Skip to main content
Fig. 3 | Genome Biology

Fig. 3

From: Genes adapt to outsmart gene-targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation

Fig. 3

Deletion of exons 2 and 3 in Rhbdf1 results in viable and healthy mice (A7). a CRISPR/Cas9-mediated deletion of exons 2 and 3 resulted in an in-frame 572-bp deletion in the Rhbdf1 gene (hereafter referred to as viable mice, Rhbdf1v/v). b The schematic shows the deleted exons and the PCR strategy to amplify the region. PCR of genomic DNA with primers flanking exons 2 and 3 shows that wildtype mice have one wildtype band (1300-bp PCR product), heterozygous mice carry a wildtype band (1300 bp) and a mutant band (728 bp), and homozygous-null mice carry one mutant band (728 bp). Samples were run in duplicate. DNA sequencing further validated an in-frame 572-bp deletion in genomic DNA (right). Also, see Additional file 1: Figure S4. c Images of representative postnatal day 15 Rhbdf1−/− mice (left) and Rhbdf1v/v mice (right). d Kaplan–Meier survival analyses showing that the lifespan of Rhbdf1v/v mice (A7) is significantly longer than that of Rhbdf1−/− mice (A5). e Body weights of Rhbdf1v/v mice compared with those of Rhbdf1−/− mice on postnatal day 21. Data represent mean ± S.D.; ***p < 0.001. f H&E-stained coronal sections of a postnatal day 21 Rhbdf1−/− brain showing prominent hemorrhages in the brain; in contrast, there was no evidence of brain hemorrhaging in Rhbdf1v/v mice. g Cardiac abnormalities were observed in Rhbdf1−/− mice but not in Rhbdf1v/v mice (right). The black rectangle illustrates cardiac fibrosis in Rhbdf1−/− mice (left). h Also, heart weight was significantly lower in Rhbdf1v/v mice than in Rhbdf1−/− mice. Data represent mean ± S.D.; ***p < 0.001. i Exome-Seq showing loss of exons 2 and 3 in Rhbdf1v/v mutant mice and not in Rhbdf1+/+ mice. The red rectangle shows the exons lost in the mutant mice. j RNA sequencing analysis of spleens isolated from Rhbdf1v/v mice indicating that CRISPR/Cas9-mediated deletion of exons 2 and 3 does not result in nonsense-mediated decay of mutant Rhbdf1v/v mRNA. Note the boxed region preceding the lost exons 2 and 3 indicates the presence of alternative transcripts. k Although the translation initiation site in exon 2 is deleted in Rhbdf1v/v mice, sequence analysis of Rhbdf1v/v cDNA revealed that the next in-frame translation initiation site (ATG) was in exon 4, which could potentially result in an N-terminally truncated protein to rescue the severe pathology of Rhbdf1−/− mice (A5). l Double digests of C-terminal Myc-DDK-tagged Rhbdf1viable and Rhbdf1non-AUG vectors using EcoRI-HF and NotI-HF restriction enzymes. m Rescue experiments in Rhbdf1−/− MEFs. RHBDF1 deficiency suppresses stimulated secretion of AREG in primary MEFs (see Additional file 1: Figure S5); hence, we performed rescue experiments to restore stimulated AREG secretion in Rhbdf1−/− MEFs (A5); Rhbdf1−/− MEFs were transiently transfected with 2 μg of either a full-length vector, or a Rhbdf1viable vector, or a Rhbdf1non-AUG vector, using Lipofectamine LTX. Forty-eight hours post-transfection, cells were stimulated overnight with either DMSO or 100 nM PMA, and cell culture supernatants were analyzed using a mouse AREG ELISA kit. Data represent mean ± S.D.; (a) significantly different from a full-length vector-transfected and DMSO-stimulated cells; (b) significantly different from a full-length vector-transfected and PMA-stimulated cells

Back to article page