Detection of LacZ expression by FACS-Gal analysis

Abstract

Rosa26-LacZ transgenic reporter mice are a very common reporter line to determine Cre activity (1). Since the disruption of target gene(s) and the expression of the Rosa26-LacZ transgene are both controlled by the same Cre recombinase, LacZ expression/activity can serve as a useful marker of the cells with targeted gene disruption. X-gal staining is a common assay used to detect LacZ+ cells in many tissues. In 1988, Nolan, et. al. (2) developed FACS-Gal analysis, a FACS-based detection of LacZ+ cells with a fluorogenic substrate, FDG, of galactosidase (LacZ enzyme). Galactosidase cleaves FDG and releases a fluorescence product FITC (3). However, it is common that LacZ signals varies from sample to sample. Here we establish a procedure to ensure reliable and consistent results for the whole hematopoietic system, with the exception of terminally differentiated erythroid cells, or red blood cells that express much lower LacZ proteins.