Fig. S2.
A knock-in strategy to generate Reg4DTR-Red mice. (A) The top boxes depict the structure of the targeting vector, the second line represents the genomic region of the Reg4 gene, and the third line is the predicted structure of the Reg4 locus following homologous recombination. Reg4 exons are shown in black boxes on the second line, and white boxes indicate the translated region of Reg4. The translation initiation codon of Reg4 was replaced with DTR-P2A-dsRed-poly(A), flanked with a floxed neo cassette and poly(A) signal. The PGK-neo cassette was removed by crossing with the Flpe mouse, which ubiquitously expresses FLP recombinase. (B) Southern blot of BamH digests showing clones 4A2, 3D11, 4C8, and wild-type clone with no insertion, using the 5′ probe shown in A. The lower band (10 kb) represents the correctly targeted ES clone containing the insertion. The WT Reg4 allele (15 kb) was present in each positive clone and wild type. (C) PCR screening of ES cells confirmed the presence of DTR. At the 5′ end, a sense primer (L3) was paired with an antisense Reg4 gene exon primer (R3). These primers amplified 447 bp from wild type (Lower). The sense primer (L3) was also paired with an antisense DTR cDNA primer (DTR-R3). These primers amplified 657 bp (Upper).