Targeted knockdown of the smc3 gene. Antisense morpholinos were designed to target the 5′ UTR (smc3UTRMO), the ATG start codon (smc3ATGMO), the splice site of exon 1, 3′ donor (smc3Splx1MO) and the splice site of exon 5, 3′ donor (smc3Splx5MO) of the smc3 mRNA. Ensembl locus ENSDARG00000019000 and sequence BC044408 were used to inform morpholino oligonucleotide design. Embryos were able to develop to 48 h.p.f. when given a 0.25-0.5 pmol dose of morpholino targeting smc3. Higher doses of 2-3 pmol caused arrest in early blastula stages. Total knockdown of Smc3 function should prevent cell division altogether. Therefore, post-blastula embryos are considered to be smc3 hypomorphs rather than smc3 nulls. (A) Representative gross morphology of Smc3 morphants at 48 hpf generated by a 0.5 pmol dose of smc3Splx1MO, compared with a wild-type embryo of the same stage (top). All morpholinos targeting smc3 generated a similar phenotype. (B) Representative anti-Smc3 immunoblot of protein obtained from embryos injected with 3 pmol smc3UTRMO. Individual embryos that had arrested at the early blastula stage were lysed and the entire sample was used for immunoblotting along with stage-matched controls treated in the same manner. The blot was cut in two and treated with anti-Smc3 antibody (Chemicon, 1:1000) or anti-α-tubulin (Sigma, 1:2000) to provide a loading control. 5 μg of protein from HT29 colon carcinoma cells was used as a positive control for the antibodies. (C) Splice-site morpholinos smc3Splx1MO and smc3Splx5MO are effective in blocking splicing of the smc3 mRNA, and show partial activity in smc3 hypomorph morphants. Agarose gel electrophoresis of PCR products obtained by RT-PCR of cDNA obtained from 24-hpf embryos. M, DNA size marker with number of base pairs indicated at left. RT-, control, morphant embryos processed without reverse transcriptase; MO, embryos injected with 2 pmol of smc3 morpholino (identity indicated at side); WT, wild-type embryos; nz171, rad21nz171 mutant embryos. White asterisks indicate bands of expected size should the intron involved at each splice junction remain unspliced. Double asterisk, band of unpredicted size. Primer sequences for RT-PCR: smc3Splx1 forward, 5′-CTGAGGAGTGTTTTGTGC-3′ and reverse, 5′-GATGACATTGTGTTTTGAGC-3′; smc3Splx5 forward, 5′-CGAGTCATTTCAGCCTTCG-3′ and reverse, 5′-TTACTGCGGGAGAATCCAG-3′.
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