Latest Articles Include:
- Building a better mouse test
- Nat Methods 8(9):697 (2011)
Nature Methods | Editorial Building a better mouse test Journal name:Nature MethodsVolume: 8,Page:697Year published:(2011)DOI:doi:10.1038/nmeth.1698Published online30 August 2011 As more mouse models are produced, researchers studying neuropsychiatric diseases will need better ways to evaluate them and more realistic assessment of the results. View full text Additional data - The author file: Greg Davis
- Nat Methods 8(9):699 (2011)
Nature Methods | This Month The author file: Greg Davis * Monya BakerJournal name:Nature MethodsVolume: 8,Page:699Year published:(2011)DOI:doi:10.1038/nmeth.1677Published online30 August 2011 Gene editing becomes faster and more facile. View full text Additional data - Points of view: Arrows
- Nat Methods 8(9):701 (2011)
Article preview View full access options Nature Methods | This Month Points of view: Arrows * Bang Wong1Journal name:Nature MethodsVolume: 8,Page:701Year published:(2011)DOI:doi:10.1038/nmeth.1676Published online30 August 2011 Arrows are one of the most commonly used graphical devices in scientific figures. In the July 2011 issue of Nature Methods alone I counted nearly 300 instances of arrows; more than half of the figures contain them. Given the widespread use of arrows, it is worthwhile to take a closer look at this privileged class of diagrammatic form and how we might benefit from its use. Arrows can be highly efficient instruments of visual communication because they guide us through complex information. Typically arrows are used to point out relevant features, order sequences of events, connect elements and indicate motion. In molecular biology, there are several conventions involving the arrow that are generally recognized (Fig. 1a). For example, an arrow with a right-angle line segment is understood as a transcription start site or promoter, and a short arrow placed parallel to a line usually indicates a PCR primer. Several other common conventions are shown in Figure 1a. But authors also use arrows to illustrate other concepts, some of which are easily understood, whereas others may be less intuitive. Figure 1: Arrows in scientific diagrams. () Well-understood conventions in molecular biology indicated by arrows. () Arrows are defined loosely by their geometric shapes and more definitely in context. () A diagram with 19 arrows used as leaders, to indicate reagent flow and to show mechanical movement. Reprinted from Nature Methods3. * Full size image (118 KB) * Figures index * Next figure In his thorough survey of diagrams Robert E. Horn documented hundreds of meanings for arrows, including metaphorical uses such as increases and decreases1. An arrow's geometric shape can tell us something about its purpose (Fig. 1b), but its meaning is refined and interpreted in context. Arrows are a special class of symbols that can have multiple meanings even when used in the same figure. A recently published figure has many arrows that are used to label parts, convey mechanical motion and show reagent flow (Fig. 1c). Figures at a glance * Figure 1: Arrows in scientific diagrams. () Well-understood conventions in molecular biology indicated by arrows. () Arrows are defined loosely by their geometric shapes and more definitely in context. () A diagram with 19 arrows used as leaders, to indicate reagent flow and to show mechanical movement. Reprinted from Nature Methods3. * Figure 2: Functional qualities of arrows. () The use of arrows versus lines as connectors suggests a certain functional relationship. () Alternatives to arrows as leader lines. () Reasonably sized arrows clearly indicate direction without being a distraction. () Trapped whitespace in 'open' arrowheads creates optical illusions that can attract unwanted attention. () Whitespace at the ends of the arrows makes them easy to discriminate from other content. () Orienting arrows in similar directions creates natural visual flow. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Methods for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Bang Wong is the creative director of the Broad Institute of the Massachusetts Institute of Technology & Harvard and an adjunct assistant professor in the Department of Art as Applied to Medicine at The Johns Hopkins University School of Medicine. Author Details * Bang Wong Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - TopFIND, a knowledgebase linking protein termini with function
- Nat Methods 8(9):703-704 (2011)
Nature Methods | Correspondence TopFIND, a knowledgebase linking protein termini with function * Philipp F Lange1, 2, 3 * Christopher M Overall1, 2, 3 * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:703–704Year published:(2011)DOI:doi:10.1038/nmeth.1669Published online07 August 2011Corrected online12 August 2011 To the Editor: We present 'termini-oriented protein function inferred database' (TopFIND; http://clipserve.clip.ubc.ca/topfind/), a knowledgebase providing integrated information on translated protein N and C termini, their formation by proteolytic processing and their amino acid modifications. TopFIND is open to data contribution from users. View full text Subject terms: * Bioinformatics * Cell Biology * Proteomics Change history * Change history * 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 Corrigendum 12 August 2011In the version of this article initially published online, the Figure 1c legend and associated text describing Figure 1c was incorrect. The panels in this figure show N-terminal amino acid occurrence after methonine removal, not all co- and post-translationally processed chains. The error has been corrected for the print, PDF and HTML versions of this article. Author information * Change history * Author information * Supplementary information Affiliations * Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada. * Philipp F Lange & * Christopher M Overall * Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada. * Philipp F Lange & * Christopher M Overall * Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada. * Philipp F Lange & * Christopher M Overall Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Christopher M Overall Author Details * Philipp F Lange Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher M Overall Contact Christopher M Overall Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Change history * Author information * Supplementary information Excel files * Supplementary Table 2 (475K) Termini inferred from protease cleavage. PDF files * Supplementary Text and Figures (3.2M) Supplementary Figures 1–6, Supplementary Table 1, Supplementary Discussion, Supplementary Methods Additional data - Making sense of chromatin states
