Okita Chiga, Sato Masae, Schroeder Timm
RIKEN Center for Developmental Biology, Kobe, 650-0047, Japan.
Biotechniques. 2004 Mar;36(3):418-22, 424. doi: 10.2144/04363ST01.
Fluorescent proteins (FPs) have revolutionized many aspects of cell biology and have become indispensable research tools. Today's increasingly complex experiments aiming to understand biological systems strongly depend on the availability of combinations of multiple FPs, which allow their distinguishable simultaneous detection in the same cell or tissue. Recently, the VENUS and DsRed. T4 FPs were described as the latest generation of yellow and red FPs. To increase the combinatorial possibilities when using these optimized FPs, we have generated and successfully tested seven new forms of VENUS and DsRed. T4 proteins with distinct subcellular localization. To facilitate their use as markers in biological experiments, bicistronic expression constructs, which have been optimized for robust expression in almost all mammalian developmental stages and cell types, were produced for the new FPs. In addition, several plasmids were created, which contain all necessary elements for inserting the reading frames of these FPs into specific gene loci in knock-in experiments without disrupting the reading frame of the endogenous gene.
荧光蛋白（FPs）彻底改变了细胞生物学的许多方面，已成为不可或缺的研究工具。如今，旨在理解生物系统的实验日益复杂，这在很大程度上依赖于多种荧光蛋白组合的可用性，这些组合能使它们在同一细胞或组织中被同时区分检测。最近，VENUS和DsRed.T4荧光蛋白被描述为最新一代的黄色和红色荧光蛋白。为了增加使用这些优化荧光蛋白时的组合可能性，我们生成并成功测试了七种具有不同亚细胞定位的VENUS和DsRed.T4蛋白新形式。为便于它们在生物学实验中用作标记，我们制备了双顺反子表达构建体，这些构建体已针对几乎所有哺乳动物发育阶段和细胞类型的强劲表达进行了优化，用于新的荧光蛋白。此外，还创建了几个质粒，其中包含在敲入实验中将这些荧光蛋白的读码框插入特定基因位点而不破坏内源基因读码框所需的所有元件。
