i sce i  (New England Biolabs)


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    Structured Review

    New England Biolabs i sce i
    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by <t>I-Sce</t> I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
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    1) Product Images from "Precise Editing of the Zebrafish Genome Made Simple and Efficient"

    Article Title: Precise Editing of the Zebrafish Genome Made Simple and Efficient

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2016.02.015

    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
    Figure Legend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Techniques Used: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction

    2) Product Images from "The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype"

    Article Title: The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006208

    Enhanced HDR of chromosomal DSBs in cell lines expressing RAD51 G151D. A. RAD51 WT and G151D were stably expressed in MCF7 cells harboring the I- Sce I reporter construct using the pRVY TET-OFF inducible expression vector. The addition of doxycycline to the media turns off exogenous RAD51 expression (repressed, abbreviated R; endogenous RAD51 protein levels only), with expression induced upon removal of DOX (induced, abbreviated I; endogenous levels + exogenous protein levels). Western blot with an antisera raised against RAD51 protein demonstrates equivalent expression of exogenous WT and G151D (I) in their respective MCF-7 DR-GFP pools (RAD51/tubulin), as well as the fold increase in expression over endogenous RAD51 (I/R). B. The percentage of GFP positive cells was measured by flow cytometry 72hrs after nucleofection with an I- Sce I expression vector. The percentage of GFP-positive cells from MCF-7 DR-GFP parental cells was normalized to 1 and the relative change of percent GFP-positive cells from MCF-7 DR-GFP RAD51 WT and G151D cells was calculated. Data are graphed as mean ± SD from 3 independent experiments ** p
    Figure Legend Snippet: Enhanced HDR of chromosomal DSBs in cell lines expressing RAD51 G151D. A. RAD51 WT and G151D were stably expressed in MCF7 cells harboring the I- Sce I reporter construct using the pRVY TET-OFF inducible expression vector. The addition of doxycycline to the media turns off exogenous RAD51 expression (repressed, abbreviated R; endogenous RAD51 protein levels only), with expression induced upon removal of DOX (induced, abbreviated I; endogenous levels + exogenous protein levels). Western blot with an antisera raised against RAD51 protein demonstrates equivalent expression of exogenous WT and G151D (I) in their respective MCF-7 DR-GFP pools (RAD51/tubulin), as well as the fold increase in expression over endogenous RAD51 (I/R). B. The percentage of GFP positive cells was measured by flow cytometry 72hrs after nucleofection with an I- Sce I expression vector. The percentage of GFP-positive cells from MCF-7 DR-GFP parental cells was normalized to 1 and the relative change of percent GFP-positive cells from MCF-7 DR-GFP RAD51 WT and G151D cells was calculated. Data are graphed as mean ± SD from 3 independent experiments ** p

    Techniques Used: Expressing, Stable Transfection, Construct, Plasmid Preparation, Western Blot, Flow Cytometry, Cytometry

    3) Product Images from "hMSH5 Regulates NHEJ and Averts Excessive Nucleotide Alterations at Repair Joints"

    Article Title: hMSH5 Regulates NHEJ and Averts Excessive Nucleotide Alterations at Repair Joints

    Journal: Genes

    doi: 10.3390/genes13040673

    hMSH5 suppresses NHEJ-mediated DSB repair. ( A ) Analysis of the hMSH5 gene alteration in cancers. Data were retrieved from cBioPortal for Cancer Genomics ( www.cbioportal.org ). The stacked column graphs summarize 10 TCGA studies, of which each study has a sample size greater than 100, with at least 5% of the sample showing hMSH5 gene alterations. NEPC, neuroendocrine prostate cancer; CCLE, cancer cell line encyclopedia. ( B ) Schematic illustration of the NHEJ reporter locus in reporter cell line 293T/#8-1 [ 49 ]. NHEJ reporter analysis of the effect of hMSH5ΔN (hMSH5 aa116-834) (Tompkins et al., 2009). The cell lines used in this test were 293T/#8-1 derivatives stably expressing hMSH5 or hMSH5ΔN. ( C ) Analysis of the effect of hMSH5 on episomal NHEJ. 293T and 293T/hMSH5 cells were transiently transfected with either the NHEJ reporter construct alone or together with I- Sce I. ( D ) Levels of I- Sce I expression in 293T and 293T/hMSH5 cells determined by immunoblotting. The transfection efficiencies of 293T and 293T/hMSH5 cells (76% and 75%, respectively) were determined by transient transfection of pEGFP-C1, while untransfected cells were used as controls. ( E ) Sequence analysis of DSB repair junctions. The NHEJ reporter plasmid, together with I- Sce I construct, was transfected into 293T and 293T/hMSH5 cells. After inducing NHEJ-mediated end-joining at the reporter locus, repair joints were recovered by PCR amplification from total DNA. Cloned PCR products were sequenced. Sequencing data were analyzed by Tatsuki’s Dot Plot to reveal nucleotide deletions at the repair junctions. Solid circles signify repair joints without any I- Sce I 3′-protruding nucleotides ( B , top ), whereas open circles denote the inclusion of at least one of the 3′-protruding nucleotides at the repair junctions. Asterisks denote a similar statistical analysis in which the outlier (deletion of 69 nts) was omitted. Error bars represent standard deviations from the means of three replicates. Statistical significance was assessed by Student’s two-tailed t -test.
    Figure Legend Snippet: hMSH5 suppresses NHEJ-mediated DSB repair. ( A ) Analysis of the hMSH5 gene alteration in cancers. Data were retrieved from cBioPortal for Cancer Genomics ( www.cbioportal.org ). The stacked column graphs summarize 10 TCGA studies, of which each study has a sample size greater than 100, with at least 5% of the sample showing hMSH5 gene alterations. NEPC, neuroendocrine prostate cancer; CCLE, cancer cell line encyclopedia. ( B ) Schematic illustration of the NHEJ reporter locus in reporter cell line 293T/#8-1 [ 49 ]. NHEJ reporter analysis of the effect of hMSH5ΔN (hMSH5 aa116-834) (Tompkins et al., 2009). The cell lines used in this test were 293T/#8-1 derivatives stably expressing hMSH5 or hMSH5ΔN. ( C ) Analysis of the effect of hMSH5 on episomal NHEJ. 293T and 293T/hMSH5 cells were transiently transfected with either the NHEJ reporter construct alone or together with I- Sce I. ( D ) Levels of I- Sce I expression in 293T and 293T/hMSH5 cells determined by immunoblotting. The transfection efficiencies of 293T and 293T/hMSH5 cells (76% and 75%, respectively) were determined by transient transfection of pEGFP-C1, while untransfected cells were used as controls. ( E ) Sequence analysis of DSB repair junctions. The NHEJ reporter plasmid, together with I- Sce I construct, was transfected into 293T and 293T/hMSH5 cells. After inducing NHEJ-mediated end-joining at the reporter locus, repair joints were recovered by PCR amplification from total DNA. Cloned PCR products were sequenced. Sequencing data were analyzed by Tatsuki’s Dot Plot to reveal nucleotide deletions at the repair junctions. Solid circles signify repair joints without any I- Sce I 3′-protruding nucleotides ( B , top ), whereas open circles denote the inclusion of at least one of the 3′-protruding nucleotides at the repair junctions. Asterisks denote a similar statistical analysis in which the outlier (deletion of 69 nts) was omitted. Error bars represent standard deviations from the means of three replicates. Statistical significance was assessed by Student’s two-tailed t -test.

    Techniques Used: Non-Homologous End Joining, Stable Transfection, Expressing, Transfection, Construct, Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Two Tailed Test

    4) Product Images from "Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants"

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190526

    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Techniques Used: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.
    Figure Legend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Techniques Used: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Selection, Marker, Homologous Recombination, Ligation, Transformation Assay, Sequencing

    5) Product Images from "The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype"

