The targeting vector contained a tetracycline-responsive tet-repressor/VP16 fusion transactivator (tTAoff) (Gossen & Bujard, PNAS 89:5547, 1992) and a neomycin-resistance gene flanked with 4.5-kb upstream and 1.3-kb downstream Pdx1 gene homology domains and was used to replace the entire coding region (both exons and the intron) of the endogenous Pdx1 locus by homologous recombination. Pdx1-tTA design Two additions helped ensure efficient production of the tTA protein and, in turn, effective levels of PDX1 from a tetO-Pdx1 transgene (see the schematic in attached Image 1): To help translation efficiency, an optimized Xenopus laevis beta-globin 5'UTR sequence (Falcone & Andrews, Mol Cell Biol 11:2656-64, 1991) was incorporated upstream of the tTA coding sequence, and the translation initiation codon and surrounding sequence was modified to conform to Kozak's rules. To help pre-mRNA processing, nuclear export and mRNA stability, a fragment of the rabbit beta-globin gene including the functional last intron and 3'UTR was added immediately downstream of the tTA stop codon. 36-nts separate the stop codon from the 5' (donor) splice junction of the beta-globin intron, well within the 50-nts maximum (Marquat et al., Curr Biol 12:R196-7, 2005) to avoid nonsense mediated destruction of the chimeric mRNA. Pdx-tTA vector construction A 4.3kb 5' flanking sequence from a Pdx1 genomic clone (a gift of C. V. E. Wright) was subcloned into XbaI/SacI sites of a modified pBS polylinker following ablation of a single XhoI site at position 2570 of the 4.3-Kb sequence by silent mutation. A fragment containing an additional (approx) 200-bp of flanking sequence and 20-bp of Pdx1 exon1 was generated by PCR from a genomic Pdx1 clone. This was fused to the 5' flanking sequence to generate the p(Pdx-pro) construct. A modified tTA gene was engineered by fusing a 50-bp double stranded oligonucleotide encoding the Xenopus beta-globin 5'UTR sequence to the tTA gene from pUHG15-1 (Gossen & Bujard, PNAS 89:5547-51, 1992) and optimizing the initiator codon by PCR mutagenesis to conform to Kozak's rules, ablating an NdeI site in the process. A 1041-bp BamHI/NruI rabbit beta-globin gene fragment was attached to the XbaI site immediately downstream of the tTA stop codon. The fragment contained, in order: a 22-bp linker, 14-bp of the 3' end of the b-globin exon 2, 573-bp of intron 2, the entire 364-bp exon 3 (including the stop codon and 98 bp of 3'UTR containing the poly A site), and 39-bp of gene flanking DNA. The 2 Kb modified XltTAbglob gene was excised via HindIII and NdeI restriction and subcloned into pMCS5 (Mobitec, Gottingen Germany) to facilitate later cloning. An approximately 1.4-Kb BglII/EcoRI fragment of 3' Pdx1 flanking sequence was subcloned from a genomic Pdx1 clone (C. V. E Wright) into BamHI/EcoRI restricted pKO V902 vector (Lexicon genetics). A Neomycin resistance positive selection cassette from pKO SelectNeo (Lexicon genetics) was then subcloned into an AscI site followed by insertion of the XltTA gene into the pKO V902 vector at the HindIII/XhoI sites. The 5' Pdx1 flanking sequence from p(Pdx-pro) was subcloned into the HpaI/HindIII sites of the pKO vector before a thymidine kinase selection cassette (pKO SelectTK - Lexicon Genetics) was inserted into the RsrII site of the final construct to generate the plasmid p(Pdx-tTA) bearing the targeting fragment.