- Nat Methods 8(9):717-722 (2011)
Nature Methods | Technology Feature Making sense of chromatin states * Monya Baker1Journal name:Nature MethodsVolume: 8,Pages:717–722Year published:(2011)DOI:doi:10.1038/nmeth.1673Published online30 August 2011 Researchers find new pieces in the puzzle of genome regulation. 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 Author Details * Monya Baker Contact Monya Baker Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - On target? Tracing zinc-finger-nuclease specificity
- Nat Methods 8(9):725-726 (2011)
Article preview View full access options Nature Methods | News and Views On target? Tracing zinc-finger-nuclease specificity * Claudio Mussolino1 * Toni Cathomen1 * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:725–726Year published:(2011)DOI:doi:10.1038/nmeth.1680Published online30 August 2011 In two independent studies, researchers experimentally test the cleavage specificity of zinc-finger nucleases across the genome. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Methods for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Claudio Mussolino and Toni Cathomen are at the Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Toni Cathomen Author Details * Claudio Mussolino Search for this author in: * NPG journals * PubMed * Google Scholar * Toni Cathomen Contact Toni Cathomen Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Beyond the rainbow: new fluorescent proteins brighten the infrared scene
- Nat Methods 8(9):726-728 (2011)
Article preview View full access options Nature Methods | News and Views Beyond the rainbow: new fluorescent proteins brighten the infrared scene * Michael Z Lin1Journal name:Nature MethodsVolume: 8,Pages:726–728Year published:(2011)DOI:doi:10.1038/nmeth.1678Published online30 August 2011 Two fluorescent proteins that emit in the far-red and infrared range for imaging applications in cells and in vivo are described. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Methods for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Michael Z. Lin is in the Departments of Pediatrics and Bioengineering, Stanford University, Stanford, California, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Michael Z Lin Author Details * Michael Z Lin Contact Michael Z Lin Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - 'MiMICing' genomic flexibility
- Nat Methods 8(9):728-729 (2011)
Article preview View full access options Nature Methods | News and Views 'MiMICing' genomic flexibility * Steven Russell1Journal name:Nature MethodsVolume: 8,Pages:728–729Year published:(2011)DOI:doi:10.1038/nmeth.1672Published online30 August 2011 A new collection of Minos transposon insertions will enhance the range and flexibility of genome engineering in Drosophila melanogaster. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Methods for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Steven Russell is in the Department of Genetics and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Steven Russell Author Details * Steven Russell Contact Steven Russell Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Three-dimensional biomaterials for the study of human pluripotent stem cells
- Nat Methods 8(9):731-736 (2011)
Nature Methods | Perspective Three-dimensional biomaterials for the study of human pluripotent stem cells * Thomas P Kraehenbuehl1, 2 * Robert Langer1 * Lino S Ferreira3, 4 * Affiliations * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:731–736Year published:(2011)DOI:doi:10.1038/nmeth.1671Published online30 August 2011 Abstract * Abstract * 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 The self-renewal and differentiation of human pluripotent stem cells (hPSCs) have typically been studied in flat, two-dimensional (2D) environments. In this Perspective, we argue that 3D model systems may be needed in addition, as they mimic the natural 3D tissue organization more closely. We survey methods that have used 3D biomaterials for expansion of undifferentiated hPSCs, directed differentiation of hPSCs and transplantation of differentiated hPSCs in vivo. View full text Subject terms: * Stem Cells * Chemistry Figures at a glance * Figure 1: Choices and challenges in the design of 3D environments for self-renewal, differentiation, in vitro culture or transplantation of hPSCs or hPSC-derived cells. * Figure 2: Three-dimensional matrices for directed hPSC differentiation. Spontaneously differentiated hESC aggregates can be seeded into scaffolds in three different ways: isolation of specific target cells by cell sorting before seeding (i), enzymatic dissociation of aggregates followed by direct seeding without purification (ii) and direct seeding into the scaffold (iii). After cell cultivation in the biomaterials, the constructs can be either implanted directly into animal models or dissociated with subsequent selection of specific target cells for implant. Author information * Abstract * Author information Affiliations * Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Thomas P Kraehenbuehl & * Robert Langer * Hoffmann-La Roche Inc., Translational Research Sciences, Nutley, New Jersey, USA. * Thomas P Kraehenbuehl * Biocant–Center of Innovation in Biotechnology, Cantanhede, Portugal. * Lino S Ferreira * Center of Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal. * Lino S Ferreira Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Robert Langer Author Details * Thomas P Kraehenbuehl Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Langer Contact Robert Langer Search for this author in: * NPG journals * PubMed * Google Scholar * Lino S Ferreira Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes
- Nat Methods 8(9):737-743 (2011)
