Fig. 1: Generation of Gabrg2-floxed mice.

a The transcript Gabrg2-003 (ENSMUST00000070735.9) was taken as an example to describe the strategy. Gabrg2 gene has 10 exons, with the ATG start codon in exon 1 and TAA stop codon in exon10. loxP sites were inserted in introns 1–2 and introns 2–3 by donor mediated homologous recombination. Exon 2 was floxed by loxP sites and can be removed via crossing with Cre-driver lines. Frameshift caused by indel mutations can destroy the Gabrg2 gene product. b Gabrg2-floxed heterozygotes or homozygotes were identified by PCR screening of tail-derived genomic DNA using the primer of 5′ preliminary screening probe, and the 158 bp or 248 bp PCR product was the wild type or homozygotes, respectively. The 158 bp and 248 bp bands present heterozygous. c The primer of 3’ preliminary screening probe was used for detecting heterozygous or homozygous Gabrg2-floxed mice. The 294 bp and 201 bp bands present heterozygous. d The loxP site and 5’ homologous arm of 5’ ssDNA were detected by D5-5 primer, and a 534 bp band was a positive clone containing the loxP site. e The loxP site and 3’ homologous arm of 3’ ssDNA were detected by D3-3 primer, and the 588 bp band was a positive clone. f The loxP site and 5’ homologous arm of 3’ ssDNA were also detected by D3-5 primer, and the 1 962 bp band was a positive clone. g The loxP site and 3’ homologous arm of 5’ ssDNA were detected by D5-3 primer, the 2 013 bp band was a positive clone. h PCR product sequencing showed that the loxP site was correctly inserted in introns 1-2. i Sequencing results indicated that the loxP site was correctly inserted in introns 2-3. TRANS 2 K Plus II maker size: 8 000, 5 000, 3 000, 2 000, 1 000, 750, 500, 250, and 100 bp; P: positive control; B6: negative control of which the template is the genomic DNA of C57BL/6 J mice; N: blank control without template.