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    TaKaRa in fusion hd cloning kit clontech takara
    Overview of <t>In-Fusion</t> enzyme-based <t>cloning</t> of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® <t>HD</t> cloning <t>kit</t> with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9
    In Fusion Hd Cloning Kit Clontech Takara, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion cloning system
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
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    TaKaRa in fusion ecodry cloning kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Ecodry Cloning Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion ecodry cloning system
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Ecodry Cloning System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion hd cloning kit w cloning enhancer
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Cloning Kit W Cloning Enhancer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa microcentrifuge spectrophotometer in fusion hd cloning plus kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    Microcentrifuge Spectrophotometer In Fusion Hd Cloning Plus Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa in fusion hd cloning plus ce kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
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    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
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    TaKaRa in fusion hd cloning system ce kit
    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent <t>cloning.</t> HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption <t>in</t> Fusarium fujikuroi created using the <t>In-Fusion</t> cloning <t>system.</t> NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.
    In Fusion Hd Cloning System Ce Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Clone Assay, CRISPR, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Expressing

    Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [ 22 ] using the In-Fusion ® HD cloning strategy as described in “ Methods ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ Methods ” and “ Results ” section for details

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [ 22 ] using the In-Fusion ® HD cloning strategy as described in “ Methods ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ Methods ” and “ Results ” section for details

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing, Construct

    Effects of HH Rz optimization on minireplicon reporter gene expression. a Sequences and predicted RNA secondary structures formed by HH Rz variants preceding the 5′ termini SYNV agRNA sequence. The cleavage triplets in HH1, HH2 and HH3, as well as the inactive GUG triplet in HHm are shown in bold letters, and the 5′ termini of SYNV leader are in red. Arrows indicate cleavage site, and the three stem structures (I, II and III) are shown in the HH1 Rz. b Analysis of RNA cleavage in vitro by three intact HH Rzs and a mutant version (HHm). The cleavage products were resolved on a polyacrylamide gel and visualized after ethidium bromide staining. All three HH Rzs efficiently cleaved the fused SYNV RNAs, resulting in disappearance of input RNAs and concomitant release of substrate strands and short ribozyme RNAs. c Semi-quantitative RT-PCR analysis of ribozyme cleavage efficiency in vivo. The MR agRNAs with 5′ terminus fusions to the HH Rz variants were produced in plant tissues via agroinfiltration mediated expression. The uncleaved transcripts were detected by RT-PCR after 28 cycles of amplification using primers annealing to the ribozyme and the SYNV 5′ termini sequence. Actin cDNA amplification products were used as a control. d and e Improved MR reporter expression activity by using optimized HH Rzs. N. benthamiana leaves were agroinfiltrated with MR constructs containing the indicated HH Rz variants and necessary supporting plasmids as described in Fig. 1 legends. Fluorescent foci were monitored under fluorescence microscope at 9 dpi ( d ) and the expression levels of GFP and RFP were evaluated by western blot analysis ( e ). Coomassie blue-stained Rubisco large subunit (Rub L) is shown to indicate equal protein loading

    Journal: Virology Journal

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

    doi: 10.1186/s12985-017-0776-7

    Figure Lengend Snippet: Effects of HH Rz optimization on minireplicon reporter gene expression. a Sequences and predicted RNA secondary structures formed by HH Rz variants preceding the 5′ termini SYNV agRNA sequence. The cleavage triplets in HH1, HH2 and HH3, as well as the inactive GUG triplet in HHm are shown in bold letters, and the 5′ termini of SYNV leader are in red. Arrows indicate cleavage site, and the three stem structures (I, II and III) are shown in the HH1 Rz. b Analysis of RNA cleavage in vitro by three intact HH Rzs and a mutant version (HHm). The cleavage products were resolved on a polyacrylamide gel and visualized after ethidium bromide staining. All three HH Rzs efficiently cleaved the fused SYNV RNAs, resulting in disappearance of input RNAs and concomitant release of substrate strands and short ribozyme RNAs. c Semi-quantitative RT-PCR analysis of ribozyme cleavage efficiency in vivo. The MR agRNAs with 5′ terminus fusions to the HH Rz variants were produced in plant tissues via agroinfiltration mediated expression. The uncleaved transcripts were detected by RT-PCR after 28 cycles of amplification using primers annealing to the ribozyme and the SYNV 5′ termini sequence. Actin cDNA amplification products were used as a control. d and e Improved MR reporter expression activity by using optimized HH Rzs. N. benthamiana leaves were agroinfiltrated with MR constructs containing the indicated HH Rz variants and necessary supporting plasmids as described in Fig. 1 legends. Fluorescent foci were monitored under fluorescence microscope at 9 dpi ( d ) and the expression levels of GFP and RFP were evaluated by western blot analysis ( e ). Coomassie blue-stained Rubisco large subunit (Rub L) is shown to indicate equal protein loading

    Article Snippet: The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan).

