human cot-1 dna Search Results


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  • 99
    Thermo Fisher human cot 1 dna
    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human <t>COT‐1</t> <t>DNA;</t> green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.
    Human Cot 1 Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore human cot1 dna
    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human <t>COT‐1</t> <t>DNA;</t> green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.
    Human Cot1 Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies human cot 1 dna
    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human <t>COT‐1</t> <t>DNA;</t> green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.
    Human Cot 1 Dna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM human cot 1 dna
    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human <t>COT‐1</t> <t>DNA;</t> green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.
    Human Cot 1 Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abbott Laboratories human cot 1 dna
    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human <t>COT‐1</t> <t>DNA;</t> green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.
    Human Cot 1 Dna, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher human cot 1 dna fluorometric qc
    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human <t>COT‐1</t> <t>DNA;</t> green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.
    Human Cot 1 Dna Fluorometric Qc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim human cot 1 dna
    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human <t>COT‐1</t> <t>DNA;</t> green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.
    Human Cot 1 Dna, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene human cot 1 dna
    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human <t>COT‐1</t> <t>DNA;</t> green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.
    Human Cot 1 Dna, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next‐generation human artificial chromosomes for Duchenne muscular dystrophy

    doi: 10.15252/emmm.201607284

    Figure Lengend Snippet: Generation of a novel HAC containing the entire human dystrophin locus by homologous recombination The scheme shows a linearised map of the vectors and the strategy used to generate DYS‐HAC2 by homologous recombination of DYS‐HAC1 (Hoshiya et al , 2009 ). pN targeting vector, which contains regions for homologous recombination (A: 3.8 kb and B: 2.6 kb, in green) and a floxable (FRT) neomycin (Neo), was used to remove extra genes (EGFP, Bsd, HPRT and Tk) on DYS‐HAC1 and to insert a floxable Neo gene. Primers designed to amplify DYS‐HAC1 or DYS‐HAC2 specific regions are highlighted in red. Phase contrast (left) and fluorescence (EGFP, right) images of DT40 cells containing DYS‐HAC1 and DYS‐HAC2. Scale bar: 50 μm. PCR analyses to discriminate between DYS‐HAC1 and DYS‐HAC2. DT40 cells: negative control. PCR panel to detect dystrophin exons in DT40(DYS‐HAC1) and DT40(DYS‐HAC2) cells. DT40 cells: negative control; human mesoangioblasts: positive control. In situ fluorescence hybridisation (FISH) analysis of DT40(DYS‐HAC2) cells. White arrowheads: DYS‐HAC2. Red: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. DT40(DYS‐HAC2) hybrid was used to transfer the DYS‐HAC2 in CHO cells (complete list in Appendix Table S1 ). FISH analyses of CHO(DYS‐HAC2)‐7 (left) and A9(DYS‐HAC2)‐9 (right) clones. White arrowheads: DYS‐HAC2. CHO(DYS‐HAC2) hybrid was used to transfer DYS‐HAC2 in A9 cells (complete list in Appendix Table S2 ). Red/purple: rhodamine‐human COT‐1 DNA; green: dystrophin FITC‐DMD‐BAC RP11‐954B16; yellow: merge. Scale bar: 5 μm. Source data are available online for this figure.

    Article Snippet: Digoxigenin‐labelled (Roche) human COT‐1 DNA (Invitrogen) and biotin‐labelled BAC DNA RP11‐954B16 (located in the Dystrophin genomic region, Children's Hospital Oakland Research Institute) were used for the detection of DYS‐HAC2 in DT40 (DYS‐HAC2), A9(DYS‐HAC2)‐9 and CHO(DYS‐HAC2)‐7 cells.

