Behaviors of Baf57Int and Baf57ΔR. (A) Western blot examining Brg/Brahma-associated factor (BAF)57 expression in total thymocytes (lanes 1 to 5, and lane 10) and purified CD4 cells (lanes 6 to 9). Samples in lanes 8 to 10 were from mice exposed to TAM. The genotype of the mice are indicated, where F, ΔR and Flp denote Baf57F, Baf57ΔR and R26FlpoER1, respectively. The bracket at the left indicates the three BAF57 mutant proteins expressed from Baf57F after deletion of floxed sequence. At least three mice of each genotype were analyzed. Shown is a representative experiment. (B) Reverse transcriptase (RT)-PCR examining BAF57 transcripts in CD4 cells expressed by the wild-type (WT) allele (lanes 1 and 3) or the mutant allele lacking exons 2 and 3 (lanes 2 and 4); the cells were isolated from Baf57+/ΔR; CD4-Cre; R26FlpoER1 and Baf57F/ΔR; CD4-Cre; R26FlpoER1 mice, respectively. RT-PCR primers targeted exons 1 and 7 (lanes 1 and 2) or 1 and 5 (lanes 3 and 4). The latter primer set yielded amplicons A to C (lanes 3 and 4, diagrammed at the bottom), which were re- amplified with a nested primer set, gel-purified, and digested with EcoN1 to verify their identities (lanes 5 to 10). There was also a nonspecific amplicon (about 310 bp, lane 3) which was not re-amplifiable (not shown). Two mice of each genotype were analyzed. Shown is a representative experiment. (C). Flow cytometric assays monitoring green fluorescent protein (GFP) expression in peripheral blood CD4, CD8 and B cells at various times after tamoxifen (TAM) injection. Three mice of each genotype were analyzed. (D) Summary of the reversion efficiencies at day 10 in CD4, CD8, and B cells, as measured by the fraction of cells that had lost GFP. Each symbol represents an individual mouse.
Chaiyachati et al. BMC Biology 2012 10:96 doi:10.1186/1741-7007-10-96