Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.
Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.
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DOI：
10.1186/1471-213x-1-4
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发表时间：
2001
影响因子：
--
通讯作者：
Costantini F
中科院分区：
文献类型：
--
作者：
Srinivas S;Watanabe T;Lin CS;William CM;Tanabe Y;Jessell TM;Costantini F
已经描述了几种Cre报告小鼠品系，其中在Cre介导的loxP侧翼转录“终止”序列的切除后，lacZ基因在表达Cre重组酶的细胞以及它们的子细胞中被打开。这些小鼠可用于细胞谱系追踪实验以及用于监测Cre转基因的表达。绿色荧光蛋白（GFP）和变体（如EYFP和ECFP）作为报告基因提供了优于lacZ的优势，因为它们可以很容易地可视化，而无需依赖于在活组织中可视化β-gal所需的重要底物。鉴于靶向遍在表达的ROSA 26基因座的一般效用，我们构建了通用ROSA 26靶向载体。然后，我们通过将EYFP或ECFP cDNA插入ROSA 26基因座，前面是loxP侧翼终止序列，产生了两个小鼠报告细胞系。通过将这些菌株与以普遍存在（β-actin-Cre）或细胞特异性（Isl 1-Cre和En 1-Cre）模式表达Cre的转基因菌株杂交来测试这些菌株。所得到的EYFP或ECFP表达模式表明，报告菌株的功能作为忠实的监测Cre活性。与现有的lacZ报告细胞系（其中lacZ表达不能在活组织中容易地检测到）相比，EYFP和ECFP报告细胞系可用于监测Cre的表达并追踪这些细胞及其后代在培养的胚胎或器官中的谱系。EYFP和ECFP的非重叠发射光谱使它们成为活组织中双标记研究的理想选择。
Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cre-mediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue. In view of the general utility of targeting the ubiquitously expressed ROSA26 locus, we constructed a generic ROSA26 targeting vector. We then generated two reporter lines of mice by inserting EYFP or ECFP cDNAs into the ROSA26 locus, preceded by a loxP-flanked stop sequence. These strains were tested by crossing them with transgenic strains expressing Cre in a ubiquitous (β-actin-Cre) or a cell-specific (Isl1-Cre and En1-Cre) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity. In contrast to existing lacZ reporter lines, where lacZ expression cannot easily be detected in living tissue, the EYFP and ECFP reporter strains are useful for monitoring the expression of Cre and tracing the lineage of these cells and their descendants in cultured embryos or organs. The non-overlapping emission spectra of EYFP and ECFP make them ideal for double labeling studies in living tissues.