restriction enzyme btsimuti  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs restriction enzyme btsimuti
    Restriction Enzyme Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme btsimuti - by Bioz Stars, 2024-05
    93/100 stars

    Images

    restriction enzyme btsimuti  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs restriction enzyme btsimuti
    Restriction Enzyme Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme btsimuti - by Bioz Stars, 2024-05
    93/100 stars

    Images

    restriction enzyme btsimuti  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs restriction enzyme btsimuti
    Restriction Enzyme Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme btsimuti - by Bioz Stars, 2024-05
    93/100 stars

    Images

    btsimuti  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs btsimuti
    Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    btsimuti - by Bioz Stars, 2024-05
    93/100 stars

    Images

    btsi muti restriction enzyme  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs btsi muti restriction enzyme
    Btsi Muti Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/btsi muti restriction enzyme/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    btsi muti restriction enzyme - by Bioz Stars, 2024-05
    93/100 stars

    Images

    pcr product  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs pcr product
    Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or <t>A136fs),</t> missense mutation <t>(K228E),</t> or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.
    Pcr Product, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr product/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr product - by Bioz Stars, 2024-05
    93/100 stars

    Images

    1) Product Images from "Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development"

    Article Title: Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0409-22.2022

    Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or A136fs), missense mutation (K228E), or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.
    Figure Legend Snippet: Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or A136fs), missense mutation (K228E), or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.

    Techniques Used: Mutagenesis, CRISPR, Generated, Sequencing, Clone Assay

    Tbr1 A136PfsX80 is a loss-of-function mutation, while K228E mutation causes TBR1 upregulation. A, B, Western blots for TBR1 in P0 cortical lysates from Tbr1 mutant mouse lines (n = 3–6 mice/genotype). β-III-Tubulin was used as loading control. Predicted molecular weight of truncated TBR1-A136PfsX80 protein is indicated but not detectable in Tbr1A136fs mutants. C, Western blots for TBR1 in adult cortical lysates from Tbr1KO and patient mutant lines (n = 3 mice per genotype). D, Schematic of Tbr1 cDNA indicating locations of Tbr1 mutations (arrowheads or bracket) and qPCR primers. Mutations are color-coded as in B. Blue shading indicates T-box coding region. See Extended Data Figure 1-2 for qPCR primer sequences. E, F, qPCR for Tbr1 using two primer sets in P0 Tbr1 mutant mouse line cortex (n = 3–6 mice per genotype). Genotypes are color coded as in B. G, H, Sanger sequencing of Tbr1 cDNA from P0 Tbr1A136fs mutant cortex. Red arrows indicate the position of mutation. Brackets indicate peaks corresponding to WT (black) and mutant (magenta) allele in Tbr1+/A136fs cDNA. Data are plotted as the mean ± SEM. Each dot represents one animal. One-way ANOVA with Tukey's multiple-comparisons test was used in B, E, and F; unpaired Student's t test was used in C. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, Not significant.
    Figure Legend Snippet: Tbr1 A136PfsX80 is a loss-of-function mutation, while K228E mutation causes TBR1 upregulation. A, B, Western blots for TBR1 in P0 cortical lysates from Tbr1 mutant mouse lines (n = 3–6 mice/genotype). β-III-Tubulin was used as loading control. Predicted molecular weight of truncated TBR1-A136PfsX80 protein is indicated but not detectable in Tbr1A136fs mutants. C, Western blots for TBR1 in adult cortical lysates from Tbr1KO and patient mutant lines (n = 3 mice per genotype). D, Schematic of Tbr1 cDNA indicating locations of Tbr1 mutations (arrowheads or bracket) and qPCR primers. Mutations are color-coded as in B. Blue shading indicates T-box coding region. See Extended Data Figure 1-2 for qPCR primer sequences. E, F, qPCR for Tbr1 using two primer sets in P0 Tbr1 mutant mouse line cortex (n = 3–6 mice per genotype). Genotypes are color coded as in B. G, H, Sanger sequencing of Tbr1 cDNA from P0 Tbr1A136fs mutant cortex. Red arrows indicate the position of mutation. Brackets indicate peaks corresponding to WT (black) and mutant (magenta) allele in Tbr1+/A136fs cDNA. Data are plotted as the mean ± SEM. Each dot represents one animal. One-way ANOVA with Tukey's multiple-comparisons test was used in B, E, and F; unpaired Student's t test was used in C. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, Not significant.