    Techniques: Expressing, Sequencing, In Vitro, Mutagenesis, Staining, Quantitative RT-PCR, In Vivo, Produced, Reverse Transcription Polymerase Chain Reaction, Amplification, Activity Assay, Construct, Fluorescence, Microscopy, Western Blot

    Efficiency of capped and HH3 Rz-processed agRNAs in supporting MR reporter gene expression. a Visualization of cells expressing GFP and RFP in N. benthamiana leaves infiltrated with agrobacteria harbouring 35S-HH3-MR or 35St-MR along with equal mixtures carrying core protein and VSR supporting plasmids. Fluorescent reporter expression was monitored at 6 and 9 dpi with an epifluorescence microscope. b Total protein extracts prepared from infiltrated leaves at 9 dpi were analysed in Western blots using anti-GFP and anti-RFP antibodies. Coomassie blue staining of the large subunit of Rubisco (Rub L) was used as loading control. c GFP and RFP mRNAs in infiltrated leaf tissues at 9 dpi were analysed by Northern blotting using probes against GFP and RFP sequences. Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used as RNA loading control. d and e The transcript levels of MR and full-length agRNAs were analysed by qRT-PCR. Total RNA samples were isolated from N. benthamiana leaves infiltrated with 35S-HH3-MR or 35St-MR ( d ), or 35S-HH3-SYNV or 35St-SYNV ( e ), together with VSR mixtures. Two pairs of primers specific for GFP and RFP were used to amplify MR cDNAs, while three pairs of N, P and G specific primers were used to amplify SYNV full-length cDNA

    Journal: Virology Journal

    Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

    doi: 10.1186/s12985-017-0776-7

    Figure Lengend Snippet: Efficiency of capped and HH3 Rz-processed agRNAs in supporting MR reporter gene expression. a Visualization of cells expressing GFP and RFP in N. benthamiana leaves infiltrated with agrobacteria harbouring 35S-HH3-MR or 35St-MR along with equal mixtures carrying core protein and VSR supporting plasmids. Fluorescent reporter expression was monitored at 6 and 9 dpi with an epifluorescence microscope. b Total protein extracts prepared from infiltrated leaves at 9 dpi were analysed in Western blots using anti-GFP and anti-RFP antibodies. Coomassie blue staining of the large subunit of Rubisco (Rub L) was used as loading control. c GFP and RFP mRNAs in infiltrated leaf tissues at 9 dpi were analysed by Northern blotting using probes against GFP and RFP sequences. Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used as RNA loading control. d and e The transcript levels of MR and full-length agRNAs were analysed by qRT-PCR. Total RNA samples were isolated from N. benthamiana leaves infiltrated with 35S-HH3-MR or 35St-MR ( d ), or 35S-HH3-SYNV or 35St-SYNV ( e ), together with VSR mixtures. Two pairs of primers specific for GFP and RFP were used to amplify MR cDNAs, while three pairs of N, P and G specific primers were used to amplify SYNV full-length cDNA

    Article Snippet: The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan).

    Techniques: Expressing, Microscopy, Western Blot, Staining, Northern Blot, Quantitative RT-PCR, Isolation

    Comparison of phenotypes and genotypes between parental ey Δ877/Δ1 strain and HR revertants. ( a ) Lateral pictures of wild type (left), parental ey Δ877/Δ1 strain (centre), and HR revertant (right). The ey Δ877/Δ1 strain shows the deformed, non-spherical compound eye (ce) phenotype. The HR revertant shows the normal eye phenotype and wild type strain. Scale bar indicates 100 μm. ( b ) Schematic illustration of genomic PCR to check the integration of attP into the Dma-ey Δ1 allele. Black thick bar indicates the Dma-ey Δ1 allele. Primers are shown by arrows. The attP knock-in results in 240 bp long PCR products, whereas no modification gives 173 bp long PCR products. ( c ) Agarose gel electrophoresis of genomic PCR products using HR revertant strains. 1 and 2 are derived from co-injection of TALEN mRNAs and targeting plasmid, whereas 3 is derived from co-injection of TALEN mRNAs and ssODN with 80 nt homology. attP knocked-in HR revertants showed 240 bp PCR products, whereas parental ey Δ877/Δ1 strain showed 173 bp PCR products. M indicates the marker DNA.

