Fig. S17

Segregation analysis of causative mutation from the multiple-inserted lines with Dox-dependent fin regeneration defects. (A) Segregation of galnt2^IM from line pIDM-A28. To segregate the causative mutation, pIDM-A28 F₁ mutants with Dox-dependent fin regeneration defects were crossed with WT. The cross containing about 50% EGFP positive offspring was selected to produce F₂ population. Left panel: Southern blot analysis of insertion copy numbers in the pool of embryos as indicated. Pool of EGFP positive F₂: The F₂ EGFP positive embryos were from the cross between the selected F₁ fish and WT; Pool of EGFP positive F₃: The F₃ EGFP positive embryos were from the cross between one of F₂ adult fish and WT. Southern blot was performed as described in Supplementary Figure 3. Right panel: PCR analysis to identify the insertion site. A pair of galnt2 intron 1 and pIDM specific primers were designed to amplify the insertion site. The DNA lysed from caudal fin of individual F₂ fish was used to do PCR analysis. The DNA from EGFP positive F₁ embryo pool was used as the positive control. The DNA from EGFP negative F₂ fish was used as the negative control. (B) Induction of the listed genes in WT zebrafish at different time points during fin regeneration as indicated. The WT caudal fin was sampled at different times after amputation and subjected to a WISH assay with the corresponding anti-sense RNA probes as indicated. (C) Segregation of ggt7l^IM from line pIDM-A3. To segregate the causative mutation, pIDM-A3 F₂ mutants with Dox-dependent fin regeneration defects were crossed with WT. The cross containing about 50% EGFP positive offspring were selected to produce F₃ population. Left panel: Southern blot analysis of insertion copy numbers in the pool of embryos as indicated. Pool of EGFP positive F₃: The F₃ EGFP positive embryos were from the cross between the selected F₂ fish and WT; Pool of EGFP positive F₄: The F₄ EGFP positive embryos were from the cross between one of F₃ adult fish and WT. Southern blot was performed as described in Supplementary Figure 3. Right panel: PCR analysis to identify the insertion site. A primer from ggt7l exon 5 was used with pIDM specific primer to amplify the insertion site. The DNA lysed from caudal fin of individual F₃ fish was used to do PCR analysis. The DNA from EGFP positive F₁ embryo pool was used as the positive control. The DNA from EGFP negative F₃ fish was used as the negative control. (D) Segregation of cry61^IM from line pIDM-E7. To segregate the causative mutation, pIDM-E7 F₂ mutants with Dox-dependent fin regeneration defects were crossed with WT. The cross containing about 50% EGFP positive offspring were selected to set up F₃ population. Left panel: Southern blot analysis of insertion copy numbers in the pool of embryos as indicated. Pool of EGFP positive F₃: The F₃ EGFP positive embryos were from the cross between the selected F₂ fish and WT; Pool of EGFP positive F₄: The F₄ EGFP positive embryos were from the cross between one of F₃ adult fish and WT. Southern blot was performed as described in Supplementary Figure 3. Right panel: PCR analysis to identify the insertion site. A primer from cry61 intron 1 was used with pIDM specific primer to amplify the insertion site. The DNA lysed from caudal fin of individual F₃ fish was used to do PCR analysis. The DNA from EGFP positive F₁ embryo pool was used as the positive control. The DNA from EGFP negative F₃ fish was used as the negative control.