    Article Title: The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006208

    Enhanced HDR of chromosomal DSBs in cell lines expressing RAD51 G151D. A. RAD51 WT and G151D were stably expressed in MCF7 cells harboring the I- Sce I reporter construct using the pRVY TET-OFF inducible expression vector. The addition of doxycycline to the media turns off exogenous RAD51 expression (repressed, abbreviated R; endogenous RAD51 protein levels only), with expression induced upon removal of DOX (induced, abbreviated I; endogenous levels + exogenous protein levels). Western blot with an antisera raised against RAD51 protein demonstrates equivalent expression of exogenous WT and G151D (I) in their respective MCF-7 DR-GFP pools (RAD51/tubulin), as well as the fold increase in expression over endogenous RAD51 (I/R). B. The percentage of GFP positive cells was measured by flow cytometry 72hrs after nucleofection with an I- Sce I expression vector. The percentage of GFP-positive cells from MCF-7 DR-GFP parental cells was normalized to 1 and the relative change of percent GFP-positive cells from MCF-7 DR-GFP RAD51 WT and G151D cells was calculated. Data are graphed as mean ± SD from 3 independent experiments ** p
    Figure Legend Snippet: Enhanced HDR of chromosomal DSBs in cell lines expressing RAD51 G151D. A. RAD51 WT and G151D were stably expressed in MCF7 cells harboring the I- Sce I reporter construct using the pRVY TET-OFF inducible expression vector. The addition of doxycycline to the media turns off exogenous RAD51 expression (repressed, abbreviated R; endogenous RAD51 protein levels only), with expression induced upon removal of DOX (induced, abbreviated I; endogenous levels + exogenous protein levels). Western blot with an antisera raised against RAD51 protein demonstrates equivalent expression of exogenous WT and G151D (I) in their respective MCF-7 DR-GFP pools (RAD51/tubulin), as well as the fold increase in expression over endogenous RAD51 (I/R). B. The percentage of GFP positive cells was measured by flow cytometry 72hrs after nucleofection with an I- Sce I expression vector. The percentage of GFP-positive cells from MCF-7 DR-GFP parental cells was normalized to 1 and the relative change of percent GFP-positive cells from MCF-7 DR-GFP RAD51 WT and G151D cells was calculated. Data are graphed as mean ± SD from 3 independent experiments ** p

    Techniques Used: Expressing, Stable Transfection, Construct, Plasmid Preparation, Western Blot, Flow Cytometry, Cytometry

    6) Product Images from "Protocols for yTREX/Tn5‐based gene cluster expression in Pseudomonas putida"

    Article Title: Protocols for yTREX/Tn5‐based gene cluster expression in Pseudomonas putida

    Journal: Microbial Biotechnology

    doi: 10.1111/1751-7915.13402

    Schematic of the gene cluster assembly in the yTREX vector. A. The yTREX vector backbone comprises replication elements and selection markers for E. coli (ori, pMB 1 origin of replication; Km R , kanamycin resistance gene) and yeast ( CEN 4 / ARS 1 , S. cerevisiae centromere region and autonomously replicating sequence; URA 3 , orotidine 5′‐phosphate decarboxylase gene) and the yTREX cassettes. L‐ yTREX (orange): oriT, origin of transfer; OE , outside end of transposon Tn5; P T 7 , T7 bacteriophage promoter, R‐ yTREX (green): tnp , Tn5 transposase gene; OE ; Tc R , tetracycline resistance gene; P T 7 . The vector is linearized by hydrolysis with restriction endonuclease I‐ Sce I, thereby exposing the partial I‐ Sce I recognition site and the sequences of the CIS (cluster integration site) at the termini. At the respective CIS 1 and CIS 2 sequences, insert fragments with appropriate homology arms to the CIS sequences and to one another can be integrated via yeast recombineering. Depiction is not drawn to scale. The complete vector sequence is available at the NCBI database (GenBank MK416190) and in the Table S1 in GenBank format. Right panel: Creation of homologous regions for recombination can generally be achieved by PCR and appropriate positioning of fully binding primers. Accordingly, designed primers can be used to re‐assemble large gene clusters in their native organization from freely defined PCR fragments (B). Alternatively, the use of primers with 5′‐elongations adding sequences to match new adjacent fragments enables re‐arrangements of genes or the addition of new parts (C). In this case, primer positions are defined by the ends of the fragments that are to be connected. Find further information under section Generation of gene cluster DNA fragments yeast assembly cloning in the yTREX vector , step 3b.
    Figure Legend Snippet: Schematic of the gene cluster assembly in the yTREX vector. A. The yTREX vector backbone comprises replication elements and selection markers for E. coli (ori, pMB 1 origin of replication; Km R , kanamycin resistance gene) and yeast ( CEN 4 / ARS 1 , S. cerevisiae centromere region and autonomously replicating sequence; URA 3 , orotidine 5′‐phosphate decarboxylase gene) and the yTREX cassettes. L‐ yTREX (orange): oriT, origin of transfer; OE , outside end of transposon Tn5; P T 7 , T7 bacteriophage promoter, R‐ yTREX (green): tnp , Tn5 transposase gene; OE ; Tc R , tetracycline resistance gene; P T 7 . The vector is linearized by hydrolysis with restriction endonuclease I‐ Sce I, thereby exposing the partial I‐ Sce I recognition site and the sequences of the CIS (cluster integration site) at the termini. At the respective CIS 1 and CIS 2 sequences, insert fragments with appropriate homology arms to the CIS sequences and to one another can be integrated via yeast recombineering. Depiction is not drawn to scale. The complete vector sequence is available at the NCBI database (GenBank MK416190) and in the Table S1 in GenBank format. Right panel: Creation of homologous regions for recombination can generally be achieved by PCR and appropriate positioning of fully binding primers. Accordingly, designed primers can be used to re‐assemble large gene clusters in their native organization from freely defined PCR fragments (B). Alternatively, the use of primers with 5′‐elongations adding sequences to match new adjacent fragments enables re‐arrangements of genes or the addition of new parts (C). In this case, primer positions are defined by the ends of the fragments that are to be connected. Find further information under section Generation of gene cluster DNA fragments yeast assembly cloning in the yTREX vector , step 3b.

    Techniques Used: Plasmid Preparation, Selection, Sequencing, Polymerase Chain Reaction, Binding Assay, Clone Assay

    7) Product Images from "The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes"

    Article Title: The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.146217

    The PAR complex promotes wound-directed migration of myeloid cells in vivo . (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I- Sce I integration sites (Sc), in the presence of I- Sce I meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry + ; PAR) and the endogenous control (Cherry − ; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); * P
    Figure Legend Snippet: The PAR complex promotes wound-directed migration of myeloid cells in vivo . (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I- Sce I integration sites (Sc), in the presence of I- Sce I meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry + ; PAR) and the endogenous control (Cherry − ; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); * P

    Techniques Used: Migration, In Vivo, Injection, Expressing, Transgenic Assay, Binding Assay, Construct, Mutagenesis, Fluorescence In Situ Hybridization

    The PAR complex promotes wound-directed migration of myeloid cells in vivo . (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I- Sce I integration sites (Sc), in the presence of I- Sce I meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry + ; PAR) and the endogenous control (Cherry − ; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); * P
    Figure Legend Snippet: The PAR complex promotes wound-directed migration of myeloid cells in vivo . (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I- Sce I integration sites (Sc), in the presence of I- Sce I meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry + ; PAR) and the endogenous control (Cherry − ; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); * P

    Techniques Used: Migration, In Vivo, Injection, Expressing, Transgenic Assay, Binding Assay, Construct, Mutagenesis, Fluorescence In Situ Hybridization

    8) Product Images from "Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks"

    Article Title: Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt872

    Assessing DNA repair pathway choice at open and closed loci. ( a ) Agarose gel showing results from digest of genomic DNA-generated amplicons with recombinant I-Sce I (denoted ‘+’ for containing recombinant I-Sce I and ‘−’ for no recombinant I-Sce I negative control). Nondigested (resistant) product is 776 bp and digested (cleaved) product is 388 bp. ( b ) Bar graph showing mean values for the amount of resistant (mutagenized) band observed over three replicates and fold loss in mutagenic product between  iRFP+/ − populations. * P
    Figure Legend Snippet: Assessing DNA repair pathway choice at open and closed loci. ( a ) Agarose gel showing results from digest of genomic DNA-generated amplicons with recombinant I-Sce I (denoted ‘+’ for containing recombinant I-Sce I and ‘−’ for no recombinant I-Sce I negative control). Nondigested (resistant) product is 776 bp and digested (cleaved) product is 388 bp. ( b ) Bar graph showing mean values for the amount of resistant (mutagenized) band observed over three replicates and fold loss in mutagenic product between iRFP+/ − populations. * P

    Techniques Used: Agarose Gel Electrophoresis, Generated, Recombinant, Negative Control

    9) Product Images from "Multi-omics profiling, in vitro and in vivo enhancer assays dissect the cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy"

    Article Title: Multi-omics profiling, in vitro and in vivo enhancer assays dissect the cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

    Journal: bioRxiv

    doi: 10.1101/2022.03.08.481329

    Schematic representation of the SED vector. The transgenesis internal control cassette is composed of the Cardiac Actin promoter (pink), driving strong expression in the somites, and the DsRed fluorescent protein (red), serving as a control for transgenesis efficiency in vivo in the F0 and the F1 embryos. The enhancer detection cassette contains a Gateway entry site (yellow), the gata2 minimal promoter (light green), and the enhanced green fluorescent protein (EGFP) reporter gene (dark green). EGFP reporter expression can be observed during early development under a fluorescent microscope. Both cassettes are flanked by insulator sequences (purple) to protect them from position effects, and together they are flanked by I- Sce I meganuclease recognition sites (blue).
    Figure Legend Snippet: Schematic representation of the SED vector. The transgenesis internal control cassette is composed of the Cardiac Actin promoter (pink), driving strong expression in the somites, and the DsRed fluorescent protein (red), serving as a control for transgenesis efficiency in vivo in the F0 and the F1 embryos. The enhancer detection cassette contains a Gateway entry site (yellow), the gata2 minimal promoter (light green), and the enhanced green fluorescent protein (EGFP) reporter gene (dark green). EGFP reporter expression can be observed during early development under a fluorescent microscope. Both cassettes are flanked by insulator sequences (purple) to protect them from position effects, and together they are flanked by I- Sce I meganuclease recognition sites (blue).

