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首页> 外文期刊>The European Journal of Neuroscience >Selective and regulated gene expression in murine Purkinje cells by in utero electroporation
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Selective and regulated gene expression in murine Purkinje cells by in utero electroporation

机译：子宫电穿孔在小鼠浦肯野细胞中的选择性和调控基因表达

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Cerebellar Purkinje cells, which convey the only output from the cerebellar cortex, play an essential role in cerebellar functions, such as motor coordination and motor learning. To understand how Purkinje cells develop and function in the mature cerebellum, an efficient method for molecularly perturbing them is needed. Here we demonstrate that Purkinje cell progenitors at embryonic day (E)11.5 could be efficiently and preferentially transfected by spatially directed in utero electroporation (IUE) with an optimized arrangement of electrodes. Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at postnatal days 25-28. By combining the L7 promoter and inducible Cre/loxP system with IUE, transgenes were expressed even more specifically in Purkinje cells and in a temporally controlled manner. We also show that three different fluorescent proteins could be simultaneously expressed, and that Bassoon, a large synaptic protein, could be expressed in the electroporated Purkinje cells. Moreover, phenotypes of staggerer mutant mice, which have a deletion in the gene encoding retinoid-related orphan receptor α (RORα1), were recapitulated by electroporating a dominant-negative form of RORα1 into Purkinje cells at E11.5. Together, these results indicate that this new IUE protocol, which allows the selective, effective and temporally regulated expression of multiple foreign genes transfected into Purkinje cell progenitors in vivo, without changing the cells' physiological characteristics, is a powerful tool for elucidating the molecular mechanisms underlying early Purkinje cell developmental events, such as dendritogenesis and migration, and synaptic plasticity in mature Purkinje cells. A new in utero electroporation (IUE) protocol has been developed to deliver genes preferentially into cerebellar Purkinje cells. IUE did not alter the physiological characteristics or normal synaptic plasticity of Purkinje cells. IUE allowed selective, effective, and temporally regulated expression of multiple foreign genes in Purkinje cells in vivo.
机译：小脑浦肯野细胞传递小脑皮层的唯一输出，在小脑功能（例如运动协调和运动学习）中发挥重要作用。为了了解浦肯野细胞如何在成熟的小脑中发育和发挥功能，需要一种有效的分子干扰方法。在这里，我们证明了在胚胎日（E）11.5时，浦肯野细胞祖细胞可以通过优化的电极排列在子宫内电穿孔（IUE）中进行空间定向而有效且优先转染。电生理分析表明，电穿孔的浦肯野细胞在出生后25-28天保持正常的膜性质，突触反应和突触可塑性。通过将L7启动子和可诱导的Cre / loxP系统与IUE结合，转基因甚至可以更特定地在Purkinje细胞中以时间控制的方式表达。我们还表明，可以同时表达三种不同的荧光蛋白，并且可以在电穿孔的Purkinje细胞中表达大的突触蛋白Bassoon。此外，通过在E11.5处将RORα1的显性负型电穿孔到Purkinje细胞中，概括了在编码类视黄醇相关的孤儿受体α（RORα1）的基因中缺失的交错突变小鼠的表型。总之，这些结果表明，这种新的IUE方案可在体内不影响细胞生理特性的情况下选择性，有效和时间调节地表达多个转染入Purkinje细胞祖细胞的外源基因，是阐明分子机制的有力工具。潜在的浦肯野早期细胞发育事件，例如树突状细胞发生和迁移，以及成熟的浦肯野细胞中的突触可塑性。已经开发了一种新的宫内电穿孔（IUE）协议，可将基因优先传递到小脑Purkinje细胞中。 IUE不会改变浦肯野细胞的生理特性或正常的突触可塑性。 IUE可以在体内Purkinje细胞中选择性，有效和在时间上调节多种外源基因的表达。

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