In vitro transcription and mRNA injections
To generate mRNA, WT and mutant forms of SMO cDNA were generated
using the mMESSAGE mMACHINE SP6 transcription kit (Thermo Fisher
Scientific). For the SMO mRNA over-expression experiments, we
used a 200 pg bolus of SMO synthetic mRNA with a 2 pg bolus ofSHH . For the rescue experiments using our K/O model, we used a 80
pg bolus of SMO synthetic mRNA. For the K/D assay, we used a 100
pg bolus of SMO synthetic mRNA with 0.1 ng bolus of bothptch1 and ptch2 MO. Each injection cocktail was injected
into fertilized one cell stage embryos. Either a gfp or control MO was
used as a negative control for over-expression and K/O experiments or
K/D experiment, respectively. Trials of appropriate dose-response were
performed before each experiment (Suppl.Fig.2 and Suppl.Tables S1,4-6).
All experimental tests of mRNA injections in this study were performed
with 50-80 embryos/injection, in triplicate and on different days.