    Techniques Used: Mutagenesis, Western Blot, Molecular Weight, Sequencing

    Homozygous K228E mutation causes ectopic TBR1 localization in cortex. A, TBR1 immunostaining in P0 Tbr1 mutant mouse line S1 cortex. B, Quantification of TBR1 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, TBR1 immunostaining and quantification in adult Tbr1KO and patient mutant line S1 cortex. E, Coimmunostaining for TBR1 and NeuN in P0 Tbr1+/+ and Tbr1K228E/K228E S1 cortex. SP, Subplate. Scale bars: A, E, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM.
    Figure Legend Snippet: Homozygous K228E mutation causes ectopic TBR1 localization in cortex. A, TBR1 immunostaining in P0 Tbr1 mutant mouse line S1 cortex. B, Quantification of TBR1 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, TBR1 immunostaining and quantification in adult Tbr1KO and patient mutant line S1 cortex. E, Coimmunostaining for TBR1 and NeuN in P0 Tbr1+/+ and Tbr1K228E/K228E S1 cortex. SP, Subplate. Scale bars: A, E, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM.

    Techniques Used: Mutagenesis, Immunostaining, Fluorescence

    K228E mutation causes distinct cortical layering defects from Tbr1 knockout and A136PfsX80. A, Hoechst nuclear stain and immunostaining for cortical layer markers CUX1 (L2–4) and CTIP2 (L5) in P0 Tbr1 mutant mouse line S1 cortex. Yellow brackets indicate abnormal cortical layers formed in homozygous mutants. B, Quantification of Hoechst, CUX1, and CTIP2 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, Hoechst, CUX1, and CTIP2 staining and quantification in adult Tbr1KO and patient line S1 cortex (n = 2–4 mice/genotype). E, CTIP2 fluorescence binned by cortical layers using fluorescence values from D. SP, Subplate. Scale bars: A, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM. Each dot represents one animal; red dots correspond to representative images. Two-way ANOVA with Šídák's multiple-comparisons test was used in E. *p < 0.05; ***p < 0.001.
    Figure Legend Snippet: K228E mutation causes distinct cortical layering defects from Tbr1 knockout and A136PfsX80. A, Hoechst nuclear stain and immunostaining for cortical layer markers CUX1 (L2–4) and CTIP2 (L5) in P0 Tbr1 mutant mouse line S1 cortex. Yellow brackets indicate abnormal cortical layers formed in homozygous mutants. B, Quantification of Hoechst, CUX1, and CTIP2 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, Hoechst, CUX1, and CTIP2 staining and quantification in adult Tbr1KO and patient line S1 cortex (n = 2–4 mice/genotype). E, CTIP2 fluorescence binned by cortical layers using fluorescence values from D. SP, Subplate. Scale bars: A, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM. Each dot represents one animal; red dots correspond to representative images. Two-way ANOVA with Šídák's multiple-comparisons test was used in E. *p < 0.05; ***p < 0.001.

    Techniques Used: Mutagenesis, Knock-Out, Staining, Immunostaining, Fluorescence

    Complementation cross shows limited functionality of K228E allele. A, TBR1, CUX1, and CTIP2 immunostaining and Hoechst nuclear stain in S1 cortex of P0 offspring from Tbr1KO and Tbr1K228E complementation cross. Yellow brackets indicate abnormal cortical layers formed in Tbr1K228E/– mice. B, Quantification of TBR1, CUX1, CTIP2, and Hoechst fluorescence intensity across the cortical mantle from A (n = 1–2 mice/genotype). C, D, TBR1, CUX1, and Hoechst staining and fluorescence quantification in S1 cortex of P3 offspring from Tbr1KO and Tbr1Δ6aa complementation cross (n = 1–5 mice/genotype). SP, Subplate. Scale bars: A and C, 200 μm. Data are plotted as the mean ± SEM.
    Figure Legend Snippet: Complementation cross shows limited functionality of K228E allele. A, TBR1, CUX1, and CTIP2 immunostaining and Hoechst nuclear stain in S1 cortex of P0 offspring from Tbr1KO and Tbr1K228E complementation cross. Yellow brackets indicate abnormal cortical layers formed in Tbr1K228E/– mice. B, Quantification of TBR1, CUX1, CTIP2, and Hoechst fluorescence intensity across the cortical mantle from A (n = 1–2 mice/genotype). C, D, TBR1, CUX1, and Hoechst staining and fluorescence quantification in S1 cortex of P3 offspring from Tbr1KO and Tbr1Δ6aa complementation cross (n = 1–5 mice/genotype). SP, Subplate. Scale bars: A and C, 200 μm. Data are plotted as the mean ± SEM.

