Triple mammalian/yeast/bacterial shuttle vectors for single and combined Lentivirus- and Sindbis virus-mediated infections of neurons
Triple mammalian/yeast/bacterial shuttle vectors for single and combined Lentivirus- and Sindbis virus-mediated infections of neurons
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DOI：
10.1007/s00438-012-0680-1
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发表时间：
2012-04-01
影响因子：
3.1
通讯作者：
Heinisch, Juergen J.
中科院分区：
文献类型：
--
作者：
Bakota, Lidia;Brandt, Roland;Heinisch, Juergen J.
如今，有多种病毒载体可用于哺乳动物细胞培养物或体内的异位基因表达。其中，辛德比斯病毒或慢病毒衍生构建体的感染通常用于解决生物学问题或用于神经元治疗中的应用。然而，克隆感兴趣的基因非常耗时，因为它依赖于限制性酶切和连接，通常是 PCR 生成的 DNA 片段，其具有由所用引物引入的合适限制性位点。我们利用酿酒酵母异常高的同源重组能力来解决这个问题，并引入了一组新的三重穿梭载体，可以在大肠杆菌、酵母和哺乳动物细胞之间进行改组。该系统允许引入很大程度上独立于载体中的靶位点的感兴趣基因。如果载体大小限制了哺乳动物细胞中的转染效率，它还允许通过 Cre 重组酶在大肠杆菌中定向重组来去除酵母选择标记。我们使用两种不同的病毒系统在哺乳动物细胞系、原代神经元和器官切片中单独和组合地展示了编码荧光蛋白（EGFP 和 mCherry）的基因的表达。
Today, a large variety of viral vectors is available for ectopic gene expression in mammalian cell cultures or in vivo. Among them, infection with Sindbis virus- or Lentivirus-derived constructs is often used to address biological questions or for applications in neuronal therapies. However, cloning of genes of interest is time consuming, since it relies on restriction and ligation, frequently of PCR-generated DNA fragments with suitable restriction sites introduced by the primers employed. We here take advantage of the unusually high capacity for homologous recombination in Saccharomyces cerevisiae to circumvent this problem, and introduce a new set of triple shuttle vectors, which can be shuffled between E. coli, yeast, and mammalian cells. The system allows the introduction of genes of interest largely independent of the target site in the vectors. It also allows the removal of the yeast selection marker by Cre-recombinase directed recombination in E. coli, if vector size limits transfection efficiency in the mammalian cells. We demonstrate the expression of genes encoding fluorescent proteins (EGFP and mCherry) both separately and in combination, using two different viral systems in mammalian cell lines, primary neurons and organotypic slices.