Figure 3

Heritable deletions in the sqt locus that result in RNA-null alleles. (a) PCR on single wild-type or sqt deletion mutant embryos (using primers indicated in Figure 2a) shows a 220 bp fragment in a sqt^sg32 locus-deletion embryo, and a 380 bp fragment in TSS deleted sqt^sg27 mutant embryo. Sometimes a larger approximately 500 bp fragment is observed in sqt^sg27/+ heterozygous embryos, but the sequence is identical to the 478 bp product. (b) Percentage of embryos with sqt mutant phenotypes in sqt ^cz35/+, sqt ^sg27/+, sqt ^sg32/+ and sqt ^sg7/+ in-crosses and mating of sqt ^cz35/+ with sqt ^sg27/+, sqt ^sg32/+ and sqt ^sg7/+. The cz35 allele is an approximately 1.9 kb insertion in sqt exon 1; the sg27 allele is a 98 bp deletion of sqt TSS sequences; sg32 allele is a whole locus deletion of sqt; the sg7 ZFN allele harbors a GGCC insertion in sqt exon 2. (c-j) DIC images of 24 h wild-type (c), sqt ^cz35/cz35 (d), sqt ^sg27/cz35 (e), sqt ^sg32/cz35 (f), sqt ^sg7/cz35 (g), sqt ^sg27/sg27 (h), sqt ^sg32/sg32 (i), and sqt ^sg7/sg7 (j) embryos; scale bar in (c), 100 μm. (k) UCSC genome browser view of the sqt locus and neighboring genomic region. (l,m) RT-PCR with primers to detect expression of sqt RNA and transcripts of neighboring genes, eif4ebp1, rnf180, and htr1ab, shows lack of sqt RNA expression in sqt ^sg27/sg27 (l) and sqt ^sg32/sg32 (m) embryos whereas all neighboring gene transcripts are expressed at wild-type levels. Actin (act) expression was used as control. In contrast, both un-spliced and spliced sqt RNA is detected in wild-type and heterozygous embryos.