    Techniques Used: Immunostaining, Staining, Fluorescence

    Summary of phenotypic findings and proposed molecular mechanisms in Tbr1 mutant mice. A, Summary of phenotypic findings in Tbr1KO line and patient mutant mouse lines Tbr1A136fs and Tbr1K228E compared with wild type. Phenotypes listed in bold text are discordant across the three mutant lines. B, Proposed molecular effects of Tbr1KO and patient mutations on the regulation of TBR1 levels in postnatal cortex. TBR1 may negatively autoregulate its expression, leading to transcriptional upregulation in Tbr1 mutant mice. Transcripts from the KO and A136fs alleles may be degraded, leading to the absence of TBR1 protein, while K228E transcripts persist and lead to high protein levels. OB, Olfactory bulb.
    Figure Legend Snippet: Summary of phenotypic findings and proposed molecular mechanisms in Tbr1 mutant mice. A, Summary of phenotypic findings in Tbr1KO line and patient mutant mouse lines Tbr1A136fs and Tbr1K228E compared with wild type. Phenotypes listed in bold text are discordant across the three mutant lines. B, Proposed molecular effects of Tbr1KO and patient mutations on the regulation of TBR1 levels in postnatal cortex. TBR1 may negatively autoregulate its expression, leading to transcriptional upregulation in Tbr1 mutant mice. Transcripts from the KO and A136fs alleles may be degraded, leading to the absence of TBR1 protein, while K228E transcripts persist and lead to high protein levels. OB, Olfactory bulb.

    Techniques Used: Mutagenesis, Expressing

    pcr product  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs pcr product
    Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or <t>A136fs),</t> missense mutation <t>(K228E),</t> or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.
    Pcr Product, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr product/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr product - by Bioz Stars, 2024-05
    93/100 stars

    Images

    1) Product Images from "Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development"

    Article Title: Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0409-22.2022

    Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or A136fs), missense mutation (K228E), or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.
    Figure Legend Snippet: Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or A136fs), missense mutation (K228E), or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.

    Techniques Used: Mutagenesis, CRISPR, Generated, Sequencing, Clone Assay

    Tbr1 A136PfsX80 is a loss-of-function mutation, while K228E mutation causes TBR1 upregulation. A, B, Western blots for TBR1 in P0 cortical lysates from Tbr1 mutant mouse lines (n = 3–6 mice/genotype). β-III-Tubulin was used as loading control. Predicted molecular weight of truncated TBR1-A136PfsX80 protein is indicated but not detectable in Tbr1A136fs mutants. C, Western blots for TBR1 in adult cortical lysates from Tbr1KO and patient mutant lines (n = 3 mice per genotype). D, Schematic of Tbr1 cDNA indicating locations of Tbr1 mutations (arrowheads or bracket) and qPCR primers. Mutations are color-coded as in B. Blue shading indicates T-box coding region. See Extended Data Figure 1-2 for qPCR primer sequences. E, F, qPCR for Tbr1 using two primer sets in P0 Tbr1 mutant mouse line cortex (n = 3–6 mice per genotype). Genotypes are color coded as in B. G, H, Sanger sequencing of Tbr1 cDNA from P0 Tbr1A136fs mutant cortex. Red arrows indicate the position of mutation. Brackets indicate peaks corresponding to WT (black) and mutant (magenta) allele in Tbr1+/A136fs cDNA. Data are plotted as the mean ± SEM. Each dot represents one animal. One-way ANOVA with Tukey's multiple-comparisons test was used in B, E, and F; unpaired Student's t test was used in C. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, Not significant.
    Figure Legend Snippet: Tbr1 A136PfsX80 is a loss-of-function mutation, while K228E mutation causes TBR1 upregulation. A, B, Western blots for TBR1 in P0 cortical lysates from Tbr1 mutant mouse lines (n = 3–6 mice/genotype). β-III-Tubulin was used as loading control. Predicted molecular weight of truncated TBR1-A136PfsX80 protein is indicated but not detectable in Tbr1A136fs mutants. C, Western blots for TBR1 in adult cortical lysates from Tbr1KO and patient mutant lines (n = 3 mice per genotype). D, Schematic of Tbr1 cDNA indicating locations of Tbr1 mutations (arrowheads or bracket) and qPCR primers. Mutations are color-coded as in B. Blue shading indicates T-box coding region. See Extended Data Figure 1-2 for qPCR primer sequences. E, F, qPCR for Tbr1 using two primer sets in P0 Tbr1 mutant mouse line cortex (n = 3–6 mice per genotype). Genotypes are color coded as in B. G, H, Sanger sequencing of Tbr1 cDNA from P0 Tbr1A136fs mutant cortex. Red arrows indicate the position of mutation. Brackets indicate peaks corresponding to WT (black) and mutant (magenta) allele in Tbr1+/A136fs cDNA. Data are plotted as the mean ± SEM. Each dot represents one animal. One-way ANOVA with Tukey's multiple-comparisons test was used in B, E, and F; unpaired Student's t test was used in C. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, Not significant.

