Sánchez-Luengo Miguel Ángel, Rovira Miguel, Serrano Manuel, Fernandez-Marcos Pablo Jose, Martinez Lola
Flow Cytometry Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, E28029, Spain.
Tumor Suppression Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, E28029, Spain.
Cytometry A. 2017 Jul;91(7):721-729. doi: 10.1002/cyto.a.23086. Epub 2017 Apr 4.
Flow cytometry is a powerful multiparametric technology, widely used for the identification, quantification, and isolation of defined populations of cells based on the expression of target proteins. It also allows for the use of surrogate reporters, either enzymatic or fluorescent, to indirectly monitor the expression of these target proteins. In this work, we optimised the dissociation protocol for the detection of the enzymatic reporter LacZ using the FACS-Gal detection system with the fluorogenic substrate FDG to compare cis- versus trans-positioned reporters efficiency. Particularly, for the FACS-Gal optimization, we studied lung and haematopoietic tissues, focusing on cell recovery, viability, FDG loading conditions and distribution of cellular populations. Reporter genes such as LacZ can be placed together with the gene of interest in the same polycistronic mRNA (in cis), or in independent alleles (in trans), which can strongly affect the correlation with the reporter readout. To address this issue, we generated a mouse model containing both types of reporters for the same gene, and compared them. Our results clearly indicate that trans-positioned reporters can be misleading, and that using a reporter gene in cis rather than trans is a much more specific method to sort for cells undergoing Cre-mediated recombination. © 2017 International Society for Advancement of Cytometry.
流式细胞术是一种强大的多参数技术，广泛用于基于靶蛋白的表达对特定细胞群体进行鉴定、定量和分离。它还允许使用酶促或荧光替代报告基因来间接监测这些靶蛋白的表达。在这项工作中，我们使用带有荧光底物FDG的FACS-Gal检测系统优化了用于检测酶促报告基因LacZ的解离方案，以比较顺式与反式定位报告基因的效率。特别是，对于FACS-Gal的优化，我们研究了肺组织和造血组织，重点关注细胞回收率、活力、FDG加载条件以及细胞群体的分布。诸如LacZ之类的报告基因可以与感兴趣的基因一起置于同一多顺反子mRNA中（顺式），或置于独立的等位基因中（反式），这可能会强烈影响与报告基因读数的相关性。为了解决这个问题，我们构建了一个针对同一基因同时包含两种类型报告基因的小鼠模型，并对它们进行了比较。我们的结果清楚地表明，反式定位的报告基因可能会产生误导，并且使用顺式而非反式报告基因是一种更特异的方法，用于分选经历Cre介导重组的细胞。© 2017国际细胞计量学促进协会。
