Generation of Csf1r-mApple reporter mice

The 7.2 kb Csf1r reporter construct previously used to generate the Csf1r-EGFP mice (26 (link)) was digested with ApaI and SalI (NEB) to remove EGFP before gel purification using the QIAquick gel extraction kit (Qiagen). Overhangs were removed with Mungbean nuclease (NEB) and DNA was purified using QIAGEN MinElute columns (Qiagen), then dephosphorylated using thermosensitive alkaline phosphatase (Promega). A construct encoding the fluorescent protein Csf1r-mApple (34 (link)) was digested with SmaI and AflII, similarly purified, and overhangs removed before both constructs were precipitated with EtOH/NaOAc and then ligated with T4 ligase (NEB) at 16°C overnight. The resulting Csf1r-mApple construct was transformed into DH5α competent cells. The Csf1r-rtTA-M2 construct utilizing the same 7.2 kb mouse Csf1r promoter region was used previously to generate Csf1r-rtTA transgenic mice (35 (link)) For generation of transgenic mice, plasmid backbones were removed by digestion with DrdI/PvuI (Csf1r-mApple, NEB) and SalI/MluI (Csf1r-rtTA, Promega/NEB) and then gel-purified using a QIAquick gel extraction kit. DNA was then further purified using AMPure XP beads (Agencourt) according to the instructions.