    Techniques Used: Mutagenesis, Western Blot, Molecular Weight, Sequencing

    Homozygous K228E mutation causes ectopic TBR1 localization in cortex. A, TBR1 immunostaining in P0 Tbr1 mutant mouse line S1 cortex. B, Quantification of TBR1 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, TBR1 immunostaining and quantification in adult Tbr1KO and patient mutant line S1 cortex. E, Coimmunostaining for TBR1 and NeuN in P0 Tbr1+/+ and Tbr1K228E/K228E S1 cortex. SP, Subplate. Scale bars: A, E, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM.
    Figure Legend Snippet: Homozygous K228E mutation causes ectopic TBR1 localization in cortex. A, TBR1 immunostaining in P0 Tbr1 mutant mouse line S1 cortex. B, Quantification of TBR1 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, TBR1 immunostaining and quantification in adult Tbr1KO and patient mutant line S1 cortex. E, Coimmunostaining for TBR1 and NeuN in P0 Tbr1+/+ and Tbr1K228E/K228E S1 cortex. SP, Subplate. Scale bars: A, E, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM.

    Techniques Used: Mutagenesis, Immunostaining, Fluorescence

    K228E mutation causes distinct cortical layering defects from Tbr1 knockout and A136PfsX80. A, Hoechst nuclear stain and immunostaining for cortical layer markers CUX1 (L2–4) and CTIP2 (L5) in P0 Tbr1 mutant mouse line S1 cortex. Yellow brackets indicate abnormal cortical layers formed in homozygous mutants. B, Quantification of Hoechst, CUX1, and CTIP2 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, Hoechst, CUX1, and CTIP2 staining and quantification in adult Tbr1KO and patient line S1 cortex (n = 2–4 mice/genotype). E, CTIP2 fluorescence binned by cortical layers using fluorescence values from D. SP, Subplate. Scale bars: A, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM. Each dot represents one animal; red dots correspond to representative images. Two-way ANOVA with Šídák's multiple-comparisons test was used in E. *p < 0.05; ***p < 0.001.
    Figure Legend Snippet: K228E mutation causes distinct cortical layering defects from Tbr1 knockout and A136PfsX80. A, Hoechst nuclear stain and immunostaining for cortical layer markers CUX1 (L2–4) and CTIP2 (L5) in P0 Tbr1 mutant mouse line S1 cortex. Yellow brackets indicate abnormal cortical layers formed in homozygous mutants. B, Quantification of Hoechst, CUX1, and CTIP2 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, Hoechst, CUX1, and CTIP2 staining and quantification in adult Tbr1KO and patient line S1 cortex (n = 2–4 mice/genotype). E, CTIP2 fluorescence binned by cortical layers using fluorescence values from D. SP, Subplate. Scale bars: A, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM. Each dot represents one animal; red dots correspond to representative images. Two-way ANOVA with Šídák's multiple-comparisons test was used in E. *p < 0.05; ***p < 0.001.

