Additional file 1 of Genes adapt to outsmart gene-targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation
Creators: name: Hosur, Vishnu
name: Low, Benjamin E.
name: Li, Daniel
name: Stafford, Grace A.
name: Kohar, Vivek
name: Shultz, Leonard D.
name: Wiles, Michael V.
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Gene-targeting strategies for generating null or conditional mutant alleles: a. In the definitive-null design, the complete coding sequence of a gene is flanked by two loxP sites, and recombination with a cre recombinase excises the floxed gene segment, creating LacZ-tagged null alleles. Loss of the complete coding sequence of the target gene ensures homozygous-null alleles. b. Targeted KO-first alleles harbor FRT sites flanking “LacZ and a neo gene,” and also harbor loxP sites flanking either an exon (exon 2, left) or specific exons chosen to be excised (exons 4 through 11, right, in this case), permitting recombination with Flp or cre recombinases and enabling conditional mutagenesis. Application of cre recombinase deletes exons flanked by two loxP sites, generating LacZ-tagged null alleles (tm1b). Application of Flp recombinase to this allele is described in the legend for 1C. c. Application of Flp recombinase to targeted KO-first alleles reverts the knockout-first alleles into conditional-ready alleles by excising lacZ and a neo gene. Subsequent recombination of conditional-ready alleles with cre recombinase generates conditional mutant alleles (tm1d). Figure S2. Sub-viability in Rhbdf1-/-Rhbdf2-/- double mutant mice (A6): a. Breeding strategy and the number of mice generated and genotyped by PCR b &amp; c. Rhbdf1-/-Rhbdf2-/- double mutant mice displayed “eyelids open at birth” phenotype (arrows) in mouse embryos harvested on embryonic day 15 (e15.5) (B) and in pups at day 2 (C). No defects were observed in Rhbdf1+/-Rhbdf2-/- littermates. Figure S3. PCR and genotyping of Rhbdf1v/v allele: Schematic showing the TRU-guides (sgRNAs 2256 and 2257) used to excise exons 2 and 3 in the Rhbdf1 gene. Also shown are the PCR primers and Taq DNA Polymerase thermocycling conditions used to genotype Rhbdf1v/v mice (A7). PCR products were sequenced to verify the 569-bp deletion. Figure S4. Histology and 5’ RACE products of Rhbdf1v/v allele: a. H&amp;E sections of Rhbdf1v/v mice displaying no abnormalities in the heart, brain, truncal skin sections, kidney, spleen, and liver tissues. b. Schematic representation of Rhbdf1v/v transcript variant with a different transcription start site. 5’ RACE identified an alternative exon (highlighted in light orange) that splices with exon 4 to reinitiate transcription from a different start site in exon 4, as opposed to using the conventional start site in exon 2. Notably, splicing of alternative exon with exon 4 is also observed in other Rhbdf1 predicted transcript variants X1, X2, X3, X4, X9, and X14. Figure S5. Stimulated secretion of AREG: Stimulated secretion of AREG in Rhbdf1+/+ and Rhbdf1-/- primary MEFs, passage one, after overnight stimulation with 100 nM PMA. Following stimulation, cell-culture supernatants were analyzed using a mouse AREG ELISA kit. Data represent mean ± S.D; ***p&lt;0.001. Figure S6. CRISPR/Cas9 Rhbdf1v/v allele (A7) reverses sub-viability in Rhbdf1-/-Rhbdf2-/- double mutant mice: a. Images of representative postnatal day 21 Rhbdf1v/+Rhbdf2-/- mice (left) and Rhbdf1v/vRhbdf2-/- mice (right). Notably, Rhbdf1v/vRhbdf2-/- mice develop a wavy hair coat (arrow). b. H&amp;E-stained sections of skin from adult Rhbdf1v/+Rhbdf2-/- and Rhbdf1v/vRhbdf2-/- mice. The Rhbdf1v/vRhbdf2-/- skin displays follicular dystrophy (arrow), hyperplasia (*), and hyperkeratosis (arrowhead), whereas that of Rhbdf1v/+Rhbdf2-/- mice does not display these phenotypes. Top panels: low magnification; bottom panels: high magnification. c. Rhbdf1v/vRhbdf2-/- double mutant mice displayed an “eyelids open at birth” phenotype in 3-day-old mouse pups (arrow). No such defects were observed in Rhbdf1v/+Rhbdf2-/- mice. Top panels: low magnification; bottom panels: high magnification. d. No cardiac or brain abnormalities were observed in either Rhbdf1v/vRhbdf2-/- double mutant mice or Rhbdf1v/+Rhbdf2-/- mice. Figure S7. Non-AUG protein coding transcripts of Rhbdf1: a. Mouse non-AUG protein-coding Rhbdf1 transcript (Top). Human non-AUG protein-coding RHBDF1 transcript. b. C-terminal Myc-DDK-tagged empty vector (lanes 1, 2), non-AUG vector (lanes 3, 4) or a positive control vector (798-bp; lanes 5, 6) were transiently expressed in 293T cells, and cell lysates were analyzed using western blotting with FLAG-specific