Canine CD117 and SCF Construct Generation

The sequence of canine CD117 and SCF was synthesized by GeneArt (Thermo Fisher Scientific, Melbourne, Australia). The CD117 sequence included a secretion signal and extracellular and transmembrane domains (NCBI Reference: NP_001003181; residues 1 to 545). This was cloned in-frame with GFP into the mammalian expression vector pEGFP-N1 (TaKaRa, Mountain View, CA, USA, discontinued), using restriction sites NheI and BamHI to create the vector pEGFP-N1-CD117-GFP. The canine SCF sequence included a secretion signal, the soluble part of the extracellular domain (NCBI Reference: NP_001012753.1; residues 1 to 191), and a 6× His-tag. This was cloned into the mammalian expression vector pcDNA3.1 (+) (Thermo Fisher Scientific), using restriction sites HindIII and XhoI to create the vector pcDNA3.1 (+)-canine-SCF. DNA for transfection was prepared using a PureLink HiPure Plasmid Maxiprep kit (Thermo Fisher Scientific). A control plasmid, pEGFP-N1-CD83-GFP, containing CD83 in place of CD117, was also prepared as described previously [18 (link)].