This allele is generated by crossing Gt(ROSA)26Sorem1(tetO-flpo*/ERT2,CAG-GFP*)Nijo line described in J:317156 with a Cre-deleter to induce the flpo expression. The targeting vector is divided into two modules (regulatory and neoantigen) separated by a 2x chicken gamma globulin (CGG) insulator element. The regulatory module expresses an inducible flpo following cre recombination. The neoantigen module expresses GFP containing the peptide antigen, which is recognized by H2-Db-restricted CD8 T cells (GP-33-41) and H2-I-Ab restricted CD4 T cells (GP67-77) following flp recombination. The vector contains from 5' to 3': the regulatory module: loxP2-Puromycin resistance cassette - loxP1- C-terminal of flpo fused to human ERT2 (amino acids 251-596) in forward orientation separated by a pA and the N-terminal of flpo/ERT2 in the reverse orientation - loxP1 - loxP2 - tetO (pTRE tight promoter)-insulator element (blocks pCAG enhancer from activating pTRE), and the neoantigen module: CAG promoter - N terminal portion of GFP - FRT - FRT3 - neoantigen with a FLAG-TAG epitope - FRT - polyA - FRT3 - splice acceptor - premature STOP (alternative reading frame) - C terminal GFP - polyA. The neoantigen is comprised of sequence from the lymphocytic meningitis virus (LCMV) glycoprotein modified such that the DNA sequence is split across three exons with exon 2 inverted and flanked by non-compatible FRT sites (F3 and FRT). The targeting vector was inserted into the Rosa26 locus. In the presence of flpo, exon 2 is inverted lining up splicing sites to allow transcription of the full-length GFP containing the peptide antigen. (J:317156)
中文
