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First published online 9 April 2008
doi: 10.1242/dev.001115

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Controlling morpholino experiments: don't stop making antisense

¹ Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254, USA.
² Wellcome Trust/CR-UK Gurdon Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

View larger version (3K): [in this window] [in a new window]   Fig. 1. Structures of conventional DNA and morpholino oligonucleotides. (A) Conventional DNA oligonucleotide. (B) Morpholino oligonucleotide. Note the six-membered morpholino ring in B and the non-ionic phosphorodiamidate link between the two rings.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Different uses of antisense morpholino oligonucleotides. (A) Inhibition of translation by an antisense morpholino oligonucleotide. The MO is targeted to sequence 5' of the translation start site and inhibits progression of the initiation complex. (B-E) Use of MOs to prevent correct splicing of X. tropicalis NRH1. (B) The first three exons of NRH1. Arrows indicate the positions of the forward (F1) and reverse (R1, R2) PCR primers used to test the efficacy of the splicing MOs. Exon 1 is 326 bp, intron 1 is 9013 bp, exon 2 is 134 bp, intron 2 is 740 bp, and exon 3 is 363 bp. If the mRNA is correctly spliced, the combination of F1 and R1 should yield no PCR product and F1 and R2 should give a product of 490 bp. (C) The +intron1 MO is designed to cause intron 1 to be retained. This would result in the first 15 amino acids of NRH1 being followed by 67 missense amino acids before a stop codon is reached. If intron 1 is retained in this way, the combination of F1 and R1 should give a PCR product of 280 bp, and F1 and R2 should yield a product in excess of 9.0 kb. (D) The exon2 MO is designed to exclude exon 2 from the mature RNA. In this case, exon 3 would no longer be in frame with exon 1, and a truncated protein consisting of 15 correct amino acids followed by 25 missense residues would be formed before a stop codon is encountered. Deletion of exon 2 in this way should cause the combination of F1 and R1 to yield no PCR product, and F1 and R2 should yield a product of 360 bp. (E) Verification of the efficacy of MOs +intron1 and exon2. Comparisons of lane 2 with lane 3, and lane 6 with lane 7, indicate that both MOs reduce levels of correctly spliced transcript by 50%. Note that the predicted 9.5 kb band in lane 4 is absent, probably because the PCR conditions do not efficiently amplify products of this size. (F) Activation of a caged morpholino. An inhibitory oligonucleotide is coupled to a specific MO through a photocleavable linker. Irradiation [10 seconds of 360 nm light (h)] cleaves the linker and allows the MO to exert its effects. B-D are redrawn, and E is reproduced, with permission, from Knapp et al. (Knapp et al., 2006). F is redrawn, with permission, from Shestopalov et al. (Shestopalov et al., 2007).

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