Hot off the presses! Nov 01 Nat Meth

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Latest Articles Include:

• Nobel thoughts
  - Nat Meth 7(11):859 (2010)
  ARTICLE NAVIGATION - ISSUE Previous November 2010, Volume 7 No 11 pp859-935 * In This Issue * Editorial * This Month * Correspondence * Research Highlights * Technology Feature * News and Views * Brief Communications * ArticlesAbout the cover In This Issue PDF - In This Issue Editorial Nobel thoughts - p859 doi:10.1038/nmeth1110-859 The community of scientists should celebrate the Nobel Prize, even if awards bestowed on one discipline are associated with another discipline. A new prize might help. Abstract - Nobel thoughts | Full Text - Nobel thoughts | PDF (65 KB) - Nobel thoughts This Month The author file: Rolf Zeller and Javier Lopez-Rios - p861 Monya Baker doi:10.1038/nmeth1110-861 Gene cutting and pasting just got a whole lot faster. Abstract - The author file: Rolf Zeller and Javier Lopez-Rios | Full Text - The author fileRolf Zeller and Javier Lopez-Rios | PDF (152 KB) - The author fileRolf Zeller and Javier Lopez-Rios Points of View: Gestalt principles (Part 1) - p863 Bang Wong doi:10.1038/nmeth1110-863 Full Text - Points of ViewGestalt principles (Part 1) | PDF (119 KB) - Points of ViewGestalt principles (Part 1) ADVERTISEMENT Correspondence Membrane molecules mobile even after chemical fixation - pp865 - 866 Kenji A K Tanaka, Kenichi G N Suzuki, Yuki M Shirai, Shusaku T Shibutani, Manami S H Miyahara, Hisae Tsuboi, Miyako Yahara, Akihiko Yoshimura, Satyajit Mayor, Takahiro K Fujiwara & Akihiro Kusumi doi:10.1038/nmeth.f.314 Full Text - Membrane molecules mobile even after chemical fixation | PDF (642 KB) - Membrane molecules mobile even after chemical fixation | Supplementary information Federal policy and the use of pluripotent stem cells - pp866 - 867 Christopher Thomas Scott, Jennifer B McCormick, Mindy C DeRouen & Jason Owen-Smith doi:10.1038/nmeth1110-866 Full Text - Federal policy and the use of pluripotent stem cells | PDF (153 KB) - Federal policy and the use of pluripotent stem cells Research Highlights Finding the trees in the forest - p869 Nicole Rusk doi:10.1038/nmeth1110-869 The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function. Abstract - Finding the trees in the forest | Full Text - Finding the trees in the forest | PDF (211 KB) - Finding the trees in the forest Protein structure gets exciting - pp870 - 871 Allison Doerr doi:10.1038/nmeth1110-870a Researchers determined the excited-state structure of a small protein using nuclear magnetic resonance spectroscopy. Abstract - Protein structure gets exciting | Full Text - Protein structure gets exciting | PDF (168 KB) - Protein structure gets exciting Many mini mind promoters - pp870 - 871 Natalie de Souza doi:10.1038/nmeth1110-870b Tools to drive restricted gene expression in the brain. Abstract - Many mini mind promoters | Full Text - Many mini mind promoters | PDF (168 KB) - Many mini mind promoters News in brief - p871 doi:10.1038/nmeth1110-871 Full Text - News in brief | PDF (143 KB) - News in brief Species collage - p872 Erika Pastrana doi:10.1038/nmeth1110-872 A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice. Abstract - Species collage | Full Text - Species collage | PDF (108 KB) - Species collage Hidden code in the protein code - p874 Monya Baker doi:10.1038/nmeth1110-874 Apparently redundant codons may not be redundant after all. Abstract - Hidden code in the protein code | Full Text - Hidden code in the protein code | PDF (83 KB) - Hidden code in the protein code Self-healing light beams - p876 Daniel Evanko doi:10.1038/nmeth1110-876 The self-reconstructing properties of Bessel beams provide healing benefits in highly scattering media. Abstract - Self-healing light beams | Full Text - Self-healing light beams | PDF (108 KB) - Self-healing light beams Technology Feature From promising to practical: tools to study networks of neurons - pp877 - 883 Monya Baker doi:10.1038/nmeth1110-877 Combinations of electrophysiology, two-photon microscopy and new tools for detecting neural activity show how neurons function in circuits. Abstract - From promising to practical: tools to study networks of neurons | Full Text - From promising to practical: tools to study networks of neurons | PDF (733 KB) - From promising to practical: tools to study networks of neurons News and Views Defining pluripotency - pp885 - 887 Martin F Pera doi:10.1038/nmeth1110-885 Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties. Full Text - Defining pluripotency | PDF (857 KB) - Defining pluripotency See also:Article by Stewart et al. DNA construction: homemade or ordered out? - pp887 - 889 Peter A Carr doi:10.1038/nmeth1110-887 Automation and optimization of DNA construction results in the efficient production of large target sequences. Full Text - DNA construction: homemade or ordered out? | PDF (214 KB) - DNA construction: homemade or ordered out? See also:Brief Communication by Gibson et al. Pacing lightly: optogenetics gets to the heart - pp889 - 891 Björn C Knollmann doi:10.1038/nmeth1110-889 Transfer of the light-activated cation channel channelrhodopsin-2 gene enables optical control of heart muscle membrane potential. Full Text - Pacing lightly: optogenetics gets to the heart | PDF (400 KB) - Pacing lightly: optogenetics gets to the heart See also:Brief Communication by Bruegmann et al. Brief Communications Dual RMCE for efficient re-engineering of mouse mutant alleles - pp893 - 895 Marco Osterwalder, Antonella Galli, Barry Rosen, William C Skarnes, Rolf Zeller & Javier Lopez-Rios doi:10.1038/nmeth.1521 An efficient one-step method for re-engineering mouse mutant alleles harboring loxP and FRT sites is reported. It may be applied to the large collection of targeted alleles from the International Knockout Mouse Consortium. Abstract - Dual RMCE for efficient re-engineering of mouse mutant alleles | Full Text - Dual RMCE for efficient re-engineering of mouse mutant alleles | PDF (565 KB) - Dual RMCE for efficient re-engineering of mouse mutant alleles | Supplementary information Optogenetic control of heart muscle in vitro and in vivo- pp897 - 900 Tobias Bruegmann, Daniela Malan, Michael Hesse, Thomas Beiert, Christopher J Fuegemann, Bernd K Fleischmann & Philipp Sasse doi:10.1038/nmeth.1512 Stimulation of the light-activated cation channel channelrhodopsin-2 can depolarize heart muscle in vitro and in vivo, resulting in precise localized stimulation and constant prolonged depolarization of genetically targeted cardiomyocytes and cardiac tissue. Abstract - Optogenetic control of heart muscle in vitro and in vivo | Full Text - Optogenetic control of heart muscle in vitro and in vivo | PDF (1,355 KB) - Optogenetic control of heart muscle in vitro and in vivo | Supplementary information See also:News and Views by Knollmann Chemical synthesis of the mouse mitochondrial genome - pp901 - 903 Daniel G Gibson, Hamilton O Smith, Clyde A Hutchison III, J Craig Venter & Chuck Merryman doi:10.1038/nmeth.1515 Using 600 oligonucleotides with 60 bases each and three enzymes, the authors assemble the entire mouse mitochondrial genome in four isothermal reactions. Abstract - Chemical synthesis of the mouse mitochondrial genome | Full Text - Chemical synthesis of the mouse mitochondrial genome | PDF (488 KB) - Chemical synthesis of the mouse mitochondrial genome | Supplementary information See also:News and Views by Carr Efficient CNS gene delivery by intravenous injection - pp905 - 907 Jean-Pierre Louboutin, Alena A Chekmasova, Elena Marusich, J Roy Chowdhury & David S Strayer doi:10.1038/nmeth.1518 Recombinant SV40 viral vectors intravenously injected into mice pretreated with mannitol effectively deliver transgenes to adult neurons in several regions of the central nervous system. Abstract - Efficient CNS gene delivery by intravenous injection | Full Text - Efficient CNS gene delivery by intravenous injection | PDF (1,239 KB) - Efficient CNS gene delivery by intravenous injection | Supplementary information De novo assembly and analysis of RNA-seq data - pp909 - 912 Gordon Robertson, Jacqueline Schein, Readman Chiu, Richard Corbett, Matthew Field, Shaun D Jackman, Karen Mungall, Sam Lee, Hisanaga Mark Okada, Jenny Q Qian, Malachi Griffith, Anthony Raymond, Nina Thiessen, Timothee Cezard, Yaron S Butterfield, Richard Newsome, Simon K Chan, Rong She, Richard Varhol, Baljit Kamoh, Anna-Liisa Prabhu, Angela Tam, YongJun Zhao, Richard A Moore, Martin Hirst, Marco A Marra, Steven J M Jones, Pamela A Hoodless & Inanc Birol doi:10.1038/nmeth.1517 The Trans-ABySS pipeline is an integrated approach for transcript assembly and analysis to identify new mRNA isoforms and structures. Abstract - De novo assembly and analysis of RNA-seq data | Full Text - De novo assembly and analysis of RNA-seq data | PDF (515 KB) - De novo assembly and analysis of RNA-seq data | Supplementary information Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples - pp913 - 915 Isaäc J Nijman, Michal Mokry, Ruben van Boxtel, Pim Toonen, Ewart de Bruijn & Edwin Cuppen doi:10.1038/nmeth.1516 By pooling barcoded genomes of thirty rats before enrichment of a 1.4-megabase target sequence, mutation discovery in 770 genes is achieved with high accuracy. Abstract - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Full Text - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | PDF (372 KB) - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Supplementary information Articles Clonal tracking of hESCs reveals differential contribution to functional assays - pp917 - 922 Morag H Stewart, Sean C Bendall, Marilyne Levadoux-Martin & Mickie Bhatia doi:10.1038/nmeth.1519 Retroviral integration is used to mark clones in human embryonic stem cell cultures and clonal distribution is assessed after functionally testing the cells with different methods. Distinct subsets of clones are detected after in vitro differentiation versus teratoma formation in vivo. Abstract - Clonal tracking of hESCs reveals differential contribution to functional assays | Full Text - Clonal tracking of hESCs reveals differential contribution to functional assays | PDF (1,421 KB) - Clonal tracking of hESCs