Nature Methods | Resource MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes * Koen J T Venken1 * Karen L Schulze1, 2 * Nele A Haelterman1 * Hongling Pan1, 2 * Yuchun He1, 2 * Martha Evans-Holm3 * Joseph W Carlson3 * Robert W Levis4 * Allan C Spradling4 * Roger A Hoskins3 * Hugo J Bellen1, 2, 5, 6 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:737–743Year published:(2011)DOI:doi:10.1038/nmeth.1662Received26 April 2011Accepted06 July 2011Published online31 July 2011 Abstract * Abstract * 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 We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaste! r toolkit. View full text Subject terms: * Genetics * Gene Expression * Genomics * Model Organisms Figures at a glance * Figure 1: The MiMIC transposon system. () MiMIC consists of two Minos inverted repeats (L and R), two inverted ΦC31 integrase attP sites (P), a gene-trap cassette consisting of a splice acceptor site (SA) followed by stop codons in all three reading frames and the EGFP coding sequence with a polyadenylation signal (pA), and the yellow+ marker. The sequence between the attP sites can be replaced via RMCE with a plasmid containing two inverted attB sites (B), resulting in two attR sites (R). () Three attB plasmids for RMCE: a correction plasmid consisting of a multiple cloning site, a gene-trap plasmid consisting of an SA fused to a downstream effector, and a protein-trap plasmid consisting of a reporter flanked by SA and splice donor site (SD). () Various MiMIC insertions in a hypothetical gene with a regulatory element (white), 5′ and 3′ untranslated regions (gray), and coding regions (black) that can be used for several applications as indicated. * Figure 2: Binary expression and lineage analysis with MiMIC insertions. () Gene-trap cassettes that incorporate genes encoding GAL4 or QF trans-activators for binary activation, and the gene encoding Flp recombinase for fate mapping. (–) Live imaging (,) and confocal microscopy analysis using an antibody to mCherry (,) of the expression domain revealed by GAL4 inserted in gogo (,) or caps (,) loci. (,) The expression of MYPT-75D revealed by GAL4 () or QF () integrated in MYPT-75D. () Live imaging of the GAL4 expression pattern revealed by GAL4 inserted in BM-40-SPARC. Scale bars, 50 μm. * Figure 3: Protein trapping with MiMIC insertions. () For each protein-trap cassette, three versions were constructed corresponding to the three intron phases (0, 1 and 2). (GGS)4, flexible peptide linker sequence encoding a Gly-Gly-Ser quadruplet tandem repeat. () Tag and multitag reporters. () A 100-kb genomic region containing CadN with all splice isoforms (CadN-RA to CadN-RL) is shown. The location of the Mi{MIC}CadNMI00393 insertion in a phase 0 coding intron is indicated. () Integration of a phase-0 EGFP-FlAsH-StrepII-3xFlag cassette (EGFP) in the indicated orientation and intron phase detected by an antibody to EGFP. L, (GGS)4 linker; R, attR; P0, splice phase 0; P1, splice phase 1; and P2, splice phase 2. Scale bars, 50 μm. * Figure 4: Expression analyses of tagged proteins. (–) Detection of different protein-trap alleles for three genes using antibodies to several encoded epitopes followed by diaminobenzidine-peroxidase (DAB) staining. Tags or multi-tag is indicated on bottom right. Expression of tagged CadN was detected during embryonic stage 15 by staining with antibodies to EGFP (), EBFP () and Dendra (). Expression of tagged Rfx was detected in stage-15 embryos with antibodies to V5 (), EBFP () and Dendra (). Expression of tagged Tutl in the ventral nerve cord of stage-15 embryos was detected with antibodies to EGFP (), mCherry () and Dendra (). (–) Fluorescence detection of different protein-trap alleles in live, stage-17 embryos expressing tagged Rhea: EGFP () mCherry () and TagRFP () signals are shown. (–) Combined localization of protein traps and endogenous proteins in stage-15 embyos for Rfx (–) and CadN (–). Shown is staining with antibodies to Rfx (), V5 () and a merged image () in an Rfx::Dendra-V5 trap line. Also shown i! s staining with antibodies to CadN (), mCherry () and a merged image () in a CadN::mCherry trap line. (–) Expression detected using protein traps: wnd mRNA detected using mRNA in situ hybridization (,); Wnd detected by staining an EGFP-FlAsH-StrepII-3xFlag trap line with an antibody to EGFP (,) and staining with an antibody to Wnd (,) during embryonic stages 11 (–) and 16 (–). Scale bars, 50 μm (–,–) and 20 μm (–). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA. * Koen J T Venken, * Karen L Schulze, * Nele A Haelterman, * Hongling Pan, * Yuchun He & * Hugo J Bellen * Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas, USA. * Karen L Schulze, * Hongling Pan, * Yuchun He & * Hugo J Bellen * Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. * Martha Evans-Holm, * Joseph W Carlson & * Roger A Hoskins * Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, Maryland, USA. * Robert W Levis & * Allan C Spradling * Department of Neuroscience, Baylor College of Medicine, Houston, Texas, USA. * Hugo J Bellen * Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, USA. * Hugo J Bellen Contributions K.J.T.V. designed the MiMIC technique and vectors, and performed all molecular biology, except for mapping of insertions. R.W.L., A.C.S., R.A.H. and H.J.B. conceived the application of MiMIC to the GDP. H.P. and Y.H. performed microinjections. K.J.T.V., H.P. and Y.H. performed fly genetics. M.E.-H. and R.A.H. mapped insertions. K.J.T.V., Y.H., M.E.-H., J.W.C., R.W.L. and R.A.H. analyzed insertion data, annotated insertions and prepared public database submissions. J.W.C. performed bioinformatic analysis. K.J.T.V., N.A.H. and H.P. verified RMCE events by PCR. K.J.T.V. and K.L.S. did staining of gene-trap events. K.L.S. and N.A.H. did staining of protein trap events. K.J.T.V., K.L.S., N.A.H. and H.J.B. analyzed expression patterns. K.J.T.V. and H.J.B. wrote the paper. R.A.H. and R.W.L. edited the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Koen J T Venken or * Hugo J Bellen Author Details * Koen J T Venken Contact Koen J T Venken Search for this author in: * NPG journals * PubMed * Google Scholar * Karen L Schulze Search for this author in: * NPG journals * PubMed * Google Scholar * Nele A Haelterman Search for this author in: * NPG journals * PubMed * Google Scholar * Hongling Pan Search for this author in: * NPG journals * PubMed * Google Scholar * Yuchun He Search for this author in: * NPG journals * PubMed * Google Scholar * Martha Evans-Holm Search for this author in: * NPG journals * PubMed * Google Scholar * Joseph W Carlson Search for this author in: * NPG journals * PubMed * Google Scholar * Robert W Levis Search for this author in: * NPG journals * PubMed * Google Scholar * Allan C Spradling Search for this author in: * NPG journals * PubMed * Google Scholar * Roger A Hoskins Search for this author in: * NPG journals * PubMed * Google Scholar * Hugo J Bellen Contact Hugo J Bellen Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (588K) Supplementary Figures 1–4, Supplementary Tables 1–6 and Supplementary Data Additional data - Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function
