Figure 1

Verification of the efficiency of injection of a splice-altering cxcl12a MO (MO^sp) and of the rescue with cxcl12a mRNA in zebrafish embryo. (A) A schematic representation of cxcl12a pre-mRNA showing its 4 exons, introns (dotted line) and the binding position of MO^sp on exon 2 to cause alternate splicing. The positions of the MO^sp detection primers (designed to obtain product smaller than full-length gene of 1521 bp), which partially spans exons 1 and 4 and the full length of exons 2 and 3, are shown. (B) RT-PCR analysis of Cxcl12a transcript confirming the splice-altering effect of MO^sp (850 ng/µL) compared to embryos injected with control MO. A low dose (85 ng/µL) MO^sp has no marked splice-altering effect, whereas MO^sp + mRNA shows rescue of wild-type (WT) product and increases its expression. M is a 100 bp ladder. A smaller PCR product (239 bp) was observed from MO^sp-injected embryos because exon 2 (118 bp) was completely skipped; the WT product was 357 bp. (C) A segment of the cxcl12a gene is shown. The underline and dotted underline show the start and stop codons, respectively. The nucleotides in bold show the coding sequence (cds), whereas the hyphens show the 118 nucleotides of exon 2 that were excised after MO^sp injection. Finally, the colored nucleotides show the positions of the forward primer (blue) on exon 1 and the reverse primer (green) on exon 4. (D) The resulting truncated transcript, which has 77 amino acids removed (dotted underline), is expected to be only 25 amino acids long (bold) and has 3 new insertions (underline). The asterisk shows the premature stop codon.