reveals differential contribution to functional assays | Supplementary information See also:News and Views by Pera Trans-SILAC: sorting out the non-cell-autonomous proteome - pp923 - 927 Oded Rechavi, Matan Kalman, Yuan Fang, Helly Vernitsky, Jasmine Jacob-Hirsch, Leonard J Foster, Yoel Kloog & Itamar Goldstein doi:10.1038/nmeth.1513 Proteins can be transferred between cells in contact, such as via trogocytosis in lymphocytes, or acquired via bacteria-host interactions during infection. A quantitative proteomics approach to identify such non-cell-autonomous proteins is described. Abstract - Trans-SILAC: sorting out the non-cell-autonomous proteome | Full Text - Trans-SILAC: sorting out the non-cell-autonomous proteome | PDF (957 KB) - Trans-SILAC: sorting out the non-cell-autonomous proteome | Supplementary information Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors - pp929 - 935 Brigitte Anliker, Tobias Abel, Sabrina Kneissl, Juraj Hlavaty, Antonio Caputi, Julia Brynza, Irene C Schneider, Robert C Münch, Helga Petznek, Roland E Kontermann, Ulrike Koehl, Ian C D Johnston, Kari Keinänen, Ulrike C Müller, Christine Hohenadl, Hannah Monyer, Klaus Cichutek & Christian J Buchholz doi:10.1038/nmeth.1514 A targeting method for lentiviral vectors relying on the use of single-chain antibodies recognizing cell-surface antigens is applied to generate lentiviral vectors specific for endothelial cells, hematopoietic progenitors and neurons. Abstract - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Full Text - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | PDF (2,053 KB) - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Supplementary information ADVERTISEMENT
• The author file: Rolf Zeller and Javier Lopez-Rios
  - Nat Meth 7(11):861 (2010)
  ARTICLE NAVIGATION - ISSUE Previous November 2010, Volume 7 No 11 pp859-935 * In This Issue * Editorial * This Month * Correspondence * Research Highlights * Technology Feature * News and Views * Brief Communications * ArticlesAbout the cover In This Issue PDF - In This Issue Editorial Nobel thoughts - p859 doi:10.1038/nmeth1110-859 The community of scientists should celebrate the Nobel Prize, even if awards bestowed on one discipline are associated with another discipline. A new prize might help. Abstract - Nobel thoughts | Full Text - Nobel thoughts | PDF (65 KB) - Nobel thoughts This Month The author file: Rolf Zeller and Javier Lopez-Rios - p861 Monya Baker doi:10.1038/nmeth1110-861 Gene cutting and pasting just got a whole lot faster. Abstract - The author file: Rolf Zeller and Javier Lopez-Rios | Full Text - The author fileRolf Zeller and Javier Lopez-Rios | PDF (152 KB) - The author fileRolf Zeller and Javier Lopez-Rios Points of View: Gestalt principles (Part 1) - p863 Bang Wong doi:10.1038/nmeth1110-863 Full Text - Points of ViewGestalt principles (Part 1) | PDF (119 KB) - Points of ViewGestalt principles (Part 1) ADVERTISEMENT Correspondence Membrane molecules mobile even after chemical fixation - pp865 - 866 Kenji A K Tanaka, Kenichi G N Suzuki, Yuki M Shirai, Shusaku T Shibutani, Manami S H Miyahara, Hisae Tsuboi, Miyako Yahara, Akihiko Yoshimura, Satyajit Mayor, Takahiro K Fujiwara & Akihiro Kusumi doi:10.1038/nmeth.f.314 Full Text - Membrane molecules mobile even after chemical fixation | PDF (642 KB) - Membrane molecules mobile even after chemical fixation | Supplementary information Federal policy and the use of pluripotent stem cells - pp866 - 867 Christopher Thomas Scott, Jennifer B McCormick, Mindy C DeRouen & Jason Owen-Smith doi:10.1038/nmeth1110-866 Full Text - Federal policy and the use of pluripotent stem cells | PDF (153 KB) - Federal policy and the use of pluripotent stem cells Research Highlights Finding the trees in the forest - p869 Nicole Rusk doi:10.1038/nmeth1110-869 The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function. Abstract - Finding the trees in the forest | Full Text - Finding the trees in the forest | PDF (211 KB) - Finding the trees in the forest Protein structure gets exciting - pp870 - 871 Allison Doerr doi:10.1038/nmeth1110-870a Researchers determined the excited-state structure of a small protein using nuclear magnetic resonance spectroscopy. Abstract - Protein structure gets exciting | Full Text - Protein structure gets exciting | PDF (168 KB) - Protein structure gets exciting Many mini mind promoters - pp870 - 871 Natalie de Souza doi:10.1038/nmeth1110-870b Tools to drive restricted gene expression in the brain. Abstract - Many mini mind promoters | Full Text - Many mini mind promoters | PDF (168 KB) - Many mini mind promoters News in brief - p871 doi:10.1038/nmeth1110-871 Full Text - News in brief | PDF (143 KB) - News in brief Species collage - p872 Erika Pastrana doi:10.1038/nmeth1110-872 A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice. Abstract - Species collage | Full Text - Species collage | PDF (108 KB) - Species collage Hidden code in the protein code - p874 Monya Baker doi:10.1038/nmeth1110-874 Apparently redundant codons may not be redundant after all. Abstract - Hidden code in the protein code | Full Text - Hidden code in the protein code | PDF (83 KB) - Hidden code in the protein code Self-healing light beams - p876 Daniel Evanko doi:10.1038/nmeth1110-876 The self-reconstructing properties of Bessel beams provide healing benefits in highly scattering media. Abstract - Self-healing light beams | Full Text - Self-healing light beams | PDF (108 KB) - Self-healing light beams Technology Feature From promising to practical: tools to study networks of neurons - pp877 - 883 Monya Baker doi:10.1038/nmeth1110-877 Combinations of electrophysiology, two-photon microscopy and new tools for detecting neural activity show how neurons function in circuits. Abstract - From promising to practical: tools to study networks of neurons | Full Text - From promising to practical: tools to study networks of neurons | PDF (733 KB) - From promising to practical: tools to study networks of neurons News and Views Defining pluripotency - pp885 - 887 Martin F Pera doi:10.1038/nmeth1110-885 Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties. Full Text - Defining pluripotency | PDF (857 KB) - Defining pluripotency See also:Article by Stewart et al. DNA construction: homemade or ordered out? - pp887 - 889 Peter A Carr doi:10.1038/nmeth1110-887 Automation and optimization of DNA construction results in the efficient production of large target sequences. Full Text - DNA construction: homemade or ordered out? | PDF (214 KB) - DNA construction: homemade or ordered out? See also:Brief Communication by Gibson et al. Pacing lightly: optogenetics gets to the heart - pp889 - 891 Björn C Knollmann doi:10.1038/nmeth1110-889 Transfer of the light-activated cation channel channelrhodopsin-2 gene enables optical control of heart muscle membrane potential. Full Text - Pacing lightly: optogenetics gets to the heart | PDF (400 KB) - Pacing lightly: optogenetics gets to the heart See also:Brief Communication by Bruegmann et al. Brief Communications Dual RMCE for efficient re-engineering of mouse mutant alleles - pp893 - 895 Marco Osterwalder, Antonella Galli, Barry Rosen, William C Skarnes, Rolf Zeller & Javier Lopez-Rios doi:10.1038/nmeth.1521 An efficient one-step method for re-engineering mouse mutant alleles harboring loxP and FRT sites is reported. It may be applied to the large collection of targeted alleles from the International Knockout Mouse Consortium. Abstract - Dual RMCE for efficient re-engineering of mouse mutant alleles | Full Text - Dual RMCE for efficient re-engineering of mouse mutant alleles | PDF (565 KB) - Dual RMCE for efficient re-engineering of mouse mutant alleles | Supplementary information Optogenetic control of heart muscle in vitro and in vivo- pp897 - 900 Tobias Bruegmann, Daniela Malan, Michael Hesse, Thomas Beiert, Christopher J Fuegemann, Bernd K Fleischmann & Philipp Sasse doi:10.1038/nmeth.1512 Stimulation of the light-activated cation channel channelrhodopsin-2 can depolarize heart muscle in vitro and in vivo, resulting in precise localized stimulation and constant prolonged depolarization of genetically targeted cardiomyocytes and cardiac tissue. Abstract - Optogenetic control of heart muscle in vitro and in vivo | Full Text - Optogenetic control of heart muscle in vitro and in vivo | PDF (1,355 KB) - Optogenetic control of heart muscle in vitro and in vivo | Supplementary information See also:News and Views by Knollmann Chemical synthesis of the mouse mitochondrial genome - pp901 - 903 Daniel G Gibson, Hamilton O Smith, Clyde A Hutchison III, J Craig Venter & Chuck Merryman doi:10.1038/nmeth.1515 Using 600 oligonucleotides with 60 bases each and three enzymes, the authors assemble the entire mouse mitochondrial genome in four isothermal reactions. Abstract - Chemical synthesis of the mouse mitochondrial genome | Full Text - Chemical synthesis of the mouse mitochondrial genome | PDF (488 KB) - Chemical synthesis of the mouse mitochondrial genome | Supplementary information See also:News and Views by Carr Efficient CNS gene delivery by intravenous injection - pp905 - 907 Jean-Pierre Louboutin, Alena A Chekmasova, Elena Marusich, J Roy Chowdhury & David S Strayer doi:10.1038/nmeth.1518 Recombinant SV40 viral vectors intravenously injected into mice pretreated with mannitol effectively deliver transgenes to adult neurons in several regions of the central nervous system. Abstract - Efficient CNS gene delivery by intravenous injection | Full Text - Efficient CNS gene delivery by intravenous injection | PDF (1,239 KB) - Efficient CNS gene delivery by intravenous injection | Supplementary information De novo assembly and analysis of RNA-seq data - pp909 - 912 Gordon Robertson, Jacqueline Schein, Readman Chiu, Richard Corbett, Matthew Field, Shaun D Jackman, Karen Mungall, Sam Lee, Hisanaga Mark Okada, Jenny Q Qian, Malachi Griffith, Anthony Raymond, Nina Thiessen, Timothee Cezard, Yaron S Butterfield, Richard Newsome, Simon K Chan, Rong She, Richard Varhol, Baljit Kamoh, Anna-Liisa Prabhu, Angela Tam, YongJun Zhao, Richard A Moore, Martin Hirst, Marco A Marra, Steven J M Jones, Pamela A Hoodless & Inanc Birol doi:10.1038/nmeth.1517 The Trans-ABySS