Knock-down of Egfl7 expression in mouse embryonic stem cells. (a) Lentiviral construct used for siRNA-mediated knock-down of Egfl7 (top), and Egfl7 gene structure (bottom) showing non-coding (unshaded) and coding (shaded) exons. The three siRNA target sequences are shown as red bars, and the location of the microRNA miR-126 is shown within intron 7. (LTR, long terminal repeat; Flap, DNA flap; H1, human H1 RNA pol III promoter; UbiC, human ubiquitin c promoter; eGFP, enhanced green fluorescent protein; WRE, woodchuck response element). (b) Verification of Egfl7 knock-down in mouse ESCs by RT-PCR (top) and western blot (bottom). (Empty, lentiviral construct alone; 1, 2, 3 (corresponding to KD1, KD2, KD3), lentivirus containing siRNA sequence; scrambled, lentivirus containing scrambled siRNA control sequence; +, HEK293 cells transfected with a His-tagged Egfl7 vector). (c) Endogenous eGFP expression in lentivirus infected undifferentiated ESCs 8 hours after plating (left two panels) and after passaging 5 times (middle two panels), and in ESC-derived EBs after 14 days of differentiation (right two panels). Magnification used 69×. (d) Quantitative PCR was carried out for Egfl7 and the microRNAs miR-126-3p and miR-126-5p. Expression was normalized to β-actin (for Egfl7) or miR-16 (for the microRNAs), and is shown relative to a value of 1.0 for the scrambled control 2 (Scr2).