    Techniques: HAC Assay, Homologous Recombination, Plasmid Preparation, Fluorescence, Polymerase Chain Reaction, Negative Control, Positive Control, In Situ, Hybridization, Fluorescence In Situ Hybridization, BAC Assay

    Generation of a novel, synthetic, multifunctional DYS ‐ HAC Schematic diagram of DYS‐HAC4, generated by integration of plasmids p17 (yellow line) and pP‐ΔHR (blue line) into DYS‐HAC2 (Fig 1 A) as shown also in Appendix Fig S4 . Four functional cassettes are present: (i) dystrophin locus (2.4 Mb) for complete genetic correction, (ii) hTERT and Bmi1 immortalising cassette under control of MA1 bidirectional promoter (Amendola et al , 2005 ) and floxed by loxP sites to be eliminated via Cre‐loxP recombination system. Excision of the immortalising cassette is monitored by EGFP expression, response to blasticidin (Bsd) resistance and sensitivity to ganciclovir (TK), (iii) codon‐optimised human dystrophin (huDYSco, 11.1 kb) under control of the Spc5‐12 promoter to increase dystrophin expression, (iv) inducible Caspase 9 (iCas9) and human MYOD‐ERT2 under a PGK promoter for controllable cell death and myogenic differentiation, respectively. PCR analyses of selected CHO(DYS‐HAC4) clones confirming the presence of DNA sequences derived from DYS‐HAC2 (DYS‐HAC backbone detected with 3′, Puro and Sk23/DMD5t primers, Cre‐lox71/loxJTZ17 recombination detected with HPRT primers, genomic dystrophin sequence detected using DYS5L/5R, DYS6L/6R DYS7L/7R and DYS8L/8R primers). PCR analyses of CHO(DYS‐HAC4) clones #19 and #20 showing the presence of all relevant novel sequences confirming insertion of plasmid p17 and pP‐ΔHR. FISH analysis of CHO(DYS‐HAC4) clone #19 showing episomal presence of DYS‐HAC4 in single copy (red: rhodamine‐human COT‐1 DNA; green: FITC‐Plasmid pP‐ΔHR containing the immortalising cassette. Scale bar: 5 μm. RT–PCR analysis showing expression of huDYSco, immortalising cassette (hTERT and Bmi1), MYOD‐ERT2 and iCaspase 9 in CHO(DYS‐HAC4) clone #19. For all PCRs, CHO‐K1 cells were used as negative control and CHO(DYS‐HAC4) parental population as positive control. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next‐generation human artificial chromosomes for Duchenne muscular dystrophy

    doi: 10.15252/emmm.201607284

    Figure Lengend Snippet: Generation of a novel, synthetic, multifunctional DYS ‐ HAC Schematic diagram of DYS‐HAC4, generated by integration of plasmids p17 (yellow line) and pP‐ΔHR (blue line) into DYS‐HAC2 (Fig 1 A) as shown also in Appendix Fig S4 . Four functional cassettes are present: (i) dystrophin locus (2.4 Mb) for complete genetic correction, (ii) hTERT and Bmi1 immortalising cassette under control of MA1 bidirectional promoter (Amendola et al , 2005 ) and floxed by loxP sites to be eliminated via Cre‐loxP recombination system. Excision of the immortalising cassette is monitored by EGFP expression, response to blasticidin (Bsd) resistance and sensitivity to ganciclovir (TK), (iii) codon‐optimised human dystrophin (huDYSco, 11.1 kb) under control of the Spc5‐12 promoter to increase dystrophin expression, (iv) inducible Caspase 9 (iCas9) and human MYOD‐ERT2 under a PGK promoter for controllable cell death and myogenic differentiation, respectively. PCR analyses of selected CHO(DYS‐HAC4) clones confirming the presence of DNA sequences derived from DYS‐HAC2 (DYS‐HAC backbone detected with 3′, Puro and Sk23/DMD5t primers, Cre‐lox71/loxJTZ17 recombination detected with HPRT primers, genomic dystrophin sequence detected using DYS5L/5R, DYS6L/6R DYS7L/7R and DYS8L/8R primers). PCR analyses of CHO(DYS‐HAC4) clones #19 and #20 showing the presence of all relevant novel sequences confirming insertion of plasmid p17 and pP‐ΔHR. FISH analysis of CHO(DYS‐HAC4) clone #19 showing episomal presence of DYS‐HAC4 in single copy (red: rhodamine‐human COT‐1 DNA; green: FITC‐Plasmid pP‐ΔHR containing the immortalising cassette. Scale bar: 5 μm. RT–PCR analysis showing expression of huDYSco, immortalising cassette (hTERT and Bmi1), MYOD‐ERT2 and iCaspase 9 in CHO(DYS‐HAC4) clone #19. For all PCRs, CHO‐K1 cells were used as negative control and CHO(DYS‐HAC4) parental population as positive control. Source data are available online for this figure.

