Mouse experiments were conducted following procedures approved by the Ethical Committee of Animal Experimentation of the University of Barcelona. Targeted replacement of Ins2 with human INS 5′ flanking sequences driving Ins2 and GFP was performed as schematized in Figure 1 (genOway). The exact sequences of these regions are provided in File S1. In brief, targeting vectors were generated with a 3.10 kb unmodified or mutated (c.C331G) 5′ flanking INS region, Ins2 exon 1, intron, and exon 2, an IRES-GFP reporter cassette inserted in the 3′ untranslated region of Ins2 exon 2, followed by a neomycin selection cassette flanked by loxP sites. This construct was flanked by 5.3 kb and 1.8 kb C57BL/6J mouse homology arms. Targeting of this vector was carried out by homologous recombination in C57BL/6J embryonic stem cells, using a diphtheria toxon A cassette for negative selection. Recombinants were verified by PCR screening and Southern blotting. Four suitable clones resulted, which were used for blastocyst injections and generation of the HIP knock-in mouse strains.-This was followed by CRE-mediated excision of the neomycin cassette in vivo, and again verified by PCR screening and Southern blotting. The sequence of the replaced region was verified by Sanger sequencing. Oligonucleotides used for genotyping are shown in Table S3.

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