适用于泛素启动子驱动荧光融合蛋白表达的门控兼容慢病毒转移载体。
Gateway-compatible lentiviral transfer vectors for ubiquitin promoter driven expression of fluorescent fusion proteins.
机构信息
Alzheimer's and Parkinson's Disease Laboratory, Brain and Mind Research Institute, The University of Sydney, 100 Mallett St., Camperdown, NSW 2050, Australia.
出版信息
Plasmid. 2010 May;63(3):155-60. doi: 10.1016/j.plasmid.2010.01.002. Epub 2010 Feb 2.
Lentiviral gene delivery has become widely used. Similarly, the Gateway cloning technology that allows restriction-independent cloning of genes into target vectors is becoming increasingly popular. Here, we have generated two Gateway-compatible lentiviral transfer vectors for expression of carboxy-terminal fluorescence tagged fusion proteins, pLVU/GFP and pLVU/RED. We used a restriction enzyme-independent PCR-based approach to introduce the carboxy-terminal fluorescence tags, EmGFP and DsRed, respectively. Both vectors combine the advantages of restriction enzyme/ligation-independent cloning using the Gateway system with a attR1-CmR-ccdB-attR2 recombination cassette, together with expression of fluorescence tagged fusion proteins driven by the robust mammalian ubiquitin C (UbC) promoter. We tested the vectors by expressing different proteins together with the carboxy-terminal fluorescence tags in 293T and SH-SY5Y cells. Both pLVU/GFP and pLVU/RED can be utilized in different experiments, including protein localization studies and live-cell in vivo imaging.
慢病毒基因传递已得到广泛应用。同样，允许基因不受限制地克隆到靶载体的 Gateway 克隆技术也越来越流行。在这里，我们生成了两个适用于表达羧基末端荧光标记融合蛋白的 Gateway 兼容慢病毒转移载体，pLVU/GFP 和 pLVU/RED。我们使用基于酶切的 PCR 方法分别引入羧基末端荧光标签 EmGFP 和 DsRed。这两个载体都结合了 Gateway 系统的限制酶/连接酶独立克隆的优点，以及带有 attR1-CmR-ccdB-attR2 重组盒的荧光标记融合蛋白的表达，由强大的哺乳动物泛素 C（UbC）启动子驱动。我们通过在 293T 和 SH-SY5Y 细胞中表达不同的蛋白质和羧基末端荧光标签来测试这些载体。pLVU/GFP 和 pLVU/RED 都可以用于不同的实验，包括蛋白质定位研究和活细胞体内成像。
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