    Article Snippet: Digoxigenin‐labelled (Roche) human COT‐1 DNA (Invitrogen) and biotin‐labelled BAC DNA RP11‐954B16 (located in the Dystrophin genomic region, Children's Hospital Oakland Research Institute) were used for the detection of DYS‐HAC2 in DT40 (DYS‐HAC2), A9(DYS‐HAC2)‐9 and CHO(DYS‐HAC2)‐7 cells.

    Techniques: HAC Assay, Generated, Functional Assay, Expressing, Polymerase Chain Reaction, Clone Assay, Derivative Assay, Sequencing, Plasmid Preparation, Fluorescence In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control

    XIST RNA cloud paints an active X chromosome in MCF7 nuclei. A) DNA FISH using the single Xq22.3 locus RP11-349A16 probe (red), and X alpha-satellite probe (green). Cell populations with three Xs display two different hybridization patterns: two Xs with a single red spot (white arrows) and a chromosome with two red signals (red arrow). B) Simultaneous detection of XIST RNA (green), X chromosome territory (blue) and Xq22.3 locus (red). The X chromosome expressing XIST has one copy of the RP11-349A16 region (merge). C) Replication timing analysis. MCF7 and HMEC were briefly labelled with BrdU and analyzed by DNA FISH using the single Xq22.3 locus RP11-349A16 probe and by BrdU immunofluorescence. The pattern of FISH staining seen in BrdU-positive cells was scored for at least 300 nuclei of each cell type: nuclei with only “singlets” are those in which no Xs has yet replicated; nuclei with “singlet/s+doublet/s” pattern contain unreplicated and replicated Xs; nuclei with only “doublets” have all replicated Xs. Both MCF7 subpopulations with two and three Xs display a synchronous replication timing. D) Characterisation of the chromatin signatures of XIST-positive X chromosome in MCF7 and HMEC, by simultaneous FISH detection of XIST RNA (red) and Cot-1 RNA (green); DAPI nuclear staining is in blue. A line scan of fluorescence intensity (white bars) is shown for both cell types. In HMEC, the scan plot revealed overlap of the DAPI and XIST RNA signals, whereas the Cot-1 RNA signal is depleted, as expected for an inactive X chromosome. On the contrary, in MCF7 cells the line scan through the XIST-positive territory shows high intensity of the Cot-1 RNA signal combined with low DAPI intensity, typical signs of euchromatin.

    Journal: PLoS ONE

    Article Title: Misbehaviour of XIST RNA in Breast Cancer Cells

    doi: 10.1371/journal.pone.0005559

    Figure Lengend Snippet: XIST RNA cloud paints an active X chromosome in MCF7 nuclei. A) DNA FISH using the single Xq22.3 locus RP11-349A16 probe (red), and X alpha-satellite probe (green). Cell populations with three Xs display two different hybridization patterns: two Xs with a single red spot (white arrows) and a chromosome with two red signals (red arrow). B) Simultaneous detection of XIST RNA (green), X chromosome territory (blue) and Xq22.3 locus (red). The X chromosome expressing XIST has one copy of the RP11-349A16 region (merge). C) Replication timing analysis. MCF7 and HMEC were briefly labelled with BrdU and analyzed by DNA FISH using the single Xq22.3 locus RP11-349A16 probe and by BrdU immunofluorescence. The pattern of FISH staining seen in BrdU-positive cells was scored for at least 300 nuclei of each cell type: nuclei with only “singlets” are those in which no Xs has yet replicated; nuclei with “singlet/s+doublet/s” pattern contain unreplicated and replicated Xs; nuclei with only “doublets” have all replicated Xs. Both MCF7 subpopulations with two and three Xs display a synchronous replication timing. D) Characterisation of the chromatin signatures of XIST-positive X chromosome in MCF7 and HMEC, by simultaneous FISH detection of XIST RNA (red) and Cot-1 RNA (green); DAPI nuclear staining is in blue. A line scan of fluorescence intensity (white bars) is shown for both cell types. In HMEC, the scan plot revealed overlap of the DAPI and XIST RNA signals, whereas the Cot-1 RNA signal is depleted, as expected for an inactive X chromosome. On the contrary, in MCF7 cells the line scan through the XIST-positive territory shows high intensity of the Cot-1 RNA signal combined with low DAPI intensity, typical signs of euchromatin.

