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    hiscript iii rt supermix for qpcr +gdna wiper  (Vazyme Biotech Co)


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    Phenotype of CTCF R567W mutant mice. (a) Genotype frequency of progeny from the mating of Ctcf mutant mice. Asterisks* indicate the number of mice that died within 30 min after birth. (b) Box and whisker plots showing the ELISA results of the growth hormone (GH) content (95% confidence interval) in blood from 3-week-old Ctcf +/+ and Ctcf +/R567W mice (n = 11 for Ctcf +/+ , n = 10 for Ctcf +/R567W ). (c) Box and whisker plots showing the <t>quantitative</t> <t>PCR</t> analysis of GH mRNA levels (95% confidence interval) in pituitaries isolated from 3-week-old Ctcf +/+ and Ctcf +/R567W mice (n = 8 for each genotype). (d) Survival curves of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W mice (n > 50 for each genotype). (e) Quantitative PCR analysis of Ctcf mRNA levels in different tissues isolated from E18.5 embryos of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W (n = 3 for each genotype). (f) Western blot analysis of CTCF protein levels in different tissues isolated from E18.5 embryos of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W . (g) Immunohistochemistry images (left) and quantitative analysis results (right) showing CTCF expression and distribution in various tissues, including the brain (cortex and hippocampus), heart, lung, liver, kidney, spleen and skeletal muscle of E18.5 mice (n = 3 for each genotype; at least 200 cells were recorded in each field from approximately 10 fields). Scale bars, 50 μm. (h) Representative H&E staining images of paraffin lung sections from Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W mice at E18.5 (left) or P0 (right). Scale bars, 200 μm. Quantitative data are presented as the mean ± SD. The p- values obtained by Chi-Squared test (a), two-tailed unpaired t -test (b and c) and one-way ANOVA with Dunnett’s multiple comparisons test (e and g) are indicated. For c, and e-h, these experiments were repeated independently <t>three</t> times with similar results. Statistically relevant data are provided in the Source Data. Uncropped gels and blots are provided in the Supplementary Information.
    Hiscript Iii Rt Supermix For Qpcr, supplied by Vazyme Biotech Co, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CTCF mutation at R567 causes developmental disorders via 3D genome rearrangement and abnormal neurodevelopment"

    Article Title: CTCF mutation at R567 causes developmental disorders via 3D genome rearrangement and abnormal neurodevelopment

    Journal: bioRxiv

    doi: 10.1101/2024.04.07.588438

    Phenotype of CTCF R567W mutant mice. (a) Genotype frequency of progeny from the mating of Ctcf mutant mice. Asterisks* indicate the number of mice that died within 30 min after birth. (b) Box and whisker plots showing the ELISA results of the growth hormone (GH) content (95% confidence interval) in blood from 3-week-old Ctcf +/+ and Ctcf +/R567W mice (n = 11 for Ctcf +/+ , n = 10 for Ctcf +/R567W ). (c) Box and whisker plots showing the quantitative PCR analysis of GH mRNA levels (95% confidence interval) in pituitaries isolated from 3-week-old Ctcf +/+ and Ctcf +/R567W mice (n = 8 for each genotype). (d) Survival curves of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W mice (n > 50 for each genotype). (e) Quantitative PCR analysis of Ctcf mRNA levels in different tissues isolated from E18.5 embryos of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W (n = 3 for each genotype). (f) Western blot analysis of CTCF protein levels in different tissues isolated from E18.5 embryos of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W . (g) Immunohistochemistry images (left) and quantitative analysis results (right) showing CTCF expression and distribution in various tissues, including the brain (cortex and hippocampus), heart, lung, liver, kidney, spleen and skeletal muscle of E18.5 mice (n = 3 for each genotype; at least 200 cells were recorded in each field from approximately 10 fields). Scale bars, 50 μm. (h) Representative H&E staining images of paraffin lung sections from Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W mice at E18.5 (left) or P0 (right). Scale bars, 200 μm. Quantitative data are presented as the mean ± SD. The p- values obtained by Chi-Squared test (a), two-tailed unpaired t -test (b and c) and one-way ANOVA with Dunnett’s multiple comparisons test (e and g) are indicated. For c, and e-h, these experiments were repeated independently three times with similar results. Statistically relevant data are provided in the Source Data. Uncropped gels and blots are provided in the Supplementary Information.
    Figure Legend Snippet: Phenotype of CTCF R567W mutant mice. (a) Genotype frequency of progeny from the mating of Ctcf mutant mice. Asterisks* indicate the number of mice that died within 30 min after birth. (b) Box and whisker plots showing the ELISA results of the growth hormone (GH) content (95% confidence interval) in blood from 3-week-old Ctcf +/+ and Ctcf +/R567W mice (n = 11 for Ctcf +/+ , n = 10 for Ctcf +/R567W ). (c) Box and whisker plots showing the quantitative PCR analysis of GH mRNA levels (95% confidence interval) in pituitaries isolated from 3-week-old Ctcf +/+ and Ctcf +/R567W mice (n = 8 for each genotype). (d) Survival curves of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W mice (n > 50 for each genotype). (e) Quantitative PCR analysis of Ctcf mRNA levels in different tissues isolated from E18.5 embryos of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W (n = 3 for each genotype). (f) Western blot analysis of CTCF protein levels in different tissues isolated from E18.5 embryos of Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W . (g) Immunohistochemistry images (left) and quantitative analysis results (right) showing CTCF expression and distribution in various tissues, including the brain (cortex and hippocampus), heart, lung, liver, kidney, spleen and skeletal muscle of E18.5 mice (n = 3 for each genotype; at least 200 cells were recorded in each field from approximately 10 fields). Scale bars, 50 μm. (h) Representative H&E staining images of paraffin lung sections from Ctcf +/+ , Ctcf +/R567W and Ctcf R567W/R567W mice at E18.5 (left) or P0 (right). Scale bars, 200 μm. Quantitative data are presented as the mean ± SD. The p- values obtained by Chi-Squared test (a), two-tailed unpaired t -test (b and c) and one-way ANOVA with Dunnett’s multiple comparisons test (e and g) are indicated. For c, and e-h, these experiments were repeated independently three times with similar results. Statistically relevant data are provided in the Source Data. Uncropped gels and blots are provided in the Supplementary Information.

