Fig. 4
Targeted KO-first targeting strategy in Rhbdf1 (A3) generates novel transcripts and N-terminally truncated functional proteins. a Schematic of the strategy used by Li et al. for generation of Rhbdf1−/− homozygous mutant mice; the Rhbdf1 KO-first allele was crossed to Cre transgenic mice to excise the floxed gene segment (exons 4–11), generating Rhbdf1−/− homozygous mutant mice (hereafter referred as viable2 mice, Rhbdf1v2/v2 mice). b Whole-exome sequencing of spleen tissue from Rhbdf1v2/v2 mice showing loss of exons 4 through 11 in Rhbdf1v2/v2 mutant mice. c RT-PCR on spleens from Rhbdf1+/+ and Rhbdf1v2/v2 mutant mice using primers to amplify exons 6 through 8, exons 7 through 10, and exons 16 and 17. Exons 4–11 are deleted in Rhbdf1v2/v2 mutant mice; hence, no amplicons were generated using either exon 6 forward and exon 8 reverse, or exon 7 forward and exon 10 reverse, primers. However, exon 16 forward and exon 17 reverse primers generated a 211-bp product. d RNA-Seq analysis of spleens from Rhbdf1v2/v2 mutant mice indicating loss of exons 4 through 11; however, there is strong evidence for mutant mRNA, as indicated by the presence of the rest of the transcript, which encodes exons 12 through 18 and is not degraded by the nonsense-mediated decay mechanism. e Schematic representation of exons and introns in the Rhbdf1v2/v2 mutant allele. 5′ RACE using a gene-specific exon 16–17 fusion primer (GSP) was used to obtain 5′ ends of the Rhbdf1v2/v2 mutant mRNA. We identified several novel mutant mRNAs with different translation initiation sites that could potentially generate N-terminally truncated RHBDF1 mutant proteins. See supplemental figures for variant protein and 5′ UTR sequences. Alternative exons are indicated as red boxes; predicted translation initiation sites are indicated by “START,” and termination codons are indicated by “STOP.” f C-terminal Myc-DDK-tagged Rhbdf1v2/v2 variant protein 1 (lanes 1, 2) or variant protein 2 (lanes 3,4), or empty vector (lanes 5, 6) were transiently expressed in 293T cells, and cell lysates were analyzed using western blotting with FLAG-specific antibody. After visualization of blots with a G:Box chemiluminescent imaging system, blots were washed, blocked in 5% nonfat dry milk, and re-probed with anti-actin antibody. g Rescue of phenotype in Rhbdf1−/− MEFs. Rhbdf1+/+ (top) and Rhbdf1−/− (bottom) MEFs were transiently transfected with 2 μg of either variant 1 or variant 2 vectors, or an empty 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.; *p < 0.05, ***p < 0.001. h Comparison of phenotypes of previously generated Rhbdf1 single mutant and Rhbdf1:Rhbdf2 double mutant mice with those of mice generated in the present study. The viability of Rhbdf1v/v, Rhbdf1v2/v2, and Rhbdf1v/vRhbdf2−/− double mutant mice demonstrates that the incomplete coding sequences not degraded by NMD have functional significance and, moreover, that targeted KO-first conditional mutagenesis (A3) or CRISPR/Cas9-mediated deletion of exon(s) (A7), in contrast to complete deletion of exons (A1 and A5), can yield gain-of-function alleles