- Nat Methods 8(9):745-752 (2011)
Nature Methods | Resource Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function * Shengli Zhao1, 9 * Jonathan T Ting1, 2, 9 * Hisham E Atallah2 * Li Qiu1 * Jie Tan3 * Bernd Gloss1, 4 * George J Augustine5, 6, 7 * Karl Deisseroth8 * Minmin Luo3 * Ann M Graybiel2 * Guoping Feng1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:745–752Year published:(2011)DOI:doi:10.1038/nmeth.1668Received17 March 2011Accepted14 July 2011Published online07 August 2011 Abstract * Abstract * 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 Optogenetic methods have emerged as powerful tools for dissecting neural circuit connectivity, function and dysfunction. We used a bacterial artificial chromosome (BAC) transgenic strategy to express the H134R variant of channelrhodopsin-2, ChR2(H134R), under the control of cell type–specific promoter elements. We performed an extensive functional characterization of the newly established VGAT-ChR2(H134R)-EYFP, ChAT-ChR2(H134R)-EYFP, Tph2-ChR2(H134R)-EYFP and Pvalb(H134R)-ChR2-EYFP BAC transgenic mouse lines and demonstrate the utility of these lines for precisely controlling action-potential firing of GABAergic, cholinergic, serotonergic and parvalbumin-expressing neuron subsets using blue light. This resource of cell type–specific ChR2(H134R) mouse lines will facilitate the precise mapping of neuronal connectivity and the dissection of the neural basis of behavior. View full text Subject terms: * Neuroscience * Genetics * Physiology * Sensors and Probes Figures at a glance * Figure 1: Functional characterization of VGAT-ChR2(H134R)-EYFP BAC transgenic mice. () Diagram of acute coronal brain slice preparation containing the cortex and representative image from the boxed region showing placement of the optic fiber in the region of recorded cortical interneurons (asterisk) (top). Scale bar, 200 μm. High-magnification infrared differential interference contrast (IR-DIC) image and EYFP fluorescence image of a layer V interneuron (bottom). Scale bars, 20 μm. () Voltage-clamp recording with stimulation by blue laser light (blue line, 26.3 mW mm−2) (top). Plot of peak steady-state photocurrent in response to indicated light (bottom). Error bars, mean ± s.e.m.; arc lamp (constant blue light), n = 12 neurons; blue laser, n = 7 neurons. () Current clamp mode recording showing firing of a single neuron in response to blue light (blue line, top left), +400 pA current injection (top right) or prolonged 20-Hz stimulation (0.52 mW mm−2, 1 ms pulse width; bottom). () Current clamp mode recording showing action potential firing in respons! e to patterned blue laser light (2.1 mW mm−2, 1 ms pulse width) (top and middle). An expanded view of initial action potential firing at 20 Hz reveals extra spikes (asterisks; bottom). () Current clamp mode recording showing action potential firing of the same interneuron to patterned blue laser light (2.1 mW mm−2, 1 ms pulse width) (top and middle). Expanded view of firing at 80 Hz is shown (bottom). () Diagram of recording configuration to test functional effect of light-induced interneuron firing in cortical microcircuits (top). Current clamp mode recording of a layer V pyramidal neuron showing hyperpolarization in response to 5-Hz blue laser light (2.1 mW mm−2, 1 ms pulse width) (bottom). () Layer V pyramidal neuron activity upon stimulation with constant blue light (top) or 50-Hz blue laser stimulation (473 nm, 2.1 mW mm−2, 1 ms pulse width) (bottom). Action potential firing was induced by constant +150 pA direct current injection. () Voltage clamp recording of! the same layer V pyramidal neuron in the absence (no drug) an! d presence of 6,7-dinitroquinoxaline-2,3-dione (DNQX), picrotoxin (Picro) and (2S)-3-(((1S)-1-(3,4-dichlorophenyl)ethyl)amino-2-hydroxypropyl)(phenylmethyl)phosphinic acid hydrochloride (CGP). Blue bars indicate 50-Hz blue laser stimulation. () Response of the same neuron to 50-Hz blue laser light (2.1 mW mm−2, 1 ms pulse width) for 1 s (top) or 10 s (bottom) during simultaneous application of GABA-A and GABA-B receptor antagonists. * Figure 2: Functional characterization of hippocampal interneurons in VGAT-ChR2(H134R)-EYFP BAC transgenic mice. () Diagram of acute coronal brain slice preparation containing the hippocampus and representative image from the boxed region showing placement of the optic fiber in the region of a recorded hippocampal interneuron (asterisk) (top). Scale bar, 200 μm. High-magnification IR-DIC and fluorescence image (EYFP) of an interneuron in the dentate gyrus molecular layer (bottom). Scale bars, 20 μm. () Example photocurrents induced by blue laser light (blue lines; 26.3 mW mm−2) in various hippocampal regions. DG, dentate gyrus. () Plot of peak steady-state photocurrent in response to blue light for interneurons in various hippocampal regions. Error bars, mean ± s.e.m.; CA1 region, n = 11 neurons; DG region, n = 7 neurons; CA3 region, n = 6 neurons. () Cell-attached recording of a dentate gyrus interneuron demonstrating firing in response to 10-s constant blue light stimulation (inset, expanded view). (,) Current clamp () and voltage clamp () mode recording of a dentate gyrus inter! neuron in response to patterned blue laser (2.1 mW mm−2, 1 ms pulse width). (,) Current clamp () and voltage clamp () mode recording of a CA1 interneuron response to patterned blue laser light (2.1 mW mm−2, 1 ms pulse