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The following 18-nucleotide oligomer phosphorothiolate-substituted ODNs, custom synthesized and purified by Oligos Etc. (Wilsonville, OR), were used in this study. Antisense ODN-1 (5′-ATCTTCATGTTTTGACGA-3′) and sense ODN-1 (5′-TCGTCAAAACATGAAGAT-3′) correspond to nucleotides +4 to +21 of the Manx cDNA sequence (11). In some experiments, a rhodamine moiety was covalently linked to the 3′ termini of antisense and sense ODN-1 to follow uptake and distribution in embryos. The control ODN was 5′-ATTTTGACTTGTGTTTCC-3′, which has a base composition similar to that of antisense ODN-1. In some experiments, we used antisense ODN-2 (5′-ATTATGATTGTTTAAATA-3′) or ODN-3 (5′-CCGAAGCAGCTCTTTCCA-3′), which correspond to nucleotides -15 to +2 and +167 to +184, respectively, in the Manx cDNA sequence (11), or a combination of the three antisense ODNs. The concentration of ODNs was determined empirically for different clutches of hybrid embryos. The standard procedure was to treat hybrids with 20 to 50 μM ODN at first cleavage (about 50 min after insemination) and incubate the embryos with the ODNs until hatching (10 to 12 hours after insemination). The morphology of the ODN-treated and control larvae was determined by light microscopy. Hybrid larvae were scored as containing restored urodele features if they contained either a brain sensory organ, a tail, or both structures. The effect of ODNs on restoration of urodele structures was confirmed by fixing larvae in 4% paraformaldehyde and examining hematoxylin and eosin-stained sections.
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This paper is dedicated to the memory of Alain Maron, whose expertise in collecting M. occulta and M. oculata was essential for these experiments. We thank J. Machula, D. Martasian, and J. Reardon for technical assistance; A. Toulmond, N. Sanséau, and L. Meijer for use of laboratory facilities at the Station Biologique; and R. Maxson for discussions on the use of antisense ODNs. Supported by NSF grants IBN 9304958 (B.J.S.) and DCB-915543 (W.R.J.) and NIH grant HD-13970 (W.R.J.).
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