    Techniques Used: Plasmid Preparation, Expressing, In Vivo, Microscopy

    10) Product Images from "Precise Editing of the Zebrafish Genome Made Simple and Efficient"

    Article Title: Precise Editing of the Zebrafish Genome Made Simple and Efficient

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2016.02.015

    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
    Figure Legend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Techniques Used: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction

    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
    Figure Legend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Techniques Used: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction

    11) Product Images from "Precise Editing of the Zebrafish Genome Made Simple and Efficient"

    Article Title: Precise Editing of the Zebrafish Genome Made Simple and Efficient

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2016.02.015

    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
    Figure Legend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Techniques Used: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction

    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
    Figure Legend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Techniques Used: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction

    12) Product Images from "Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants"

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190526

    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Techniques Used: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.
    Figure Legend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Techniques Used: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Selection, Marker, Homologous Recombination, Ligation, Transformation Assay, Sequencing

    13) Product Images from "The Role of Blm Helicase in Homologous Recombination, Gene Conversion Tract Length, and Recombination Between Diverged Sequences in Drosophilamelanogaster"

    Article Title: The Role of Blm Helicase in Homologous Recombination, Gene Conversion Tract Length, and Recombination Between Diverged Sequences in Drosophilamelanogaster

    Journal: Genetics

    doi: 10.1534/genetics.117.300285

    DmBlm impacts DSB repair pathway usage. (A) I- Sce I heat shock-induced DSB repair events in a DmBlm N1/D2 null mutant background (red; n = 94) compared to DmBlm N1 heterozygote controls (blue; n = 125). Results shown are averages and SEM of individual male germline events compiled from five independent experiments. * P
    Figure Legend Snippet: DmBlm impacts DSB repair pathway usage. (A) I- Sce I heat shock-induced DSB repair events in a DmBlm N1/D2 null mutant background (red; n = 94) compared to DmBlm N1 heterozygote controls (blue; n = 125). Results shown are averages and SEM of individual male germline events compiled from five independent experiments. * P

    Techniques Used: Mutagenesis

    DR- white and DR- white.mu DSB repair assays. (A) The DR- white assay contains two nonfunctional direct repeats of the white gene. The first repeat, Sce.white , is nonfunctional due to the insertion of an I- Sce I recognition sequence into the wild-type Sac I recognition sequence of white cDNA resulting in a defective white gene. The second repeat, iwhite , is nonfunctional due to 5′ and 3′ truncations and serves as a homologous donor sequence for repair. DR- white is targeted using the attB sequence (blue) and integration is confirmed using yellow ( y + ) transgene expression. DR- white flies are crossed with flies containing an I- Sce I transgene, in which expression results in DSB formation at the I- Sce I recognition sequence. Repair events are observed by crossing these males to y w tester females; progeny of this cross represent single DSB repair events of the male germline. One of three phenotypes will result depending on the repair. (i) White-eyed progeny ( y + w − ) suggest no DSB, intersister HR, or repair by NHEJ with processing, resulting in loss of the I- Sce I recognition sequence. NHEJ with processing can be identified through molecular analysis. (ii) Repair by HR results in restoration of the wild-type Sac I site from the iwhite donor sequence and a red-eyed fly ( y + w + ). (iii) Yellow-bodied, white-eyed ( y − w − ) progeny indicates repair by SSA, mitotic crossover event (indistinguishable from SSA), or an aberrant repair event that impedes y + expression, such as a deletion into the y + transgene. (B) The DR- white.mu assay includes the incorporation of 28 silent polymorphisms on the iwhite.mu donor sequence, resulting in a 1.4% increase of sequence divergence between the two direct repeats. HR gene conversion of each of the polymorphisms varies from one repair product to the next (indicated by “?”), and can be determined by molecular analyses. DR- white ; Direct Repeat of white ; DR- white.mu ; Direct Repeat of white with mutations ; DSB, double-strand break; HR, homologous recombination; NHEJ, nonhomologous end-joining; SSA, single-strand annealing.
    Figure Legend Snippet: DR- white and DR- white.mu DSB repair assays. (A) The DR- white assay contains two nonfunctional direct repeats of the white gene. The first repeat, Sce.white , is nonfunctional due to the insertion of an I- Sce I recognition sequence into the wild-type Sac I recognition sequence of white cDNA resulting in a defective white gene. The second repeat, iwhite , is nonfunctional due to 5′ and 3′ truncations and serves as a homologous donor sequence for repair. DR- white is targeted using the attB sequence (blue) and integration is confirmed using yellow ( y + ) transgene expression. DR- white flies are crossed with flies containing an I- Sce I transgene, in which expression results in DSB formation at the I- Sce I recognition sequence. Repair events are observed by crossing these males to y w tester females; progeny of this cross represent single DSB repair events of the male germline. One of three phenotypes will result depending on the repair. (i) White-eyed progeny ( y + w − ) suggest no DSB, intersister HR, or repair by NHEJ with processing, resulting in loss of the I- Sce I recognition sequence. NHEJ with processing can be identified through molecular analysis. (ii) Repair by HR results in restoration of the wild-type Sac I site from the iwhite donor sequence and a red-eyed fly ( y + w + ). (iii) Yellow-bodied, white-eyed ( y − w − ) progeny indicates repair by SSA, mitotic crossover event (indistinguishable from SSA), or an aberrant repair event that impedes y + expression, such as a deletion into the y + transgene. (B) The DR- white.mu assay includes the incorporation of 28 silent polymorphisms on the iwhite.mu donor sequence, resulting in a 1.4% increase of sequence divergence between the two direct repeats. HR gene conversion of each of the polymorphisms varies from one repair product to the next (indicated by “?”), and can be determined by molecular analyses. DR- white ; Direct Repeat of white ; DR- white.mu ; Direct Repeat of white with mutations ; DSB, double-strand break; HR, homologous recombination; NHEJ, nonhomologous end-joining; SSA, single-strand annealing.

    Techniques Used: Sequencing, Expressing, Non-Homologous End Joining, Homologous Recombination

    14) Product Images from "A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish"

    Article Title: A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish

    Journal: Biology

    doi: 10.3390/biology10080777

    Workflow to generate double maternal mutants for dvl2 and dvl3a genes. ( A ) Two cloning steps to generate transgenic vectors containing eight sgRNA expression cassettes. By Golden Gate ligation, four sgRNA expression cassettes are cloned tandemly into pGGDestISceIEG-4sgRNA backbone, generating pGGDestISceIEG-4sgRNA ( dvl2 ) and pGGDestISceIEG-4sgRNA ( dvl3a ). The four sgRNAs targeting dvl3a were then amplified from the pGGDestISceIEG-4sgRNA ( dvl3a ) plasmid and inserted into the Acc 65I site in pGGDestISceIEG-4sgRNA ( dvl2 ) vector via T5 exonuclease-dependent assembly (TEDA). ( B ) The resultant pGGDestISceIEG-8sgRNA ( dvl2 ; dvl3a ) was coinjected with I- Sce I into embryos spawned by a wild-type female and a Tg( zpc : zcas9 ) homozygous male. The mosaic female with germ-line transmission was outcrossed to produce GFP-positive embryos, which were then genotyped and phenotyped for double maternal mutations.
    Figure Legend Snippet: Workflow to generate double maternal mutants for dvl2 and dvl3a genes. ( A ) Two cloning steps to generate transgenic vectors containing eight sgRNA expression cassettes. By Golden Gate ligation, four sgRNA expression cassettes are cloned tandemly into pGGDestISceIEG-4sgRNA backbone, generating pGGDestISceIEG-4sgRNA ( dvl2 ) and pGGDestISceIEG-4sgRNA ( dvl3a ). The four sgRNAs targeting dvl3a were then amplified from the pGGDestISceIEG-4sgRNA ( dvl3a ) plasmid and inserted into the Acc 65I site in pGGDestISceIEG-4sgRNA ( dvl2 ) vector via T5 exonuclease-dependent assembly (TEDA). ( B ) The resultant pGGDestISceIEG-8sgRNA ( dvl2 ; dvl3a ) was coinjected with I- Sce I into embryos spawned by a wild-type female and a Tg( zpc : zcas9 ) homozygous male. The mosaic female with germ-line transmission was outcrossed to produce GFP-positive embryos, which were then genotyped and phenotyped for double maternal mutations.

