Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Generation of Slc12a2 knock in mouse strains. ( A ) Nucleotides of wild-type ( + ), Em1 and Em2 alleles at the acceptor site of the exon 21 region of Slc12a2 . (B) RT-PCR of cochlear and cerebellar samples of Slc12a2 Em2 mice at age 4 weeks (4 W). Genotypes and the sizes of the transcripts are shown on the top and right. M; molecular weight marker. (C) ABR thresholds of Slc12a2 Em2 mice at 4 W. Arrows indicate no response to the maximum sound. ***; p < 10 − 6 , **; p < 0.01, *; p < 0.05.

Article Snippet: Slc12a2 Em1 and Slc12a2 Em2 mouse strains generated in this study are deposited to RIKEN BioResource Research Center.

Techniques: Knock-In, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Histological study of Slc12a2 Em2 cochlea. Hematoxylin-eosin staining of cochlear ducts of Slc12a2 Em2/+ ( A-C , G-I , M-O ) and Slc12a2 Em2/Em2 ( D-F , J-L , P-R ) mice at P1 ( A-F ), P12 ( G-L ) and 4 W ( M-R ). Arrows indicate the stria vascularis. Asterisc indicates partial loss of the lateral wall. Scale bars = 50 μm. ( S-U ) Estimated cell density in the eosinophilic StV areas at 4 W. N = 4. GER, greater epithelial ridge; RM, Reissner’s membrane; SM, scala media; StV, stria vascularis.

Article Snippet: Slc12a2 Em1 and Slc12a2 Em2 mouse strains generated in this study are deposited to RIKEN BioResource Research Center.

Techniques: Staining, Membrane

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Reconstructed nano-CT images of Slc12a2 Em2 cochlea. Slc12a2 Em2/+ ( A , C ) and Slc12a2 Em2/Em2 ( B , D ) cochleae at P28. ( C , D ) Visualization of StVs at basal turns (magenta). ( E-G ) Lengths of StV lined with spiral ligament at the apical ( E ), middle ( F ), and basal ( G ) turns ( N = 3, two images per turn). Small arrows indicate non-homogenous brightness in the areas of spiral ganglion neurons. Scale bars = 100 μm.

Article Snippet: Slc12a2 Em1 and Slc12a2 Em2 mouse strains generated in this study are deposited to RIKEN BioResource Research Center.

Techniques:

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Slc12a2 signals in the Slc12a2 Em2 cochlea. Paraffin sections of Slc12a2 Em2/+ ( A , C-E ) and Slc12a2 Em2/Em2 ( B , F-H ) cochleae at P1 ( A , B ) and 4 W (C-H ) were immunostained with anti-SLC12A2 antiserum. Arrows indicate StV. SLF; spiral ligament fibrocytes. Scale bars = 50 μm ( A-C , E , F , H ) and 10 μm ( D , G ).

Article Snippet: Slc12a2 Em1 and Slc12a2 Em2 mouse strains generated in this study are deposited to RIKEN BioResource Research Center.

Techniques:

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Changes of gene expression in the whole cochleae of Slc12a2 Em2/Em2 mice. ( A ) Heatmap of genes differentially expressed in the whole cochleae of Slc12a2 Em2/Em2 and Slc12a2 +/+ mice. Red and green indicate higher or lower expression levels in the Slc12a2 Em2/Em2 cochlea . ( B ) Volcano plot of the differentially expressed genes. Genes subjected to qRT-PCR (Supplemental Fig. 7) are indicated. Red and blue dots indicate genes with higher- or lower-expression in Slc12a2 Em2/Em2 cochleae. ( C-E ) Levels of expression of Cldn9 in the developing Slc12a2 Em2 cochleae. N = 4.

Article Snippet: Slc12a2 Em1 and Slc12a2 Em2 mouse strains generated in this study are deposited to RIKEN BioResource Research Center.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Cldn9 signals in the Slc12a2 Em2 cochlea at 4 W. Cldn9 signals in the lateral wall ( A-D ) and strial surface preparations ( E-J ) of the cochleae of Slc12a2 Em2/+ ( A , B , E-G ) and Slc12a2 Em2/Em2 ( C , D , H-J ) mice. The signals in ( A-D ) are enhanced and not comparable to those in ( E-J ). ( E-J ) Cldn9 signals counterstained with Hoechsst 33,258 (Nucleus) in the auditory epithelium. Asterisks indicate predicted positions of hair cells. Scale bar = 50 μm.

