源于胚胎干细胞（ES）在体外，这些属性正在紧张的调查，以设计细胞替代疗法。在这里，我们报告了一种新的小鼠ES细胞（mESC）线，窝藏的遗传元素，让RV介导的跨突触电路跟踪在ES细胞衍生的神经元和它们的突触网络的一代。为了促进跨突触追踪，我们通过基因靶向将编码tdTomato、狂犬病G糖蛋白和禽类TVA受体的cDNA引入ROSA 26位点来设计新的报告等位基因。我们证明了这些新的胚胎干细胞高效分化为功能性神经元，显示其功能性连接与原代神经元培养的免疫组织化学和电生理记录证明，并显示其作为源细胞的神经元网络在体外和体内的突触前示踪的能力。总之，我们的数据突出了使用基因工程干细胞研究突触和电路形成的基本机制的潜力，明确识别突触前输入到感兴趣的神经元群体。
Transsynaptic circuit tracing using genetically modified Rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it has become possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem (ES) cells in vitro, and these properties are under intense investigation towards devising cell replacement therapies. Here, we report the generation of a novel mouse ES cell (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ES cell-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to functionally connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest.
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影响因子：
3.7
作者：
Cui YF;Xu JC;Hargus G;Jakovcevski I;Schachner M;Bernreuther C
通讯作者：
Bernreuther C
影响因子：
12.4
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
4
作者：
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影响因子：
14.8
作者：
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通讯作者：
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