Fig. 1

Targeting vector and breeding strategy for generation of myeloid cell specific GPR109A CKO (GPR109A^flox/flox/LysM-Cre⁺) mouse model. (A) GPR109A CDS was cloned into pcDNA3.1 vector whose cloning site is flanked by LoxP regions. Floxed GPR109A region was incorporated into mouse embryotic stem cell allele via electroporation and homologous recombination. Embryotic stem cells carrying floxed GPR109A CDS were injected into C57BL/6 embryos which were carried to term. Adult mice carrying floxed GPR109A CDS were backcrossed to develop a stable mouse line carrying a conditional GPR109A CKO allele. (B) Female GPR109A^flox/flox mice were crossed with male LysM-Cre⁺ mice to give offspring with either GPR109A^flox/+/LysM-Cre⁺ or GPR109A^flox/+/LysM-Cre^- genotypes. Male GPR109A^flox/+/LysM-Cre⁺ and female GPR109A^flox/+/LysM-Cre^- offspring were crossed to give Cre⁺, f/f and CKO genotypes as well as others needed for this study. (C) Genotyping was performed on mouse tails using PCR followed by agarose gel electrophoresis (1.5%). Primers were designed to detect the presence (350 bp) or absence (400 bp) of floxed regions flanking GPR109A CDS as well as presence of LysM-Cre (750 bp) or Cre (350 bp) in DNA isolated from mouse tail samples.