Article Snippet: Slc12a2 Em1 and Slc12a2 Em2 mouse strains generated in this study are deposited to RIKEN BioResource Research Center.

Techniques:

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Characterization of the Slc12a2 Em1 mouse. ( A ) RT-PCR of samples from the cochleae and cerebella of Slc12a2 Em1 mice at 4 W. Genotypes are shown at the top. ( B ) ABR thresholds of Slc12a2 Em1/+ and Slc12a2 Em1/Em1 mice at 4 W. ( C , D ) Partial nucleotide sequence of Slc12a2 (NM_009194.3) ( C ) and partial electropherogram of the longer RT-PCR transcript ( D ) in Slc12a2 Em1/Em1 cochleae (asterisk in A ). Nucleotides mapped to exons 20, 21, and 22 are in green, red, and blue, respectively. Spliced out nucleotides in the Em1 allele are highlighted in gray. Predicted amino acid residues are shown below the sequences.

Article Snippet: Slc12a2 Em1 and Slc12a2 Em2 mouse strains generated in this study are deposited to RIKEN BioResource Research Center.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Minigene assay of exon 21 of Slc12a2 . ( A ) Minigene structures. Raus sarcoma virus long terminal repeat (LTR), exon2 and 3 of rat insulin-2 ( rnIns2 ), M13 forward sequencing primer (M13F) and M13 reverse primer (M13R), partial genomic region of wild-type or Em2 allele of Slc12a2 including exon 21, and polyA signal are shown. Numbers in blue indicate the nucleotide lengths (bp) of the Slc12a2 genomic region inserted into the vector. Gray arrows indicate primer sets to amplify reporter transcripts. ( B ) RT-PCR to detect reporter transcripts in HEK293T cells. Inserted alleles with nucleotide at c.2912 are indicated above the image of the RT-PCR transcripts. Lane 7; 100 bp ladder marker.

Article Snippet: Slc12a2 Em1 and Slc12a2 Em2 mouse strains generated in this study are deposited to RIKEN BioResource Research Center.

Techniques: Mini Gene Assay, Virus, Sequencing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Generation of Slc12a2 knock in mouse strains. ( A ) Nucleotides of wild-type ( + ), Em1 and Em2 alleles at the acceptor site of the exon 21 region of Slc12a2 . (B) RT-PCR of cochlear and cerebellar samples of Slc12a2 Em2 mice at age 4 weeks (4 W). Genotypes and the sizes of the transcripts are shown on the top and right. M; molecular weight marker. (C) ABR thresholds of Slc12a2 Em2 mice at 4 W. Arrows indicate no response to the maximum sound. ***; p < 10 − 6 , **; p < 0.01, *; p < 0.05.

Article Snippet: Slc12a2 KI mice were created on an FVB/NJcl (FVB/N, Japan CLEA, Japan) genetic background using the CRISPR/Cas9 system with Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies).

Techniques: Knock-In, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Histological study of Slc12a2 Em2 cochlea. Hematoxylin-eosin staining of cochlear ducts of Slc12a2 Em2/+ ( A-C , G-I , M-O ) and Slc12a2 Em2/Em2 ( D-F , J-L , P-R ) mice at P1 ( A-F ), P12 ( G-L ) and 4 W ( M-R ). Arrows indicate the stria vascularis. Asterisc indicates partial loss of the lateral wall. Scale bars = 50 μm. ( S-U ) Estimated cell density in the eosinophilic StV areas at 4 W. N = 4. GER, greater epithelial ridge; RM, Reissner’s membrane; SM, scala media; StV, stria vascularis.

Article Snippet: Slc12a2 KI mice were created on an FVB/NJcl (FVB/N, Japan CLEA, Japan) genetic background using the CRISPR/Cas9 system with Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies).

Techniques: Staining, Membrane

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Reconstructed nano-CT images of Slc12a2 Em2 cochlea. Slc12a2 Em2/+ ( A , C ) and Slc12a2 Em2/Em2 ( B , D ) cochleae at P28. ( C , D ) Visualization of StVs at basal turns (magenta). ( E-G ) Lengths of StV lined with spiral ligament at the apical ( E ), middle ( F ), and basal ( G ) turns ( N = 3, two images per turn). Small arrows indicate non-homogenous brightness in the areas of spiral ganglion neurons. Scale bars = 100 μm.

Article Snippet: Slc12a2 KI mice were created on an FVB/NJcl (FVB/N, Japan CLEA, Japan) genetic background using the CRISPR/Cas9 system with Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies).