    Techniques Used: Mutagenesis, Knock-Out, Staining, Immunostaining, Fluorescence

    Complementation cross shows limited functionality of K228E allele. A, TBR1, CUX1, and CTIP2 immunostaining and Hoechst nuclear stain in S1 cortex of P0 offspring from Tbr1KO and Tbr1K228E complementation cross. Yellow brackets indicate abnormal cortical layers formed in Tbr1K228E/– mice. B, Quantification of TBR1, CUX1, CTIP2, and Hoechst fluorescence intensity across the cortical mantle from A (n = 1–2 mice/genotype). C, D, TBR1, CUX1, and Hoechst staining and fluorescence quantification in S1 cortex of P3 offspring from Tbr1KO and Tbr1Δ6aa complementation cross (n = 1–5 mice/genotype). SP, Subplate. Scale bars: A and C, 200 μm. Data are plotted as the mean ± SEM.
    Figure Legend Snippet: Complementation cross shows limited functionality of K228E allele. A, TBR1, CUX1, and CTIP2 immunostaining and Hoechst nuclear stain in S1 cortex of P0 offspring from Tbr1KO and Tbr1K228E complementation cross. Yellow brackets indicate abnormal cortical layers formed in Tbr1K228E/– mice. B, Quantification of TBR1, CUX1, CTIP2, and Hoechst fluorescence intensity across the cortical mantle from A (n = 1–2 mice/genotype). C, D, TBR1, CUX1, and Hoechst staining and fluorescence quantification in S1 cortex of P3 offspring from Tbr1KO and Tbr1Δ6aa complementation cross (n = 1–5 mice/genotype). SP, Subplate. Scale bars: A and C, 200 μm. Data are plotted as the mean ± SEM.

    Techniques Used: Immunostaining, Staining, Fluorescence

    Summary of phenotypic findings and proposed molecular mechanisms in Tbr1 mutant mice. A, Summary of phenotypic findings in Tbr1KO line and patient mutant mouse lines Tbr1A136fs and Tbr1K228E compared with wild type. Phenotypes listed in bold text are discordant across the three mutant lines. B, Proposed molecular effects of Tbr1KO and patient mutations on the regulation of TBR1 levels in postnatal cortex. TBR1 may negatively autoregulate its expression, leading to transcriptional upregulation in Tbr1 mutant mice. Transcripts from the KO and A136fs alleles may be degraded, leading to the absence of TBR1 protein, while K228E transcripts persist and lead to high protein levels. OB, Olfactory bulb.
    Figure Legend Snippet: Summary of phenotypic findings and proposed molecular mechanisms in Tbr1 mutant mice. A, Summary of phenotypic findings in Tbr1KO line and patient mutant mouse lines Tbr1A136fs and Tbr1K228E compared with wild type. Phenotypes listed in bold text are discordant across the three mutant lines. B, Proposed molecular effects of Tbr1KO and patient mutations on the regulation of TBR1 levels in postnatal cortex. TBR1 may negatively autoregulate its expression, leading to transcriptional upregulation in Tbr1 mutant mice. Transcripts from the KO and A136fs alleles may be degraded, leading to the absence of TBR1 protein, while K228E transcripts persist and lead to high protein levels. OB, Olfactory bulb.

    Techniques Used: Mutagenesis, Expressing

    restriction enzymes btsimuti  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs restriction enzymes btsimuti
    Restriction Enzymes Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzymes btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction enzymes btsimuti - by Bioz Stars, 2024-05
    93/100 stars

    Images

    btsimuti  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs btsimuti
    Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    btsimuti - by Bioz Stars, 2024-05
    93/100 stars

    Images

    btsi muti  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs btsi muti
    Btsi Muti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/btsi muti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    btsi muti - by Bioz Stars, 2024-05
    93/100 stars

    Images

    btsi muti  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    New England Biolabs btsi muti
    Btsi Muti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/btsi muti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    btsi muti - by Bioz Stars, 2024-05
    93/100 stars

    Images

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    New England Biolabs restriction enzyme btsimuti
    Restriction Enzyme Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme btsimuti - by Bioz Stars, 2024-05
    93/100 stars
      Buy from Supplier

    93
    New England Biolabs btsimuti
    Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    btsimuti - by Bioz Stars, 2024-05
    93/100 stars
      Buy from Supplier

    93
    New England Biolabs btsi muti restriction enzyme
    Btsi Muti Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/btsi muti restriction enzyme/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    btsi muti restriction enzyme - by Bioz Stars, 2024-05
    93/100 stars
      Buy from Supplier

    93
    New England Biolabs pcr product
    Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or <t>A136fs),</t> missense mutation <t>(K228E),</t> or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.
    Pcr Product, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr product/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr product - by Bioz Stars, 2024-05
    93/100 stars
      Buy from Supplier

    93
    New England Biolabs restriction enzymes btsimuti
    Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or <t>A136fs),</t> missense mutation <t>(K228E),</t> or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.
    Restriction Enzymes Btsimuti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzymes btsimuti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction enzymes btsimuti - by Bioz Stars, 2024-05
    93/100 stars
      Buy from Supplier

    93
    New England Biolabs btsi muti
    Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or <t>A136fs),</t> missense mutation <t>(K228E),</t> or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.
    Btsi Muti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/btsi muti/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    btsi muti - by Bioz Stars, 2024-05
    93/100 stars
      Buy from Supplier

    Image Search Results


    Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or A136fs), missense mutation (K228E), or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.