width). () Current clamp mode recording of a CA3 interneuron showing sustained action potential firing in response to repeated bouts of constant blue light. () Diagram of recording configuration to test functional effect of light-induced interneuron firing in the CA3c subfield circuitry (left) and voltage clamp recording of a CA3c pyramidal neuron demonstrating outward current in response to blue light (right). * Figure 3: Functional characterization of ChAT-ChR2(H134R)-EYFP line 6 BAC transgenic mice. () Diagram of acute coronal brain slice containing the dorsal striatum and a representative image from the boxed region showing placement of the optic fiber in the region of a recorded neuron (top). Scale bar, 200 μm. IR-DIC and EYFP fluorescence image of a recorded striatal cholinergic neuron (bottom). Scale bars, 20 μm. () Voltage clamp recording demonstrating inward current induced by blue laser light (26.3 mW mm−2) (top). Summary plot of peak steady-state photocurrent in response to blue light (bottom). Error bars, mean ± s.e.m.; arc lamp, n = 5 neurons; blue laser, n = 5 neurons. () Action potential firing in response to patterned blue laser light (5.21 mW mm−2, 5 ms pulse width). Asterisks indicate extra spikes. A small hyperpolarizing current injection was applied to silence basal firing. () Cell-attached recording of firing in response to patterned blue laser light (2.1 mW mm−2, 5 ms pulse width). Light stimuli were delivered on top of basal tonic firing. * Figure 4: In vivo striatal electrophysiology for ChAT-ChR2(H134R)-EYFP line 6 BAC transgenic mice. () Raster (top) and spike-density histograms (bottom) of a striatal cholinergic neuron in response to a single pulse of blue laser light (10 mW, 18 ms pulse width, blue arrow) over repeated trials. () Spike-density histogram of a striatal cholinergic neuron in response to 40 s of 30-Hz blue laser light stimulation (10 mW, 18 ms pulse width). () Spike-density histogram of a striatal cholinergic neuron showing rapid response (1–2 ms) to blue laser light (10 mW, 18 ms pulse width, blue arrow). () Bar graph of putative medium spiny neuron firing rate in response to 40 s of 30-Hz blue laser light stimulation (10 mW, 18 ms pulse width). Error bars, mean ± s.e.m.; n = 20 units, P < 0.01). * Figure 5: Functional characterization of TPH2-ChR2(H134R)-EYFP BAC transgenic mice. () Diagram of acute coronal brainstem slice preparation and representative image from the boxed region showing placement of the optic fiber in the region of a recorded neuron (top). Scale bars, 200 μm. IR-DIC and EYFP fluorescence image of a recorded serotonergic neuron in the dorsal raphe nucleus (bottom). Scale bars, 20 μm. () Voltage clamp recording of a serotonergic neuron demonstrating inward current induced by blue laser light (26.3 mW mm−2) (top). Plot of peak steady-state photocurrent in response to blue light delivered as indicated (bottom). Error bars, mean ± s.e.m.; arc lamp, n = 11 neurons; blue laser, n = 9 neurons. () Current clamp mode recording showing action potential firing in response to blue laser light (26.3 mW mm−2) (top) or in response to +100 pA current injection (bottom). (–) Current clamp mode () and voltage clamp () recording of responses to patterned blue laser light (26.3 mW mm−2, 5 ms pulse width). Asterisks indicate missed action pot! entials. A small hyperpolarizing current injection was applied (,) to silence basal firing. (,) Cell-attached recording of a serotonergic neuron under prolonged constant blue light stimulation () or to repeated bouts of blue light (). () Summary plot of baseline and blue light-induced firing rates. AP, action potential. Error bars, mean ± s.e.m.; laser-off and laser-on conditions, n = 5 neurons; **P < 0.01 (one-tailed paired t-test). * Figure 6: Functional characterization of Pvalb-ChR2(H134R)-EYFP BAC transgenic mice. () Diagram of a coronal brain slice containing the TRN and representative image from the boxed region showing placement of the optic fiber (top) and EYFP fluorescence (bottom) in the region of recorded neurons. Scale bars, 200 μm. () Extracellular field recordings of a putative single TRN neuron that was silent at rest in response to blue laser light at 10.5 mW mm−2 (top) and 26.3 mW mm−2 (middle), or delivered from a mercury arc lamp (bottom). () Extracellular field recording of action potential firing in response to patterned blue laser light (26.3 mW mm−2, 5 ms pulse width). Asterisks indicate initial burst firing. () Expanded view of the bottom recording in . () Diagram of an acute brain slice containing the cerebellum and representative slice image from the boxed region showing placement of the optic fiber in the region of a recorded neuron in the Purkinje cell layer (top). Scale bar, 200 μm. IR-DIC image and EYFP fluorescence image of a recorded Purkinje cell (! bottom). EYFP fluorescence was not easily detected in the Purkinje cell somata owing to saturating EYFP fluorescence of the Purkinje cell dendrites in the adjacent molecular layer. Scale bars, 20 μm. () Voltage clamp recording demonstrating inward current induced by blue laser light (26.3 mW mm−2) (top). Summary plot of peak steady-state photocurrent in response to blue light delivered as indicated (bottom). Error bars, mean ± s.e.m.; arc lamp, n = 5 neurons; blue laser, n = 10 neurons. () Cell-attached recordings demonstrating potentiation of baseline firing in response to blue laser light at 1.05 mW mm−2 (top), 26.3 mW mm−2 (middle) and 157.9 mW mm−2 (bottom). () Current clamp mode recording showing firing in response to blue light (top) or to +400 pA current injection (bottom). (,) Voltage clamp () and current clamp mode () recordings demonstrating responses to patterned blue laser light (26.3 mW mm−2, 5 ms pulse width). Asterisks indicate initial doublet fir! ing. A small hyperpolarizing current injection was applied to ! silence basal firing. () Cell-attached recording of action potential firing from a single neuron in response to patterned blue laser light (26.3 mW mm−2, 5-ms pulse width). Asterisks indicate doublet firing. This recorded Purkinje cell was silent at rest. