Col1a1^{tm7(tetO-Pou5f1,-Klf4,-Sox2,-mCherry)Hoch}

A targeting vector was designed to insert a FRT-flanked PGK-Neo-pA into the 3' UTR of the Col1a1 locus. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were identified. One of these ES cells clones, C10, was re-targeted to insert an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a beta-globin intron, polyA signal and PGK-puromycin cassette, all inserted downstream of the Gt(ROSA)26Sor promoter. Correctly targeted ES cells were identified. One of these ES cell clones, KH2, was selected and the FRT-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus was replaced by co-injection of a tetOP-OKSM "flip-in plasmid" and a CAG-Flpe plasmid. The tetOP-OKSmC "flip-in plasmid" contained a splice acceptor-double polyA sequence and the tetracycline responsive element (TRE, tetOP, or tetO) upstream of the three mouse reprogramming genes Pou5f1, Klf4, and Sox2, and an enhanced red fluorescence sequence, mCherry. Internal ribosome entry site (IRES) sequences were placed between the coding sequences for Klf4, Sox2, and mCherry. Expression of Pou5f1, Klf4, and Sox2 was linked by self-cleaving peptides that mediate ribosomal skipping (F2A [from foot-and-mouth disease virus] and E2A [from equine rhinitis A virus], respectively). (J:284696)