Techniques:

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Slc12a2 signals in the Slc12a2 Em2 cochlea. Paraffin sections of Slc12a2 Em2/+ ( A , C-E ) and Slc12a2 Em2/Em2 ( B , F-H ) cochleae at P1 ( A , B ) and 4 W (C-H ) were immunostained with anti-SLC12A2 antiserum. Arrows indicate StV. SLF; spiral ligament fibrocytes. Scale bars = 50 μm ( A-C , E , F , H ) and 10 μm ( D , G ).

Article Snippet: Slc12a2 KI mice were created on an FVB/NJcl (FVB/N, Japan CLEA, Japan) genetic background using the CRISPR/Cas9 system with Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies).

Techniques:

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Changes of gene expression in the whole cochleae of Slc12a2 Em2/Em2 mice. ( A ) Heatmap of genes differentially expressed in the whole cochleae of Slc12a2 Em2/Em2 and Slc12a2 +/+ mice. Red and green indicate higher or lower expression levels in the Slc12a2 Em2/Em2 cochlea . ( B ) Volcano plot of the differentially expressed genes. Genes subjected to qRT-PCR (Supplemental Fig. 7) are indicated. Red and blue dots indicate genes with higher- or lower-expression in Slc12a2 Em2/Em2 cochleae. ( C-E ) Levels of expression of Cldn9 in the developing Slc12a2 Em2 cochleae. N = 4.

Article Snippet: Slc12a2 KI mice were created on an FVB/NJcl (FVB/N, Japan CLEA, Japan) genetic background using the CRISPR/Cas9 system with Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Cldn9 signals in the Slc12a2 Em2 cochlea at 4 W. Cldn9 signals in the lateral wall ( A-D ) and strial surface preparations ( E-J ) of the cochleae of Slc12a2 Em2/+ ( A , B , E-G ) and Slc12a2 Em2/Em2 ( C , D , H-J ) mice. The signals in ( A-D ) are enhanced and not comparable to those in ( E-J ). ( E-J ) Cldn9 signals counterstained with Hoechsst 33,258 (Nucleus) in the auditory epithelium. Asterisks indicate predicted positions of hair cells. Scale bar = 50 μm.

Article Snippet: Slc12a2 KI mice were created on an FVB/NJcl (FVB/N, Japan CLEA, Japan) genetic background using the CRISPR/Cas9 system with Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies).

Techniques:

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Characterization of the Slc12a2 Em1 mouse. ( A ) RT-PCR of samples from the cochleae and cerebella of Slc12a2 Em1 mice at 4 W. Genotypes are shown at the top. ( B ) ABR thresholds of Slc12a2 Em1/+ and Slc12a2 Em1/Em1 mice at 4 W. ( C , D ) Partial nucleotide sequence of Slc12a2 (NM_009194.3) ( C ) and partial electropherogram of the longer RT-PCR transcript ( D ) in Slc12a2 Em1/Em1 cochleae (asterisk in A ). Nucleotides mapped to exons 20, 21, and 22 are in green, red, and blue, respectively. Spliced out nucleotides in the Em1 allele are highlighted in gray. Predicted amino acid residues are shown below the sequences.

Article Snippet: Slc12a2 KI mice were created on an FVB/NJcl (FVB/N, Japan CLEA, Japan) genetic background using the CRISPR/Cas9 system with Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Scientific Reports

Article Title: Complete omission of exon 21 from Slc12a2 transcripts in mice results in hearing loss

doi: 10.1038/s41598-025-99827-7

Figure Lengend Snippet: Minigene assay of exon 21 of Slc12a2 . ( A ) Minigene structures. Raus sarcoma virus long terminal repeat (LTR), exon2 and 3 of rat insulin-2 ( rnIns2 ), M13 forward sequencing primer (M13F) and M13 reverse primer (M13R), partial genomic region of wild-type or Em2 allele of Slc12a2 including exon 21, and polyA signal are shown. Numbers in blue indicate the nucleotide lengths (bp) of the Slc12a2 genomic region inserted into the vector. Gray arrows indicate primer sets to amplify reporter transcripts. ( B ) RT-PCR to detect reporter transcripts in HEK293T cells. Inserted alleles with nucleotide at c.2912 are indicated above the image of the RT-PCR transcripts. Lane 7; 100 bp ladder marker.

Article Snippet: Slc12a2 KI mice were created on an FVB/NJcl (FVB/N, Japan CLEA, Japan) genetic background using the CRISPR/Cas9 system with Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies).

Techniques: Mini Gene Assay, Virus, Sequencing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Marker