Fig. 2

Morpholino knockdown of rap1b is specific. A: Schematic of the rap1b gene showing exons in blue, introns in black, and the approximate position of morpholino binding sites in green. The S17N mutation generating dominant-negative rap1b is indicated with a red star. B: Agarose gel showing 100-bp ladder with 500- and 600-bp bands marked, PCR product of wild-type rap1b cDNA, and PCR product of wild-type and altered splice product generated via splice-blocking morpholino (MO2) for rap1b. As confirmed by sequencing, the altered splice-product lacks the second exon and is 69 bp smaller than the wild-type rap1b cDNA. This disruption of splicing results in deletion of 23 amino acids from the predicted protein. C: Lateral view of DIC image for 12–15-somite-stage embryo injected with both rap1b MO1 and rap1b DN mRNA (3 experiments). For rap1b MO1+MO2 injections, 71.4% were affected (3 experiments, n = 248) (data not shown). Anterior is left.