    Journal: Scientific Reports

    Article Title: TALEN-mediated homologous recombination in Daphnia magna

    doi: 10.1038/srep18312

    Figure Lengend Snippet: Comparison of phenotypes and genotypes between parental ey Δ877/Δ1 strain and HR revertants. ( a ) Lateral pictures of wild type (left), parental ey Δ877/Δ1 strain (centre), and HR revertant (right). The ey Δ877/Δ1 strain shows the deformed, non-spherical compound eye (ce) phenotype. The HR revertant shows the normal eye phenotype and wild type strain. Scale bar indicates 100 μm. ( b ) Schematic illustration of genomic PCR to check the integration of attP into the Dma-ey Δ1 allele. Black thick bar indicates the Dma-ey Δ1 allele. Primers are shown by arrows. The attP knock-in results in 240 bp long PCR products, whereas no modification gives 173 bp long PCR products. ( c ) Agarose gel electrophoresis of genomic PCR products using HR revertant strains. 1 and 2 are derived from co-injection of TALEN mRNAs and targeting plasmid, whereas 3 is derived from co-injection of TALEN mRNAs and ssODN with 80 nt homology. attP knocked-in HR revertants showed 240 bp PCR products, whereas parental ey Δ877/Δ1 strain showed 173 bp PCR products. M indicates the marker DNA.

    Article Snippet: Finally, the attP fragments were cloned into the TALEN-targeted site of the pCR-BluntII-Dma-ey1.5kb by the In-Fusion PCR cloning kit (Clontech, California, USA).

    Techniques: Polymerase Chain Reaction, Knock-In, Modification, Agarose Gel Electrophoresis, Derivative Assay, Injection, Plasmid Preparation, Marker

    Establishment and use of Dma-lig4 deficient mutant by CRISPR/Cas. ( a ) Schematic gene structure of Dma-lig4 . Dma-lig4 gene is annotated to consist of 8 exons (open boxes). Putative catalytic domain-encoding regions are highlighted in yellow (adenylation domain) and light green (oligonucleotide binding fold domain). gRNA-targeted sites are shown by triangles. Primers used for RT-PCR are shown by black arrows. Scale bar indicates 0.2 kb. ( b ) PAGE analyses around gRNA #2 target site of putative Dma-lig4 mutants. The genomic region surrounding gRNA #2 target site was amplified from genome DNAs of randomly selected 18 G1 lines. The original size of the PCR products should be 145 bp, indicated by black arrows. Thus, different patterns of band migration indicate the various in-del formation on the gRNA #2 targeted site. Asterisks (*) show the putative monoallelic and/or biallelic mutants. M indicates the marker DNA. ( c ) Genotype of Dma-lig4 biallelic mutant strain. Deduced amino acid sequences of wild type or Dma-lig4 mutant #14 are shown under the genome DNA sequences. gRNA #2 targeted sequences and Protospacer Adjacent Motif (PAM) are lined. Especially, gRNA #2 targeted sequences are highlighted in bold. Lower case indicates the introduced in-del mutations into mutant #14 genome. As shown in red, a premature termination codon was introduced into both Dma-lig4 alleles in the mutant #14 genome. ( d , e ) PAGE analyses of G1 offspring from TALEN and targeting plasmid injection using ey Δ877/Δ1 strain with or without Dma-ey deficiency (d, e, respectively). To estimate the germ line in-del mutation rate, the genomic region surrounding the TALEN targeted site was amplified from genome DNAs of randomly selected 24 G1 lines. Original size of the PCR products should be 173 bp, indicated by black arrows. Thus, different patterns of band migration indicate the various in-del formations on TALEN targeted site. Asterisks (*) show the putative in-del introduced lines. M indicates the marker DNA.