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Shengli Zhao & * Jonathan T Ting Affiliations * Department of Neurobiology, Duke University Medical Center, Durham, North Carolina, USA. * Shengli Zhao, * Jonathan T Ting, * Li Qiu, * Bernd Gloss & * Guoping Feng * McGovern Institute for Brain Research and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Jonathan T Ting, * Hisham E Atallah, * Ann M Graybiel & * Guoping Feng * National Institute of Biological Sciences, Beijing, China. * Jie Tan & * Minmin Luo * Duke NeuroTransgenic Laboratory, Duke University Medical Center, Durham, North Carolina, USA. * Bernd Gloss * Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul, Republic of Korea. * George J Augustine * Program in Neuroscience and Behavioral Disorders, Duke–National University of Singapore Graduate Medical School, Singapore. * George J Augustine * Agency for Science, Technology and Research, Duke–National University of Singapore Neuroscience Research Partnership, Singapore. * George J Augustine * Department of Bioengineering, Stanford University, California, USA. * Karl Deisseroth Contributions G.F., K.D. and G.J.A. initiated the project. K.D. provided ChR2(H134R) DNA constructs. S.Z., L.Q. and B.G. generated the ChR2 BAC transgenic founder lines. S.Z. and L.Q. screened the founder lines. S.Z. performed all confocal imaging experiments. J.T.T. performed electrophysiological recordings, and analyzed and interpreted acute-brain-slice experiments for all mouse lines. J.T. performed electrophysiological recordings, and M.L. and J.T. analyzed and interpreted acute brain slice experiments on ChAT-ChR2(H134R)-EYFP line 6 and VGAT-ChR2(H134R)-EYFP line 8 mice. H.E.A. performed in vivo electrophysiology, and H.E.A. and A.M.G. analyzed and interpreted in vivo electrophysiology data on ChAT-ChR2(H134R)-EYFP line 6 mice. J.T.T. and G.F. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Guoping Feng Author Details * Shengli Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan T Ting Search for this author in: * NPG journals * PubMed * Google Scholar * Hisham E Atallah Search for this author in: * NPG journals * PubMed * Google Scholar * Li Qiu Search for this author in: * NPG journals * PubMed * Google Scholar * Jie Tan Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd Gloss Search for this author in: * NPG journals * PubMed * Google Scholar * George J Augustine Search for this author in: * NPG journals * PubMed * Google Scholar * Karl Deisseroth Search for this author in: * NPG journals * PubMed * Google Scholar * Minmin Luo Search for this author in: * NPG journals * PubMed * Google Scholar * Ann M Graybiel Search for this author in: * NPG journals * PubMed * Google Scholar * Guoping Feng Contact Guoping Feng Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–15, Supplementary Tables 1–3 Additional data - High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases
- Nat Methods 8(9):753-755 (2011)
Nature Methods | Brief Communication High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases * Fuqiang Chen1 * Shondra M Pruett-Miller1 * Yuping Huang1 * Monika Gjoka1 * Katarzyna Duda2 * Jack Taunton3 * Trevor N Collingwood1 * Morten Frodin2 * Gregory D Davis1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:753–755Year published:(2011)DOI:doi:10.1038/nmeth.1653Received04 April 2011Accepted27 June 2011Published online17 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions. View full text Subject terms: * Molecular Biology * Molecular Engineering * Genetics * Genomics Figures at a glance * Figure 1: ssODN design and genome editing at the human RSK2 locus. () The schematic shows a 125-mer ssODN (RSK2-125) donor DNA used to incorporate three mutation types into the RSK2 locus: a silent cytosine to adenine (C to A) mutation to create a silent BamHI site, a codon conversion (TGC to GTT) to create the desired cysteine to valine change in the resulting protein and a ZFN-blocking mutation (ZBM) for each ZFN arm (ZFN-L and ZFN-R). () Acrylamide gel separation of amplified and BamHI-digested genomic DNA from pooled K562 cells transfected with the indicated constructs or encoding the indicated proteins and collected 2 d after nucleofection. The frequency of BamHI cleavage was quantified by densitometry. Each lane represents pooled cells from an independent transfection event. M, DNA marker (Sigma). () Immunoblots probing the kinase activity of the RSK2 C436V mutant in K562 cells with wild-type or ssODN-mutated RSK2 C436V (three clones) treated with fmk and phorbol 12-myristate 13-acetate (PMA) to stimulate ERK that activates RSK2 as in! dicated. Cell extracts were immunoprecipitated with antibodies to RSK2 and the precipitates immunoblotted with antibodies to active phosphorylated hydrophobic motif of RSK2 (anti-pHM RSK2) that detect active RSK2 or to total RSK2 (anti-RSK2). Pre-precipitation cell extracts were immunoblotted with antibodies to phosphoERK (anti-pERK) that detect active ERK or to total ERK (anti-ERK). * Figure 2: Deletion of chromosomal segments using ssODNs and ZFNs at the human AAVS1 locus. () General ssODN design rules for deletion of chromosomal segments relative to the ZFN cut site. Sequence distal to the ZFN cleavage site (purple) and DNA sequence containing the ZFN half-site farthest from the distal deletion sequence (green) are shown. () ssODN sequence used to delete 5 kb upstream of the AAVS1 ZFN cut site. The ZFN binding half-site is underlined. () Agarose gel separation of amplified genomic DNA from K562 cells transfected with the following constructs 2 d after nucleofection (1, ssODN plus construct encoding ZFN; 2, ssODN only; and 3, construct encoding ZFN only; see Supplementary Note 2 for ssODN sequence). The expected fragment sizes of the wild-type and deletion alleles are indicated. PCR fragments greater than 1.5 kb were not detected under the experimental conditions. M, DNA marker (Sigma); *3′ deletion from the ZFN cut site; **5′ and 3′ deletion off the ZFN cut site by transfecting both d5-AAVS1-0.1kb and d3-AAVS1-0.1kb deletion ssODNs; Sup! plementary Note 2); all other lanes are 5′ deletions from the cut site. Author information * Author information * Supplementary information Affiliations * Sigma-Aldrich Biotechnology, St. Louis, Missouri, USA. * Fuqiang Chen, * Shondra M Pruett-Miller, * Yuping Huang, * Monika Gjoka, * Trevor N Collingwood & * Gregory D Davis * Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark. * Katarzyna Duda & * Morten Frodin * Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA. * Jack Taunton Contributions F.C., S.M.P.