Fig. 2 S2

Dnmt3bb.1 morpholino phenocopies TALEN-induced mutant defects.

(a) Schematic diagram showing the splice acceptor morpholino target site at the dnmt3bb.1 intron 3-Exon 4 boundary (red line) and positions of primers used to detect splicing changes (blue arrows). (b) RT-PCR gel image showing loss of correctly spliced dnmt3bb.1 transcripts in splice acceptor morpholino-injected animals. (c-f) WISH of 36 hpf trunks (c,d) and 72 hpf tails (e,f) from control (c,e) and dnmt3bb.1 (d,f) morpholino injected animals, probed for cmyb. cmyb is expressed in dnmt3bb.1 morphants at 36 hpf (arrows) but the expression is lost by 72 hpf (arrowheads). (g-j) WISH of 72 hpf tails probed for l-plastin (g,i) and 5 dpf heads probed for rag1 (h,j) from control (g,h) or dnmt3bb.1 (i,j) morpholino-injected animals. (k-n) WISH of 5 dpf embryos probed for gata1 expression in control (k,m) and dnmt3bb.1 (l,n) morpholino injected embryos. m,n, higher magnification images of highlighted areas from k,l. (o) Quantitative RT-PCR analysis at 5 dpf lck, ikaros and eGFPtranscript levels in WT siblings (red columns), dnmt3bb.1 mutants (green columsn), control morpholino (blue columns) or dnmt3bb.1 (yellow columns) morpholino-injected animals. Transcript levels are normalized to the reference gene elf1α and to levels in controls. (p,q) WISH of 5 dpf embryos probed for ikaros expression in control (p) and dnmt3bb.1 (q) morpholino injected embryos. Expression of l-plastin, rag1, gata1 and ikaros is present in controls (arrows) but reduced in dnmt3bb.1 morpholino-injected animals (arrowheads). (r,s) Confocal images of 5 dpf Tg(lck:gfp) embryos injected with control (q) or dnmt3bb.1 morpholinos. Scale bars 50 µm in d, 100 µm in f,i,j,n,p, 25 µm in r.