    Journal: Scientific Reports

    Article Title: TALEN-mediated homologous recombination in Daphnia magna

    doi: 10.1038/srep18312

    Figure Lengend Snippet: Establishment and use of Dma-lig4 deficient mutant by CRISPR/Cas. ( a ) Schematic gene structure of Dma-lig4 . Dma-lig4 gene is annotated to consist of 8 exons (open boxes). Putative catalytic domain-encoding regions are highlighted in yellow (adenylation domain) and light green (oligonucleotide binding fold domain). gRNA-targeted sites are shown by triangles. Primers used for RT-PCR are shown by black arrows. Scale bar indicates 0.2 kb. ( b ) PAGE analyses around gRNA #2 target site of putative Dma-lig4 mutants. The genomic region surrounding gRNA #2 target site was amplified from genome DNAs of randomly selected 18 G1 lines. The original size of the PCR products should be 145 bp, indicated by black arrows. Thus, different patterns of band migration indicate the various in-del formation on the gRNA #2 targeted site. Asterisks (*) show the putative monoallelic and/or biallelic mutants. M indicates the marker DNA. ( c ) Genotype of Dma-lig4 biallelic mutant strain. Deduced amino acid sequences of wild type or Dma-lig4 mutant #14 are shown under the genome DNA sequences. gRNA #2 targeted sequences and Protospacer Adjacent Motif (PAM) are lined. Especially, gRNA #2 targeted sequences are highlighted in bold. Lower case indicates the introduced in-del mutations into mutant #14 genome. As shown in red, a premature termination codon was introduced into both Dma-lig4 alleles in the mutant #14 genome. ( d , e ) PAGE analyses of G1 offspring from TALEN and targeting plasmid injection using ey Δ877/Δ1 strain with or without Dma-ey deficiency (d, e, respectively). To estimate the germ line in-del mutation rate, the genomic region surrounding the TALEN targeted site was amplified from genome DNAs of randomly selected 24 G1 lines. Original size of the PCR products should be 173 bp, indicated by black arrows. Thus, different patterns of band migration indicate the various in-del formations on TALEN targeted site. Asterisks (*) show the putative in-del introduced lines. M indicates the marker DNA.

    Article Snippet: Finally, the attP fragments were cloned into the TALEN-targeted site of the pCR-BluntII-Dma-ey1.5kb by the In-Fusion PCR cloning kit (Clontech, California, USA).

    Techniques: Mutagenesis, CRISPR, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Migration, Marker, Plasmid Preparation, Injection

    Expression of OsPIN2 rescues the phenotype of the Arabidopsis pin2 mutant. (A) The phenotypes of 10-d-old Col-0, Atpin2 , and Atpin2p:OsPIN2/Atpin2 transgenic lines. (B) Molecular characterization of Col-0, Atpin2 , and three independent Atpin2p:OsPIN2/Atpin2 transgenic lines using PCR. Primers are listed in Supplementary Table S1 . (C) Expression levels of AtPIN2 and OsPIN2 in the transgenic lines using RT-PCR. (D) Kinetics of root reorientation of Col-0, Atpin2 , and Atpin2p:OsPIN2/Atpin2 transgenic lines (R1-1): 4-day-old seedlings were placed horizontally and the root angle was measured at time points as indicated. Data are means ±SD ( n =20). (E) Fluorescence of DR5:GFP in Col-0, Atpin2 , and Atpin2p:OsPIN2/Atpin2 line R1-1; scale bars =50 μm.

    Journal: Journal of Experimental Botany

    Article Title: LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice

    doi: 10.1093/jxb/erx427

    Figure Lengend Snippet: Expression of OsPIN2 rescues the phenotype of the Arabidopsis pin2 mutant. (A) The phenotypes of 10-d-old Col-0, Atpin2 , and Atpin2p:OsPIN2/Atpin2 transgenic lines. (B) Molecular characterization of Col-0, Atpin2 , and three independent Atpin2p:OsPIN2/Atpin2 transgenic lines using PCR. Primers are listed in Supplementary Table S1 . (C) Expression levels of AtPIN2 and OsPIN2 in the transgenic lines using RT-PCR. (D) Kinetics of root reorientation of Col-0, Atpin2 , and Atpin2p:OsPIN2/Atpin2 transgenic lines (R1-1): 4-day-old seedlings were placed horizontally and the root angle was measured at time points as indicated. Data are means ±SD ( n =20). (E) Fluorescence of DR5:GFP in Col-0, Atpin2 , and Atpin2p:OsPIN2/Atpin2 line R1-1; scale bars =50 μm.

