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crispr cas9 technology  (Macrogen)

 
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    Macrogen crispr cas9 technology
    ChRO1 deficiency and condensate disruption induce a muscle atrophic phenotype. ( A ) Strategy for generating ChRO1 KO (ChRO1−/−) mice using <t>CRISPR–Cas9</t> genome editing targeting two gRNA sites to delete ChRO1 promoter, exon1, and part of intron1 region. ( B ) qRT-RCR quantification of ChRO1a and ChRO1b expression in gastrocnemius from 13-months-old-male mice WT and KO mice (ChRO1+/+ and ChRO1−/−). ( C ) qRT-PCR analysis of atrophic genes and satellite RNAs in gastrocnemius muscle from WT and ChRO1 KO mice. ( D ) Representative wheat germ agglutinin staining (left) and myofiber CSA (μm 2 ) quantification (right) of indicated skeletal muscle tissues from WT and ChRO1 KO mice. n ≥ 500 fibers analyzed per tissue. Scale bars, 40 μm. ( E ) Representative images (left) and quantification (right) of heterochromatin foci in gastrocnemius muscle from WT and ChRO1 KO mice. Scale bars, 1 μm. n = 50 nuclei. ( F ) Immunostaining for DAXX and H3K9me3 in gastrocnemius muscle of WT and ChRO1 KO mice. n = 50 nuclei. Scale bars, 2 μm. ( G ) qRT-PCR analysis of Atrogin1 (left) and Murf1 (right) in C2C12 MT differentiated for 5 days followed by 1,6-HD treatment at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Expression normalized to Gapdh and shown as fold change relative to untreated control for each time point. ( H ) Western blot analysis of MyHC protein levels in C2C12 MT treated with 1,6-HD at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Immunofluorescence staining of MyHC (AF488, green) and nuclei (DAPI, blue) ( I ) and quantification of myotube diameters [( J ), n = 100 cells] of C2C12 MT treated with 1,6-HD at indicated concentrations. ( K ) Quantification of myotube diameters of C2C12 MT treated with 2,5-HD or 1,6-HD at indicated concentration for 24 h. n = 45 cells. Statistical analyses, box plot elements, and data normalization procedures are detailed in the “Materials and methods” section.
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    1) Product Images from "Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity"

    Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkag168

    ChRO1 deficiency and condensate disruption induce a muscle atrophic phenotype. ( A ) Strategy for generating ChRO1 KO (ChRO1−/−) mice using CRISPR–Cas9 genome editing targeting two gRNA sites to delete ChRO1 promoter, exon1, and part of intron1 region. ( B ) qRT-RCR quantification of ChRO1a and ChRO1b expression in gastrocnemius from 13-months-old-male mice WT and KO mice (ChRO1+/+ and ChRO1−/−). ( C ) qRT-PCR analysis of atrophic genes and satellite RNAs in gastrocnemius muscle from WT and ChRO1 KO mice. ( D ) Representative wheat germ agglutinin staining (left) and myofiber CSA (μm 2 ) quantification (right) of indicated skeletal muscle tissues from WT and ChRO1 KO mice. n ≥ 500 fibers analyzed per tissue. Scale bars, 40 μm. ( E ) Representative images (left) and quantification (right) of heterochromatin foci in gastrocnemius muscle from WT and ChRO1 KO mice. Scale bars, 1 μm. n = 50 nuclei. ( F ) Immunostaining for DAXX and H3K9me3 in gastrocnemius muscle of WT and ChRO1 KO mice. n = 50 nuclei. Scale bars, 2 μm. ( G ) qRT-PCR analysis of Atrogin1 (left) and Murf1 (right) in C2C12 MT differentiated for 5 days followed by 1,6-HD treatment at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Expression normalized to Gapdh and shown as fold change relative to untreated control for each time point. ( H ) Western blot analysis of MyHC protein levels in C2C12 MT treated with 1,6-HD at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Immunofluorescence staining of MyHC (AF488, green) and nuclei (DAPI, blue) ( I ) and quantification of myotube diameters [( J ), n = 100 cells] of C2C12 MT treated with 1,6-HD at indicated concentrations. ( K ) Quantification of myotube diameters of C2C12 MT treated with 2,5-HD or 1,6-HD at indicated concentration for 24 h. n = 45 cells. Statistical analyses, box plot elements, and data normalization procedures are detailed in the “Materials and methods” section.
    Figure Legend Snippet: ChRO1 deficiency and condensate disruption induce a muscle atrophic phenotype. ( A ) Strategy for generating ChRO1 KO (ChRO1−/−) mice using CRISPR–Cas9 genome editing targeting two gRNA sites to delete ChRO1 promoter, exon1, and part of intron1 region. ( B ) qRT-RCR quantification of ChRO1a and ChRO1b expression in gastrocnemius from 13-months-old-male mice WT and KO mice (ChRO1+/+ and ChRO1−/−). ( C ) qRT-PCR analysis of atrophic genes and satellite RNAs in gastrocnemius muscle from WT and ChRO1 KO mice. ( D ) Representative wheat germ agglutinin staining (left) and myofiber CSA (μm 2 ) quantification (right) of indicated skeletal muscle tissues from WT and ChRO1 KO mice. n ≥ 500 fibers analyzed per tissue. Scale bars, 40 μm. ( E ) Representative images (left) and quantification (right) of heterochromatin foci in gastrocnemius muscle from WT and ChRO1 KO mice. Scale bars, 1 μm. n = 50 nuclei. ( F ) Immunostaining for DAXX and H3K9me3 in gastrocnemius muscle of WT and ChRO1 KO mice. n = 50 nuclei. Scale bars, 2 μm. ( G ) qRT-PCR analysis of Atrogin1 (left) and Murf1 (right) in C2C12 MT differentiated for 5 days followed by 1,6-HD treatment at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Expression normalized to Gapdh and shown as fold change relative to untreated control for each time point. ( H ) Western blot analysis of MyHC protein levels in C2C12 MT treated with 1,6-HD at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Immunofluorescence staining of MyHC (AF488, green) and nuclei (DAPI, blue) ( I ) and quantification of myotube diameters [( J ), n = 100 cells] of C2C12 MT treated with 1,6-HD at indicated concentrations. ( K ) Quantification of myotube diameters of C2C12 MT treated with 2,5-HD or 1,6-HD at indicated concentration for 24 h. n = 45 cells. Statistical analyses, box plot elements, and data normalization procedures are detailed in the “Materials and methods” section.