    Journal: The Journal of Neuroscience

    Article Title: Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development

    doi: 10.1523/JNEUROSCI.0409-22.2022

    Figure Lengend Snippet: Generation of mouse lines carrying Tbr1 mutations. A, Schematic of Tbr1 gene structure (left) and predicted protein products (right) for Tbr1 mutant mouse lines used in this study: published Tbr1KO line replacing exons 2-3 with a neomycin cassette (Bulfone et al., 1998) and three CRISPR-generated lines carrying a frameshift mutation (A136PfsX80 or A136fs), missense mutation (K228E), or in-frame deletion of 6 aa (E348_P353del or Δ6aa). See Extended Data Figure 1-1 for reported human mutations within the in-frame deletion site of Tbr1Δ6aa mice. For Tbr1 gene, blue boxes indicate T-box coding sequence, gray boxes indicate other coding sequence, and white boxes indicate untranslated regions. B, Multiple sequence alignment of TBR1 mutation sites across vertebrate species. C, Pairwise alignment of predicted frameshift regions of human and mouse TBR1-A136PfsX80 proteins. D, Multiple sequence alignment of K228 and E348_P353 sites across mouse T-box family proteins. E, Sanger sequencing of genomic or TOPO-cloned DNA showing CRISPR-generated Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutations. See Extended Data Figure 1-2 for CRISPR oligonucleotide and Sanger primer sequences. F, Dorsal view of brains from postnatal day 0 Tbr1A136fs, Tbr1K228E, and Tbr1Δ6aa mutant mice and wild-type littermates. Arrowheads indicate underdeveloped olfactory bulbs in Tbr1A136fs/A136fs and Tbr1K228E/K228E mice. Scale bar, 1 mm.

    Article Snippet: Tbr1 A136fs and Tbr1 K228E mice were genotyped using PCR amplification of the mutation-containing genomic region followed by restriction enzyme digest of the PCR product (BtsCI for A136fs, BtsIMutI for K228E, New England Biolabs).

    Techniques: Mutagenesis, CRISPR, Generated, Sequencing, Clone Assay

    Tbr1 A136PfsX80 is a loss-of-function mutation, while K228E mutation causes TBR1 upregulation. A, B, Western blots for TBR1 in P0 cortical lysates from Tbr1 mutant mouse lines (n = 3–6 mice/genotype). β-III-Tubulin was used as loading control. Predicted molecular weight of truncated TBR1-A136PfsX80 protein is indicated but not detectable in Tbr1A136fs mutants. C, Western blots for TBR1 in adult cortical lysates from Tbr1KO and patient mutant lines (n = 3 mice per genotype). D, Schematic of Tbr1 cDNA indicating locations of Tbr1 mutations (arrowheads or bracket) and qPCR primers. Mutations are color-coded as in B. Blue shading indicates T-box coding region. See Extended Data Figure 1-2 for qPCR primer sequences. E, F, qPCR for Tbr1 using two primer sets in P0 Tbr1 mutant mouse line cortex (n = 3–6 mice per genotype). Genotypes are color coded as in B. G, H, Sanger sequencing of Tbr1 cDNA from P0 Tbr1A136fs mutant cortex. Red arrows indicate the position of mutation. Brackets indicate peaks corresponding to WT (black) and mutant (magenta) allele in Tbr1+/A136fs cDNA. Data are plotted as the mean ± SEM. Each dot represents one animal. One-way ANOVA with Tukey's multiple-comparisons test was used in B, E, and F; unpaired Student's t test was used in C. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, Not significant.