pipeline is an integrated approach for transcript assembly and analysis to identify new mRNA isoforms and structures. Abstract - De novo assembly and analysis of RNA-seq data | Full Text - De novo assembly and analysis of RNA-seq data | PDF (515 KB) - De novo assembly and analysis of RNA-seq data | Supplementary information Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples - pp913 - 915 Isaäc J Nijman, Michal Mokry, Ruben van Boxtel, Pim Toonen, Ewart de Bruijn & Edwin Cuppen doi:10.1038/nmeth.1516 By pooling barcoded genomes of thirty rats before enrichment of a 1.4-megabase target sequence, mutation discovery in 770 genes is achieved with high accuracy. Abstract - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Full Text - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | PDF (372 KB) - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Supplementary information Articles Clonal tracking of hESCs reveals differential contribution to functional assays - pp917 - 922 Morag H Stewart, Sean C Bendall, Marilyne Levadoux-Martin & Mickie Bhatia doi:10.1038/nmeth.1519 Retroviral integration is used to mark clones in human embryonic stem cell cultures and clonal distribution is assessed after functionally testing the cells with different methods. Distinct subsets of clones are detected after in vitro differentiation versus teratoma formation in vivo. Abstract - Clonal tracking of hESCs reveals differential contribution to functional assays | Full Text - Clonal tracking of hESCs reveals differential contribution to functional assays | PDF (1,421 KB) - Clonal tracking of hESCs reveals differential contribution to functional assays | Supplementary information See also:News and Views by Pera Trans-SILAC: sorting out the non-cell-autonomous proteome - pp923 - 927 Oded Rechavi, Matan Kalman, Yuan Fang, Helly Vernitsky, Jasmine Jacob-Hirsch, Leonard J Foster, Yoel Kloog & Itamar Goldstein doi:10.1038/nmeth.1513 Proteins can be transferred between cells in contact, such as via trogocytosis in lymphocytes, or acquired via bacteria-host interactions during infection. A quantitative proteomics approach to identify such non-cell-autonomous proteins is described. Abstract - Trans-SILAC: sorting out the non-cell-autonomous proteome | Full Text - Trans-SILAC: sorting out the non-cell-autonomous proteome | PDF (957 KB) - Trans-SILAC: sorting out the non-cell-autonomous proteome | Supplementary information Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors - pp929 - 935 Brigitte Anliker, Tobias Abel, Sabrina Kneissl, Juraj Hlavaty, Antonio Caputi, Julia Brynza, Irene C Schneider, Robert C Münch, Helga Petznek, Roland E Kontermann, Ulrike Koehl, Ian C D Johnston, Kari Keinänen, Ulrike C Müller, Christine Hohenadl, Hannah Monyer, Klaus Cichutek & Christian J Buchholz doi:10.1038/nmeth.1514 A targeting method for lentiviral vectors relying on the use of single-chain antibodies recognizing cell-surface antigens is applied to generate lentiviral vectors specific for endothelial cells, hematopoietic progenitors and neurons. Abstract - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Full Text - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | PDF (2,053 KB) - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Supplementary information ADVERTISEMENT
• Points of View: Gestalt principles (Part 1)
  - Nat Meth 7(11):863 (2010)
  ARTICLE NAVIGATION - ISSUE Previous November 2010, Volume 7 No 11 pp859-935 * In This Issue * Editorial * This Month * Correspondence * Research Highlights * Technology Feature * News and Views * Brief Communications * ArticlesAbout the cover In This Issue PDF - In This Issue Editorial Nobel thoughts - p859 doi:10.1038/nmeth1110-859 The community of scientists should celebrate the Nobel Prize, even if awards bestowed on one discipline are associated with another discipline. A new prize might help. Abstract - Nobel thoughts | Full Text - Nobel thoughts | PDF (65 KB) - Nobel thoughts This Month The author file: Rolf Zeller and Javier Lopez-Rios - p861 Monya Baker doi:10.1038/nmeth1110-861 Gene cutting and pasting just got a whole lot faster. Abstract - The author file: Rolf Zeller and Javier Lopez-Rios | Full Text - The author fileRolf Zeller and Javier Lopez-Rios | PDF (152 KB) - The author fileRolf Zeller and Javier Lopez-Rios Points of View: Gestalt principles (Part 1) - p863 Bang Wong doi:10.1038/nmeth1110-863 Full Text - Points of ViewGestalt principles (Part 1) | PDF (119 KB) - Points of ViewGestalt principles (Part 1) ADVERTISEMENT Correspondence Membrane molecules mobile even after chemical fixation - pp865 - 866 Kenji A K Tanaka, Kenichi G N Suzuki, Yuki M Shirai, Shusaku T Shibutani, Manami S H Miyahara, Hisae Tsuboi, Miyako Yahara, Akihiko Yoshimura, Satyajit Mayor, Takahiro K Fujiwara & Akihiro Kusumi doi:10.1038/nmeth.f.314 Full Text - Membrane molecules mobile even after chemical fixation | PDF (642 KB) - Membrane molecules mobile even after chemical fixation | Supplementary information Federal policy and the use of pluripotent stem cells - pp866 - 867 Christopher Thomas Scott, Jennifer B McCormick, Mindy C DeRouen & Jason Owen-Smith doi:10.1038/nmeth1110-866 Full Text - Federal policy and the use of pluripotent stem cells | PDF (153 KB) - Federal policy and the use of pluripotent stem cells Research Highlights Finding the trees in the forest - p869 Nicole Rusk doi:10.1038/nmeth1110-869 The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function. Abstract - Finding the trees in the forest | Full Text - Finding the trees in the forest | PDF (211 KB) - Finding the trees in the forest Protein structure gets exciting - pp870 - 871 Allison Doerr doi:10.1038/nmeth1110-870a Researchers determined the excited-state structure of a small protein using nuclear magnetic resonance spectroscopy. Abstract - Protein structure gets exciting | Full Text - Protein structure gets exciting | PDF (168 KB) - Protein structure gets exciting Many mini mind promoters - pp870 - 871 Natalie de Souza doi:10.1038/nmeth1110-870b Tools to drive restricted gene expression in the brain. Abstract - Many mini mind promoters | Full Text - Many mini mind promoters | PDF (168 KB) - Many mini mind promoters News in brief - p871 doi:10.1038/nmeth1110-871 Full Text - News in brief | PDF (143 KB) - News in brief Species collage - p872 Erika Pastrana doi:10.1038/nmeth1110-872 A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice. Abstract - Species collage | Full Text - Species collage | PDF (108 KB) - Species collage Hidden code in the protein code - p874 Monya Baker doi:10.1038/nmeth1110-874 Apparently redundant codons may not be redundant after all. Abstract - Hidden code in the protein code | Full Text - Hidden code in the protein code | PDF (83 KB) - Hidden code in the protein code Self-healing light beams - p876 Daniel Evanko doi:10.1038/nmeth1110-876 The self-reconstructing properties of Bessel beams provide healing benefits in highly scattering media. Abstract - Self-healing light beams | Full Text - Self-healing light beams | PDF (108 KB) - Self-healing light beams Technology Feature From promising to practical: tools to study networks of neurons - pp877 - 883 Monya Baker doi:10.1038/nmeth1110-877 Combinations of electrophysiology, two-photon microscopy and new tools for detecting neural activity show how neurons function in circuits. Abstract - From promising to practical: tools to study networks of neurons | Full Text - From promising to practical: tools to study networks of neurons | PDF (733 KB) - From promising to practical: tools to study networks of neurons News and Views Defining pluripotency - pp885 - 887 Martin F Pera doi:10.1038/nmeth1110-885 Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties. Full Text - Defining pluripotency | PDF (857 KB) - Defining pluripotency See also:Article by Stewart et al. DNA construction: homemade or ordered out? - pp887 - 889 Peter A Carr doi:10.1038/nmeth1110-887 Automation and optimization of DNA construction results in the efficient production of large target sequences. Full Text - DNA construction: homemade or ordered out? | PDF (214 KB) - DNA construction: homemade or ordered out? See also:Brief Communication by Gibson et al. Pacing lightly: optogenetics gets to the heart - pp889 - 891 Björn C Knollmann doi:10.1038/nmeth1110-889 Transfer of the light-activated cation channel channelrhodopsin-2 gene enables optical control of heart muscle membrane potential. Full Text - Pacing lightly: optogenetics gets to the heart | PDF (400 KB) - Pacing lightly: optogenetics gets to the heart See also:Brief Communication by Bruegmann et al. Brief Communications Dual RMCE for efficient re-engineering of mouse mutant alleles - pp893 - 895 Marco Osterwalder, Antonella Galli, Barry Rosen, William C Skarnes, Rolf Zeller & Javier Lopez-Rios doi:10.1038/nmeth.1521 An efficient one-step method for re-engineering mouse mutant alleles harboring loxP and FRT sites is reported. It may be applied to the large collection of targeted alleles from the International Knockout Mouse Consortium. Abstract - Dual RMCE for efficient re-engineering of mouse mutant alleles | Full Text - Dual RMCE for efficient re-engineering of mouse mutant alleles | PDF (565 KB) - Dual RMCE for efficient re-engineering of mouse mutant alleles | Supplementary information Optogenetic control of heart muscle in vitro and in vivo- pp897 - 900 Tobias Bruegmann, Daniela Malan, Michael Hesse, Thomas Beiert, Christopher J Fuegemann, Bernd K Fleischmann & Philipp Sasse doi:10.1038/nmeth.1512 Stimulation of the light-activated cation channel channelrhodopsin-2 can depolarize heart muscle in vitro and in vivo, resulting in precise localized stimulation and constant prolonged depolarization of genetically targeted cardiomyocytes and cardiac tissue. Abstract - Optogenetic control of heart muscle in vitro and in vivo | Full Text - Optogenetic control of heart muscle in vitro and in vivo | PDF (1,355 KB) - Optogenetic control of heart muscle in vitro and in vivo | Supplementary information See also:News and Views by Knollmann Chemical synthesis of the mouse mitochondrial genome - pp901 - 903 Daniel G Gibson, Hamilton O Smith, Clyde A Hutchison III, J Craig Venter & Chuck Merryman doi:10.1038/nmeth.1515 Using 600 oligonucleotides with 60 bases each and three enzymes, the authors assemble the entire mouse mitochondrial genome in four isothermal reactions. Abstract - Chemical synthesis of the mouse mitochondrial genome | Full Text - Chemical synthesis of the mouse mitochondrial genome | PDF (488 KB) - Chemical synthesis of the mouse mitochondrial genome | Supplementary information See also:News and Views by Carr Efficient CNS gene delivery by intravenous injection - pp905 - 907 Jean-Pierre Louboutin, Alena A Chekmasova, Elena Marusich, J Roy Chowdhury & David S Strayer doi:10.1038/nmeth.1518 Recombinant SV40 viral vectors intravenously injected into mice pretreated with mannitol effectively deliver transgenes to adult neurons in several regions of the central nervous system. Abstract - Efficient CNS gene delivery by intravenous injection | Full Text - Efficient CNS gene delivery by intravenous injection | PDF (1,239 KB) - Efficient CNS gene delivery by intravenous injection | Supplementary information De novo assembly and analysis of RNA-seq data - pp909 - 912 Gordon Robertson, Jacqueline Schein, Readman Chiu, Richard Corbett, Matthew Field, Shaun D Jackman, Karen Mungall, Sam Lee, Hisanaga Mark Okada, Jenny Q Qian, Malachi Griffith, Anthony Raymond, Nina Thiessen, Timothee Cezard, Yaron S Butterfield, Richard Newsome, Simon K Chan, Rong She, Richard Varhol, Baljit Kamoh, Anna-Liisa Prabhu, Angela Tam, YongJun Zhao, Richard A Moore, Martin Hirst, Marco A Marra, Steven J M Jones, Pamela A Hoodless & Inanc Birol doi:10.1038/nmeth.1517 The Trans-ABySS pipeline is an integrated approach for transcript assembly and analysis to identify new mRNA isoforms and structures. Abstract - De novo assembly and analysis of RNA-seq data | Full Text - De novo assembly and analysis of RNA-seq data | PDF (515 KB) - De novo assembly and analysis of RNA-seq data | Supplementary information Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples - pp913 - 915 Isaäc J Nijman, Michal Mokry, Ruben van Boxtel, Pim Toonen, Ewart de Bruijn & Edwin Cuppen doi:10.1038/nmeth.1516 By pooling barcoded genomes of thirty rats before enrichment of a 1.4-megabase target sequence, mutation discovery in 770 genes is achieved with high accuracy. Abstract - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Full Text - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | PDF (372 KB) - Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples | Supplementary information Articles Clonal tracking of hESCs reveals differential contribution to functional assays - pp917 - 922 Morag H Stewart, Sean C Bendall, Marilyne Levadoux-Martin & Mickie Bhatia doi:10.1038/nmeth.1519 Retroviral integration is used to mark clones in human embryonic stem cell cultures and clonal distribution is assessed after functionally testing the cells with different methods. Distinct subsets of clones are detected after in vitro differentiation versus teratoma formation in vivo. Abstract - Clonal tracking of hESCs reveals differential contribution to functional assays | Full Text - Clonal tracking of hESCs reveals differential contribution to functional assays | PDF (1,421 KB) - Clonal tracking of hESCs reveals differential contribution to functional assays | Supplementary information See also:News and Views by Pera Trans-SILAC: sorting out the non-cell-autonomous proteome - pp923 - 927 Oded Rechavi, Matan Kalman, Yuan Fang, Helly Vernitsky, Jasmine Jacob-Hirsch, Leonard J Foster, Yoel Kloog & Itamar Goldstein doi:10.1038/nmeth.1513 Proteins can be transferred between cells in contact, such as via trogocytosis in lymphocytes, or acquired via bacteria-host interactions during infection. A quantitative proteomics approach to identify such non-cell-autonomous proteins is described. Abstract - Trans-SILAC: sorting out the non-cell-autonomous proteome | Full Text - Trans-SILAC: sorting out the non-cell-autonomous proteome | PDF (957 KB) - Trans-SILAC: sorting out the non-cell-autonomous proteome | Supplementary information Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors - pp929 - 935 Brigitte Anliker, Tobias Abel, Sabrina Kneissl, Juraj Hlavaty, Antonio Caputi, Julia Brynza, Irene C Schneider, Robert C Münch, Helga Petznek, Roland E Kontermann, Ulrike Koehl, Ian C D Johnston, Kari Keinänen, Ulrike C Müller, Christine Hohenadl, Hannah Monyer, Klaus Cichutek & Christian J Buchholz doi:10.1038/nmeth.1514 A targeting method for lentiviral vectors relying on the use of single-chain antibodies recognizing cell-surface antigens is applied to generate lentiviral vectors specific for endothelial cells, hematopoietic progenitors and neurons. Abstract - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Full Text - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | PDF (2,053 KB) - Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors | Supplementary information ADVERTISEMENT
• Membrane molecules mobile even after chemical fixation
  - Nat Meth 7(11):865-866 (2010)
  Nature Methods | Editorial Nobel thoughts Journal name:Nature MethodsVolume: 7 ,Page:859Year published:(2010)DOI:doi:10.1038/nmeth1110-859Published online28 October 2010 The community of scientists should celebrate the Nobel Prize, even if awards bestowed on one discipline are associated with another discipline. A new prize might help. View full text Additional data
• Federal policy and the use of pluripotent stem cells
  - Nat Meth 7(11):866-867 (2010)
  Nature Methods | This Month The author file: Rolf Zeller and Javier Lopez-Rios * Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:861Year published:(2010)DOI:doi:10.1038/nmeth1110-861Published online28 October 2010 Gene cutting and pasting just got a whole lot faster. View full text Additional data
• Finding the trees in the forest
  - Nat Meth 7(11):869 (2010)
  Gestalt principles of perception are theories proposed by German psychologists in the 1920s to explain how people organize visual information1. Gestalt is a German word meaning shape or form.
• Protein structure gets exciting
  - Nat Meth 7(11):870-871 (2010)
  Nature Methods | Correspondence Membrane molecules mobile even after chemical fixation * Kenji A K Tanaka1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Kenichi G N Suzuki2, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuki M Shirai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shusaku T Shibutani1 Search for this author in: * NPG journals * PubMed * Google Scholar * Manami S H Miyahara1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hisae Tsuboi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Miyako Yahara1 Search for this author in: * NPG journals * PubMed * Google Scholar * Akihiko Yoshimura3 Search for this author in: * NPG journals * PubMed * Google Scholar * Satyajit Mayor4 Search for this author in: * NPG journals * PubMed * Google Scholar * Takahiro K Fujiwara5 Search for this author in: * NPG journals * PubMed * Google Scholar * Akihiro Kusumi1, 5akusumi@frontier.kyoto-u.ac.jp Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:865–866Year published:(2010)DOI:doi:10.1038/nmeth.f.314Published online03 October 2010 To the Editor: Fixation of cells and tissues is the critical first step for histochemical or cytochemical investigations1, 2. Recent efforts to visualize lipid rafts by immunofluorescence and immunoelectron microscopy3, particularly in nonstimulated cells, have yielded varied results, raising a question about the efficacy of chemical cross-linking fixation protocols for blocking the lateral diffusion of membrane molecules and thus their antibody-induced clustering3, 4. In a literature search we did not find any systematic investigation of this issue. Here we investigated whether the lateral diffusion of both raft-associated and non–raft-associated molecules is blocked upon chemical cross-linking and treatment with cold methanol in human T24 cells. We used single fluorescent molecule tracking, which allowed us to directly observe the variation in molecular immobilization after fixation (Fig. 1a, Supplementary Figs. 1 and 2, Supplementary Videos 1 and 2, and Supplementary Methods). View full text Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Primary authors * These authors contributed equally to this work. * Kenji A K Tanaka & * Kenichi G N Suzuki Affiliations * Membrane Mechanisms Project, International Cooperative Research Project, Japan Science and Technology Agency (JST), Institute for Integrated Cell-Material Sciences (iCeMS) and Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan. * Kenji A K Tanaka, * Yuki M Shirai, * Shusaku T Shibutani, * Manami S H Miyahara, * Hisae Tsuboi, * Miyako Yahara & * Akihiro Kusumi * Precursory Research for Embryonic Science and Technology, JST, iCeMS, Kyoto University, Kyoto, Japan. * Kenichi G N Suzuki * Department of Microbiology and Immunology, School of Medicine, Keio University and Core Research for Evolutional Science and Technology, JST, Tokyo, Japan. * Akihiko Yoshimura * National Centre for Biological Sciences, Bangalore, India. * Satyajit Mayor * Center for Meso-Bio Single-Molecule Imaging, iCeMS, Kyoto University, Kyoto, Japan. * Takahiro K Fujiwara & * Akihiro Kusumi Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Akihiro Kusumi (akusumi@frontier.kyoto-u.ac.jp) Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (3M) A typical video clip showing the diffusion of single Halo-GPI molecules on the apical plasma membrane at 37 °C. Before chemical cross-linking (untreated, left), and after treatment with 4% PFA for 30 min (center) or with 4% PFA + 0.1% GA for 30 min (right). * Supplementary Video 2 (2M) A typical video clip showing the diffusion of single Halo–H-Ras molecules on the apical plasma membrane at 37 °C. Before chemical cross-linking (untreated, left), and after treatment with 4% PFA for 30 min (center) or with 4% PFA + 0.1% GA for 30 min (right). PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–6, Supplementary Methods Additional data
• Many mini mind promoters
  - Nat Meth 7(11):870-871 (2010)
  In March 2009, US President Barack H. Obama ended eight years of restrictions on federal funding for human embryonic stem cell (hESC) research1.