    Techniques Used: Clone Assay, Transgenic Assay, Expressing, Ligation, Amplification, Plasmid Preparation, Transmission Assay

    15) Product Images from "DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function"

    Article Title: DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2009.03.004

    Repair of I-Sce 1 induced breaks (a) Single-strand annealing assay. A schematic of the P ], in double-stranded format. The construct carries an I-Sce I recognition site (I-site, white rectangle) flanked by a partial w gene of 3.5 kb (white arrow) and a wild-type w gene of 4.5 kb (red arrow); arrowheads represent P element ends. Expression of I-Sce I leads to cutting at the I-site to produce a DSB. Repair by SSA requires resection through both copies of the w gene, followed by annealing of complementary sequences, trimming, and ligation. The product has only a partial w gene, resulting in white eyes. (b) SSA frequency. The percentage of progeny that inherited a chromosome on which repair occurred by SSA was calculated for each of 20 male parents. Bars show the mean percentages, with error bars showing SEM. There was no significant different between wild-type and nbs 1 / nbs SM9 larvae. (c) Distribution of repair events recovered. SSA, deletion, and imprecise NHEJ frequencies were determined as described in Materials and Methods. Bars show the relative distribution of repair events from SSA and from imprecise NHEJ; no deletions were detected among repair events from these genotypes. There was no significant difference in SSA and imprecise NHEJ between wild-type and nbs 1 / nbs SM9 larvae.
    Figure Legend Snippet: Repair of I-Sce 1 induced breaks (a) Single-strand annealing assay. A schematic of the P ], in double-stranded format. The construct carries an I-Sce I recognition site (I-site, white rectangle) flanked by a partial w gene of 3.5 kb (white arrow) and a wild-type w gene of 4.5 kb (red arrow); arrowheads represent P element ends. Expression of I-Sce I leads to cutting at the I-site to produce a DSB. Repair by SSA requires resection through both copies of the w gene, followed by annealing of complementary sequences, trimming, and ligation. The product has only a partial w gene, resulting in white eyes. (b) SSA frequency. The percentage of progeny that inherited a chromosome on which repair occurred by SSA was calculated for each of 20 male parents. Bars show the mean percentages, with error bars showing SEM. There was no significant different between wild-type and nbs 1 / nbs SM9 larvae. (c) Distribution of repair events recovered. SSA, deletion, and imprecise NHEJ frequencies were determined as described in Materials and Methods. Bars show the relative distribution of repair events from SSA and from imprecise NHEJ; no deletions were detected among repair events from these genotypes. There was no significant difference in SSA and imprecise NHEJ between wild-type and nbs 1 / nbs SM9 larvae.

    Techniques Used: Construct, Expressing, Ligation, Non-Homologous End Joining

    16) Product Images from "Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes"

    Article Title: Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

    Journal: The Plant Journal

    doi: 10.1111/tpj.14450

    Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .
    Figure Legend Snippet: Fast‐track recombineering using I‐ Sce I insertion cassettes. (a) Schematic presentation of N‐ and C‐terminal KmR and SpR gene‐linked I‐ Sce I cassettes (Figure S4 ) designed for replacement of start and stop codons of target genes with coding regions of GFP, mCherry and PIPL (His 18 StrepII‐HA) epitope. (b) The work flow of fast‐track recombineering is illustrated schematically by the replacement of stop codons of CYCH and H3.1 genes (Figure S2 f,g), which are carried by BACs with KmR markers. The BAC harbouring the target gene is transformed into the recombineering host SW102 and verified by PCR amplification of a segment of target gene with primers flanking its stop codon (green arrowheads). In the first step of recombineering (1), the C–GFPstop‐SpR I‐ Sce I cassette (Figure S4 ) is PCR amplified with primers carrying 50‐nt flanks of the stop codon (red and blue bars) and the cassette DNA fragment (2.07 kb) is transformed into SW102 harbouring the target BAC. Transformants are selected for the SpR marker of the I‐ Sce I cassette and verified by colony PCR with the gene‐specific primers. The PCR will detect BACs both with and without cassette insertions (2.07 kb + space between the primers versus distance between the gene‐specific primers). In the second step (2), the target gene carrying the I‐ Sce I cassette insertion replacing its stop codon is moved by gap‐repair into the pGAPBRHyg (or pGAPBRKm, Figure S5 ) binary vector. pGAPBRHyg is linearized with Bam HI, phosphatase treated (see Experimental Procedures for necessary control step), and PCR amplified with primers that carry 50 nt flanks of BAC sequences designed for transfer into plants linked to the modified target gene (Figure S2 f,g). The purified linear pGAPBRHyg is transformed into SW102 (BAC:GFPstop‐SpR). Following selection of AmpR transformants, plasmid DNA is prepared and transformed into E. coli DH10B to purify the pGAPBRHyg clones from the resident BACs. In the third step (3), the pGAPBRHyg clone is fingerprinted with restriction enzymes, cleaved by I‐ Sce I, self‐ligated and transformed into E. coli DH10B. AmpR transformants are screened for the loss of SpR marker and subjected to verification by sequencing the junction of modified plant gene in pGAPBRHyg using the gene‐specific primers. Finally, the construct is transferred by conjugation from E. coli into Agrobacterium for plant transformation as described in Figure 1 .

    Techniques Used: SPR Assay, Flow Cytometry, BAC Assay, Transformation Assay, Polymerase Chain Reaction, Amplification, Marker, Plasmid Preparation, Modification, Purification, Selection, Clone Assay, Sequencing, Construct, Conjugation Assay

    17) Product Images from "Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection"

    Article Title: Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection

    Journal: BMC Research Notes

    doi: 10.1186/s13104-015-1241-6

    A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro R puromycin-resistance gene, Hyg R hygromycin-resistance gene, Neo R neomycin-resistance gene, βgeo lacZ / Neo R , EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox 71 and lox P sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an att B sequence. The four att B sequences att B4, att B1, att B2 and att B3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c ), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I- Sce I site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with att B-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments ( see text for details). SA splice acceptor site, drug R drug-resistance gene, Km R kanamycin-resistance gene, Amp R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “ Methods ” for details.
    Figure Legend Snippet: A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro R puromycin-resistance gene, Hyg R hygromycin-resistance gene, Neo R neomycin-resistance gene, βgeo lacZ / Neo R , EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox 71 and lox P sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an att B sequence. The four att B sequences att B4, att B1, att B2 and att B3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c ), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I- Sce I site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with att B-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments ( see text for details). SA splice acceptor site, drug R drug-resistance gene, Km R kanamycin-resistance gene, Amp R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “ Methods ” for details.

    Techniques Used: Construct, Clone Assay, Plasmid Preparation, Sequencing, Derivative Assay, Polymerase Chain Reaction, Amplification, Flow Cytometry, Marker

    18) Product Images from "ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining"

    Article Title: ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

    Journal: Cell Cycle

    doi: 10.4161/cc.9.14.12363

    Suppression of in vivo MMEJ after Mirin-treatment or knocking down Mre11. (A) Schematic representation of the pMMEJ plasmid used to assess in vivo MMEJ repair. The pMMEJ plasmid was derived from the pEGFP-C3 plasmid by an insertion of 35 bp within the ORF of the wtEGFP gene. This creates an I- Sce I megaendonuclease recognition site flanked by two 5 bp microhomologies. The repair of a linearized pMMEJ plasmid by MMEJ reconstitutes the wtEGFP gene and allows expression of EGFP. (B) WI-38VA13 (CF) and AT5BIVA (AT) cells were transfected with either linear or circular pMMEJ and with a mCherry transfection control plasmid. Repair was allowed and cells were analyzed for fluorescent protein expression by flow cytometry. Cells treated with 25 mM Mirin or in which Mre11 was knocked down by transduction with lentivirus encoding Mre11 shRNA (Mre11 sh3 or Mre11 sh4) were also analyzed. Cells transduced with empty vector or virus-free transduction medium were included as controls. Repair through MMEJ is represented by %EGFP expression; %EGFP expression = (number of cells expressing both EGFP and mCherry/number of cells expressing mCherry) × 100.
    Figure Legend Snippet: Suppression of in vivo MMEJ after Mirin-treatment or knocking down Mre11. (A) Schematic representation of the pMMEJ plasmid used to assess in vivo MMEJ repair. The pMMEJ plasmid was derived from the pEGFP-C3 plasmid by an insertion of 35 bp within the ORF of the wtEGFP gene. This creates an I- Sce I megaendonuclease recognition site flanked by two 5 bp microhomologies. The repair of a linearized pMMEJ plasmid by MMEJ reconstitutes the wtEGFP gene and allows expression of EGFP. (B) WI-38VA13 (CF) and AT5BIVA (AT) cells were transfected with either linear or circular pMMEJ and with a mCherry transfection control plasmid. Repair was allowed and cells were analyzed for fluorescent protein expression by flow cytometry. Cells treated with 25 mM Mirin or in which Mre11 was knocked down by transduction with lentivirus encoding Mre11 shRNA (Mre11 sh3 or Mre11 sh4) were also analyzed. Cells transduced with empty vector or virus-free transduction medium were included as controls. Repair through MMEJ is represented by %EGFP expression; %EGFP expression = (number of cells expressing both EGFP and mCherry/number of cells expressing mCherry) × 100.

