Extended Data Figure 3. Characterization of jam1a MOex7-injected embryos.

a, RT-PCR results from jam1a MOex7-injected embryos. cDNA from embryos uninjected or injected with various doses of jam1a MOex7 (100 – 500μM) was subjected to RT-PCR analysis using specific primers, which amplify from exon 4 to 10 of jam1a. ef1a was used as a control. The expected size of PCR products in uninjected embryos is 746 base pairs (bp) (black arrow). Exon 7 (108bp)-skipped products were detected in jam1a MOex7-injected embryos (red arrow). The dose of 300μM was used in this study. b, Exon 7-skipped products were verified by sequencing. The dotted line indicates the junctions between exon 6 and exon 7 (uninjected, upper panel) or exon 8 (jam1a MOex7, lower panel). c, A schematic diagram of the mRNA splicing in jam1a MOex7-injected embryos. The red bar indicates the binding site of jam1a MOex7. d, Schematic diagrams of Jam1a protein in wild type (left) or jam1a MOex7-injected embryos (right). Since exon 7 encodes the transmembrane domain (TM), jam1a MOex7-injected embryos express a mutant protein lacking the TM. Sp, signal peptide; Ig, immunoglobulin-like domain; PBD, PDZ-binding domain. e, The relative expression of wild type jam1a mRNA in uninjected or jam1a MOex7 (300μM)-injected embryos at 24hpf. The reverse primer was designed in exon 7. Error bars, s.d. f-i, The expression of cmyb and rag1 in uninjected or jam1a MOex7-injected embryos. Arrowheads indicate the dorsal aorta (f, g) or the thymus (h, i). Data are representative of two independent experiments with two different clutches of embryos (a, e-i).