    Article Snippet: Then the cDNA (2119 bp) of OsPIN2 was amplified by PCR using the primers OsPIN2 -cDNA-F (containing the Kpn I restriction site) and OsPIN2 -cDNA-R (containing the Kpn I restriction site) and inserted into the pCAMIBA1300 vector containing the promoter of AtPIN2 at the Kpn I site using In-Fusion™ PCR Cloning Kits (Takara Clontech, Japan).

    Techniques: Expressing, Mutagenesis, Transgenic Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Fluorescence

    MTA1 transcriptionally suppresses p21 WAF1 by recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. A and B , Western blot ( A ) or qPCR ( B ) analysis of p21 WAF1 expression in the MTA1 +/+ or MTA1 −/− MEFs. The relative fold-increase of p21 WAF1 mRNA levels in the MTA1 −/− MEFs relative to the wild-type controls are shown in B . Each value represents the mean ± S.E. of three independent experiments. C , MTA1 +/+ or MTA1 −/− MEFs were transfected with the pGL3 -p21 WAF1 luciferase reporter plasmid as well as the pCMVβ vector carrying the β-galactosidase gene as an internal control for transfection efficiency. After 36 h of transfection, cells were lysed and luciferase activities in triplicate samples were determined. Luciferase values (relative light units) were calculated by dividing the luciferase activity by the β-galactosidase activity and normalized to controls. Values are reported as the fold-increase in the MTA1 −/− MEFs over the wild-type controls and represent the mean ± S.E. from three independent transfection experiments. D , ChIP analysis of recruitment of MTA1 on the p21 WAF1 promoter. MTA1 +/+ or MTA1 −/− MEFs were treated with 1% formaldehyde to cross-link histones to DNA, lysed by sonication, and immunoprecipitated ( IP ) with anti-MTA1 antibody or IgG control. The immunoprecipitates were washed, the DNA was eluted from the beads, and purified DNA was subjected to PCR. E , double ChIP analysis of recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. The first ChIP was carried out with anti-MTA1 antibody, followed by a second ChIP with anti-HDAC2 antibody. F , ChIP analysis of recruitment of the p53 protein on the p21 WAF1 promoter using MTA1 +/+ or MTA1 −/− MEFs. G , ChIP analysis of recruitment of the MTA1 protein on the p21 WAF1 promoter using p53 +/+ or p53 −/− MEFs. Error bars represent S.E.

    Journal: The Journal of Biological Chemistry

    Article Title: Revelation of p53-independent Function of MTA1 in DNA Damage Response via Modulation of the p21WAF1-Proliferating Cell Nuclear Antigen Pathway *

    doi: 10.1074/jbc.M109.079095

    Figure Lengend Snippet: MTA1 transcriptionally suppresses p21 WAF1 by recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. A and B , Western blot ( A ) or qPCR ( B ) analysis of p21 WAF1 expression in the MTA1 +/+ or MTA1 −/− MEFs. The relative fold-increase of p21 WAF1 mRNA levels in the MTA1 −/− MEFs relative to the wild-type controls are shown in B . Each value represents the mean ± S.E. of three independent experiments. C , MTA1 +/+ or MTA1 −/− MEFs were transfected with the pGL3 -p21 WAF1 luciferase reporter plasmid as well as the pCMVβ vector carrying the β-galactosidase gene as an internal control for transfection efficiency. After 36 h of transfection, cells were lysed and luciferase activities in triplicate samples were determined. Luciferase values (relative light units) were calculated by dividing the luciferase activity by the β-galactosidase activity and normalized to controls. Values are reported as the fold-increase in the MTA1 −/− MEFs over the wild-type controls and represent the mean ± S.E. from three independent transfection experiments. D , ChIP analysis of recruitment of MTA1 on the p21 WAF1 promoter. MTA1 +/+ or MTA1 −/− MEFs were treated with 1% formaldehyde to cross-link histones to DNA, lysed by sonication, and immunoprecipitated ( IP ) with anti-MTA1 antibody or IgG control. The immunoprecipitates were washed, the DNA was eluted from the beads, and purified DNA was subjected to PCR. E , double ChIP analysis of recruitment of the MTA1-HDAC2 complex onto the p21 WAF1 promoter. The first ChIP was carried out with anti-MTA1 antibody, followed by a second ChIP with anti-HDAC2 antibody. F , ChIP analysis of recruitment of the p53 protein on the p21 WAF1 promoter using MTA1 +/+ or MTA1 −/− MEFs. G , ChIP analysis of recruitment of the MTA1 protein on the p21 WAF1 promoter using p53 +/+ or p53 −/− MEFs. Error bars represent S.E.