antibody. Blots were washed, blocked in 5% nonfat dry milk, and re-probed with anti-actin antibody. Figure S8. Retention of En2 splice acceptor site in the Rhbdf1v2/v2 allele:a. mRNA expression of the Rhbdf1 gene assessed by RT-qPCR using SYBR Green RT-PCR master mix and, exon 16 forward and exon 17 reverse primers. There is no difference in the mRNA levels of Rhbdf1 between Rhbdf1+/+ and Rhbdf1v2/v2 mice. b. Schematic representation of Rhbdf1v2/v2 allele indicating the presence of En2 splice acceptor site (Extra) between exons 3 and 12. RT-PCR of spleens from Rhbdf1v2/v2 mutant mice using primers in exon 3 and exon 12. Exon 3 forward (2817 For) and exon 12 reverse (2818 Rev) primers generated a 324-bp product, instead of an expected 209-bp product due to splicing of exons 3 and 12 together. c. Sanger’s sequencing indicated the presence of En2 splice acceptor site (Extra) flanked by exons 3 and 12. d. Schematic representation of the targeted KO-first allele used to generate ES cell clones displaying the splice acceptor site downstream of exon 3. e. Transcription and translation from the conventional start site in exon 2 can yield a 10.8 kDa truncated protein owing to a stop codon in the splice acceptor site. Figure S9. 5’ RACE identifies alternative exon upstream of the conventional exon 1: a. Schematic representation of the Rhbdf1 KO-first allele used to generate ES cell clones displaying an alternative exon 1 (arrow) upstream of the conventional exon 1. b. Schematic representation of the Rhbdf1 genomic DNA displaying the alternative exon 1 (arrow) identified by 5’ RACE. The DNA sequence of the alternative exon 1 is also shown. Figure S10. Translation initiation from alternative exon 1 in the Rhbdf1v2/v2 allele could generate a truncated protein: a. Schematic representation of an Rhbdf1v2/v2 splice variant displaying transcription initiation from the alternative exon 1, which could result in a 285-bp transcript and a 12.3 kDa truncated protein. b. Primers (2827, 2818, and 2826) used to sequence verify splicing of alternative exon 1 with exon 2 (top), and En2 splice acceptor site (En2 SA) with exon 12 (bottom). Figure S11. Splicing of alternative exon 1 with exon 4 generates an N-terminally truncated variant 1 protein: a. Schematic representation of an Rhbdf1v2/v2 splice variant displaying transcription initiation from the exon 14, which could result in a 801-bp transcript and a 32.77 kDa N-terminally truncated protein (variant 1). b. RT-PCR of spleens from Rhbdf1v2/v2 mutant mice using primers (2827 and 2814) in alternative exon 1 and exon 15 resulted in a 430-bp amplicon. Sanger’s sequence demonstrated splicing of alternative exon 1 with En2 SA. Notably, Rhbdf1v/v mutant mice resulted in an amplicon with primers 2827 and 2814, suggesting the use of alternative exon 1. However, higher molecular weight PCR fragments in Rhbdf1v/v mutant mice can be attributed to the presence of exons 4 through 11, which are excised in Rhbdf1v2/v2 mutant mice. Thermocycling conditions using Taq DNA Polymerase with standard buffer are also shown. Figure S12. Splicing of alternative exon 1 with exon 4 can also generate an N-terminally truncated variant 2 protein: a. Schematic representation of an Rhbdf1v2/v2 splice variant displaying transcription initiation from the exon 15, which could result in a 717-bp splice variant and a 29.6 kDa N-terminally truncated protein (variant 2). b. RT-PCR of spleens from Rhbdf1v2/v2 mutant mice using primers (2827 and 2814) in alternative exon 1 and exon 15 also resulted in a 639-bp amplicon. Sanger’s sequence demonstrated splicing of En2 SA with exon 12. Figure S13. Double digests with NEB's restriction enzymes: Double digests of C-terminal Myc-DDK-tagged vectors (1 μg DNA) with NEB's restriction enzymes EcoRI-HF and NotI-HF. Figure S14. Unpredicted reinitiation of transcription and translation: Our data strongly suggests that mice harboring homozygous definitive-null or homozygous targeted KO-first alleles are homozygous null, however, mice harboring homozygous KO-first mutant alleles tm1b and tm1d can be null or gain-of-function or hypomorphic mice (arrows) owing to unpredicted reinitiation of transcription and translation. Figure S15. PCR and genotyping of Rhbdf1 and Rhbdf2 KOMP alleles: a. PCR primers used to genotype Rhbdf1-/- mice (A5) generated in the present study. Thermocycling conditions using Taq DNA Polymerase with standard buffer. b. Schematic showing the PCR strategy and the primers used to genotype Rhbdf2-/- mice. Thermocycling conditions using Taq DNA Polymerase with standard buffer. PCR products were purified and sequence verified.