Supplementary Figure 5: Maternal expression of dzip1l, efficacy of the dzip1l-splice morpholinos and generation of the dzip1l-deletion allele. | Nature Genetics

Supplementary Figure 5: Maternal expression of dzip1l, efficacy of the dzip1l-splice morpholinos and generation of the dzip1l-deletion allele.

From: Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease

Supplementary Figure 5

(a) The maternal expression of dzip1l mRNA at the 64-cell stage of zebrafish embryonic development. Zygotic expression is shown for the 10 somite stage. acta1b expression was used as loading control. (b) Splice morpholino (e5i5MO) efficiently interferes with dzip1l pre-mRNA splicing. RT-PCR with primers binding to exon2 and exon10, expected band of about 650 bp is visible from cDNA of wild-type embryos (arrow). A smaller band of 350 bp (arrowhead) is visible from cDNA of the morphants. Sequencing of this band showed deletion of exon 5-7. The lower band (asterisk) is a non-specific band. acta1b expression was used as loading control. (c), Splice morpholino (i1e2MO) interference with dzip1l pre-mRNA splicing. RT-PCR with primers binding to exon1 and exon3, expected band of about 680 bp is visible from cDNA of wild-type embryos (arrow) and significantly reduced in cDNA of morphant embryos. Wild-type control band was confirmed by sequencing, lower bands are unspecific. (d) Splice morpholino (i4e5MO) interferes with dzip1l pre-mRNA splicing. RT-PCR with primers binding to exon2 and exon10, expected 870 bp band is visible in cDNA of wild-type embryos and missing in morphant embryos (arrow). eef1a1l1 expression was used as loading control. (e) Schematic diagram of CRISPR/Cas9 mediated genomic modification of the dzip1l locus. Two guide RNAs, targeting exon2 and exon13, were designed to excise the intervening genomic DNA, to create a deletion allele. (f) PCR screening of CRISPR/Cas9 mediated dzip1l deletion among F0 founder fish with primers binding to exon2 and exon14. Fish 1, 2 and 3 did not carry the dzip1l deletion allele. Fish 4 and 5 were identified as dzip1l deletion allele carriers with the expected PCR product of 350 bp (arrow). Sequencing of this band showed the expected deletion. (g) Three otoliths in the otic vesicle of a 24 hpf maternal-zygotic dzip1l mutant embryo (n = 10 embryos).

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