Generation of FoxP3-GFP-hCre BAC Transgenic Mice

The GFP-hCre cDNA fragment was inserted immediately downstream of the FoxP3 ATG translational start site by homologous recombination into a 188-kb mouse BAC (from the C57BL/6 genome; clone RP23-143D8) carrying the intact FoxP3 gene, as previously described (29 (link)). We used a codon-optimized “humanized” Cre (hCre) to improve translational efficiency in eukaryotic cells. An EGFP-hcre-Frt-Neo-Frt cassette was provided by N. Killeen (UCSF, San Francisco, CA). The modified BAC was purified using cesium chloride gradient ultracentrifugation and microinjected by the UCSF Transgenic/Targeted Mutagenesis Core Facility into the pronuclei of nonobese diabetic mouse embryos to generate FoxP3-GFP-hCre BAC Tg mice. Mice carrying the GFP-hCre transgene were screened by FACS analysis using peripheral blood cells. Several founder mice were generated and screened for GFP expression and lack of functional FoxP3 protein, as indicated by an inability of the transgene to rescue the FoxP3 KO mice. Dicer^lox/lox (14 (link)) mice and ROSA26R-YFP reporter mice (16 (link)) have been previously described. All mice were housed and bred under specific pathogen-free conditions at the UCSF Animal Barrier Facility. All animal experiments were approved by the Institutional Animal Care and Use Committee of UCSF.