    Article Snippet: Mutant mouse p21WAF1 promoter lacking the MTA1 binding region was constructed by cloning the PCR product amplified from the mouse p21WAF1 promoter (region from −1860 to −632) using the forward primer (5′-TAGCCCGGGCTCGAGAGATATCCGTTCGTTCAAACTAAGACTCC-3′) and reverse primer (5′-CCGGAATGCCAAGCTTGAGGCACGAGGGGCGTTACAGGTTCAA-3′), and then cloned into a pGL3 basic vector after digesting with XhoI and HindIII using the Clontech Infusion PCR cloning kit (Clontech).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Purification, Polymerase Chain Reaction

    Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent cloning. HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption in Fusarium fujikuroi created using the In-Fusion cloning system. NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.

    Journal: The Plant Pathology Journal

    Article Title: Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    doi: 10.5423/PPJ.OA.12.2015.0263

    Figure Lengend Snippet: Schematic representation of the construction of a gene replacement cassette. (A) Construction of a disruption and replacement cassette using restriction enzyme- and ligase-dependent cloning. HPH , hygromycin B phosphotransferase . (B) Construct used for FUM1 (8.1 kb) disruption in Fusarium fujikuroi created using the In-Fusion cloning system. NPTII , neomycin phosphotransferase . * The marks indicate clones exhibiting expected results. (C) Comparison of the construction systems used in these experiments for targeted gene deletion in fungi.

    Article Snippet: Primer design for Fusarium cyclin C1 gene (FCC1 ) and FUM1 deletions To generate target gene deletion mutants using the In-Fusion® HD Cloning system (Clontech Laboratories Inc., Mountain View, CA, USA), primers were designed using a primer design tool developed by In-Fusion® HD Cloning.

    Techniques: Clone Assay, Construct

    Fusarium cyclin C1 ( FCC1 ) disruption in the Fusarium fujikuroi strain B14 using the In-Fusion cloning system. (A) Strategies for FCC1 disruption by homologous recombination. (B) Amplification of the three primary products: the 5′ flanking region of FCC1 , neomycin phosphotransferase ( NPTII ), and the 3′ flanking region of FCC1 . (C) Verification of the constructs prior to target gene disruption. (D) PCR screening results from the selected transformants using specific primers. P, positive control. (E) Southern blot analysis of wild-type and fcc1 mutants.

    Journal: The Plant Pathology Journal

    Article Title: Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    doi: 10.5423/PPJ.OA.12.2015.0263

    Figure Lengend Snippet: Fusarium cyclin C1 ( FCC1 ) disruption in the Fusarium fujikuroi strain B14 using the In-Fusion cloning system. (A) Strategies for FCC1 disruption by homologous recombination. (B) Amplification of the three primary products: the 5′ flanking region of FCC1 , neomycin phosphotransferase ( NPTII ), and the 3′ flanking region of FCC1 . (C) Verification of the constructs prior to target gene disruption. (D) PCR screening results from the selected transformants using specific primers. P, positive control. (E) Southern blot analysis of wild-type and fcc1 mutants.

    Article Snippet: Primer design for Fusarium cyclin C1 gene (FCC1 ) and FUM1 deletions To generate target gene deletion mutants using the In-Fusion® HD Cloning system (Clontech Laboratories Inc., Mountain View, CA, USA), primers were designed using a primer design tool developed by In-Fusion® HD Cloning.

    Techniques: Clone Assay, Homologous Recombination, Amplification, Construct, Polymerase Chain Reaction, Positive Control, Southern Blot