    Techniques Used: In Vivo, Plasmid Preparation, Derivative Assay, Expressing, Transfection, Flow Cytometry, Cytometry, Transduction, shRNA

    19) Product Images from "Precise Editing of the Zebrafish Genome Made Simple and Efficient"

    Article Title: Precise Editing of the Zebrafish Genome Made Simple and Efficient

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2016.02.015

    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
    Figure Legend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Techniques Used: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction

    20) Product Images from "The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes"

    Article Title: The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.146217

    The PAR complex promotes wound-directed migration of myeloid cells in vivo . (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I- Sce I integration sites (Sc), in the presence of I- Sce I meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry + ; PAR) and the endogenous control (Cherry − ; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); * P
    Figure Legend Snippet: The PAR complex promotes wound-directed migration of myeloid cells in vivo . (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I- Sce I integration sites (Sc), in the presence of I- Sce I meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry + ; PAR) and the endogenous control (Cherry − ; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); * P

    Techniques Used: Migration, In Vivo, Injection, Expressing, Transgenic Assay, Binding Assay, Construct, Mutagenesis, Fluorescence In Situ Hybridization

    The PAR complex promotes wound-directed migration of myeloid cells in vivo . (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I- Sce I integration sites (Sc), in the presence of I- Sce I meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry + ; PAR) and the endogenous control (Cherry − ; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); * P
    Figure Legend Snippet: The PAR complex promotes wound-directed migration of myeloid cells in vivo . (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I- Sce I integration sites (Sc), in the presence of I- Sce I meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry + ; PAR) and the endogenous control (Cherry − ; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); * P

    Techniques Used: Migration, In Vivo, Injection, Expressing, Transgenic Assay, Binding Assay, Construct, Mutagenesis, Fluorescence In Situ Hybridization

    21) Product Images from "Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection"

    Article Title: Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection

    Journal: BMC Research Notes

    doi: 10.1186/s13104-015-1241-6

    A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro R puromycin-resistance gene, Hyg R hygromycin-resistance gene, Neo R neomycin-resistance gene, βgeo lacZ / Neo R , EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox 71 and lox P sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an att B sequence. The four att B sequences att B4, att B1, att B2 and att B3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c ), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I- Sce I site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with att B-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments ( see text for details). SA splice acceptor site, drug R drug-resistance gene, Km R kanamycin-resistance gene, Amp R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “ Methods ” for details.
    Figure Legend Snippet: A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro R puromycin-resistance gene, Hyg R hygromycin-resistance gene, Neo R neomycin-resistance gene, βgeo lacZ / Neo R , EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox 71 and lox P sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an att B sequence. The four att B sequences att B4, att B1, att B2 and att B3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c ), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I- Sce I site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with att B-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments ( see text for details). SA splice acceptor site, drug R drug-resistance gene, Km R kanamycin-resistance gene, Amp R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “ Methods ” for details.

    Techniques Used: Construct, Clone Assay, Plasmid Preparation, Sequencing, Derivative Assay, Polymerase Chain Reaction, Amplification, Flow Cytometry, Marker

    22) Product Images from "DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function"

    Article Title: DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2009.03.004

    Repair of I-Sce 1 induced breaks (a) Single-strand annealing assay. A schematic of the P ], in double-stranded format. The construct carries an I-Sce I recognition site (I-site, white rectangle) flanked by a partial w gene of 3.5 kb (white arrow) and a wild-type w gene of 4.5 kb (red arrow); arrowheads represent P element ends. Expression of I-Sce I leads to cutting at the I-site to produce a DSB. Repair by SSA requires resection through both copies of the w gene, followed by annealing of complementary sequences, trimming, and ligation. The product has only a partial w gene, resulting in white eyes. (b) SSA frequency. The percentage of progeny that inherited a chromosome on which repair occurred by SSA was calculated for each of 20 male parents. Bars show the mean percentages, with error bars showing SEM. There was no significant different between wild-type and nbs 1 / nbs SM9 larvae. (c) Distribution of repair events recovered. SSA, deletion, and imprecise NHEJ frequencies were determined as described in Materials and Methods. Bars show the relative distribution of repair events from SSA and from imprecise NHEJ; no deletions were detected among repair events from these genotypes. There was no significant difference in SSA and imprecise NHEJ between wild-type and nbs 1 / nbs SM9 larvae.
    Figure Legend Snippet: Repair of I-Sce 1 induced breaks (a) Single-strand annealing assay. A schematic of the P ], in double-stranded format. The construct carries an I-Sce I recognition site (I-site, white rectangle) flanked by a partial w gene of 3.5 kb (white arrow) and a wild-type w gene of 4.5 kb (red arrow); arrowheads represent P element ends. Expression of I-Sce I leads to cutting at the I-site to produce a DSB. Repair by SSA requires resection through both copies of the w gene, followed by annealing of complementary sequences, trimming, and ligation. The product has only a partial w gene, resulting in white eyes. (b) SSA frequency. The percentage of progeny that inherited a chromosome on which repair occurred by SSA was calculated for each of 20 male parents. Bars show the mean percentages, with error bars showing SEM. There was no significant different between wild-type and nbs 1 / nbs SM9 larvae. (c) Distribution of repair events recovered. SSA, deletion, and imprecise NHEJ frequencies were determined as described in Materials and Methods. Bars show the relative distribution of repair events from SSA and from imprecise NHEJ; no deletions were detected among repair events from these genotypes. There was no significant difference in SSA and imprecise NHEJ between wild-type and nbs 1 / nbs SM9 larvae.

    Techniques Used: Construct, Expressing, Ligation, Non-Homologous End Joining

    23) Product Images from "Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals"

    Article Title: Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084379

    Structure and confirmation of the 1S and 2S GFP transgenes. ( A ) For each construct schematic, the numbers of bases are indicated to show the lengths of homology between the two as well as the relative positions of the engineered I- Sce I restriction sites. The 3’UTR sequences of the two constructs do not share homology and are indicated as a hatched box of 1270 bp for 1S-GFP and a grey box of 535 bp for 2S-GFP; these non-homologous sequences allow for PCR amplification specific to each transgene. Nested PCR primer pairs used for verification of intact construct sequences and for analysis of GFP+ hematopoietic colonies are indicated. Primers 1F-4R followed by 2F-3R amplify sequence flanking the I- Sce I site in 1S-GFP. Primers 1F-7R followed by 5F-6R amplify sequence flanking the I- Sce I site in 2S-GFP. ( B ) Southern blotting to estimate copy number utilized a GFP ORF DNA fragment of 3.1 kb and diluted to pg amounts that approximated 0, 0.2, 1.0, and 5.0 copies per genome spiked into 10µg non-transgenic mouse DNA. Genomic DNA from single transgenic mice (either 1S-GFP or 2S-GFP) was digested with restriction endonucleases within the GFP promoter and ORF of both transgenes to yield a 3.1 kb fragment. Band intensities are consistent with 4-5 copies of 1S-GFP and 2-4 copies of 2S-GFP, and were confirmed with Q-PCR data on the same samples (data not shown). ( C ) PCR reactions flanking each DSB site in the two GFP constructs confirm intact I-SceI recognition sites. Nested PCR as described in Materials amplified each transgene shown in the left side lane of each image. Digestion with I- Sce I endonuclease produced the expected sizes indicated in the middle lane of each image. Right side Marker lane PhiX.
    Figure Legend Snippet: Structure and confirmation of the 1S and 2S GFP transgenes. ( A ) For each construct schematic, the numbers of bases are indicated to show the lengths of homology between the two as well as the relative positions of the engineered I- Sce I restriction sites. The 3’UTR sequences of the two constructs do not share homology and are indicated as a hatched box of 1270 bp for 1S-GFP and a grey box of 535 bp for 2S-GFP; these non-homologous sequences allow for PCR amplification specific to each transgene. Nested PCR primer pairs used for verification of intact construct sequences and for analysis of GFP+ hematopoietic colonies are indicated. Primers 1F-4R followed by 2F-3R amplify sequence flanking the I- Sce I site in 1S-GFP. Primers 1F-7R followed by 5F-6R amplify sequence flanking the I- Sce I site in 2S-GFP. ( B ) Southern blotting to estimate copy number utilized a GFP ORF DNA fragment of 3.1 kb and diluted to pg amounts that approximated 0, 0.2, 1.0, and 5.0 copies per genome spiked into 10µg non-transgenic mouse DNA. Genomic DNA from single transgenic mice (either 1S-GFP or 2S-GFP) was digested with restriction endonucleases within the GFP promoter and ORF of both transgenes to yield a 3.1 kb fragment. Band intensities are consistent with 4-5 copies of 1S-GFP and 2-4 copies of 2S-GFP, and were confirmed with Q-PCR data on the same samples (data not shown). ( C ) PCR reactions flanking each DSB site in the two GFP constructs confirm intact I-SceI recognition sites. Nested PCR as described in Materials amplified each transgene shown in the left side lane of each image. Digestion with I- Sce I endonuclease produced the expected sizes indicated in the middle lane of each image. Right side Marker lane PhiX.