• News in brief
  - Nat Meth 7(11):871 (2010)
  Nature Methods | Research Highlights Finding the trees in the forest * Nicole Rusk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:869Year published:(2010)DOI:doi:10.1038/nmeth1110-869Published online28 October 2010 The integration of quantitative proteomics and analysis by machine learning yields a refined list of proteins involved in chromosome function. View full text Subject terms: * Cell Biology Additional data
• Species collage
  - Nat Meth 7(11):872 (2010)
  Nature Methods | Research Highlights Protein structure gets exciting * Allison Doerr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Pages:870–871Year published:(2010)DOI:doi:10.1038/nmeth1110-870aPublished online28 October 2010 Researchers determined the excited-state structure of a small protein using nuclear magnetic resonance spectroscopy. View full text Subject terms: * Structural biology Additional data
• Hidden code in the protein code
  - Nat Meth 7(11):874 (2010)
  Nature Methods | Research Highlights Many mini mind promoters * Natalie de Souza Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Pages:870–871Year published:(2010)DOI:doi:10.1038/nmeth1110-870bPublished online28 October 2010 Tools to drive restricted gene expression in the brain. View full text Subject terms: * Gene expression Additional data
• Self-healing light beams
  - Nat Meth 7(11):876 (2010)
  Molecular engineering Imaging Epigenetics Bioinformatics Biophysics
• From promising to practical: tools to study networks of neurons
  - Nat Meth 7(11):877-883 (2010)
  Nature Methods | Research Highlights Species collage * Erika Pastrana Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:872Year published:(2010)DOI:doi:10.1038/nmeth1110-872Published online28 October 2010 A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice. View full text Subject terms: * Stem Cells Additional data
• Defining pluripotency
  - Nat Meth 7(11):885-887 (2010)
  Nature Methods | Research Highlights Hidden code in the protein code * Monya Baker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:874Year published:(2010)DOI:doi:10.1038/nmeth1110-874Published online28 October 2010 Apparently redundant codons may not be redundant after all. View full text Subject terms: * Biochemistry Additional data
• DNA construction: homemade or ordered out?
  - Nat Meth 7(11):887-889 (2010)
  Nature Methods | Research Highlights Self-healing light beams * Daniel Evanko Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Page:876Year published:(2010)DOI:doi:10.1038/nmeth1110-876Published online28 October 2010 The self-reconstructing properties of Bessel beams provide healing benefits in highly scattering media. View full text Subject terms: * Microscopy Additional data
• Pacing lightly: optogenetics gets to the heart
  - Nat Meth 7(11):889-891 (2010)
  Nature Methods | Technology Feature From promising to practical: tools to study networks of neurons * Monya Baker1techfeatures@nature.com Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MethodsVolume: 7 ,Pages:877–883Year published:(2010)DOI:doi:10.1038/nmeth1110-877Published online28 October 2010 Combinations of electrophysiology, two-photon microscopy and new tools for detecting neural activity show how neurons function in circuits. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Monya Baker is technology editor for Nature and Nature Methods Corresponding author Correspondence to: * Monya Baker (techfeatures@nature.com) Additional data
• Dual RMCE for efficient re-engineering of mouse mutant alleles
  - Nat Meth 7(11):893-895 (2010)
  Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties.
• Optogenetic control of heart muscle in vitro and in vivo
  - Nat Meth 7(11):897-900 (2010)
  Automation and optimization of DNA construction results in the efficient production of large target sequences.
• Chemical synthesis of the mouse mitochondrial genome
  - Nat Meth 7(11):901-903 (2010)
  Transfer of the light-activated cation channel channelrhodopsin-2 gene enables optical control of heart muscle membrane potential.
• Efficient CNS gene delivery by intravenous injection
  - Nat Meth 7(11):905-907 (2010)
  Nature Methods | Brief Communication Dual RMCE for efficient re-engineering of mouse mutant alleles * Marco Osterwalder1 Search for this author in: * NPG journals * PubMed * Google Scholar * Antonella Galli1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Barry Rosen2 Search for this author in: * NPG journals * PubMed * Google Scholar * William C Skarnes2 Search for this author in: * NPG journals * PubMed * Google Scholar * Rolf Zeller1rolf.zeller@unibas.ch Search for this author in: * NPG journals * PubMed * Google Scholar * Javier Lopez-Rios1javier.lopez-rios@unibas.ch Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 7 ,Pages:893–895Year published:(2010)DOI:doi:10.1038/nmeth.1521Received13 April 2010Accepted20 September 2010Published online17 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We have developed dual recombinase-mediated cassette exchange (dRMCE) to efficiently re-engineer the thousands of available conditional alleles in mouse embryonic stem cells. dRMCE takes advantage of the wild-type loxP and FRT sites present in these conditional alleles and in many gene-trap lines. dRMCE is a scalable, flexible tool to introduce tags, reporters and mutant coding regions into an endogenous locus of interest in an easy and highly efficient manner. View full text Figures at a glance * Figure 1: The principle of dRMCE to re-engineer mouse conditional alleles. () Schematic of the target locus shows the configuration of a conditional mouse allele with a genomic region flanked by two loxP sites and an outside selection cassette flanked by two FRT sites. Upon transfection, the combination of iCre- and Flpo-mediated recombination in cis results in a deleted allele flanked by single loxP and FRT sites, which serves as a 'docking site' for insertion of the replacement vector. ex, exon. () Schematic representation of replacement in the Smad4 locus by dRMCE. The target locus is a Smad4 conditional allele (Smad4f) with a promoterless selection cassette. Co-transfection of the pDIRE and pDREV-1 plasmids induces replacement, probably through production of the Smad4− deleted allele as intermediate. Correct trans insertion of the replacement vector results in the Smad4YFP allele. F1–F4 and R1–R3 denote primers used for PCR screening of colonies (see Supplementary Table 2 for sequences). H2B-Venus, YFP fusion protein with histone 2B; lacZ! , β-galactosidase coding region; neo, neomycin resistance coding region; puro, puromycin resistance cassette; rox, Dre recombinase target sites11; SA, splice acceptor; T, autocleavable T2A peptide coding region14. () PCR screening reveals a large number of clones with correct 3′ and 5′ replacement (69%). Col, colony; 3′ recombination, 5′ recombination, correct replacement at the 3′ and 5′ ends, respectively. () The parental Smad4f cells are β-galactosidase positive. (,) Clones with correct replacement (Smad4YFP) lack β-galactosidase activity but show YFP fluorescence. () Micrograph shows single cells expressing the H2B-Venus fusion protein engineered by dRMCE. Scale bars: 100 μm (–), 5 μm (). * Figure 2: dRMCE for efficient modification of difficult-to-target loci. () Schematic shows the conditional Hand2 allele (Hand2f) used as a target locus for insertion of a Flag epitope tag into the Hand2 coding region. After co-transfection of the replacement vector (pRVH2) and pDIRE plasmid into heterozygous Hand2f mouse embryonic stem cells, dRMCE-mediated replacement results in the Hand2Flag allele. The PGK-hygro (hygromycin resistance gene) selection cassette is flanked by the attB and attP target sites for excision by the φC31 recombinase. F5–F7 and R5–R7 denote primers used for PCR screening and genotyping. E, EcoRV site required to detect correct 5′ replacement by combining PCR amplification with an EcoRV restriction digestion. () PCR screening at both ends of the locus identified Hand2 colonies with correct replacement (13%). Scheme at right shows PCR fragment patterns indicative of particular genomic configurations. 3′ recombination, 5′ recombination, correct replacement at the 3′ and 5′ ends, respectively. A, B, primers t! o amplify a region serving as positive control (see Supplementary Table 2 for sequences). () Gels show germline transmission of the Hand2Flag allele (lanes 1, 5, 6). Above, PCR analysis to detect Hand2Flag allele. Below, PCR detection of wild-type allele. () In situ detection of Hand2 transcripts in a wild-type (Hand2+/+; left) and Hand2Flag/Flag (right) mouse embryo at embryonic day 10.5. Note expression in the posterior limb bud mesenchyme (arrow), branchial arches (black arrowhead) and heart (white arrowhead). Scale bar, 500 μm. Author information * Author information * Supplementary information Affiliations * Developmental Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland. * Marco Osterwalder, * Antonella Galli, * Rolf Zeller & * Javier Lopez-Rios * The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. * Barry Rosen & * William C Skarnes * Present address: Department of Medicine and Genetics & Development, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, USA. * Antonella Galli Contributions M.O., J.L.-R. and R.Z. conceived and designed the experiments. M.O., J.L.-R. and B.R. designed and constructed the dRMCE tool-kit vectors. B.R. and W.C.S. provided the IKMC mouse embryonic stem cell lines. M.O., A.G., J.L.-R. and B.R. performed the experiments. J.L.-R., W.C.S. and R.Z. wrote the paper. Competing financial interests A dRMCE patent is pending. Corresponding authors Correspondence to: * Javier Lopez-Rios (javier.lopez-rios@unibas.ch) or * Rolf Zeller (rolf.zeller@unibas.ch) Supplementary information * Author information * Supplementary information Zip files * Supplementary Data (32K) Vector sequences in GenBank format. PDF files * Supplementary Text and Figures (8M) Supplementary Figures 1–5, Supplementary Tables 1 and 2 Additional data
• De novo assembly and analysis of RNA-seq data
  - Nat Meth 7(11):909-912 (2010)
  Nature Methods | Brief Communication Optogenetic control of heart muscle in vitro and in vivo * Tobias Bruegmann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniela Malan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Hesse1 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Beiert1 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J Fuegemann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd K Fleischmann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Philipp Sasse1philipp.sasse@uni-bonn.de Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:897–900Year published:(2010)DOI:doi:10.1038/nmeth.1512Received09 June 2010Accepted30 August 2010Published online03 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Electrical stimulation is the standard technique for exploring electrical behavior of heart muscle, but this approach has considerable technical limitations. Here we report expression of the light-activated cation channel channelrhodopsin-2 for light-induced stimulation of heart muscle in vitro and in mice. This method enabled precise localized stimulation and constant prolonged depolarization of cardiomyocytes and cardiac tissue resulting in alterations of pacemaking, Ca2+ homeostasis, electrical coupling and arrhythmogenic spontaneous extrabeats. View full text Figures at a glance * Figure 1: Generation and characterization of ChR2-expressing cardiomyocytes in vitro. (,) Immunostainings showing ESCs expressing the classical stem cell marker Oct4 in the nucleus (, red) as well as cardiomyocytes in embryoid bodies expressing α-actinin (, red) overlaid with fluorescence images of the native EYFP signal (green; membrane bound). Nuclei are shown in blue. Scale bar, 20 μm. () Frequency analysis of spontaneously beating embryoid bodies upon pulsed light stimulation at 100 beats per minute (bpm) (blue dashed line; 20 ms, 0.6 mW mm−2; ) and continuous light stimulation (blue bar; 30 s, 0.6 mW mm−2; ). Shown