|
Fig. S7 Knockdown of Ve-cadherin using a splice-directed MO. (A) Intron/exon structure between exon 8 and exon 11 in the ve-cadherin gene showing the location of the morpholino and the RT-PCR primers (P1-P5) used for molecular confirmation. (B) RT-PCR results. Wild-type embryos are designated with a 0. RT-PCR reactions from embryos injected with 10 ng of the ve-cadherin MO are labeled with a 10. Each lane represents one embryo (1 dpf). P1/P2 primers were used as a control and amplify a similar product (148 bp) for all conditions. P3/P4 primers amplify an expected product for a properly spliced ve-cadherin transcript in wild-type embryos (347 bp). By contrast, RT-PCR from embryos injected with the ve-cadherin MO amplifies two aberrant splicing products (alternative splicing 1 and 2 in C; 104 bp and 301 bp, respectively). P5/P4 primers amplify an RT-PCR product of the expected size from wild-type embryos (112 bp). In embryos injected with 10 ng of the ve-cadherin MO, a smaller RT-PCR product (66 bp) is observed. (C) The effect of ve-cadherin MO on ve-cadherin splicing. The ve-cadherin MO caused two alternative splicing products. Alternative splicing 1 results in complete loss of exon 10 and the following exons. Alternative splicing 2 causes partial loss of exon 10 and complete loss of the following exons because of a cryptic splice site within exon 10. PCR product sequences were confirmed by sequence analysis. Primers used in PCR analysis of ve-cadherin splicing products were as follows: P1, 5′-agcctgaaggttctggacat-3′; P2, 5′-gtaaagcgaaacctgcctga-3′; P3, 5′-caggcaggtttcgctttact-3′; P4, 5′-ctttttgatagcgccgtctc-3′; and P5, 5′-gtcctacgcaaggactggaa-3′.