Discovery of novel fluorescent reporter genes

新型荧光报告基因的发现

• 批准号: 6795657
• 负责人: MING ZHAO
• 金额: $ 14.98万
• 依托单位: ANTICANCER, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6795657
• 关键词: aquatic organism; athymic mouse; charge coupled device camera; communicable diseases; fluorescence; fluorescence microscopy; fluorescence spectrometry; gene expression; green fluorescent proteins; metastasis; proteolysis; reporter genes; saltwater environment; site directed mutagenesis; technology /technique development; two dimensional gel electrophoresis; whole body imaging /scanning

DESCRIPTION (provided by applicant): The green fluorescent protein (GFP) from Aequorea victoria has been developed into a highly versatile reporter gene. Another family of fluorescent proteins, including Discosoma Red (DsRed), has been cloned from corals (4). This was followed by Anemonia asRed (5) and HcRed (6). We have developed GFP as an in vivo whole-body imaging reporter for cancer and metastasis, gene expression, and infectious disease. In these applications, the red emission is of special importance for minimizing background emission and scattering in vivo as well as for FRET (fluorescence resonance energy transfer) analysis. High extinction coefficients, quantum yield and the monomeric state of proteins are very important parameters for the above-mentioned applications. In contrast to GFP, which has only a small tendency to dimerize, the new proteins from corals have a pronounced tendency to form oligomers, e.g. tetramers are observed for DsRed, or even higher aggregates (11,12). Extinction coefficients and quantum yields are also relatively low for red proteins (4-6) and for the newly developed monomeric DsRed (17). Thus, there is an important need for new reporters that are monomeric, and therefore good candidates for fusion to other genes with high extinction coefficients and long wavelength emission for in vivo use. Discovery of novel fluorescent proteins has previously used potential sequence and structural homology of beta-strands or stretches between beta-strands of novel proteins with those of GFP from A. victoria (4- 6). These primers, used to clone the new fluorescence proteins, code for surface amino acids. Despite the significant success of this strategy in discovery of novel fluorescent proteins in corals and related organisms, the approach based on sequence homology has serious limitations. To overcome this limitation, a discovery strategy initially based on separation of candidate proteins from selected marine organisms, by 2-D electrophoresis and then analyzed by fluorescence spectroscopy will be developed. The first step is to localize novel fluorescent proteins from murine organisms by spectral and protein analysis of the total body of the marine organism. After separation of candidate proteins by 2-D gel electrophresis, and fluorescence spectroscopy, the proteins will be sequenced and cloned by novel primer design and PCR. The second step is to generate variants of the reporters by mutagenesis of the novel proteins. The goal is to identify novel fluorescent proteins with emission peaks greater than 620 nm, high extinction coefficients, and high quantum yields. Thorough evaluation of the novel fluorescent proteins will be carried out by whole-body imaging in mice with tumor cells expressing these genes using an orthotopic model of lung cancer growing on the lung and metastasized to other organs. Characterization will also include immunogenicity and proteolytic stability of the new fluorescent protein. Reporters with spectral properties that can be used for whole-body imaging of tumors in the lung and their metastases will be candidates for further development for numerous applications of multi-color imaging.

描述（由申请人提供）：来自维多利亚水母的绿色荧光蛋白（GFP）已被开发为高度通用的报告基因。另一个荧光蛋白家族，包括Discosoma Red（DsRed），已经从珊瑚中克隆出来（4）。其次是Anemonia asRed（5）和HcRed（6）。我们已经开发了GFP作为癌症和转移、基因表达和感染性疾病的体内全身成像报告物。在这些应用中，红色发射对于最小化背景发射和体内散射以及FRET（荧光共振能量转移）分析特别重要。高消光系数、量子产率和蛋白质的单体状态是上述应用的非常重要的参数。与GFP相比，其仅具有较小的二聚化倾向，来自珊瑚的新蛋白具有形成寡聚体的明显倾向，例如，对于DsRed观察到四聚体，或甚至更高的聚集体（11，12）。红色蛋白（4-6）和新开发的单体DsRed（17）的消光系数和量子产率也相对较低。因此，非常需要单体的新报告基因，因此是与体内使用的具有高消光系数和长波长发射的其他基因融合的良好候选物。新型荧光蛋白的发现之前已经使用了新型蛋白质的β链或β链之间的延伸与来自A的GFP的潜在序列和结构同源性。维多利亚（4- 6）。这些引物用于克隆新的荧光蛋白，编码表面氨基酸。尽管这种策略在珊瑚和相关生物中发现新的荧光蛋白方面取得了重大成功，但基于序列同源性的方法具有严重的局限性。为了克服这一局限性，将制定一种发现战略，首先通过二维电泳从选定的海洋生物中分离候选蛋白质，然后通过荧光光谱法进行分析。第一步是通过光谱和蛋白质分析的海洋生物体的整体定位新的荧光蛋白从小鼠生物体。在通过2-D凝胶电泳和荧光光谱分离候选蛋白质之后，将通过新的引物设计和PCR对蛋白质进行测序和克隆。第二步是通过新型蛋白质的突变产生报告基因的变体。目标是识别发射峰大于620 nm、消光系数高和量子产率高的新型荧光蛋白。新的荧光蛋白的全面评估将进行全身成像的肿瘤细胞表达这些基因的小鼠使用原位模型的肺癌生长在肺和转移到其他器官。表征还将包括新荧光蛋白的免疫原性和蛋白水解稳定性。可用于肺部肿瘤及其转移的全身成像的具有光谱特性的报告者将成为进一步开发多色成像的许多应用的候选者。