Figure 4

Higher concentration of pX330-Satb2-2129 efficiently disrupts the Satb2 gene.

(a) Layer 2/3 neurons were co-transfected with pCAG-EGFP and pX330-Satb2 (2.5 mg/ml) using in utero electroporation at E15.5. Coronal sections were prepared at P4 and immunostained with anti-Satb2 antibody (red), anti-EGFP antibody (green) and Hoechst 33342 (blue). Arrows indicate normal Satb2 expression in EGFP-positive neurons transfected with a pX330 control plasmid. Arrowheads indicate a dramatic reduction of Satb2 expression in EGFP-positive neurons transfected with pX330-Satb2-2129. Scale bar = 20 μm. (b) Histogram of the expression levels of Satb2 in transfected neurons. The average Satb2 signal intensity in EGFP-positive neurons transfected with a pX330 control vector was set as Satb2 expression level 100. The number of neurons with Satb2 expression level 100-110 and that of neurons with Satb2 expression level <10 were also shown in (c) and (d), respectively. (c) The number of neurons with normal levels of Satb2 expression was markedly reduced by pX330-Satb2-2129 in the central (Cent) and peripheral (Peri) regions of the EGFP-positive cortical area. (d) The number of neurons with no Satb2 expression was greatly increased by pX330-Satb2-2129 in the central and peripheral regions of the EGFP-positive cortical area. *p < 0.05; **p < 0.01; N.S., not significant; Welch’s t-test (n = 4 pups for each condition). Error bars indicate SD. (e) A coronal section showing central (Cent) and peripheral (Peri) regions of the EGFP-positive cortical area used in (c,d). The area inside the box (asterisk) in the left panel was magnified and shown in the right panel.