Schematic illustrations of IL-R1 wild-type (top) and restore knock-in (bottom) alleles. Top, Relevant sequence of IL-1R1 gene containing exons IX, X, and XI. Short arm for homologous recombination is designed from intron VIII to intron IX. Long arm for homologous recombination is designed from exon XI to the 3′ noncoding region. In the restore allele, knock-in Target I and knock-in Target II are inserted into the IL-1R1 locus. Target I DNA segment contains an En2 sequence, a destabilization domain (DD), and 2 polyA signal sequence followed by a neocassette. Target I segment is flanked by two loxP sites. Knock-in Target II is placed at the stop codon of the IL-1R1 sequence. The original stop codon is replaced by 3HA-STOP-IRES-tdTomato-STOP. Positions of the probes for Southern blot analysis are shown with the red arrows. The 5′ probe and 3′ probe detects a 15 kb fragment and an 8.7 kb fragment respectively in the restore allele after SacI digestion, but a 22.4 kb fragment in the wild-type. TM, Transmembrane; 5′-Prb, 5′ probe; 3′-Prb, 3′ probe; En2 SA, Engrailed-2 splice acceptor sequence; 2PolyA, 2PolyA sequence; neo, neocassette; 3HAs, 3HA tag sequence; IRES, IRES sequence; tdTomato, tdTomato sequence; SacI, restriction endonuclease SacI cleavage site.