    Journal: The Journal of Neuroscience

    Article Title: Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development

    doi: 10.1523/JNEUROSCI.0409-22.2022

    Figure Lengend Snippet: Tbr1 A136PfsX80 is a loss-of-function mutation, while K228E mutation causes TBR1 upregulation. A, B, Western blots for TBR1 in P0 cortical lysates from Tbr1 mutant mouse lines (n = 3–6 mice/genotype). β-III-Tubulin was used as loading control. Predicted molecular weight of truncated TBR1-A136PfsX80 protein is indicated but not detectable in Tbr1A136fs mutants. C, Western blots for TBR1 in adult cortical lysates from Tbr1KO and patient mutant lines (n = 3 mice per genotype). D, Schematic of Tbr1 cDNA indicating locations of Tbr1 mutations (arrowheads or bracket) and qPCR primers. Mutations are color-coded as in B. Blue shading indicates T-box coding region. See Extended Data Figure 1-2 for qPCR primer sequences. E, F, qPCR for Tbr1 using two primer sets in P0 Tbr1 mutant mouse line cortex (n = 3–6 mice per genotype). Genotypes are color coded as in B. G, H, Sanger sequencing of Tbr1 cDNA from P0 Tbr1A136fs mutant cortex. Red arrows indicate the position of mutation. Brackets indicate peaks corresponding to WT (black) and mutant (magenta) allele in Tbr1+/A136fs cDNA. Data are plotted as the mean ± SEM. Each dot represents one animal. One-way ANOVA with Tukey's multiple-comparisons test was used in B, E, and F; unpaired Student's t test was used in C. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, Not significant.

    Article Snippet: Tbr1 A136fs and Tbr1 K228E mice were genotyped using PCR amplification of the mutation-containing genomic region followed by restriction enzyme digest of the PCR product (BtsCI for A136fs, BtsIMutI for K228E, New England Biolabs).

    Techniques: Mutagenesis, Western Blot, Molecular Weight, Sequencing

    Homozygous K228E mutation causes ectopic TBR1 localization in cortex. A, TBR1 immunostaining in P0 Tbr1 mutant mouse line S1 cortex. B, Quantification of TBR1 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, TBR1 immunostaining and quantification in adult Tbr1KO and patient mutant line S1 cortex. E, Coimmunostaining for TBR1 and NeuN in P0 Tbr1+/+ and Tbr1K228E/K228E S1 cortex. SP, Subplate. Scale bars: A, E, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM.

    Journal: The Journal of Neuroscience

    Article Title: Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development

    doi: 10.1523/JNEUROSCI.0409-22.2022

    Figure Lengend Snippet: Homozygous K228E mutation causes ectopic TBR1 localization in cortex. A, TBR1 immunostaining in P0 Tbr1 mutant mouse line S1 cortex. B, Quantification of TBR1 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, TBR1 immunostaining and quantification in adult Tbr1KO and patient mutant line S1 cortex. E, Coimmunostaining for TBR1 and NeuN in P0 Tbr1+/+ and Tbr1K228E/K228E S1 cortex. SP, Subplate. Scale bars: A, E, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM.

    Article Snippet: Tbr1 A136fs and Tbr1 K228E mice were genotyped using PCR amplification of the mutation-containing genomic region followed by restriction enzyme digest of the PCR product (BtsCI for A136fs, BtsIMutI for K228E, New England Biolabs).

    Techniques: Mutagenesis, Immunostaining, Fluorescence

    K228E mutation causes distinct cortical layering defects from Tbr1 knockout and A136PfsX80. A, Hoechst nuclear stain and immunostaining for cortical layer markers CUX1 (L2–4) and CTIP2 (L5) in P0 Tbr1 mutant mouse line S1 cortex. Yellow brackets indicate abnormal cortical layers formed in homozygous mutants. B, Quantification of Hoechst, CUX1, and CTIP2 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, Hoechst, CUX1, and CTIP2 staining and quantification in adult Tbr1KO and patient line S1 cortex (n = 2–4 mice/genotype). E, CTIP2 fluorescence binned by cortical layers using fluorescence values from D. SP, Subplate. Scale bars: A, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM. Each dot represents one animal; red dots correspond to representative images. Two-way ANOVA with Šídák's multiple-comparisons test was used in E. *p < 0.05; ***p < 0.001.

    Journal: The Journal of Neuroscience

    Article Title: Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development

    doi: 10.1523/JNEUROSCI.0409-22.2022

    Figure Lengend Snippet: K228E mutation causes distinct cortical layering defects from Tbr1 knockout and A136PfsX80. A, Hoechst nuclear stain and immunostaining for cortical layer markers CUX1 (L2–4) and CTIP2 (L5) in P0 Tbr1 mutant mouse line S1 cortex. Yellow brackets indicate abnormal cortical layers formed in homozygous mutants. B, Quantification of Hoechst, CUX1, and CTIP2 fluorescence intensity across the cortical mantle from A (n = 2–4 mice/genotype). C, D, Hoechst, CUX1, and CTIP2 staining and quantification in adult Tbr1KO and patient line S1 cortex (n = 2–4 mice/genotype). E, CTIP2 fluorescence binned by cortical layers using fluorescence values from D. SP, Subplate. Scale bars: A, 200 μm; C, 500 μm. Data are plotted as the mean ± SEM. Each dot represents one animal; red dots correspond to representative images. Two-way ANOVA with Šídák's multiple-comparisons test was used in E. *p < 0.05; ***p < 0.001.