are representative examples of six experiments. () Membrane potential recording of a ChR2-EYFP–expressing cardiomyocyte upon stimulation with blue light (20 mW mm−2) for durations indicated by blue bars. A representative example of five experiments is shown. () Cytosolic Ca2+ imaging traces directly after termination of ChR2 stimulation (47 mW mm−2) for 20 ms (black), 200 ms, 500 ms or 800 ms (red) (durations are indicated by bars ! below the traces). Shown is a representative example of nine experiments. * Figure 2: Expression and function of ChR2 in ventricular cardiomyocytes from CAG-ChR2 mice. () Fluorescence image of the native membrane-bound ChR2-EYFP signal (green) overlaid with α-actinin immunostaining (red) in cardiomyocytes of the ventricle and colocalization with the t-tubulus system (inset). Nuclei are shown in blue. Scale bars, 20 μm. () Inward currents evoked at a holding potential of −40 mV by light stimulation at 0.09, 0.18, 0.45 and 1.75 mW mm−2 (from top to bottom). Monoexponential fit to measure the time constant of decay is shown in red. pA, picoampere; pF, picofarad. () Relationship between light intensity and peak or steady-state currents (holding potential was −40 mV). Error bars, s.d. (n = 7 cells). () Current (I) and voltage (membrane potenial) relationship of light-induced steady-state currents. Error bars, s.d. (n = 7 cells). () Repetitive action potential generation by 1-ms light pulses (blue bars) of 0.91 mW mm−2. () Stimulation-response diagram with percentages of cardiomyocytes showing a 1:1 light pulse to action potential coup! ling depending on the light intensity and duration of light pulses (data for a minimum of 13 cells were used to generate each data point). () Action potential generation by light pulses (10 ms; light blue line) of different intensities in a representative single cell (), and analysis of the delay to action potential threshold and peak (; error bars, s.d.; n = 5 cells). * Figure 3: Light-induced stimulation of ChR2-expressing hearts in vivo. () Pulsed light stimulation (illumination area, 38 mm2; 10 ms, 2.8 mW mm−2, blue) of the right atrium () or three indicated ventricular areas () and parallel recordings of the electrocardiogram (black). () Strength-duration curve for threshold of 1:1 coupling in atria (n = 5) and ventricles (n = 4) at 450 beats per min (illumination area, 2.0 mm2). () Electrocardiogram recording (black traces) during pulsed (20 ms) light stimulations of the left ventricle in an area of 0.8 mm2 (1.1 mW mm−2, top) or 0.05 mm2 (7.2 mW mm−2, bottom). () Continuous light stimulation (blue bar, 3.9 mW mm−2) of a left ventricular area (0.2 mm2) and recording of the electrocardiogram (black). Author information * Author information * Supplementary information Affiliations * Institute of Physiology I, Life and Brain Center, University of Bonn, Bonn, Germany. * Tobias Bruegmann, * Daniela Malan, * Michael Hesse, * Thomas Beiert, * Christopher J Fuegemann, * Bernd K Fleischmann & * Philipp Sasse Contributions T. Bruegmann, B.K.F. and P.S. designed the study and prepared the manuscript. T. Bruegmann, D.M., T. Beiert and P.S. performed experiments and analyzed data. C.J.F. and M.H. generated the transgenic mice. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Philipp Sasse (philipp.sasse@uni-bonn.de) Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (3M) Video of a spontaneously beating embryoid body with ChR2-expressing cardiomyocytes. Light stimulation (100 ms, 7.1 mW mm−2) is indicated by a blue box in right upper corner. Recording and display frame rate is 20 frames s−1. PDF files * Supplementary Text and Figures (972K) Supplementary Figures 1–6, Supplementary Note 1 Additional data
• Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples
  - Nat Meth 7(11):913-915 (2010)
  Nature Methods | Brief Communication Chemical synthesis of the mouse mitochondrial genome * Daniel G Gibson1dgibson@jcvi.org Search for this author in: * NPG journals * PubMed * Google Scholar * Hamilton O Smith2 Search for this author in: * NPG journals * PubMed * Google Scholar * Clyde A Hutchison III2 Search for this author in: * NPG journals * PubMed * Google Scholar * J Craig Venter1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Chuck Merryman1cmerryman@jcvi.org Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 7 ,Pages:901–903Year published:(2010)DOI:doi:10.1038/nmeth.1515Received26 May 2010Accepted08 September 2010Published online10 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.3-kilobase mouse mitochondrial genome from 600 overlapping 60-mers. View full text Figures at a glance * Figure 1: Schematic demonstrating assembly of the synthetic mouse mitochondrial genome. The 60-base oligos (red lines) were assembled in groups of eight into seventy-five 284-bp cassettes (red arrows). These were joined in sets of five to produce fifteen 1.2-kb assemblies (blue arrows) and then again in sets of five to produce three 5.6-kb assemblies (green arrows). These three fragments were recombined into a complete 16.5-kb genome (orange arrow), which includes a 221-bp repeat. NotI restriction sites (N, black lines) were designed to release the 284-bp cassettes from the pUC19 vector (gray lines). * Figure 2: Summary of results for obtaining the 75 sequence-verified first-stage assemblies. (,) The 75 reactions of eight oligos each were pooled and transformed into E. coli () or individually transformed (). The number of correct clones obtained for each segment (1–75) is shown. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions GenBank * NC_005089 Author information * Accession codes * Author information * Supplementary information Affiliations * The J. Craig Venter Institute, Rockville, Maryland, USA. * Daniel G Gibson & * Chuck Merryman * The J. Craig Venter Institute, San Diego, California, USA. * Hamilton O Smith & * Clyde A Hutchison III Contributions D.G.G. and C.M. designed research, performed research, analyzed data and wrote the paper. H.O.S., C.A.H. III and J.C.V. designed research and analyzed data. Competing financial interests J.C.V. is chief executive officer and co-chief scientific officer of Synthetic Genomics, Inc (SGI). H.O.S. is co-chief scientific officer and a member of the board of directors of SGI. C.A.H. III is chairman of the SGI Scientific Advisory Board. J.C.V., H.O.S. and C.A.H. III hold SGI stock. Corresponding authors Correspondence to: * Daniel G Gibson (dgibson@jcvi.org) or * Chuck Merryman (cmerryman@jcvi.org) Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–6, Supplementary Tables 1–15, Supplementary Note 1 Additional data
• Clonal tracking of hESCs reveals differential contribution to functional assays
  - Nat Meth 7(11):917-922 (2010)
  Nature Methods | Brief Communication Efficient CNS gene delivery by intravenous injection * Jean-Pierre Louboutin1jplouboutin@hotmail.com Search for this author in: * NPG journals * PubMed * Google Scholar * Alena A Chekmasova1 Search for this author in: * NPG journals * PubMed * Google Scholar * Elena Marusich1 Search for this author in: * NPG journals * PubMed * Google Scholar * J Roy Chowdhury2 Search for this author in: * NPG journals * PubMed * Google Scholar * David S Strayer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:905–907Year published:(2010)DOI:doi:10.1038/nmeth.1518Received19 May 2010Accepted13 September 2010Published online17 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We administered recombinant SV40-derived viral vectors (rSV40s) intravenously to mice with or without prior intraperitoneal injection of mannitol to deliver transgenes to the central nervous system (CNS). We detected transgene-expressing cells (mainly neurons) most prominently in the cortex and spinal cord; prior intraperitoneal mannitol injection increased CNS gene delivery tenfold. Intravenous injection of rSV40s, particularly with mannitol pretreatment, resulted in extensive expression of multiple transgenes throughout the CNS. View full text Figures at a glance * Figure 1: AU1 expression from a transgene after intravenous injection of recombinant SV40 virus. () AU1 immunostaining in coronal cryostat sections of brains of mice injected intravenously with SV(RevM10.AU1) or control SV(BUGT), with or without prior intraperitoneal administration of mannitol. The motor cortex is shown. Scale bar corresponds to 60 μm in the top three images and 30 μm in the bottom image. (–) Percentages of AU1-positive cells in the indicated brain regions after indicated treatments (), with different doses of intravenously injected vector with prior mannitol treatment () and after different numbers of injections of intravenous vector with prior mannitol treatment of mice (). Error bars, ± s.e.m. (n = 5 mice for each treatment group). **P < 0.001. In and , unless indicated on the plots, P < 0.05 between the different doses () and between the numbers of injections (). * Figure 2: Transgene-expressing cells were mostly neurons. () Cryostat sections of mice brains (motor cortex is shown) immunostained for AU1 and stained with NeuroTrace one month after intravenous injection with the indicated vectors with or without prior intraperitoneal mannitol injection. Arrows point to neurons expressing AU1. Insets show higher-magnification images. Scale bar corresponds to 60 μm in large images and 15 μm in insets. () Higher magnification of another field of the cryostat section shown in the bottom image in . (–) Representative images of sections from mice injected with SV(RevM10.AU1) and prior intraperitoneal mannitol administration, immunostained for NeuN and AU1 (), Iba1 and AU1 (), and GFAP and AU1 (). Five mice were examined in each treatment group. Data are representative of three experiments. Scale bars, 10 μm (), 20 μm (), 25 μm () and 40 μm (). * Figure 3: Distribution of AU1 expression in the brain. () Image of a cryostat section through the cingulate cortex of mice one month after injection of viral vectors with prior mannitol treatment. Sections were labeled with DAPI (left) and immunostained for AU1 (right). M1, primary motor cortex; M2, secondary motor cortex; CG, cingulate cortex; PrL, prelimbic cortex; IL, infralimbic cortex; Pir, piriform cortex; LO, lateral orbital cortex; and AI, agranular insular cortex. () Images showing transgene expression (AU1, green) in the indicated brain areas overlaid with NeuroTrace staining (red). DG, dentate gyrus; Mol, molecular layer of the dentate gyrus; ML, molecular layer of the cerebellum; PCL, Purkinje cell layer; and GCL, granule cell layer. () Percentages of total cells (DAPI-stained) and of neurons (NeuroTrace-stained) expressing AU1 in the indicated areas of the brain. Error bars, s.e.m. (n = 5 mice in each treatment group). Scale bars, 240 μm () and 30 μm (). * Figure 4: Expression of transgene-encoded AU1 in the spinal cord. () Cryostat section through upper thoracic spinal cord of mice one month after injection of viral vectors with prior mannitol treatment. Section was labeled with DAPI (left) and immunostained for AU1 (right). DH, dorsal horn; VH, ventral horn; and CC, central canal. Scale bar corresponds to 240 μm in large images and 120 μm in insets. () Images of AU1-expressing cells in the upper thoracic cord under the indicated conditions. Scale bar, 60 μm. () AU1-immunolabeled and NeuroTrace-stained sections through the thoracic and lumbar spinal cord. The shown scale bar corresponds to 60 μm for the top images and 45 μm for the middle and bottom images. Some AU1-expressing neurons had the morphology and size of sensory or motor neurons (arrowheads), others of interneurons (arrows). Data are representative of three experiments with five mice in each group. (–) Percentages of total cells (DAPI-stained) and of neurons (NeuroTrace (NT)-stained) expressing the transgenes SOD1 (), GPx1! () and Flag () in different areas of the brain. Error bars, s.e.m. (n = 5 mice in each group). Author information * Author information * Supplementary information Affiliations * Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. * Jean-Pierre Louboutin, * Alena A Chekmasova, * Elena Marusich & * David S Strayer * Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA. * J Roy Chowdhury Contributions J.-P.L. designed and performed experiments, processed and analyzed data, and wrote the paper. A.A.C. performed experiments and designed vectors. E.M. designed vectors. J.R.C. analyzed data and provided collaboration and grant support. D.S.S. coordinated the project and helped to write the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jean-Pierre Louboutin (jplouboutin@hotmail.com) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (18M) Supplementary Figures 1–4 Additional data
