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a – c Detection of frameshift and in-frame mutations using the DSB reporter. a Schematic representation of the DSB reporter system, in which a gRNA targets the tyrosine residue within the TagBFP chromophore, potentially disrupting fluorescence. b Diagram of the reporter construct, where TagBFP is fused to the C-terminus of Actb in mouse embryonic stem (ES) cells. “LNK” denotes a linker sequence; the same notation is used in panels “ d ”, “ e ”, and “ g ”. c Deep sequencing analysis of genome-edited populations after plasmid transfection expressing Cas9 and gRNA. “NC” represents non-genome-edited control cells, while “Bulk” represents genome-edited but unsorted cells. The proportion of frame-shift (red) and in-frame (black) mutations is shown. Data are presented as mean ± SD. ( n = 3 independent experiments). d – f The targeted insertion reporter differentiates between knock-in and EJ-TI. d Schematic representation of the targeted insertion (TI) reporter system. A lacZ–mCherry cassette was inserted into Actb using CRISPR/Cas9, driven by a splice acceptor to enable expression alongside Actb . e Diagram illustrating the distinction between knock-in and EJ-TI events. The donor vector introduces mEGFP upstream of mCherry. Knock-in results in the expression of both mEGFP and mCherry, whereas EJ-TI leads to mEGFP expression without mCherry. f PCR analysis of the fluorescence-sorted cell populations. Wild-type (WT) bands were detected in the mCherry single-positive and double-negative fractions. Knock-in-specific bands were observed in the double-positive fraction, whereas EJ-TI bands were detected in the mEGFP single-positive fraction. g, h Establishment of a triple-reporter system for the simultaneous detection of DSB and targeted insertion events. g Schematic representation of the system integrating DSB and TI reporters. h Flow cytometric analysis of genome editing outcomes in triple-reporter ES cells after transfection of plasmid vectors both expressing Cas9 and gRNA, and <t>pReporter–Donor.</t> DSB events are identified by the loss of TagBFP fluorescence (TagBFP-negative fraction). The restriction of knock-in and EJ-TI events to the TagBFP-negative population confirms the coordination between DSB and TI reporters. The illustrations of scissors and Cas9 were created with BioRender.com.
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a – c Detection of frameshift and in-frame mutations using the DSB reporter. a Schematic representation of the DSB reporter system, in which a gRNA targets the tyrosine residue within the TagBFP chromophore, potentially disrupting fluorescence. b Diagram of the reporter construct, where TagBFP is fused to the C-terminus of Actb in mouse embryonic stem (ES) cells. “LNK” denotes a linker sequence; the same notation is used in panels “ d ”, “ e ”, and “ g ”. c Deep sequencing analysis of genome-edited populations after plasmid transfection expressing Cas9 and gRNA. “NC” represents non-genome-edited control cells, while “Bulk” represents genome-edited but unsorted cells. The proportion of frame-shift (red) and in-frame (black) mutations is shown. Data are presented as mean ± SD. ( n = 3 independent experiments). d – f The targeted insertion reporter differentiates between knock-in and EJ-TI. d Schematic representation of the targeted insertion (TI) reporter system. A lacZ–mCherry cassette was inserted into Actb using CRISPR/Cas9, driven by a splice acceptor to enable expression alongside Actb . e Diagram illustrating the distinction between knock-in and EJ-TI events. The donor vector introduces mEGFP upstream of mCherry. Knock-in results in the expression of both mEGFP and mCherry, whereas EJ-TI leads to mEGFP expression without mCherry. f PCR analysis of the fluorescence-sorted cell populations. Wild-type (WT) bands were detected in the mCherry single-positive and double-negative fractions. Knock-in-specific bands were observed in the double-positive fraction, whereas EJ-TI bands were detected in the mEGFP single-positive fraction. g, h Establishment of a triple-reporter system for the simultaneous detection of DSB and targeted insertion events. g Schematic representation of the system integrating DSB and TI reporters. h Flow cytometric analysis of genome editing outcomes in triple-reporter ES cells after transfection of plasmid vectors both expressing Cas9 and gRNA, and <t>pReporter–Donor.</t> DSB events are identified by the loss of TagBFP fluorescence (TagBFP-negative fraction). The restriction of knock-in and EJ-TI events to the TagBFP-negative population confirms the coordination between DSB and TI reporters. The illustrations of scissors and Cas9 were created with BioRender.com.
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a – c Detection of frameshift and in-frame mutations using the DSB reporter. a Schematic representation of the DSB reporter system, in which a gRNA targets the tyrosine residue within the TagBFP chromophore, potentially disrupting fluorescence. b Diagram of the reporter construct, where TagBFP is fused to the C-terminus of Actb in mouse embryonic stem (ES) cells. “LNK” denotes a linker sequence; the same notation is used in panels “ d ”, “ e ”, and “ g ”. c Deep sequencing analysis of genome-edited populations after plasmid transfection expressing Cas9 and gRNA. “NC” represents non-genome-edited control cells, while “Bulk” represents genome-edited but unsorted cells. The proportion of frame-shift (red) and in-frame (black) mutations is shown. Data are presented as mean ± SD. ( n = 3 independent experiments). d – f The targeted insertion reporter differentiates between knock-in and EJ-TI. d Schematic representation of the targeted insertion (TI) reporter system. A lacZ–mCherry cassette was inserted into Actb using CRISPR/Cas9, driven by a splice acceptor to enable expression alongside Actb . e Diagram illustrating the distinction between knock-in and EJ-TI events. The donor vector introduces mEGFP upstream of mCherry. Knock-in results in the expression of both mEGFP and mCherry, whereas EJ-TI leads to mEGFP expression without mCherry. f PCR analysis of the fluorescence-sorted cell populations. Wild-type (WT) bands were detected in the mCherry single-positive and double-negative fractions. Knock-in-specific bands were observed in the double-positive fraction, whereas EJ-TI bands were detected in the mEGFP single-positive fraction. g, h Establishment of a triple-reporter system for the simultaneous detection of DSB and targeted insertion events. g Schematic representation of the system integrating DSB and TI reporters. h Flow cytometric analysis of genome editing outcomes in triple-reporter ES cells after transfection of plasmid vectors both expressing Cas9 and gRNA, and <t>pReporter–Donor.</t> DSB events are identified by the loss of TagBFP fluorescence (TagBFP-negative fraction). The restriction of knock-in and EJ-TI events to the TagBFP-negative population confirms the coordination between DSB and TI reporters. The illustrations of scissors and Cas9 were created with BioRender.com.
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a – c Detection of frameshift and in-frame mutations using the DSB reporter. a Schematic representation of the DSB reporter system, in which a gRNA targets the tyrosine residue within the TagBFP chromophore, potentially disrupting fluorescence. b Diagram of the reporter construct, where TagBFP is fused to the C-terminus of Actb in mouse embryonic stem (ES) cells. “LNK” denotes a linker sequence; the same notation is used in panels “ d ”, “ e ”, and “ g ”. c Deep sequencing analysis of genome-edited populations after plasmid transfection expressing Cas9 and gRNA. “NC” represents non-genome-edited control cells, while “Bulk” represents genome-edited but unsorted cells. The proportion of frame-shift (red) and in-frame (black) mutations is shown. Data are presented as mean ± SD. ( n = 3 independent experiments). d – f The targeted insertion reporter differentiates between knock-in and EJ-TI. d Schematic representation of the targeted insertion (TI) reporter system. A lacZ–mCherry cassette was inserted into Actb using CRISPR/Cas9, driven by a splice acceptor to enable expression alongside Actb . e Diagram illustrating the distinction between knock-in and EJ-TI events. The donor vector introduces mEGFP upstream of mCherry. Knock-in results in the expression of both mEGFP and mCherry, whereas EJ-TI leads to mEGFP expression without mCherry. f PCR analysis of the fluorescence-sorted cell populations. Wild-type (WT) bands were detected in the mCherry single-positive and double-negative fractions. Knock-in-specific bands were observed in the double-positive fraction, whereas EJ-TI bands were detected in the mEGFP single-positive fraction. g, h Establishment of a triple-reporter system for the simultaneous detection of DSB and targeted insertion events. g Schematic representation of the system integrating DSB and TI reporters. h Flow cytometric analysis of genome editing outcomes in triple-reporter ES cells after transfection of plasmid vectors both expressing Cas9 and gRNA, and pReporter–Donor. DSB events are identified by the loss of TagBFP fluorescence (TagBFP-negative fraction). The restriction of knock-in and EJ-TI events to the TagBFP-negative population confirms the coordination between DSB and TI reporters. The illustrations of scissors and Cas9 were created with BioRender.com.

