Fluorescein utp

An Msx1 1.2kb XhoI-XbaI cDNA fragment was subcloned into pSP72. Msx2 full-length cDNA was cloned into pBSKII(+) between BamHI and EcoRI sites. The Blimp1 cDNA construct is a kind gift from Dr. Saitou at RIKEN Kobe Institute, Japan (Ohinata et al., 2005 (link)). The Fragilis cDNA probe was obtained from Dr. Surani at Wellcome Trust/Cancer Research UK Institute (Saitou et al., 2002 (link)). The Wnt5a probe was generously provided by Dr. SK Dey at Cincinnati Children’s Hospital Medical Center (Daikoku et al., 2011 (link)). A 700bp Fibronectin cDNA probe was generated by PCR with forward 5′-AGA TGA CTC ATG CTT TGA CCC-3′ and reverse 5′-TGC TGA AGC TGA GAA CAT GGC-3′ primers. Ribonucleotide probes were synthesized by PCR with either Fluorescein-UTP or Digoxygenin-UTP (Roche Applied Science). Whole-mount in situ hybridization was performed largely based on the method reported by Hogan and visualized by BM-purple substrate (Roche Applied Science) (Hogan et al., 1994 ). Prior to sectioning, embryos were fixed overnight in 4% PFA/PBS and embedded in HistoPrep (Fisher). In situ hybridization on frozen sections was performed as previously described and fluorophore-conjugated Tyramide (TSA™PLUS, Perkin Elmer) was used to amplify the signal (Ting et al., 2009 (link)).

Transcription and analysis of RNA transcripts were carried out as described previously (14 (link)). When a T7 transcription was followed by a SP6 transcription, the T7 transcription was stopped by 2 mM competitive dsDNA (5'-GAAATTAATACGACTCACTATA-3')(16 (link),17 (link)). Then 0.02 mM fluorescein-UTP (Roche) and 2 U/ml SP6 RNA polymerase (Fermentas, Thermo Scientific) were added and the samples were incubated at 37°C for 60 min, followed by a treatment with 0.04 U/ml DNase I (Fermentas, Thermo Scientific) at 37°C for 15 min and an extraction with phenol/chloroform. RNA products were denatured and resolved on an 8% denaturing polyacrylamide gel.