Fig. 3

Morpholino (MO) knockdown of Thsd7a in zebrafish embryos. A: The genomic structure of thsd7a showing the effect of MO1 injection on mRNA splicing. Relative positions of primers used for reverse transcription polymerase chain reaction (RT-PCR) are shown. B: MO1 injection disrupted the accurate mRNA splicing. RT-PCR products using the F1/R1-1 primer pair reveal the level of accurate mRNA splicing. Products using the F1/R1-2 primer pair show the level of mRNA containing the retained intron. Beta-actin (bactin) was amplified as an internal control. A total of 2 ng of MO1/ msMO1 were injected. C: Real-time quantification PCR (RTq-PCR) was performed to quantify the efficacy of thsd7a MO1 knockdown in zebrafish embryos. Relative expression levels of thsd7a transcript in msMO1- and MO1-injected embryos were plotted as mean ± SEM. The 2 ng of MO1/ msMO1 were injected. D: MO2 targets the splice junction of the twelfth exon. Relative positions of primers used for RT-PCR are shown. E: Amplification product using cDNA derived from msMO2 embryos showed the expected 977 base pair fragment. MO2 injection resulted in a shorter PCR product of 712 base pairs. The 18 ng of MO2/ msMO2 were injected.