    Techniques Used: Construct, Polymerase Chain Reaction, Amplification, Nested PCR, Sequencing, Southern Blot, Transgenic Assay, Mouse Assay, Produced, Marker

    24) Product Images from "Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines"

    Article Title: Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006445

    The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.
    Figure Legend Snippet: The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.

    Techniques Used: Selection, Marker, FACS, Transfection, BAC Assay, Electroporation, Incubation, Fluorescence, Flow Cytometry, Cytometry

    25) Product Images from "An Enhanced Gene Targeting Toolkit for Drosophila: Golic+"

    Article Title: An Enhanced Gene Targeting Toolkit for Drosophila: Golic+

    Journal: Genetics

    doi: 10.1534/genetics.114.173716

    Comparison between different gene targeting strategies. Ends-out GT steps are generation of linear donor DNA, homologous recombination between donor DNA and targeted sequences, and recovery of correct GT. (A) For the Golic heat-shock strategy, the donor is first inserted in the genome as a P -element transgene and then released (flip-out and linearization) in larval primordial germ cells by heat-shock-induced expression of FLP and I- Sce I. Targeting occurs rarely through endogenous homologous recombination machinery. Candidates, possibly carrying the same GT event due to later clonal expansion, are recovered based on the mini-white eye marker in between the 5′ and 3′ homology arms. (B) For embryo microinjection, donor DNA is injected together with the corresponding sequence-specific nuclease to boost GT in embryonic pole cells. Clonal expansion can again lead to multiple offspring carrying identical GT. (C) In Golic+, donor DNA is not released from the transgene until the birth of each cystoblast (CB) from the ovarian germline stem cells, guaranteeing independent GT among candidates. Ends-out GT in CBs requires DNA double-strand breaks made by CRISPR/Cas, and recovery of correct GT is facilitated by a repressor-based lethality selection. The CB-specific induction of FLP, I- Sce I, and Cas9 depends on bamP-GAL4 ; guide RNA for CRISPR/Cas is broadly expressed with the dU6 promoter; strong candidates are recovered based on inheritance of a repressor, miRNA against rCD2, to rescue the pupal lethality caused by nSyb-driven riTS-Rac1 V12 .
    Figure Legend Snippet: Comparison between different gene targeting strategies. Ends-out GT steps are generation of linear donor DNA, homologous recombination between donor DNA and targeted sequences, and recovery of correct GT. (A) For the Golic heat-shock strategy, the donor is first inserted in the genome as a P -element transgene and then released (flip-out and linearization) in larval primordial germ cells by heat-shock-induced expression of FLP and I- Sce I. Targeting occurs rarely through endogenous homologous recombination machinery. Candidates, possibly carrying the same GT event due to later clonal expansion, are recovered based on the mini-white eye marker in between the 5′ and 3′ homology arms. (B) For embryo microinjection, donor DNA is injected together with the corresponding sequence-specific nuclease to boost GT in embryonic pole cells. Clonal expansion can again lead to multiple offspring carrying identical GT. (C) In Golic+, donor DNA is not released from the transgene until the birth of each cystoblast (CB) from the ovarian germline stem cells, guaranteeing independent GT among candidates. Ends-out GT in CBs requires DNA double-strand breaks made by CRISPR/Cas, and recovery of correct GT is facilitated by a repressor-based lethality selection. The CB-specific induction of FLP, I- Sce I, and Cas9 depends on bamP-GAL4 ; guide RNA for CRISPR/Cas is broadly expressed with the dU6 promoter; strong candidates are recovered based on inheritance of a repressor, miRNA against rCD2, to rescue the pupal lethality caused by nSyb-driven riTS-Rac1 V12 .

    Techniques Used: Homologous Recombination, Expressing, Marker, Injection, Sequencing, CRISPR, Selection

    26) Product Images from "Double-Strand Break Repair Assays Determine Pathway Choice and Structure of Gene Conversion Events in Drosophila melanogaster"

    Article Title: Double-Strand Break Repair Assays Determine Pathway Choice and Structure of Gene Conversion Events in Drosophila melanogaster

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.113.010074

    DR- white.mu determines gene conversion tract direction and length. (A) DR- white.mu is similar to DR- white ( Figure 2 ), except it contains 28 silent polymorphisms along the length of the iwhite donor sequence (not to scale). For a list of the polymorphisms and exact location, see Table S1 . After I- Sce I expression and cleavage, homologous recombination using iwhite.mu as the donor sequence results in restoration of the wild-type Sac I sequence and white+ phenotype. Gene conversion tracts include at least the Sac I site (gray) and may or may not include polymorphisms to the left or right of the break (indicated by “?”). To analyze changes to the donor sequence, iwhite was amplified with primers DR- white 3 and iwhite .a (3, iwa) and sequenced. (B) To determine gene conversion direction and length, Sce.white was amplified from y+ w+ isolates with primers 1.3 and 1a, and then sequenced for conversion to the polymorphisms of the iwhite.mu donor sequence. Minimal tract lengths of 41 y+ w+ HR repair events are shown, including the last polymorphism converted. Distance converted to the left and to the right of the Sac I site (0) is given.
    Figure Legend Snippet: DR- white.mu determines gene conversion tract direction and length. (A) DR- white.mu is similar to DR- white ( Figure 2 ), except it contains 28 silent polymorphisms along the length of the iwhite donor sequence (not to scale). For a list of the polymorphisms and exact location, see Table S1 . After I- Sce I expression and cleavage, homologous recombination using iwhite.mu as the donor sequence results in restoration of the wild-type Sac I sequence and white+ phenotype. Gene conversion tracts include at least the Sac I site (gray) and may or may not include polymorphisms to the left or right of the break (indicated by “?”). To analyze changes to the donor sequence, iwhite was amplified with primers DR- white 3 and iwhite .a (3, iwa) and sequenced. (B) To determine gene conversion direction and length, Sce.white was amplified from y+ w+ isolates with primers 1.3 and 1a, and then sequenced for conversion to the polymorphisms of the iwhite.mu donor sequence. Minimal tract lengths of 41 y+ w+ HR repair events are shown, including the last polymorphism converted. Distance converted to the left and to the right of the Sac I site (0) is given.

