Step 2: Insertion of fluorescent protein reporter genes. (A) Diagrams showing the shuttle vectors containing different spectral FP variants for the specified genes and the two step recombination methodology. Homology arms for Ibsp (Arms A & B), Dmp1 (Arms C & D) and Trap (Arms E & F) were cloned into pLD53-Topaz, pLD53-mCherry, and pLD53-ECFP. Recombination was selected for using ampicillin plus gentamicin or spectinomycin. Sucrose resolution was carried out to remove unwanted vector sequences including ampicillin resistance. (B) Diagram of ECFP insertion into TRAP. (C) ECFP insertion introduced a unique Not1 site verifying integration into GM-Trap. (D) Diagram of Topaz insertion into Ibsp followed by mCherry insertion into Dmp1. (E) Cla1 restriction digestion of Dmp1/Ibsp subcloned fragment at progressive stages of reporter gene insertion. The insertion of mCherry introduces an additional Cla1 site. (F) Diagram of diagnostic PCR carried out with primers flanking the homology arms and fluorescent protein coding regions for all three genes. (G) PCR amplification comparing original BAC clones to fluorescent protein reporter BAC clones. PCR product sizes without reporter gene insertion were 1426 bp for Dmp1, 1569 bp for Ibsp, and 1450 bp for Trap. PCR product sizes with reporter gene insertion were 2559 bp for Dmp1, 2299 bp for Ibsp, and 2545 bp for Trap.