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Addgene inc pcas9 mcherry frame 1
Pcas9 Mcherry Frame 1, supplied by Addgene inc, 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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pcas9 mcherry frame 1 - by Bioz Stars, 2024-07
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Addgene inc pcas9 mcherry frame 1 plasmid
Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional <t>CRISPR-Cas9</t> method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.
Pcas9 Mcherry Frame 1 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcas9 mcherry frame 1 plasmid/product/Addgene inc
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcas9 mcherry frame 1 plasmid - by Bioz Stars, 2024-07
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1) Product Images from "Tuft cells act as regenerative stem cells in the human intestine"

Article Title: Tuft cells act as regenerative stem cells in the human intestine

Journal: bioRxiv

doi: 10.1101/2024.03.17.585165

Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional CRISPR-Cas9 method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.
Figure Legend Snippet: Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional CRISPR-Cas9 method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.

Techniques Used: Over Expression, Expressing, Generated, CRISPR, Knock-Out, Flow Cytometry, Fluorescence, Cell Differentiation


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Addgene inc pcas9 mcherry frame 1
Pcas9 Mcherry Frame 1, supplied by Addgene inc, 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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Promega pcas9 mcherry frame 1
Pcas9 Mcherry Frame 1, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcas9 mcherry frame 1/product/Promega
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pcas9 mcherry frame 1 - by Bioz Stars, 2024-07
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Addgene inc pcas9 mcherry frame 1 addgene
Pcas9 Mcherry Frame 1 Addgene, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcas9 mcherry frame 1 addgene/product/Addgene inc
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pcas9 mcherry frame 1 addgene - by Bioz Stars, 2024-07
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Addgene inc pcas9 mcherry frame 1

Pcas9 Mcherry Frame 1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcas9 mcherry frame 1/product/Addgene inc
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcas9 mcherry frame 1 - by Bioz Stars, 2024-07
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1) Product Images from "Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression"

Article Title: Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression

Journal: Current Biology

doi: 10.1016/j.cub.2023.01.010


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Techniques Used: Recombinant, Sequencing, Expressing, Plasmid Preparation, Software, Imaging


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Addgene inc pcas9 mcherry frame 1 addgene
Pcas9 Mcherry Frame 1 Addgene, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcas9 mcherry frame 1 addgene/product/Addgene inc
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pcas9 mcherry frame 1 addgene - by Bioz Stars, 2024-07
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Addgene inc frame selector plasmid pcas9 mcherry frame 1
Frame Selector Plasmid Pcas9 Mcherry Frame 1, supplied by Addgene inc, 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/frame selector plasmid pcas9 mcherry frame 1/product/Addgene inc
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frame selector plasmid pcas9 mcherry frame 1 - by Bioz Stars, 2024-07
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Addgene inc pcas9 mcherry frame 1
Pcas9 Mcherry Frame 1, supplied by Addgene inc, 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/pcas9 mcherry frame 1/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcas9 mcherry frame 1 - by Bioz Stars, 2024-07
93/100 stars

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Addgene inc pcas9 mcherry frame 1
Pcas9 Mcherry Frame 1, supplied by Addgene inc, 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/pcas9 mcherry frame 1/product/Addgene inc
Average 93 stars, based on 1 article reviews
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pcas9 mcherry frame 1 - by Bioz Stars, 2024-07
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    Addgene inc pcas9 mcherry frame 1
    Pcas9 Mcherry Frame 1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcas9 mcherry frame 1/product/Addgene inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcas9 mcherry frame 1 - by Bioz Stars, 2024-07
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    Addgene inc pcas9 mcherry frame 1 plasmid
    Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional <t>CRISPR-Cas9</t> method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.
    Pcas9 Mcherry Frame 1 Plasmid, supplied by Addgene inc, 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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    Promega pcas9 mcherry frame 1
    Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional <t>CRISPR-Cas9</t> method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.
    Pcas9 Mcherry Frame 1, supplied by Promega, 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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    Addgene inc pcas9 mcherry frame 1 addgene
    Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional <t>CRISPR-Cas9</t> method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.
    Pcas9 Mcherry Frame 1 Addgene, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcas9 mcherry frame 1 addgene/product/Addgene inc
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    Addgene inc frame selector plasmid pcas9 mcherry frame 1
    Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional <t>CRISPR-Cas9</t> method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.
    Frame Selector Plasmid Pcas9 Mcherry Frame 1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional CRISPR-Cas9 method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.