    Techniques Used: Sequencing, Expressing, Homologous Recombination, Amplification

    DR- white and DR- white.mu measure repair of an induced DSB. (A) To analyze repair of an inducible chromosomal DSB, an I- Sce I recognition sequence is inserted into the wild-type Sac I recognition sequence of white cDNA, resulting in a defective white sequence ( Sce.white ; black). The second white sequence is defective because of 5′ and 3′ truncations ( iwhite ; gray). Integration of DR- white is targeted using the attB sequence and followed with the yellow ( y +) transgene (not to scale). Embryos and larvae containing both DR- white and a heat-shock–inducible I- Sce I transgene are heat-shocked and crossed to y w females to score individual germline repair events. (B) After I- Sce I cleavage, three phenotypes associated with DSB repair outcomes occur. (i) Noncrossover intrachromosomal HR occurs with gene conversion of the I- Sce I sequence to wild-type Sac I sequence (conversion shown in gray), resulting in white+ recombinants. (ii) Retention of the y+ w− parental phenotype occurs after intersister HR, NHEJ without processing, no DSB, or NHEJ with processing. The latter can be identified by amplification of Sce.white with primers DR- white 1 and DR- white 1a (1, 1a), followed by in vitro cleavage of the PCR product with both I- Sce I and Sac I. Junctions of NHEJ with processing events are analyzed by sequencing Sce.white PCR products. (iii) SSA results from extensive resection and annealing of direct repeats and loss of intervening y + sequence. These events are confirmed by 2.0-kb amplification across DR- white with primers DR- white 1.3 and DR- white 4a (1.3, 4a). Phenotypes of the DSB repair events and status of DSB break site sequence are given for all outcomes.
    Figure Legend Snippet: DR- white and DR- white.mu measure repair of an induced DSB. (A) To analyze repair of an inducible chromosomal DSB, an I- Sce I recognition sequence is inserted into the wild-type Sac I recognition sequence of white cDNA, resulting in a defective white sequence ( Sce.white ; black). The second white sequence is defective because of 5′ and 3′ truncations ( iwhite ; gray). Integration of DR- white is targeted using the attB sequence and followed with the yellow ( y +) transgene (not to scale). Embryos and larvae containing both DR- white and a heat-shock–inducible I- Sce I transgene are heat-shocked and crossed to y w females to score individual germline repair events. (B) After I- Sce I cleavage, three phenotypes associated with DSB repair outcomes occur. (i) Noncrossover intrachromosomal HR occurs with gene conversion of the I- Sce I sequence to wild-type Sac I sequence (conversion shown in gray), resulting in white+ recombinants. (ii) Retention of the y+ w− parental phenotype occurs after intersister HR, NHEJ without processing, no DSB, or NHEJ with processing. The latter can be identified by amplification of Sce.white with primers DR- white 1 and DR- white 1a (1, 1a), followed by in vitro cleavage of the PCR product with both I- Sce I and Sac I. Junctions of NHEJ with processing events are analyzed by sequencing Sce.white PCR products. (iii) SSA results from extensive resection and annealing of direct repeats and loss of intervening y + sequence. These events are confirmed by 2.0-kb amplification across DR- white with primers DR- white 1.3 and DR- white 4a (1.3, 4a). Phenotypes of the DSB repair events and status of DSB break site sequence are given for all outcomes.

    Techniques Used: Sequencing, Non-Homologous End Joining, Amplification, In Vitro, Polymerase Chain Reaction

    27) Product Images from "Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks"

    Article Title: Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt872

    Assessing DNA repair pathway choice at open and closed loci. ( a ) Agarose gel showing results from digest of genomic DNA-generated amplicons with recombinant I-Sce I (denoted ‘+’ for containing recombinant I-Sce I and ‘−’ for no recombinant I-Sce I negative control). Nondigested (resistant) product is 776 bp and digested (cleaved) product is 388 bp. ( b ) Bar graph showing mean values for the amount of resistant (mutagenized) band observed over three replicates and fold loss in mutagenic product between  iRFP+/ − populations. * P
    Figure Legend Snippet: Assessing DNA repair pathway choice at open and closed loci. ( a ) Agarose gel showing results from digest of genomic DNA-generated amplicons with recombinant I-Sce I (denoted ‘+’ for containing recombinant I-Sce I and ‘−’ for no recombinant I-Sce I negative control). Nondigested (resistant) product is 776 bp and digested (cleaved) product is 388 bp. ( b ) Bar graph showing mean values for the amount of resistant (mutagenized) band observed over three replicates and fold loss in mutagenic product between iRFP+/ − populations. * P

    Techniques Used: Agarose Gel Electrophoresis, Generated, Recombinant, Negative Control

    28) Product Images from "A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish"

    Article Title: A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish

    Journal: Biology

    doi: 10.3390/biology10080777

    Workflow to generate double maternal mutants for dvl2 and dvl3a genes. ( A ) Two cloning steps to generate transgenic vectors containing eight sgRNA expression cassettes. By Golden Gate ligation, four sgRNA expression cassettes are cloned tandemly into pGGDestISceIEG-4sgRNA backbone, generating pGGDestISceIEG-4sgRNA ( dvl2 ) and pGGDestISceIEG-4sgRNA ( dvl3a ). The four sgRNAs targeting dvl3a were then amplified from the pGGDestISceIEG-4sgRNA ( dvl3a ) plasmid and inserted into the Acc 65I site in pGGDestISceIEG-4sgRNA ( dvl2 ) vector via T5 exonuclease-dependent assembly (TEDA). ( B ) The resultant pGGDestISceIEG-8sgRNA ( dvl2 ; dvl3a ) was coinjected with I- Sce I into embryos spawned by a wild-type female and a Tg( zpc : zcas9 ) homozygous male. The mosaic female with germ-line transmission was outcrossed to produce GFP-positive embryos, which were then genotyped and phenotyped for double maternal mutations.
    Figure Legend Snippet: Workflow to generate double maternal mutants for dvl2 and dvl3a genes. ( A ) Two cloning steps to generate transgenic vectors containing eight sgRNA expression cassettes. By Golden Gate ligation, four sgRNA expression cassettes are cloned tandemly into pGGDestISceIEG-4sgRNA backbone, generating pGGDestISceIEG-4sgRNA ( dvl2 ) and pGGDestISceIEG-4sgRNA ( dvl3a ). The four sgRNAs targeting dvl3a were then amplified from the pGGDestISceIEG-4sgRNA ( dvl3a ) plasmid and inserted into the Acc 65I site in pGGDestISceIEG-4sgRNA ( dvl2 ) vector via T5 exonuclease-dependent assembly (TEDA). ( B ) The resultant pGGDestISceIEG-8sgRNA ( dvl2 ; dvl3a ) was coinjected with I- Sce I into embryos spawned by a wild-type female and a Tg( zpc : zcas9 ) homozygous male. The mosaic female with germ-line transmission was outcrossed to produce GFP-positive embryos, which were then genotyped and phenotyped for double maternal mutations.

    Techniques Used: Clone Assay, Transgenic Assay, Expressing, Ligation, Amplification, Plasmid Preparation, Transmission Assay

    29) Product Images from "Precise Editing of the Zebrafish Genome Made Simple and Efficient"

    Article Title: Precise Editing of the Zebrafish Genome Made Simple and Efficient

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2016.02.015

    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
    Figure Legend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Techniques Used: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction

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    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by <t>I-Sce</t> I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p
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    Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Journal: Developmental cell

    Article Title: Precise Editing of the Zebrafish Genome Made Simple and Efficient

    doi: 10.1016/j.devcel.2016.02.015

    Figure Lengend Snippet: Reporter knock-in/knock-out alleles at the kcnh6a locus (A) Schematic representation of the genomic structure of the kcnh6a gene, indicating the kcnh6a-int1 TALEN target, and the structures of the donor DNAs (composed in pKH4 vector), highlighting the reporter coding sequences (colored) and translation/transcription termination signal sequences (grey) that are introduced by the donor. Left and right homology arms are bordered by I-Sce I recognition sites in head-to-head orientation (red arrows). Diagnostic primers are depicted: the rP1/kR1 pair specifically amplifies edited alleles, whereas the kF3/kR1 pair amplifies edited and unedited forms of the kcnh6a gene. (B) In vivo I-Sce I-digestion of donor plasmids stimulates genome editing. Individual embryos were injected with kcnh6a (eGFP) or kcnh6a (mCherry) donor DNAs with or without the I-Sce I meganuclease. Edited alleles were detected by PCR with diagnostic primers. (C) Genome editing is enhanced following I-Sce I digestion of donor plasmids, performed either in vivo or in vitro , prior to injection. Zygotes were injected with TALEN RNA and donor plasmid DNA mixed with differing amounts of I-Sce I enzyme on ice until injection (no pre-digestion) or digested in vitro prior to injection (pre-digestion). As a control, in vitro -digested donor plasmid was injected alone. The fraction of edited alleles (detected with the rP1/kR1 primer pair) relative to total kcnh6a alleles (detected with the kF3/kR1 primer pair) present in injected 2 dpf embryos was determined by qPCR. The relative recombination efficiency was determined by normalizing to 1.0 the mean fraction of edited alleles following injection of TALEN RNA and undigested donor plasmid DNA. For each condition, six individual embryos were analyzed (circles) and the mean relative recombination efficiency is indicated (horizontal dash). Unpaired t-test analysis indicated that in vivo or in vitro digestion of donor DNA with 1mU enzyme significantly stimulated the production of edited alleles as compared with untreated donor DNA (p

    Article Snippet: For co-injection with I-Sce I, 0.25mU or 1mU I-Sc eI enzyme was mixed with 50pg donor DNA and 100pg total TALEN mRNA in 0.5× I-Sce I buffer (NEB).