    Techniques Used: Disruption, CRISPR, Expressing, Quantitative RT-PCR, Staining, Immunostaining, Concentration Assay, Control, Western Blot, Immunofluorescence



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    ChRO1 deficiency and condensate disruption induce a muscle atrophic phenotype. ( A ) Strategy for generating ChRO1 KO (ChRO1−/−) mice using CRISPR–Cas9 genome editing targeting two gRNA sites to delete ChRO1 promoter, exon1, and part of intron1 region. ( B ) qRT-RCR quantification of ChRO1a and ChRO1b expression in gastrocnemius from 13-months-old-male mice WT and KO mice (ChRO1+/+ and ChRO1−/−). ( C ) qRT-PCR analysis of atrophic genes and satellite RNAs in gastrocnemius muscle from WT and ChRO1 KO mice. ( D ) Representative wheat germ agglutinin staining (left) and myofiber CSA (μm 2 ) quantification (right) of indicated skeletal muscle tissues from WT and ChRO1 KO mice. n ≥ 500 fibers analyzed per tissue. Scale bars, 40 μm. ( E ) Representative images (left) and quantification (right) of heterochromatin foci in gastrocnemius muscle from WT and ChRO1 KO mice. Scale bars, 1 μm. n = 50 nuclei. ( F ) Immunostaining for DAXX and H3K9me3 in gastrocnemius muscle of WT and ChRO1 KO mice. n = 50 nuclei. Scale bars, 2 μm. ( G ) qRT-PCR analysis of Atrogin1 (left) and Murf1 (right) in C2C12 MT differentiated for 5 days followed by 1,6-HD treatment at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Expression normalized to Gapdh and shown as fold change relative to untreated control for each time point. ( H ) Western blot analysis of MyHC protein levels in C2C12 MT treated with 1,6-HD at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Immunofluorescence staining of MyHC (AF488, green) and nuclei (DAPI, blue) ( I ) and quantification of myotube diameters [( J ), n = 100 cells] of C2C12 MT treated with 1,6-HD at indicated concentrations. ( K ) Quantification of myotube diameters of C2C12 MT treated with 2,5-HD or 1,6-HD at indicated concentration for 24 h. n = 45 cells. Statistical analyses, box plot elements, and data normalization procedures are detailed in the “Materials and methods” section.

    Journal: Nucleic Acids Research

    Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

    doi: 10.1093/nar/gkag168

    Figure Lengend Snippet: ChRO1 deficiency and condensate disruption induce a muscle atrophic phenotype. ( A ) Strategy for generating ChRO1 KO (ChRO1−/−) mice using CRISPR–Cas9 genome editing targeting two gRNA sites to delete ChRO1 promoter, exon1, and part of intron1 region. ( B ) qRT-RCR quantification of ChRO1a and ChRO1b expression in gastrocnemius from 13-months-old-male mice WT and KO mice (ChRO1+/+ and ChRO1−/−). ( C ) qRT-PCR analysis of atrophic genes and satellite RNAs in gastrocnemius muscle from WT and ChRO1 KO mice. ( D ) Representative wheat germ agglutinin staining (left) and myofiber CSA (μm 2 ) quantification (right) of indicated skeletal muscle tissues from WT and ChRO1 KO mice. n ≥ 500 fibers analyzed per tissue. Scale bars, 40 μm. ( E ) Representative images (left) and quantification (right) of heterochromatin foci in gastrocnemius muscle from WT and ChRO1 KO mice. Scale bars, 1 μm. n = 50 nuclei. ( F ) Immunostaining for DAXX and H3K9me3 in gastrocnemius muscle of WT and ChRO1 KO mice. n = 50 nuclei. Scale bars, 2 μm. ( G ) qRT-PCR analysis of Atrogin1 (left) and Murf1 (right) in C2C12 MT differentiated for 5 days followed by 1,6-HD treatment at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Expression normalized to Gapdh and shown as fold change relative to untreated control for each time point. ( H ) Western blot analysis of MyHC protein levels in C2C12 MT treated with 1,6-HD at indicated concentration (%) for varying durations (1.5, 5.5, or 24 h). Immunofluorescence staining of MyHC (AF488, green) and nuclei (DAPI, blue) ( I ) and quantification of myotube diameters [( J ), n = 100 cells] of C2C12 MT treated with 1,6-HD at indicated concentrations. ( K ) Quantification of myotube diameters of C2C12 MT treated with 2,5-HD or 1,6-HD at indicated concentration for 24 h. n = 45 cells. Statistical analyses, box plot elements, and data normalization procedures are detailed in the “Materials and methods” section.

    Article Snippet: The CRISPR/Cas9 technology was used by Macrogen Inc. (Seoul, Korea) to produce ChRO1 knockout (KO) mice.

    Techniques: Disruption, CRISPR, Expressing, Quantitative RT-PCR, Staining, Immunostaining, Concentration Assay, Control, Western Blot, Immunofluorescence