FIG. 2.

Generation of Cybr knockout, GFP knockin mice. (A) The endogenous Cybr locus (upper panel) and targeting vector (lower panel) are illustrated. Part of exon 2 and the entire third exon are deleted, and a stop codon is engineered in exon 4 of the recombined allele. In addition, EGFP is expressed as a fusion protein with the N-terminal part of Cybr encoded by exon 1 and part of exon 2 under the endogenous Cybr promoter. Panel B, upper panel, shows that homologous recombination was confirmed by Southern blotting. Lack of Cybr mRNA expression was confirmed in lymphocytes from homozygous mice by real-time PCR using primers and probe that amplify at the exon 5/exon 6 boundary outside of the targeted region of the gene. Panel B, lower panel, shows that lymphocytes were stimulated in vitro under Th1-polarizing conditions for optimal Cybr expression. Error bars indicate standard deviations. (C) Cybr promoter activity was tracked using EGFP expression by flow cytometry in double-positive (DP) thymocytes that were either unstimulated (unstim.) or stimulated (stim.) with anti-CD3 and anti-CD28. Activated thymocytes expressing CD69 had high levels of Cybr promoter activity, as measured by GFP expression.