• Trans-SILAC: sorting out the non-cell-autonomous proteome
  - Nat Meth 7(11):923-927 (2010)
  Nature Methods | Brief Communication De novo assembly and analysis of RNA-seq data * Gordon Robertson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline Schein1 Search for this author in: * NPG journals * PubMed * Google Scholar * Readman Chiu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Corbett1 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew Field1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shaun D Jackman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Karen Mungall1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sam Lee2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hisanaga Mark Okada1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jenny Q Qian1 Search for this author in: * NPG journals * PubMed * Google Scholar * Malachi Griffith1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony Raymond1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nina Thiessen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Timothee Cezard1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Yaron S Butterfield1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Newsome1 Search for this author in: * NPG journals * PubMed * Google Scholar * Simon K Chan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rong She1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Varhol1 Search for this author in: * NPG journals * PubMed * Google Scholar * Baljit Kamoh1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna-Liisa Prabhu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Angela Tam1 Search for this author in: * NPG journals * PubMed * Google Scholar * YongJun Zhao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard A Moore1 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Hirst1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marco A Marra1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Steven J M Jones1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pamela A Hoodless2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Inanc Birol1ibirol@bcgsc.ca Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:909–912Year published:(2010)DOI:doi:10.1038/nmeth.1517Received18 June 2010Accepted13 September 2010Published online10 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We describe Trans-ABySS, a de novo short-read transcriptome assembly and analysis pipeline that addresses variation in local read densities by assembling read substrings with varying stringencies and then merging the resulting contigs before analysis. Analyzing 7.4 gigabases of 50-base-pair paired-end Illumina reads from an adult mouse liver poly(A) RNA library, we identified known, new and alternative structures in expressed transcripts, and achieved high sensitivity and specificity relative to reference-based assembly methods. View full text Figures at a glance * Figure 1: Representation of transcripts and contigs across assemblies. () Distributions of normalized mean transcript coverage from read-to-genome alignments and assembly k-mer length, for unmerged contigs from assemblies for every other k value between 26 and 50 bp (left to right, with the curve for each k value in a different color). Results are shown for all Ensembl v54 mouse transcripts (gray), and for contigs that cover at least 80% of the transcript's total exon length. Inset, distribution of transcripts for each each k value. () Result of contig merging for main contigs from assemblies with k values of 26–50 bp. 'Buried' contigs are those with an exact sequence match within a longer 'parent' contig from another assembly. 'Untouched' contigs have no sequence match in another assembly. * Figure 2: Performance comparisons between ABySS and reference-based transcriptome analysis tools. () Number of Ensembl v54 transcripts reconstructed to 80% by ABySS, Cufflinks and Scripture by a single contig as a function of mean read coverage, C. () Intron-level sensitivity and specificity of Trans-ABySS, Cufflinks, Scripture and TopHat, relative to all 298,893 nonredundant introns from UCSC genome browser, RefSeq, Ensembl and AceView transcript models. Tophat split-read alignments are shown as a curve for intron support levels ranging from 1 to 100 reads. Alignments of Trans-ABySS de novo contigs, and reference-based Tophat Cufflinks and Scripture generated contigs, are represented as points, each of which represents a set of contigs. Two points are shown for ABySS: non-reference-based filtering with (light blue) or without (dark blue) contig-level splice-site filtering. Author information * Author information * Supplementary information Affiliations * Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada. * Gordon Robertson, * Jacqueline Schein, * Readman Chiu, * Richard Corbett, * Matthew Field, * Shaun D Jackman, * Karen Mungall, * Hisanaga Mark Okada, * Jenny Q Qian, * Malachi Griffith, * Anthony Raymond, * Nina Thiessen, * Timothee Cezard, * Yaron S Butterfield, * Richard Newsome, * Simon K Chan, * Rong She, * Richard Varhol, * Baljit Kamoh, * Anna-Liisa Prabhu, * Angela Tam, * YongJun Zhao, * Richard A Moore, * Martin Hirst, * Marco A Marra, * Steven J M Jones & * Inanc Birol * Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada. * Sam Lee & * Pamela A Hoodless * Department of Medical Genetics, University of British Columbia, Vancouver, Canada. * Marco A Marra, * Steven J M Jones & * Pamela A Hoodless * Present address: University of Edinburgh, Edinburgh, UK. * Timothee Cezard Contributions G.R. and J.S. wrote the paper. J.S., G.R. and K.M. reviewed predictions and recommended analysis methods. G.R. coordinated analysis and validation. B.K., A.-L.P. and A.T. constructed libraries under the supervision of YJ.Z. S.L. generated biological material and performed RT-PCR validation. R.A.M. supervised sequencing activities. Y.S.B., T.C., R. Corbett, R. Chiu, M.F., M.G., J.Q.Q., R.N., H.M.O., N.T., R.V., S.K.C. and R.S. developed analysis methods and code and performed analyses. R. Corbett and R. Chiu performed comparisons with reference-based methods. S.D.J. develops and maintains ABySS and generated the ABySS assemblies. A.R. contributed algorithms and code for ABySS. M.A.M., S.J.M.J. and P.A.H. directed research. S.J.M.J. suggested analysis methods. YJ.Z. and M.H. developed the WTSS protocol. J.S. supervised activities. P.A.H. supervised validation. I.B. developed ABySS and Trans-ABySS and directed bioinformatics work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Inanc Birol (ibirol@bcgsc.ca) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–21, Supplementary Tables 1–4, Supplementary Note Additional data
• Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors
  - Nat Meth 7(11):929-935 (2010)
  Nature Methods | Brief Communication Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples * Isaäc J Nijman1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michal Mokry1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ruben van Boxtel1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pim Toonen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ewart de Bruijn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Edwin Cuppen1, 2e.cuppen@hubrecht.eu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 7 ,Pages:913–915Year published:(2010)DOI:doi:10.1038/nmeth.1516Received02 June 2010Accepted06 September 2010Published online17 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Targeted genomic enrichment followed by next-generation DNA sequencing has dramatically increased efficiency of mutation-discovery efforts. We describe a protocol for genomic enrichment of pooled barcoded samples in a single assay that increases experimental flexibility and efficiency. We screened 770 genes (1.4 megabases) in thirty N-ethyl-N-nitrosourea (ENU)-mutagenized rats and identified known variants at >96% sensitivity as well as new mutations at a false positive rate < 1 in 8 megabases. View full text Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE22024 Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Isaäc J Nijman, * Michal Mokry & * Ruben van Boxtel Affiliations * Hubrecht Institute, Developmental Biology and Stem Cell Research, Royal Netherlands Academy of Arts and Sciences and the University Medical Center Utrecht, Utrecht, The Netherlands. * Isaäc J Nijman, * Michal Mokry, * Ruben van Boxtel, * Pim Toonen, * Ewart de Bruijn & * Edwin Cuppen * Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands. * Edwin Cuppen Contributions R.v.B., I.J.N., M.M. and E.C. designed the experiments. R.v.B. and P.T. performed rat ENU mutagenesis. M.M. performed library preparation and genomic enrichments. E.d.B. performed SOLiD sequencing. I.J.N. analyzed the data. R.v.B. performed capillary sequencing reconfirmation experiments. I.J.N. and E.C. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Edwin Cuppen (e.cuppen@hubrecht.eu) Supplementary information * Accession codes * Author information * Supplementary information Excel files * Supplementary Table 1 (2M) List of rat genes targeted in the next-generation reverse genetic. * Supplementary Table 6 (116K) Complete list of polymorphic positions identified in the next-generation reverse genetics screen. Zip files * Supplementary Software (4k) Custom SNP filtering PERL script. PDF files * Supplementary Text and Figures (564K) Supplementary Figures 1–2 and Supplementary Tables 2–5 Additional data

Hot off the presses! Jul 01 Nat Meth

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Latest Articles Include:

• Bioclouds
  - Nat Meth 7(7):481 (2010)
  Understanding how cloud computing can serve the scientific community is a research question in its own right and one that researchers—with the help of funders—should address.
• The author file: Vivek Jayaraman
  - Nat Meth 7(7):483 (2010)
  Fly brains light the way for neurobehavioral circuits.
• Primer-initiated sequence synthesis to detect and assemble structural variants
  Massouras A Hens K Gubelmann C Uplekar S Decouttere F Rougemont J Cole ST Deplancke B - Nat Meth 7(7):485-486 (2010)
  To the Editor: Structural variants constitute the largest portion of nucleotide variation in genomes, yet their comprehensive characterization based on high-throughput sequencing technologies is still challenging. Here we present primer-initiated sequence synthesis for genomes (PrInSeS-G), a software tool that detects and assembles sequence variants (1 base pair (bp) to |[sim]|10 kilobases (kb)) from single- or paired-end short reads.
• Nano-imaging of membrane topography affects interpretations in cell biology
  Jalink K van Rheenen J - Nat Meth 7(7):486 (2010)
  To the Editor: In their correspondence in Nature Methods, Adler et al. claim to present the first high-resolution topography of plasma membranes in living cells. Using hopping-probe ion conductance microscopy (HPICM), they show that the plasma membrane contains many subresolution membrane folds and point out serious implications for single-particle tracking studies.
• Advent of synthetic life
  - Nat Meth 7(7):487 (2010)
  Researchers synthesized the first functional synthetic bacterial genome; repeating this feat with different bacteria will require further methodological development.
• A toolset for the proficient geneticist
  - Nat Meth 7(7):488-489 (2010)
  New strategies expand the genetic toolkit for transgene expression, lineage tracing and mosaic analysis of gene function in flies and mammalian cells.
