Extended Data Fig. 1: Generation of a conditional KO allele of Insm1.
From: Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1
a, We generated a targeting construct in which the sole exon of Insm1 (green rectangle, with the coding sequence in dark green and the UTRs in light green) has a loxP site (purple triangle) inserted in a poorly conserved area of its 5′UTR and another loxP site downstream of the Insm1 gene. The construct also incorporates a neomycin resistance cassette (NEO, blue) surrounded by Frt sites (red triangles) and a thymidine kinase cassette (HSV-TK; orange), which are used to select for recombination events after gene targeting. b, We screened 439 clones and identified 5 recombinants (1 non-recombinant wild type, B5, and 3 recombinants, B6, E10 and B3, are shown) with PCR using primers indicated in a (arrows). The expected size for the wild-type allele using primers WT to R2 is 6,163 bp. The expected size for recombinant clones using CKO-reverse is 6,145 bp. c, Selected embryonic stem (ES) cell clones were additionally screened for homologous recombination upstream of the first loxP site by Southern blotting after digestion with SpeI and using the 5′ probe indicated in a6. Southern blotting was performed twice. The expected band sizes for wild-type and conditional KO alleles are 18,162 bp and 14,333 bp, respectively. From one of these clones (B3) we generated first chimeric mice and then mice with floxed alleles of Insm1 (obtained by crossing the chimaeras with mice expressing the FlpE recombinase (B6-Tg(CAG-FLPe)36, which deleted the NEO cassette flanked by FRT sites). Homozygous Insm1F/F mice are viable, demonstrating that the loxP insertions do not interfere with the vital functions of Insm1 and hence may be used for its conditional ablation. Co-expression with Cre recombinase generates an Insm1 KO allele lacking its entire coding sequence.
