Srinivas S, Watanabe T, Lin C S, William C M, Tanabe Y, Jessell T M, Costantini F
Department of Genetics and Development, Columbia University, New York, USA.
BMC Dev Biol. 2001;1:4. doi: 10.1186/1471-213x-1-4. Epub 2001 Mar 27.
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 beta-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 (beta-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.
已经描述了几种小鼠Cre报告基因品系，其中在表达Cre重组酶的细胞及其子代细胞中，loxP侧翼的转录“终止”序列经Cre介导切除后，lacZ基因被开启。这些小鼠可用于细胞谱系追踪实验以及监测Cre转基因的表达。绿色荧光蛋白（GFP）及其变体如EYFP和ECFP作为报告基因比lacZ具有优势，因为无需借助在活组织中可视化β-半乳糖苷酶所需的活性底物就能轻松观察到它们。
鉴于靶向普遍表达的ROSA26位点的广泛用途，我们构建了一个通用的ROSA26靶向载体。然后通过将EYFP或ECFP cDNA插入ROSA26位点，其前面带有loxP侧翼的终止序列，生成了两个小鼠报告基因品系。通过将这些品系与以普遍（β-肌动蛋白-Cre）或细胞特异性（Isl1-Cre和En1-Cre）模式表达Cre的转基因品系杂交进行测试。所得的EYFP或ECFP表达模式表明报告基因品系可作为Cre活性的可靠监测指标。
与现有的lacZ报告基因品系不同，在活组织中不易检测到lacZ表达，而EYFP和ECFP报告基因品系可用于监测Cre的表达，并追踪培养的胚胎或器官中这些细胞及其后代的谱系。EYFP和ECFP不重叠的发射光谱使其成为活组织双重标记研究的理想选择。
