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).