    Journal: bioRxiv

    Article Title: Tuft cells act as regenerative stem cells in the human intestine

    doi: 10.1101/2024.03.17.585165

    Figure Lengend Snippet: Transcription factor knock outs and overexpression in human ileum organoids. a, Expression of POU2F3 across human healthy adult intestine tissue scRNA-seq dataset as in 1a. Left: Dot color relates to mean expression values and dot size to fraction of expressing cells. Right: dot color indicates log normalized expression. b, Genotypes of clonal transcriptional factor knock outs generated from human ileum AVIL-Clover reporter organoids. Homozygous knock outs of POU2F3 , HOXB8 , TCF7 , SPIB , SOX9 and heterozygous knock outs of ZFHX3 , RUNX1 , PROX1 are generated using base editing (C to T) technology to induce stop codon (TAG, TAA) within exons. HMX2 -/- and GFI1B -/- lines are generated by using conventional CRISPR-Cas9 method to induce frameshift. c, Quantification of AVIL + cell frequency in heterozygous knock out organoid lines by flow cytometry. Each dot is one well. One of 3 independent experiments is shown, see Supplementary Information . WT measurements are pooled from 4 experiments. Error bars indicate SE. FDR-adjusted two-sided Student’s t-test against the WT levels. d, Fluorescence images of differentiated AVIL-Clover organoids, depicting AVIL (green), POU2F3 (red) and DAPI (blue) from 2 donors (donors 1, 4). e, Schematics of the experimental set-up for (f-g) . ATOH1-inducible human ileum organoids were expanded for 4 days, then differentiated in tuft cell medium without DAPT, with or without a doxycycline pulse. f, Quantification of AVIL + cell frequency in ATOH1-inducible organoids (as in e ) by flow cytometry. Each dot is a well. One of 3 independent experiments on donor 1 was shown, see Supplementary Information . Error bars indicate SE. Two-sided Student’s t- test. g, qPCR quantification of ATOH1 expression and intestinal epithelial lineage markers. Each dot is a well. One of 2 independent experiments was shown, Supplementary Information . h, Genotype of clonal ATOH1 knock outs generated from human ileum organoids. i, Representative flow cytometric analysis (left) and quantification (right) of KIT + cell frequency in ATOH1 knock out organoids. Organoids were differentiated for 7 days in depicted media. Each dot is a well. Results are pooled from two ATOH1 knockout clonal lines from donor 1 (for 3 additional experiments, see Supplementary Information ). Error bars indicate SE. Two-sided Student’s t-test. j, qPCR quantification of tuft-1-4 characteristic genes in KIT + cells sorted from WT and ATOH1 -knockout organoids. Organoids were differentiated for 7 days in tuft cell differentiation medium with IL-4/IL-13- removal of DAPT. Each dot is a technical replicate. n= 3 wells pooled from two ATOH1 knockout lines. Error bars indicate SE. Diff: human tuft cell differentiation medium; TA: Transit-Amplifying Cells; EEC: Enteroendocrine cells; WT: wildtype; SE: standard error. *P < 0.05; **P< 0.01; ***P< 0.001.

    Article Snippet: For electroporation, 2.5 μg sgRNA plasmid (BPK1520, Addgene #65777), 7.5 μg pCMV_AncBE4max_P2A_GFP plasmid (for base-editing, Addgene #112100) or pCAS9- mCherry-Frame +1 plasmid (for conventional CRISPR-Cas9, Addgene #66940), together with 10 μg PiggyBac transposon system (5 μg transposase + 5 μg hygromycin resistance containing transposon) were co-electroporated into human duodenum, ileum and colon AVIL -Clover reporter organoids.

    Techniques: Over Expression, Expressing, Generated, CRISPR, Knock-Out, Flow Cytometry, Fluorescence, Cell Differentiation