• Where do you come from?
  - Nat Meth 7(7):488-489 (2010)
  Live-cell time-lapse imaging of somatic cells undergoing reprogramming raises interesting questions about the mechanism of the process.
• News in brief
  - Nat Meth 7(7):489 (2010)
  Genomics The splicing code, decoded For many years, researchers have attempted to define the combinatorial rules that control alternative splicing. Barash et al. now report a code, implemented in a web tool, that predicts tissue-specific alternative splicing with high accuracy.
• Robotics on the nanoscale
  - Nat Meth 7(7):490 (2010)
  Nanoscale robots can be programmed to walk a DNA origami track.
• Searching for mismatches in a vast genomic landscape
  - Nat Meth 7(7):492 (2010)
  Raw data of millions of sequences used to assemble the reference genomes of ten organisms are analyzed in search of mismatches indicative of editing events. Findings include candidate sites for in vivo DNA and RNA editing, and a common sequencing error.
• Faster, sharper electron microscopy
  - Nat Meth 7(7):494 (2010)
  A technique combining laser and electron pulses is used to achieve nanometer and femtosecond resolution in biological imaging.
• Next-generation sequencing: adjusting to data overload
  Baker M - Nat Meth 7(7):495-499 (2010)
  To keep pace with accelerating sequencing machines, genomics researchers clean house and move toward the cloud.
• Illuminating eukaryotic transcription start sites
  Stamatoyannopoulos JA - Nat Meth 7(7):501-503 (2010)
  The initiation of transcription by RNA polymerase II is a defining event in eukaryotic gene regulation and represents the convergence of multiple cis- and trans-acting regulatory mechanisms. Genes are conventionally held to have a single canonical promoter, which in some cases may be accompanied by alternative promoters located at some distance (typically >500 base pairs (bp)) from the canonical promoter and one another.
• Watching the fly brain in action
  Clark DA de Vries SE Clandinin TR - Nat Meth 7(7):505-506 (2010)
  How does the brain transform sensory input into behavioral output? A strong approach to answering this question is to monitor activity from neurons in awake, behaving animals responding to sensory stimuli. Most of these experiments have been done in primates and rodents, and have identified brain regions and neural pathways involved in perception, sensorimotor integration and decision-making, among others.
• The inside view on plant growth
  Spalding EP - Nat Meth 7(7):506-507 (2010)
  Plant growth is a fine example of how a system in flux, such as a river, nonetheless presents a constant structure. From one day to the next, a plant meristem looks more or less the same despite being the site of prodigious cell division.
• Proteome-wide analysis of protein carboxy termini: C terminomics
  Schilling O Barré O Huesgen PF Overall CM - Nat Meth 7(7):508-511 (2010)
  As proteome-wide C-terminal sequence analysis has been largely intractable, we developed a polymer-based enrichment approach to profile protein C-terminal peptides by mass spectrometry and identified hundreds of C-terminal peptides in the Escherichia coli proteome. We isotopically labeled GluC protease–digested and undigested samples and identified GluC substrates and their cleavage sites by quantification of neo–C-terminal peptides. Our method thus enables global annotation of protein C-terminal posttranslational modifications, including proteolytic truncations.
• Complementary positional proteomics for screening substrates of endo- and exoproteases
  Van Damme P Staes A Bronsoms S Helsens K Colaert N Timmerman E Aviles FX Vandekerckhove J Gevaert K - Nat Meth 7(7):512-515 (2010)
  We describe a positional proteomics approach to simultaneously analyze N- and C-terminal peptides and used it to screen for human protein substrates of granzyme B and carboxypeptidase A4 in human cell lysates. This approach allowed comprehensive proteome studies, and we report the identification of 965 database-annotated protein C termini, 334 neo–C termini resulting from granzyme B processing and 16 neo–C termini resulting from carboxypeptidase A4 processing.
• Simultaneous intracellular chloride and pH measurements using a GFP-based sensor
  Arosio D Ricci F Marchetti L Gualdani R Albertazzi L Beltram F - Nat Meth 7(7):516-518 (2010)
  Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concentration in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.
• Estimating prion concentration in fluids and tissues by quantitative PMCA
  Chen B Morales R Barria MA Soto C - Nat Meth 7(7):519-520 (2010)
  Prions, the proteinaceous infectious agent responsible for prion diseases, can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA) technology. Here we describe a quantitative PMCA procedure to calculate the concentration of very low levels of prions in biological samples. Using this procedure, we determined the quantities of misfolded prion protein (PrPSc) in brain, spleen, blood and urine of scrapie-affected hamsters.
• A paired-end sequencing strategy to map the complex landscape of transcription initiation
  - Nat Meth 7(7):521-527 (2010)
  Recent studies using high-throughput sequencing protocols have uncovered the complexity of mammalian transcription by RNA polymerase II, helping to define several initiation patterns in which transcription start sites (TSSs) cluster in both narrow and broad genomic windows. Here we describe a paired-end sequencing strategy, which enables more robust mapping and characterization of capped transcripts. We used this strategy to explore the transcription initiation landscape in the Drosophila melanogaster embryo. Extending the previous findings in mammals, we found that fly promoters exhibited distinct initiation patterns, which were linked to specific promoter sequence motifs. Furthermore, we identified many 5′ capped transcripts originating from coding exons; our analyses support that they are unlikely the result of alternative TSSs, but rather the product of post-transcriptional modifications. We demonstrated paired-end TSS analysis to be a powerful method to uncover ! the transcriptional complexity of eukaryotic genomes.
• Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan
  Plessy C Bertin N Takahashi H Simone R Salimullah M Lassmann T Vitezic M Severin J Olivarius S Lazarevic D Hornig N Orlando V Bell I Gao H Dumais J Kapranov P Wang H Davis CA Gingeras TR Kawai J Daub CO Hayashizaki Y Gustincich S Carninci P - Nat Meth 7(7):528-534 (2010)
  Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5′ ends that vary in size. Methods to identify the 5′ ends of transcripts will facilitate the discovery of new promoters and 5′ ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano–cap analysis of gene expression (nanoCAGE), a method that captures the 5′ ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5′ ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome.
• Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior
  Seelig JD Chiappe ME Lott GK Dutta A Osborne JE Reiser MB Jayaraman V - Nat Meth 7(7):535-540 (2010)
  Drosophila melanogaster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball's motion is tracked at high resolution and can be treated as a proxy for the fly's own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in horizontal-system neurons correlated with robust optomotor behavior during walking. Our technique allows both behavior and physiology in identified neurons to be monitored in a genetic model organism with an extensive repertoire of walking behaviors.
• cnvHap: an integrative population and haplotype–based multiplatform model of SNPs and CNVs
  Coin LJ Asher JE Walters RG El-Sayed Moustafa JS de Smith AJ Sladek R Balding DJ Froguel P Blakemore AI - Nat Meth 7(7):541-546 (2010)
  Although genome-wide association studies have uncovered single-nucleotide polymorphisms (SNPs) associated with complex disease, these variants account for a small portion of heritability. Some contribution to this 'missing heritability' may come from copy-number variants (CNVs), in particular rare CNVs; but assessment of this contribution remains challenging because of the difficulty in accurately genotyping CNVs, particularly small variants. We report a population-based approach for the identification of CNVs that integrates data from multiple samples and platforms. Our algorithm, cnvHap, jointly learns a chromosome-wide haplotype model of CNVs and cluster-based models of allele intensity at each probe. Using data for 50 French individuals assayed on four separate platforms, we found that cnvHap correctly detected at least 14% more deleted and 50% more amplified genotypes than PennCNV or QuantiSNP, with an 82% and 115% improvement for aberrations containing <10 probes! . Combining data from multiple platforms additionally improved sensitivity.
• Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution
  Fernandez R Das P Mirabet V Moscardi E Traas J Verdeil JL Malandain G Godin C - Nat Meth 7(7):547-553 (2010)
  Quantitative information on growing organs is required to better understand morphogenesis in both plants and animals. However, detailed analyses of growth patterns at cellular resolution have remained elusive. We developed an approach, multiangle image acquisition, three-dimensional reconstruction and cell segmentation–automated lineage tracking (MARS-ALT), in which we imaged whole organs from multiple angles, computationally merged and segmented these images to provide accurate cell identification in three dimensions and automatically tracked cell lineages through multiple rounds of cell division during development. Using these methods, we quantitatively analyzed Arabidopsis thaliana flower development at cell resolution, which revealed differential growth patterns of key regions during early stages of floral morphogenesis. Lastly, using rice roots, we demonstrated that this approach is both generic and scalable.
• Enhanced neuronal RNAi in C. elegans using SID-1
  Calixto A Chelur D Topalidou I Chen X Chalfie M - Nat Meth 7(7):554-559 (2010)
  We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons. This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. Mutations in the lin-15b and lin-35 genes enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes. Neuronal expression of sid-1 decreased nonneuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(–) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation.
• Probabilistic density maps to study global endomembrane organization
  Schauer K Duong T Bleakley K Bardin S Bornens M Goud B - Nat Meth 7(7):560-566 (2010)
  We developed a computational imaging approach that describes the three-dimensional spatial organization of endomembranes from micromanipulation-normalized mammalian cells with probabilistic density maps. Applied to several well-known marker proteins, this approach revealed the average steady-state organization of early endosomes, multivesicular bodies or lysosomes, endoplasmic reticulum exit sites, the Golgi apparatus and Golgi-derived transport carriers in crossbow-shaped cells. The steady-state organization of each tested endomembranous population was well-defined, unique and in some cases depended on the cellular adhesion geometry. Density maps of all endomembrane populations became stable when pooling several tens of cells only and were reproducible in independent experiments, allowing construction of a standardized cell model. We detected subtle changes in steady-state organization induced by disruption of the cellular cytoskeleton, with statistical significance o! bserved for just 20 cells. Thus, combining micropatterning with construction of endomembrane density maps allows the systematic study of intracellular trafficking determinants.
• Corrigendum: A photoconvertible reporter of the ubiquitin-proteasome system in vivo
  - Nat Meth 7(7):567 (2010)
  Introduction Nat. Methods 7, 473–478 (2010); published online 9 May 2010; corrected after print 17 June 2010. ADVERTISEMENT In the version of this paper originally published, a reference to previous work on the use of Dendra2 as a reporter for protein stability in cultured cells should have been included (ref. 35). The error has been corrected in the PDF and HTML versions of the article.