Figure 2

Characterization of the Er81 promoter. (A) Diagram to the left indicates the relative size of deletion constructs used in the reporter assay. The horizontal lines represent deleted fragments of the putative Er81 promoter. Diagram to the right shows the corresponding reporter activities of the indicated deletion constructs in primary cortical cell cultures, expressed as mean ± standard deviation (error bars) in each assay. The asterisk masks the construct, p131 with the highest luciferase activity among other deletion constructs. (B) Multiple sequence alignment of the mouse, rat, chimpanzee, and human Er81 genes was performed using the ClustalW algorithm implemented in the eShadow web application [55]. The x- and y-axes indicate the distance (in base pairs) to the starting codon (0) and percentage variation, respectively. The resulting alignment indicates a highly conserved sequence of approximate 2 kb upstream of the Er81 gene for all species examined. Schematic representation of the vector used to generate a Cre-transgenic mouse line in which the expression of Er81 is driven by the identified 2-kb Er81 promoter sequence. The Er81 promoter was subcloned upstream of the β-globin minimal promoter (minPr) in a plasmid containing a DNA fragment encoding Cre [53]. This construct allows the Er81 promoter to simultaneously drive expression of Cre via the β-globin minimal promoter, and a GFP reporter sequence via an IRES sequence. (C) After crossing with Gtrosa26^tm1Sho reporter mice [27], LacZ staining of E16.5 forebrains isolated from double-transgenic Er81Cre Gtrosa26 mice showed recombination in the L5, and the VZ of cortex, striatum, piriform cortex, and olfactory bulb (C, cross section; D, sagittal section). CP, Cortical plate; Hi, Hippocampus; L5, Layer 5; OB, Olfactory bulb; Pir, Piriform cortex; Str, Striatum; VZ, Ventricular zone.