Zebrafish microinjections
Gene Tools, LLC (http://www.gene-tools.com/) designed the morpholino (MO). Antisense MOs (GeneTools) were microinjected into fertilized one-cell stage embryos according to standard protocols (Nasevicius & Ekker, 2000). The sequences of the rapgef1a translation-blocking and splice-blocking morpholinos were 5′-TGTCTATTTTCCCAGACATCTTGCT-3′ (ATG-MO) and 5′-GACGCTTAAAAACATTTTACCTGCT-3′ (E2I2-MO), respectively. The sequence for the standard control morpholino was 5’-CCTCTTACCTCAGTTACAATTTATA-3’ (Gene Tools). The amount of the MOs used for injection was as follows: Control-MO and E2I2-MO, 1 ng per embryo; ATG-MO, 2ng per embryo. Total RNA was extracted from 30 to 50 embryos per group in TriPure Isolation Reagent (Roche) according to the manufacturer’s instructions. RNA was reverse transcribed using the PrimeScript RT reagent Kit with gDNA Eraser (Takara). Primers spanningrapgef1a exon 1 (forward primer: 5‘-CCACCAGAACAACCCGTAAA-3’) and exon 3 (reverse primer: 5‘-ATTCACACCCTCCAGCATTAC-3’) were used for RT-PCR analysis for confirmation of the efficacy of the E2I2-MO. The primer ef1α sequences used as the internal control were 5‘-GGAAATTCGAGACCAGCAAATAC-3’ (forward) and 5‘-GATACCAGCCTCAAACTCACC-3’ (reverse). For rescue experiments, 2ng rapgef1a-E2I2-MO was co-injected with 50 pg pcDNA3.1 containing human RAPGEF1 (nonmutant and mutant) cDNA per embryo respectively. The coding region of the wild-type or mutant human RAPGEF1 was synthesized by Sangon Biotech and subcloned into pcDNA3.1 vector (Invitrogen).