    Article Snippet: Tbr1 A136fs and Tbr1 K228E mice were genotyped using PCR amplification of the mutation-containing genomic region followed by restriction enzyme digest of the PCR product (BtsCI for A136fs, BtsIMutI for K228E, New England Biolabs).

    Techniques: Mutagenesis, Knock-Out, Staining, Immunostaining, Fluorescence

    Complementation cross shows limited functionality of K228E allele. A, TBR1, CUX1, and CTIP2 immunostaining and Hoechst nuclear stain in S1 cortex of P0 offspring from Tbr1KO and Tbr1K228E complementation cross. Yellow brackets indicate abnormal cortical layers formed in Tbr1K228E/– mice. B, Quantification of TBR1, CUX1, CTIP2, and Hoechst fluorescence intensity across the cortical mantle from A (n = 1–2 mice/genotype). C, D, TBR1, CUX1, and Hoechst staining and fluorescence quantification in S1 cortex of P3 offspring from Tbr1KO and Tbr1Δ6aa complementation cross (n = 1–5 mice/genotype). SP, Subplate. Scale bars: A and C, 200 μm. Data are plotted as the mean ± SEM.

    Journal: The Journal of Neuroscience

    Article Title: Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development

    doi: 10.1523/JNEUROSCI.0409-22.2022

    Figure Lengend Snippet: Complementation cross shows limited functionality of K228E allele. A, TBR1, CUX1, and CTIP2 immunostaining and Hoechst nuclear stain in S1 cortex of P0 offspring from Tbr1KO and Tbr1K228E complementation cross. Yellow brackets indicate abnormal cortical layers formed in Tbr1K228E/– mice. B, Quantification of TBR1, CUX1, CTIP2, and Hoechst fluorescence intensity across the cortical mantle from A (n = 1–2 mice/genotype). C, D, TBR1, CUX1, and Hoechst staining and fluorescence quantification in S1 cortex of P3 offspring from Tbr1KO and Tbr1Δ6aa complementation cross (n = 1–5 mice/genotype). SP, Subplate. Scale bars: A and C, 200 μm. Data are plotted as the mean ± SEM.

    Article Snippet: Tbr1 A136fs and Tbr1 K228E mice were genotyped using PCR amplification of the mutation-containing genomic region followed by restriction enzyme digest of the PCR product (BtsCI for A136fs, BtsIMutI for K228E, New England Biolabs).

    Techniques: Immunostaining, Staining, Fluorescence

    Summary of phenotypic findings and proposed molecular mechanisms in Tbr1 mutant mice. A, Summary of phenotypic findings in Tbr1KO line and patient mutant mouse lines Tbr1A136fs and Tbr1K228E compared with wild type. Phenotypes listed in bold text are discordant across the three mutant lines. B, Proposed molecular effects of Tbr1KO and patient mutations on the regulation of TBR1 levels in postnatal cortex. TBR1 may negatively autoregulate its expression, leading to transcriptional upregulation in Tbr1 mutant mice. Transcripts from the KO and A136fs alleles may be degraded, leading to the absence of TBR1 protein, while K228E transcripts persist and lead to high protein levels. OB, Olfactory bulb.

    Journal: The Journal of Neuroscience

    Article Title: Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development

    doi: 10.1523/JNEUROSCI.0409-22.2022

    Figure Lengend Snippet: Summary of phenotypic findings and proposed molecular mechanisms in Tbr1 mutant mice. A, Summary of phenotypic findings in Tbr1KO line and patient mutant mouse lines Tbr1A136fs and Tbr1K228E compared with wild type. Phenotypes listed in bold text are discordant across the three mutant lines. B, Proposed molecular effects of Tbr1KO and patient mutations on the regulation of TBR1 levels in postnatal cortex. TBR1 may negatively autoregulate its expression, leading to transcriptional upregulation in Tbr1 mutant mice. Transcripts from the KO and A136fs alleles may be degraded, leading to the absence of TBR1 protein, while K228E transcripts persist and lead to high protein levels. OB, Olfactory bulb.

    Article Snippet: Tbr1 A136fs and Tbr1 K228E mice were genotyped using PCR amplification of the mutation-containing genomic region followed by restriction enzyme digest of the PCR product (BtsCI for A136fs, BtsIMutI for K228E, New England Biolabs).

    Techniques: Mutagenesis, Expressing