    Techniques: Knock-In, Knock-Out, Plasmid Preparation, Diagnostic Assay, In Vivo, Injection, Polymerase Chain Reaction, In Vitro, Real-time Polymerase Chain Reaction

    Enhanced HDR of chromosomal DSBs in cell lines expressing RAD51 G151D. A. RAD51 WT and G151D were stably expressed in MCF7 cells harboring the I- Sce I reporter construct using the pRVY TET-OFF inducible expression vector. The addition of doxycycline to the media turns off exogenous RAD51 expression (repressed, abbreviated R; endogenous RAD51 protein levels only), with expression induced upon removal of DOX (induced, abbreviated I; endogenous levels + exogenous protein levels). Western blot with an antisera raised against RAD51 protein demonstrates equivalent expression of exogenous WT and G151D (I) in their respective MCF-7 DR-GFP pools (RAD51/tubulin), as well as the fold increase in expression over endogenous RAD51 (I/R). B. The percentage of GFP positive cells was measured by flow cytometry 72hrs after nucleofection with an I- Sce I expression vector. The percentage of GFP-positive cells from MCF-7 DR-GFP parental cells was normalized to 1 and the relative change of percent GFP-positive cells from MCF-7 DR-GFP RAD51 WT and G151D cells was calculated. Data are graphed as mean ± SD from 3 independent experiments ** p

    Journal: PLoS Genetics

    Article Title: The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype

    doi: 10.1371/journal.pgen.1006208

    Figure Lengend Snippet: Enhanced HDR of chromosomal DSBs in cell lines expressing RAD51 G151D. A. RAD51 WT and G151D were stably expressed in MCF7 cells harboring the I- Sce I reporter construct using the pRVY TET-OFF inducible expression vector. The addition of doxycycline to the media turns off exogenous RAD51 expression (repressed, abbreviated R; endogenous RAD51 protein levels only), with expression induced upon removal of DOX (induced, abbreviated I; endogenous levels + exogenous protein levels). Western blot with an antisera raised against RAD51 protein demonstrates equivalent expression of exogenous WT and G151D (I) in their respective MCF-7 DR-GFP pools (RAD51/tubulin), as well as the fold increase in expression over endogenous RAD51 (I/R). B. The percentage of GFP positive cells was measured by flow cytometry 72hrs after nucleofection with an I- Sce I expression vector. The percentage of GFP-positive cells from MCF-7 DR-GFP parental cells was normalized to 1 and the relative change of percent GFP-positive cells from MCF-7 DR-GFP RAD51 WT and G151D cells was calculated. Data are graphed as mean ± SD from 3 independent experiments ** p

    Article Snippet: Signals of the +I-Sce I and–I-Sce I bands were quantified using Quantity One software version 4.6.5.

    Techniques: Expressing, Stable Transfection, Construct, Plasmid Preparation, Western Blot, Flow Cytometry, Cytometry

    hMSH5 suppresses NHEJ-mediated DSB repair. ( A ) Analysis of the hMSH5 gene alteration in cancers. Data were retrieved from cBioPortal for Cancer Genomics ( www.cbioportal.org ). The stacked column graphs summarize 10 TCGA studies, of which each study has a sample size greater than 100, with at least 5% of the sample showing hMSH5 gene alterations. NEPC, neuroendocrine prostate cancer; CCLE, cancer cell line encyclopedia. ( B ) Schematic illustration of the NHEJ reporter locus in reporter cell line 293T/#8-1 [ 49 ]. NHEJ reporter analysis of the effect of hMSH5ΔN (hMSH5 aa116-834) (Tompkins et al., 2009). The cell lines used in this test were 293T/#8-1 derivatives stably expressing hMSH5 or hMSH5ΔN. ( C ) Analysis of the effect of hMSH5 on episomal NHEJ. 293T and 293T/hMSH5 cells were transiently transfected with either the NHEJ reporter construct alone or together with I- Sce I. ( D ) Levels of I- Sce I expression in 293T and 293T/hMSH5 cells determined by immunoblotting. The transfection efficiencies of 293T and 293T/hMSH5 cells (76% and 75%, respectively) were determined by transient transfection of pEGFP-C1, while untransfected cells were used as controls. ( E ) Sequence analysis of DSB repair junctions. The NHEJ reporter plasmid, together with I- Sce I construct, was transfected into 293T and 293T/hMSH5 cells. After inducing NHEJ-mediated end-joining at the reporter locus, repair joints were recovered by PCR amplification from total DNA. Cloned PCR products were sequenced. Sequencing data were analyzed by Tatsuki’s Dot Plot to reveal nucleotide deletions at the repair junctions. Solid circles signify repair joints without any I- Sce I 3′-protruding nucleotides ( B , top ), whereas open circles denote the inclusion of at least one of the 3′-protruding nucleotides at the repair junctions. Asterisks denote a similar statistical analysis in which the outlier (deletion of 69 nts) was omitted. Error bars represent standard deviations from the means of three replicates. Statistical significance was assessed by Student’s two-tailed t -test.

    Journal: Genes

    Article Title: hMSH5 Regulates NHEJ and Averts Excessive Nucleotide Alterations at Repair Joints

    doi: 10.3390/genes13040673

    Figure Lengend Snippet: hMSH5 suppresses NHEJ-mediated DSB repair. ( A ) Analysis of the hMSH5 gene alteration in cancers. Data were retrieved from cBioPortal for Cancer Genomics ( www.cbioportal.org ). The stacked column graphs summarize 10 TCGA studies, of which each study has a sample size greater than 100, with at least 5% of the sample showing hMSH5 gene alterations. NEPC, neuroendocrine prostate cancer; CCLE, cancer cell line encyclopedia. ( B ) Schematic illustration of the NHEJ reporter locus in reporter cell line 293T/#8-1 [ 49 ]. NHEJ reporter analysis of the effect of hMSH5ΔN (hMSH5 aa116-834) (Tompkins et al., 2009). The cell lines used in this test were 293T/#8-1 derivatives stably expressing hMSH5 or hMSH5ΔN. ( C ) Analysis of the effect of hMSH5 on episomal NHEJ. 293T and 293T/hMSH5 cells were transiently transfected with either the NHEJ reporter construct alone or together with I- Sce I. ( D ) Levels of I- Sce I expression in 293T and 293T/hMSH5 cells determined by immunoblotting. The transfection efficiencies of 293T and 293T/hMSH5 cells (76% and 75%, respectively) were determined by transient transfection of pEGFP-C1, while untransfected cells were used as controls. ( E ) Sequence analysis of DSB repair junctions. The NHEJ reporter plasmid, together with I- Sce I construct, was transfected into 293T and 293T/hMSH5 cells. After inducing NHEJ-mediated end-joining at the reporter locus, repair joints were recovered by PCR amplification from total DNA. Cloned PCR products were sequenced. Sequencing data were analyzed by Tatsuki’s Dot Plot to reveal nucleotide deletions at the repair junctions. Solid circles signify repair joints without any I- Sce I 3′-protruding nucleotides ( B , top ), whereas open circles denote the inclusion of at least one of the 3′-protruding nucleotides at the repair junctions. Asterisks denote a similar statistical analysis in which the outlier (deletion of 69 nts) was omitted. Error bars represent standard deviations from the means of three replicates. Statistical significance was assessed by Student’s two-tailed t -test.

    Article Snippet: Purified DNA was digested by I-Sce I (New England Biolabs, Ipswich, MA, USA) to eliminate uncut or rejoined I-SceI sites.

    Techniques: Non-Homologous End Joining, Stable Transfection, Expressing, Transfection, Construct, Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Two Tailed Test

    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Multigene constructs were released from Level 1 vector with I-Sce I (NEB).

    Techniques: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Design of Plant X-tender expression vectors. Vector pCAMBIA 1300 (A) or Gateway vectors (pK7WG, pH7WG or pB7WG) (B) were used as a backbone. (A) I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette was introduced into the MCS region of pCAMBIA1300 by overlap-based cloning methods after backbone digestion with Bam HI and Hin dIII to obtain pCAMBIA_ASX. (B) T35S–AttR2– ccd B–AttR1 cassette was released from the Gateway plasmid backbone by digestion with Xba I and Sac I and replaced with a I- Sce I–A0– Hin dIII– ccd B– Hin dIII–B0–I- Sce I cassette by overlap-based cloning methods to obtain pK7WG_ASX, pH7WG_ASX or pB7WG_ASX. MCS: multiple cloning site, A0/B0: homology regions, Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Spec: selection marker conferring spectinomycin resistance in E . coli and A . tumefaciens , Hyg: selection marker conferring hygromycin resistance in plants, R: selection marker conferring resistance in plants (kanamycin resistance in pK7WG, hygromycin resistance in pH7WG, herbicide glufosinate-ammonium resistance in pB7WG), LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, Hin dIII, I- Sce I, Bam HI, Xba I, Sac I: restriction enzyme recognition sites, AttR1/AttR2: Gateway cloning recombination sites, T35S: cauliflower mosaic virus CaMV 35S terminator, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method.

    Article Snippet: Multigene constructs were released from Level 1 vector with I-Sce I (NEB).

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Selection, Marker, Ligation

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Multigene constructs were released from Level 1 vector with I-Sce I (NEB).

    Techniques: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Article Snippet: Multigene constructs were released from Level 1 vector with I-Sce I (NEB).

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Selection, Marker, Homologous Recombination, Ligation, Transformation Assay, Sequencing