    Article Snippet: Cot-1 probe was obtained labeling 100 ng of Cot-1 DNA (1 mg/ml, Invitrogen) by Prime-It Fluor, Fluorescence labeling Kit with FITC-dUTP.

    Techniques: Fluorescence In Situ Hybridization, Hybridization, Expressing, Immunofluorescence, Staining, Fluorescence

    The human Xi is partially reactivated upon cell fusion-mediated reprogramming. ( a ) Expression of human Xi transcripts examined by allele-specific RNA-seq in female human fibroblasts (clone b1) before (day 0) and after (day 5/6) mESC-fusion. Data points show Xi expression of 113 genes along the X chromosome ( x axis) where genes that escape XCI before reprogramming (blue closed circles),, or that are sensitive to reactivation following fusion (day 5 green open circles; day 6 green closed circles) are highlighted. Xi expression is represented as the ratio of reads overlapping the minor (Xi) allele versus the total of both alleles (Xi+Xa) and result from two biological replicates. Genes were classified accordingly to Xi expression ratio and significance 62 , as detailed in the Methods. ( b ) RNA-FISH analyses of candidate reactivation-refractory ( HDAC8 ) and reactivation-sensitive ( RP11-706O15.1 , WWC3 ) genes in human fibroblasts before (day 0) and after mESC-fusion (day 5). Histogram plots show the percentage of human nuclei (heterokaryon/hybrid cells; n = 44/83, 33/74, 48/81 for HDAC8 , RP11-706O15.1 and WWC3 , respectively) with one, two, or > 2 punctate RNA signals. Representative confocal images showing mono-allelic expression of WWC3 (red) in human nuclei before (day 0) and bi-allelic expression after (day 5) mESC-fusion (arrowed) where mouse nuclei were discriminated using mCotI probe (green). Arrows indicate transcribed loci (that is, punctate RNA signals). Scale bar, 5 μm. ( c ) Confocal images of hFxmESC heterokaryons where either one (top) or two (lower) transcribed X chromosomes (arrowed) were evident. Mouse Cot1 probe identifies mouse (ESC-derived) nuclei. X chr RNA paints were pre-treated with human Cot1 DNA to diminish the detection of repeat-rich elements 63 within the transcribed X chromosome domains. Xa* marks the smaller (and Xa the larger) domain. Scale bar, 5 μm. ( d ) Box plots showing the volume of the RNA (top) and DNA (bottom) X domains detected by sequential RNA/DNA FISH in hFxmESC expressing one (single domain) or two (two domains) X chromosomes (shown in Supplementary Fig. 5b ). Significant volume differences are highlighted ( P values) and were estimated by Wilcoxon signed-rank test or Mann–Whitney test, for the comparison of Xa/Xa* and Xa/Xi, respectively. Data represent at least 50 cells in each graph. See also Supplementary Fig. 5 .