-M., Y.H., M.G. and K.D. performed experiments. G.D.D., F.C., S.M.P.-M. and M.F. designed experiments. G.D.D., T.N.C., M.F., F.C., S.M.P.-M. and J.T. wrote the paper. Competing financial interests F.C., S.M.P.-M., Y.H., M.G., T.N.C. and G.D.D. are employees of Sigma-Aldrich Corp. Corresponding author Correspondence to: * Gregory D Davis Author Details * Fuqiang Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Shondra M Pruett-Miller Search for this author in: * NPG journals * PubMed * Google Scholar * Yuping Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Monika Gjoka Search for this author in: * NPG journals * PubMed * Google Scholar * Katarzyna Duda Search for this author in: * NPG journals * PubMed * Google Scholar * Jack Taunton Search for this author in: * NPG journals * PubMed * Google Scholar * Trevor N Collingwood Search for this author in: * NPG journals * PubMed * Google Scholar * Morten Frodin Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory D Davis Contact Gregory D Davis Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (70K) Supplementary Figures 1–18, Supplementary Tables 1–2, Supplementary Notes 1–3 Additional data - Deep and fast live imaging with two-photon scanned light-sheet microscopy
- Nat Methods 8(9):757-760 (2011)
Nature Methods | Brief Communication Deep and fast live imaging with two-photon scanned light-sheet microscopy * Thai V Truong1, 3 * Willy Supatto1, 2, 3 * David S Koos1 * John M Choi1 * Scott E Fraser1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MethodsVolume: 8,Pages:757–760Year published:(2011)DOI:doi:10.1038/nmeth.1652Received20 October 2010Accepted09 June 2011Published online17 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology. View full text Subject terms: * Imaging * Model Organisms * Microscopy * Physiology Figures at a glance * Figure 1: Optical setup and quantitative analysis of penetration depth. (,) Schematic of SPIM: sample is illuminated with near-infrared or visible light (magenta) for 2P- or 1P-excitation, focused with two low-numerical-aperture microscope objective lenses, and the fluorescence signal (green) is detected orthogonally (z direction) by a charge-coupled device (CCD) camera (not shown) through a high-numerical-aperture water-immersion objective lens (). The illumination light sheet (x-y plane) is generated by laser beam scanning in the y direction (). () Schematic showing the 3D geometry of the illumination and detection light paths for the three imaging modalities compared in this study. (,) Quantitative analysis of the z depth () and x depth () penetration performance of the three imaging modalities. The useful contrasts were calculated for individual image x-y and y-z image slices from 3D datasets similar to those presented in Figure 2d–f, averaged over six embryo samples for each imaging modality. * Figure 2: High imaging depth of 2P-SPIM compared with 1P-SPIM and 2P-LSM in 3D imaging of fly embryos with GFP-labeled nuclei. (–) x-y image slices of stage-13 embryos, obtained using the indicated imaging modalities at z = ~50 μm from embryo surface. Sim., simultaneous bidirectional illumination; seq., sequential bidirectional illumination. (–) y-z image slices of stage-13 embryos at x = ~90 μm from embryo surface (middle of the embryo). MI, monodirectional illumination. Arrows in indicate a deep cell at x = ~90 μm and z = ~70 μm (magnified images at bottom with color map enhancing the contrast). In schematics, light red and gray planes denote the light sheet and the computational slice of the 3D dataset, respectively; green arrow denotes the signal detection direction, and magenta and blue arrows denote the illumination directions. A, anterior; P, posterior; D, dorsal; and V, ventral. (–) x-z image slices of stage-5 embryos. Arrowheads denote the midpoint. () Shown are 3D renderings of live fly embryos at various developmental stages imaged with 2P-SPIM, with simultaneous bidirectional i! llumination. Scale bars, 50 μm. * Figure 3: Non-photodamaging 4D imaging of fly development with 2P-SPIM. () Analysis of developmental stages of embryos imaged using high laser power. Embryos 1–6 were constantly illuminated with total excitation power of 200 mW, and embryos 7–8 with 150 mW, for ~18 h from pre-gastrulation (stage 4–5) until hatching (stage 17). All embryos were scanned through the light sheet at 10 μm s−1 over their entire depth (190 μm) every 20 s, with z-stack imaging taken at 10 frames s−1. Gastrulation onset served as a time reference (3 h of development) to synchronize the sequences. () Relative fluorescence signal, summed over the entire z-stacks, averaged over embryos 1–6 and normalized to the signal at the onset of gastrulation (gray line at 1), is plotted as a function of time. Error bars, s.d. for these 6 embryos. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Thai V Truong & * Willy Supatto Affiliations * California Institute of Technology, Beckman Institute, Pasadena, California, USA. * Thai V Truong, * Willy Supatto, * David S Koos, * John M Choi & * Scott E Fraser * Laboratory for Optics and Biosciences, Ecole Polytechnique, Centre National de la Recherche Scientifique, Institut de la Sante et de la Recherche Medicale, Palaiseau, France. * Willy Supatto Contributions T.V.T., W.S. and S.E.F. conceived and designed the project with consultation from D.S.K. and J.M.C.; T.V.T. designed and assembled the instrumentation; J.M.C. provided help with instrument software control. T.V.T. and W.S. imaged flies; T.V.T. imaged zebrafish; T.V.T. and D.S.K. imaged mice; W.S. and T.V.T. performed image reconstruction and analysis, and designed the figures; and T.V.T., W.S. and S.E.F. wrote the paper. Competing financial interests T.V.T., W.S., D.S.K., J.M.C. and S.E.F. have submitted a patent on the technology of multiphoton light-sheet microscopy (US patent application 12/915,921; international patent application PCT/US2010/054760). Corresponding authors Correspondence to: * Thai V Truong or * Scott E Fraser Author Details * Thai V Truong Contact Thai V Truong Search for this author in: * NPG