Knockdown of erbB1a using morpholinos. For knock-down of erbB1a, we used a morpholino targeted to a splice site. To test the activity of this morpholino over time, primers spanning the targeted intron-exon boundary were designed. (A) PCR performed on cDNA from morphant zebrafish larvae at different time points reveals morpholino efficiency. (B) A working morpholino causes splice events to fail at the targeted splice site (red) resulting in larger PCR product. Genomic DNA contamination of the cDNA causes a third, larger, product as the amplicon spans two introns on the genomic template. This analysis reveals that the used morpholino only prevents efficient splicing of erbB1a RNA before 48hpf, causing equal amounts of spliced vs. morphant RNA at later stages. Primers used for PCR: 5′-CCACCAACA TCGACTCCTTT-3′; 5′-AAACCTTGAGGTCATCCGAG-3′. Morpholino sequence: 5′-AAATGCTCTTCCTCACCCTCTGAAT-3′.
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