Sp6 polymerase

Digoxigenin-labelled antisense riboprobes were prepared for Gfp, Fgf8 and Hoxa13. Gfp (Aequorea victoria) is the full-length open reading frame, cloned into the pGEM T easy vector (Promega) and transcribed with SP6 polymerase (Roche); Fgf8 (Gallus gallus) is the full-length open reading frame cloned into pBlueScript and transcribed with T7 polymerase (Roche); Hoxa13 (Gallus gallus) is a partial clone (80–290 of a 290 amino acid protein) in pBlueScript and transcribed with T3 polymerase (Roche). Briefly, for in situ hybridization in tissue sections, the samples were fixed overnight in 4% PFA, dehydrated, cleared, embedded in paraffin and sectioned at 7 μm. Consecutive sections were placed on separate slides to be analysed with different probes. The sections were de-paraffined, rehydrated, mildly digested with proteinase K (10 μg ml⁻¹ for 10 min) and hybridized overnight. Sections were then washed with decreasing concentrations of SSC in 50% formamide (at 65 °C), and then blocked at room temperature with 10% sheep serum before overnight incubation at 4 °C in the standard anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche,1:2,000). Finally, the staining reaction was carried out with NBT/BCIP and allowed to develop for the desired time.

Mab-Mmp1 was identified from genome and transcriptome sequences. A genomic fragment (MK090468) was cloned after PCR amplification from the locus, a cDNA fragment after amplification through 5’-RACE (MK090469). Double-stranded RNA (dsRNA) was synthesized as described (Urbansky et al., 2016 (link)); Mab-Mmp1 dsRNA comprised pos. +103 to +1167 of the genomic fragment (pos. one refers to first nucleotide in ORF) and included a 57 bp intron at pos. +575. Guide RNAs for a knock-out of Mab-Mmp1 were designed using CCTop as CRISPR/Cas9 target online predictor (Stemmer et al., 2015 (link)). Three single guide RNAs (sgRNAs) were designed to target the following positions (pos. one refers to first nucleotide in ORF): sgRNA1, 5’-TGCAGAGCGTATCTCTTT, pos +404 to +387; sgRNA2, 5’-CGTGGACTATTGATTGTC, pos +710 to +693; sgRNA3, 5’-TCGGCAACCGAGTTTTCA, pos +898 to+881. Guide RNAs as well as Cas9 mRNA were synthesized as described (Stemmer et al., 2015 (link)).
Mab-bsg was identified from genome and transcriptome sequences. A fragment encompassing the full open reading frame was PCR amplified and used in a Gibson Assembly to generate a 3’ fusion with eGFP in a pSP expression vector (pSP-Mab-bsg-eGFP). RNA was in vitro transcribed using SP6 Polymerase (Roche), capping and polyA-tailing was performed using ScriptCap Cap 1 Capping System and Poly(A) Polymerase Tailing Kit (CellScript).

One-color WISH was performed as previously described [30 (link)], with modifications according to [1 (link),31 (link)]. Whole-mount FISH was performed based on [6 ] and the optimized FISH according to [8 (link)]. Digoxigenin-labeled myoD [GenBank:NM_131262] and cxcr4b [GenBank:NM_131834] probes were synthetized using T7 or Sp6 polymerase, respectively, according to the manufacturer’s protocol (Roche, Basel, Switzerland).