    Techniques Used: Mutagenesis, Whisker Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Isolation, Western Blot, Immunohistochemistry, Expressing, Staining, Two Tailed Test

    Generation of CTCF R567W hESC lines and the phenotype of cortical organoids. (a) Schematic diagram of the CRISPR/Cas9 knock-in CTCF R567W mutation in human H1 ESCs. The underline indicates the sgRNA targeting sequence ( CTCF exon 9) and the mutation site R567W. The mutated base, PAM sequence NGG and enzyme site for identification are highlighted in red. (b) Gel images showing enzyme digestion of PCR products and cDNA amplification (aa 469-664) of the hESC mutation region. (c) Sanger sequencing results showing successful generation of CTCF +/R567W and CTCF R567W/R567W hESCs. The underline indicates the mutation base C > T and restriction enzyme digestion site. The sequences of each allele are listed below. The black dashed lines represent exon/intron boundaries. (d) Sanger sequencing results of cDNA (region: aa 469-664) from CTCF +/R567W and CTCF R567W/R567W hESCs. (e) RT-qPCR analysis of the expression of CTCF and pluripotency genes in CTCF +/+ , CTCF +/R567W and CTCF R567W/R567W hESCs ( CTCF +/+ and CTCF +/R567W : 3 clones, CTCF R567W/R567W : 2 clones). (f) Western blot analysis of CTCF protein levels in CTCF +/+ , CTCF +/R567W and CTCF R567W/R567W hESCs. (g) Immunofluorescence for the neuronal marker MAP2, dorsal forebrain neural progenitor marker SOX2, and CPN marker SATB2 in CTCF +/+ , CTCF +/R567W and CTCF R567W/R567W brain organoids at 1 month and 3 months after derivation. Scale bars: whole organoids (1 month), 200 μm; higher magnification (right column), 50 μm; others (3 months), 100 μm. CPN, callosal projection neurons. (h, i) RT-qPCR analysis of the expression of excitatory (h) and inhibitory (i) neuronal markers in CTCF +/+ and CTCF +/R567W organoids on day 35 (n = 2 clones for each genotype). Quantitative data are presented as the mean ± SD. The p- values obtained by one-way ANOVA with Dunnett’s multiple comparisons test (e) and two-tailed unpaired t -test (h and i) are indicated. For e-i, these experiments were repeated three times independently with similar results. Statistically relevant data are provided in the Source Data. The uncropped gels and blots are provided in the Supplementary Information.
    Figure Legend Snippet: Generation of CTCF R567W hESC lines and the phenotype of cortical organoids. (a) Schematic diagram of the CRISPR/Cas9 knock-in CTCF R567W mutation in human H1 ESCs. The underline indicates the sgRNA targeting sequence ( CTCF exon 9) and the mutation site R567W. The mutated base, PAM sequence NGG and enzyme site for identification are highlighted in red. (b) Gel images showing enzyme digestion of PCR products and cDNA amplification (aa 469-664) of the hESC mutation region. (c) Sanger sequencing results showing successful generation of CTCF +/R567W and CTCF R567W/R567W hESCs. The underline indicates the mutation base C > T and restriction enzyme digestion site. The sequences of each allele are listed below. The black dashed lines represent exon/intron boundaries. (d) Sanger sequencing results of cDNA (region: aa 469-664) from CTCF +/R567W and CTCF R567W/R567W hESCs. (e) RT-qPCR analysis of the expression of CTCF and pluripotency genes in CTCF +/+ , CTCF +/R567W and CTCF R567W/R567W hESCs ( CTCF +/+ and CTCF +/R567W : 3 clones, CTCF R567W/R567W : 2 clones). (f) Western blot analysis of CTCF protein levels in CTCF +/+ , CTCF +/R567W and CTCF R567W/R567W hESCs. (g) Immunofluorescence for the neuronal marker MAP2, dorsal forebrain neural progenitor marker SOX2, and CPN marker SATB2 in CTCF +/+ , CTCF +/R567W and CTCF R567W/R567W brain organoids at 1 month and 3 months after derivation. Scale bars: whole organoids (1 month), 200 μm; higher magnification (right column), 50 μm; others (3 months), 100 μm. CPN, callosal projection neurons. (h, i) RT-qPCR analysis of the expression of excitatory (h) and inhibitory (i) neuronal markers in CTCF +/+ and CTCF +/R567W organoids on day 35 (n = 2 clones for each genotype). Quantitative data are presented as the mean ± SD. The p- values obtained by one-way ANOVA with Dunnett’s multiple comparisons test (e) and two-tailed unpaired t -test (h and i) are indicated. For e-i, these experiments were repeated three times independently with similar results. Statistically relevant data are provided in the Source Data. The uncropped gels and blots are provided in the Supplementary Information.

    Techniques Used: CRISPR, Knock-In, Mutagenesis, Sequencing, Amplification, Quantitative RT-PCR, Expressing, Clone Assay, Western Blot, Immunofluorescence, Marker, Two Tailed Test

    hiscript iii rt supermix for qpcr  (Vazyme Biotech Co)


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