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首页> 外文期刊>Neurochemistry International: The International Journal for the Rapid Publication of Critical Reviews, Preliminary and Original Research Communications in Neurochemistry >Gene transfer into Purkinje cells using herpesviral amplicon vectors in cerebellar cultures.
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Gene transfer into Purkinje cells using herpesviral amplicon vectors in cerebellar cultures.

机译：在小脑培养物中使用疱疹病毒扩增子载体将基因转移到浦肯野细胞中。

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摘要

Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.
机译：浦肯野细胞在感觉运动协调中起着至关重要的作用，因为它们是小脑皮层中唯一的输出投射神经元，并且在大多数脊髓小脑共济失调中受到影响。由于它们的大尺寸和大量分支的树突状乔木，它们在中枢神经系统中脱颖而出。然而，对浦肯野细胞的分子和细胞研究常常因难以维持这些细胞在培养中而受到阻碍。在这里，我们报告了一种简单，可靠且可重现的方法，可使用木瓜蛋白酶消化和半限定培养基从第16天小鼠胚胎中获得富含Purkinje的混合小脑细胞培养物，该培养物的组成约为20％Purkinje细胞，其中70％为非-Purkinje神经元和10％的神经胶质细胞。我们证明使用单纯疱疹病毒1（HSV-1）衍生的载体可以有效地将基因转移到浦肯野细胞（以及其他小脑种群）。实际上，可以转导多达50％的Purkinje细胞，基因表达可以持续至少14天。结果，该程序允许在培养的浦肯野神经元上进行功能基因表达研究。为了证明这一点，我们表明糖原合酶激酶3的显性负型的表达保护浦肯野神经元免受磷脂酰肌醇-3激酶化学抑制剂触发的细胞死亡。总而言之，我们已经建立了富含可浦肯野细胞的可再现且可靠的小脑细胞培养物，从而能够使用疱疹病毒载体进行基因转移研究。

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