Fig. S4

Fig. S4

Knockdown of insulin via insulin-a morpholino (insaMO). (A) Diagram illustrating insa pre-mRNA structure in wild type and insaMO-injected cells. insaMO targets the exon 2-intron 2 boundary of preproinsulin-a mRNA and generates two nonsense alternative splicing products. Primers complementary to exon 1 and exon 3 amplify the differentially spliced cDNA products. (B) Agarose gel showing PCR amplified insa cDNA products; mRNA was extracted from control and insaMO-injected embryos at 2 days post fertilization (dpf). Note that a single PCR product with size of 420 bp was detected in control embryos but two aberrant PCR products—520 and 360 bp—were detected in insaMO-injected embryos. β actin was used as internal control. (C) The PCR products in control and insaMO-injected embryos were extracted, cloned into pJet vector, and sequenced. insaMO-injected embryos showed two aberrant splice products: Product 1 included intronic sequence; Product 2 showed partial deletion of exon 2. (D) Predicted peptide sequence of control and two aberrant splice products. (E) No overt gross embryonic phenotypes resulted from injection of increasing doses of insaMO ranging from 2 ng to 8 ng.