Journal: Communications Biology

Article Title: ATM Inhibition Enhances Knock-in Efficiency by Suppressing AAV-Induced Activation of Apoptotic Pathways

doi: 10.1038/s42003-026-09604-z

Figure Lengend Snippet: a – c Detection of frameshift and in-frame mutations using the DSB reporter. a Schematic representation of the DSB reporter system, in which a gRNA targets the tyrosine residue within the TagBFP chromophore, potentially disrupting fluorescence. b Diagram of the reporter construct, where TagBFP is fused to the C-terminus of Actb in mouse embryonic stem (ES) cells. “LNK” denotes a linker sequence; the same notation is used in panels “ d ”, “ e ”, and “ g ”. c Deep sequencing analysis of genome-edited populations after plasmid transfection expressing Cas9 and gRNA. “NC” represents non-genome-edited control cells, while “Bulk” represents genome-edited but unsorted cells. The proportion of frame-shift (red) and in-frame (black) mutations is shown. Data are presented as mean ± SD. ( n = 3 independent experiments). d – f The targeted insertion reporter differentiates between knock-in and EJ-TI. d Schematic representation of the targeted insertion (TI) reporter system. A lacZ–mCherry cassette was inserted into Actb using CRISPR/Cas9, driven by a splice acceptor to enable expression alongside Actb . e Diagram illustrating the distinction between knock-in and EJ-TI events. The donor vector introduces mEGFP upstream of mCherry. Knock-in results in the expression of both mEGFP and mCherry, whereas EJ-TI leads to mEGFP expression without mCherry. f PCR analysis of the fluorescence-sorted cell populations. Wild-type (WT) bands were detected in the mCherry single-positive and double-negative fractions. Knock-in-specific bands were observed in the double-positive fraction, whereas EJ-TI bands were detected in the mEGFP single-positive fraction. g, h Establishment of a triple-reporter system for the simultaneous detection of DSB and targeted insertion events. g Schematic representation of the system integrating DSB and TI reporters. h Flow cytometric analysis of genome editing outcomes in triple-reporter ES cells after transfection of plasmid vectors both expressing Cas9 and gRNA, and pReporter–Donor. DSB events are identified by the loss of TagBFP fluorescence (TagBFP-negative fraction). The restriction of knock-in and EJ-TI events to the TagBFP-negative population confirms the coordination between DSB and TI reporters. The illustrations of scissors and Cas9 were created with BioRender.com.

Article Snippet: To prepare dsDNA, the pReporter–donor plasmid was digested with HindIII and AflII (NEB).

Techniques: Residue, Fluorescence, Construct, Sequencing, Plasmid Preparation, Transfection, Expressing, Control, Knock-In, CRISPR