    Journal: Nature Communications

    Article Title: Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming

    doi: 10.1038/ncomms12354

    Figure Lengend Snippet: The human Xi is partially reactivated upon cell fusion-mediated reprogramming. ( a ) Expression of human Xi transcripts examined by allele-specific RNA-seq in female human fibroblasts (clone b1) before (day 0) and after (day 5/6) mESC-fusion. Data points show Xi expression of 113 genes along the X chromosome ( x axis) where genes that escape XCI before reprogramming (blue closed circles),, or that are sensitive to reactivation following fusion (day 5 green open circles; day 6 green closed circles) are highlighted. Xi expression is represented as the ratio of reads overlapping the minor (Xi) allele versus the total of both alleles (Xi+Xa) and result from two biological replicates. Genes were classified accordingly to Xi expression ratio and significance 62 , as detailed in the Methods. ( b ) RNA-FISH analyses of candidate reactivation-refractory ( HDAC8 ) and reactivation-sensitive ( RP11-706O15.1 , WWC3 ) genes in human fibroblasts before (day 0) and after mESC-fusion (day 5). Histogram plots show the percentage of human nuclei (heterokaryon/hybrid cells; n = 44/83, 33/74, 48/81 for HDAC8 , RP11-706O15.1 and WWC3 , respectively) with one, two, or > 2 punctate RNA signals. Representative confocal images showing mono-allelic expression of WWC3 (red) in human nuclei before (day 0) and bi-allelic expression after (day 5) mESC-fusion (arrowed) where mouse nuclei were discriminated using mCotI probe (green). Arrows indicate transcribed loci (that is, punctate RNA signals). Scale bar, 5 μm. ( c ) Confocal images of hFxmESC heterokaryons where either one (top) or two (lower) transcribed X chromosomes (arrowed) were evident. Mouse Cot1 probe identifies mouse (ESC-derived) nuclei. X chr RNA paints were pre-treated with human Cot1 DNA to diminish the detection of repeat-rich elements 63 within the transcribed X chromosome domains. Xa* marks the smaller (and Xa the larger) domain. Scale bar, 5 μm. ( d ) Box plots showing the volume of the RNA (top) and DNA (bottom) X domains detected by sequential RNA/DNA FISH in hFxmESC expressing one (single domain) or two (two domains) X chromosomes (shown in Supplementary Fig. 5b ). Significant volume differences are highlighted ( P values) and were estimated by Wilcoxon signed-rank test or Mann–Whitney test, for the comparison of Xa/Xa* and Xa/Xi, respectively. Data represent at least 50 cells in each graph. See also Supplementary Fig. 5 .

    Article Snippet: Briefly, 5 μl of concentrated X paint was mixed with 5 μg of human Cot1 DNA (Life Technologies), denatured for 7 min at 75 °C and pre-annealed for 30 min at 37 °C in order to deplete the probe of Cot1 repeats before hybridizing with cells overnight at 37 °C.

    Techniques: Expressing, RNA Sequencing Assay, Fluorescence In Situ Hybridization, Derivative Assay, MANN-WHITNEY

    hMSCs are retained and proliferate in the right ventricle (RV). Double immunofluorescence staining of RV sections for (A) 4′-6-diamidino-2-phenylindole (DAPI) for nuclear staining (blue) and Ki67 (green), (B) CD105 (red) from PV-banded pigs 24 days after intracoronary injection of hMSCs. (C) Merged sections show co-localization of CD105 and Ki67, indicating proliferation of hMSCs. Arrows show positive immunostaining. Representative images are shown. FISH was performed on RV sections to confirm the presence of human cells. (E) Human control hybridized with combined green (human) and red (pig) species-specific Cot-1 FISH probe. (F) Two adjacent human (daughter) cells in the final stages of cell division (telophase/cytokinesis). (G) An individual human cell (green) and (H) a group of human cells (green) are seen in the RV. Representative images are shown.

    Journal: Cytotherapy

    Article Title: Persistence and proliferation of human mesenchymal stromal cells in the right ventricular myocardium after intracoronary injection in a large animal model of pulmonary hypertension

    doi: 10.1016/j.jcyt.2017.03.002

    Figure Lengend Snippet: hMSCs are retained and proliferate in the right ventricle (RV). Double immunofluorescence staining of RV sections for (A) 4′-6-diamidino-2-phenylindole (DAPI) for nuclear staining (blue) and Ki67 (green), (B) CD105 (red) from PV-banded pigs 24 days after intracoronary injection of hMSCs. (C) Merged sections show co-localization of CD105 and Ki67, indicating proliferation of hMSCs. Arrows show positive immunostaining. Representative images are shown. FISH was performed on RV sections to confirm the presence of human cells. (E) Human control hybridized with combined green (human) and red (pig) species-specific Cot-1 FISH probe. (F) Two adjacent human (daughter) cells in the final stages of cell division (telophase/cytokinesis). (G) An individual human cell (green) and (H) a group of human cells (green) are seen in the RV. Representative images are shown.

    Article Snippet: A two-color species-differentiation fluorescence in situ hybridization (FISH) probe was generated by nick translation of species-specific repeated DNA sequences: human Cot-1 (Invitrogen) was labeled with SpectrumGreen, and porcine ID Block-It Cot-1 (Empire Genomics) was labeled with SpectrumOrange.

