Fig. 1

Generation of the DHPS deficiency zebrafish model. (A) The first three exons and introns of the dhps gene and selected targeting sites of two antisense morpholino oligomers and PCR primers are shown, as well as the predicted amplicon lengths. (B) RT-PCR analysis confirmed the predicted lengths of the dhps mRNA amplicons from Wt, Ctrl MO and dhps E2I2 MO larvae with two biological repeats. While the length of the PCR amplicon covering the first three exons of dhps mRNA is 375 bp (b) in Wt, Ctrl MO, and dhps E2I2 MO larvae, in the latter two, additional bands appear (a and c). Appearance of the 172-bp amplicon (c), indicating deletion of exon 2 from the dhps mRNA, and reduction in amount of the 375-bp amplicon (b), together confirm partial knockdown of dhps. Appearance of an approximately 450-bp amplicon (a) in dhps E2I2 MO larvae may be due to partial activation of a cryptic splice site induced by antisense blockage of the E2I2 splice site. (C) Analysis of protein expression in dhps knockdown zebrafish. Zebrafish embryos (2 dpf) with knockdown by dhps E2I2 MO or dhps AUG MO were analyzed by Western blot for expression of DHPS, eIF5A^TOTAL, eIF5A^HYP, and total protein (as visualized by REVERT™). (D) Densitometric data for the relative expression of DHPS, eIF5A^TOTAL, and eIF5A^HYP, normalized on the basis of total protein as quantified by REVERT™ staining