    Techniques: Double Immunofluorescence Staining, Staining, Injection, Immunostaining, Fluorescence In Situ Hybridization

    AR FISH on normal female metaphase spread slides. Slides were hybridized with TAMRA labeled AR probe with and without repeats and hybridized with and without the presence of C 0 t -1 in the hybmix. ( A ) RF AR probe hybridized with 25× excess C 0 t -1 DNA. ( B ) RC AR probe hybridized with 25× excess C 0 t -1 DNA. ( C ) RF AR probe hybridized without blocking DNA. ( D ) RC AR probe hybridized without blocking DNA. The two histograms show the line intensity profiles of the lines in the pictures. The top histogram shows the profiles of the lines from image A and B to which C 0 t -1 DNA was added and the bottom histograms show the profiles of the lines in image C and D to which no C 0 t -1 DNA was added.

    Journal: Nucleic Acids Research

    Article Title: Construction of repeat-free fluorescence in situ hybridization probes

    doi: 10.1093/nar/gkr1123

    Figure Lengend Snippet: AR FISH on normal female metaphase spread slides. Slides were hybridized with TAMRA labeled AR probe with and without repeats and hybridized with and without the presence of C 0 t -1 in the hybmix. ( A ) RF AR probe hybridized with 25× excess C 0 t -1 DNA. ( B ) RC AR probe hybridized with 25× excess C 0 t -1 DNA. ( C ) RF AR probe hybridized without blocking DNA. ( D ) RC AR probe hybridized without blocking DNA. The two histograms show the line intensity profiles of the lines in the pictures. The top histogram shows the profiles of the lines from image A and B to which C 0 t -1 DNA was added and the bottom histograms show the profiles of the lines in image C and D to which no C 0 t -1 DNA was added.

    Article Snippet: Fifty nanogram of this material was added to a 40× excess of C 0 t -1 DNA (Invitrogen, Carlsbad, CA, USA, cat. 15279-011) in 300 mM NaCl in a total volume of 10 µl and denatured for 10 min at 95°C.

    Techniques: Fluorescence In Situ Hybridization, Labeling, Blocking Assay

    AR FISH on paraffin-embedded MCF-7 cells. Five micrometer thick fixed paraffin-embedded MCF7 cells were hybridized with TAMRA labeled AR probe with and without repeats and hybridized with and without the presence of C 0 t -1 in the hybmix. ( A ) RF AR probe hybridized with 25× excess C 0 t -1 DNA. ( B ) RC AR probe hybridized with 25× excess C 0 t -1 DNA. ( C ) RF AR probe hybridized without blocking DNA. ( D ) RC AR probe hybridized without blocking DNA. The two histograms show the line intensity profiles of the lines in the pictures. The top histogram shows the profiles of the lines from image A and B to which C 0 t -1 DNA was added and the bottom histograms show the profiles of the lines in image C and D to which no C 0 t -1 DNA was added.

    Journal: Nucleic Acids Research

    Article Title: Construction of repeat-free fluorescence in situ hybridization probes

    doi: 10.1093/nar/gkr1123

    Figure Lengend Snippet: AR FISH on paraffin-embedded MCF-7 cells. Five micrometer thick fixed paraffin-embedded MCF7 cells were hybridized with TAMRA labeled AR probe with and without repeats and hybridized with and without the presence of C 0 t -1 in the hybmix. ( A ) RF AR probe hybridized with 25× excess C 0 t -1 DNA. ( B ) RC AR probe hybridized with 25× excess C 0 t -1 DNA. ( C ) RF AR probe hybridized without blocking DNA. ( D ) RC AR probe hybridized without blocking DNA. The two histograms show the line intensity profiles of the lines in the pictures. The top histogram shows the profiles of the lines from image A and B to which C 0 t -1 DNA was added and the bottom histograms show the profiles of the lines in image C and D to which no C 0 t -1 DNA was added.

    Article Snippet: Fifty nanogram of this material was added to a 40× excess of C 0 t -1 DNA (Invitrogen, Carlsbad, CA, USA, cat. 15279-011) in 300 mM NaCl in a total volume of 10 µl and denatured for 10 min at 95°C.

    Techniques: Fluorescence In Situ Hybridization, Labeling, Blocking Assay