journals * PubMed * Google Scholar * Willy Supatto Search for this author in: * NPG journals * PubMed * Google Scholar * David S Koos Search for this author in: * NPG journals * PubMed * Google Scholar * John M Choi Search for this author in: * NPG journals * PubMed * Google Scholar * Scott E Fraser Contact Scott E Fraser Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (618K) Animated depiction of the bidirectional scanned light sheet. * Supplementary Video 2 (3M) A 3D view of high and near-isotropic spatial resolution obtained in early fly embryo (stage 5) using 2P-SPIM. A 3D rendering of the raw images obtained with 2P-SPIM (same data as shown in Supplementary Fig. 4a,b,g) using only linear contrast adjustment and without additional image filtering. The 3D rendering uses maximum intensity projection and shows the homogeneous spatial resolution in both x-y and x-z directions. It illustrates that 2P-SPIM can resolve nuclei almost around the entire embryo, even though the imaging was collected from only one view (from the ventral side along the z direction). Grid spacing in movie, 100 μm. * Supplementary Video 3 (10M) 2P-SPIM imaging of entire fly development without photodamage. Representative movie shows 3D-rendered views of the time lapse imaging carried out to test the photodamage of 2P-SPIM imaging with high laser power over the entire fly development. Time-lapse started at stage 5 before gastrulation and ended ~18 h later when the embryo hatched and crawled away. Analysis of these time-lapse data, showing normal development of the embryos, is presented in Figure 3 and Supplementary Figure 7. Embryo anterior pole is to the left. Scale bar, 100 μm. * Supplementary Video 4 (6M) Comparison of multiview imaging with 2P-SPIM and 1P-SPIM: z-stack view. Data correspond to Supplementary Figure 6. Scale bar, 50 μm. * Supplementary Video 5 (8M) Comparison of multiview imaging with 2P-SPIM and 1P-SPIM: rotating 3D view. Data correspond to Supplementary Figure 6. Grid spacing in movie, 100 μm. * Supplementary Video 6 (2M) A 3D reconstruction of entire fly embryo from multiview 2P-SPIM dataset. A 3D rendering after stitching z-stacks acquired from two opposing views as presented in Supplementary Figure 6. Computational cuts show internal structures. Grid spacing in movie, 100 μm. * Supplementary Video 7 (2M) Fast non-phototoxic 2P-SPIM imaging of live zebrafish beating heart. Transgenic zebrafish with GFP-labeled endocardium, at 5.4 d after fertilization, was imaged at 70 frames s–1, with field of view of 400 pixels × 400 pixels. The heart valve leaflets, and their fast motions, can be seen at the center of the field of view. Analysis of movie, showing lack of phototoxicity, is in Supplementary Figure 8. PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–9, Supplementary Table 1, Supplementary Results 1–5, Supplementary Discussion 1–4 Additional data - Bayesian community-wide culture-independent microbial source tracking
- Nat Methods 8(9):761-763 (2011)
Nature Methods | Brief Communication Bayesian community-wide culture-independent microbial source tracking * Dan Knights1 * Justin Kuczynski2 * Emily S Charlson3, 4 * Jesse Zaneveld2 * Michael C Mozer1 * Ronald G Collman3 * Frederic D Bushman3 * Rob Knight5, 6 * Scott T Kelley7 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:761–763Year published:(2011)DOI:doi:10.1038/nmeth.1650Received10 February 2011Accepted02 June 2011Published online17 July 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Contamination is a critical issue in high-throughput metagenomic studies, yet progress toward a comprehensive solution has been limited. We present SourceTracker, a Bayesian approach to estimate the proportion of contaminants in a given community that come from possible source environments. We applied SourceTracker to microbial surveys from neonatal intensive care units (NICUs), offices and molecular biology laboratories, and provide a database of known contaminants for future testing. View full text Subject terms: * Bioinformatics * Genomics * Microbiology Figures at a glance * Figure 1: Comparison of SourceTracker and other models. Indicated models estimate the proportions of two source environments in simulated samples, as the degree of overlap between the environments was varied from a Jensen-Shannon divergence of 0 (completely identical and thus impossible to disambiguate) to 1 (completely non-overlapping and thus trivial to disambiguate). The coefficients of determination (R2) of the estimated proportions are plotted. Each point represents the mean R2 for three trials of 100 samples each; error bars show s.e.m. (n = 3). * Figure 2: SourceTracker proportion estimates for a subset of sink samples. (–) Source environment proportions for three sink samples estimated using SourceTracker and 45 training samples from each source environment: mean proportions for 100 draws from Gibbs sampling (), data for the same samples, including s.d. of the proportion estimates (), and visualization of the 100 Gibbs draws; each column shows the mixture from one draw, with columns ordered to keep similar mixtures together (. * Figure 3: Relative abundance of common contaminating operational taxonomic units (OTUs). SourceTracker may assign a different source environment to each observation (sequence) of an OTU in the sink samples. These ten OTU-source pairs had the highest average relative abundance across sink environments, excluding the unknown source. The legend gives the genus-level taxonomic classification14 of the OTU, the OTU identifier and the source environment assigned to these observations of the OTU. Note that the OTU classified as Enterobacter, a lineage commonly seen in the gut, was more prevalent in the skin training samples than the gut training samples. Author information * Author information * Supplementary information Affiliations * Department of Computer Science, University of Colorado, Boulder, Colorado, USA. * Dan Knights & * Michael C Mozer * Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, USA. * Justin Kuczynski & * Jesse Zaneveld * Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Emily S Charlson, * Ronald G Collman & * Frederic D Bushman * Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Emily S Charlson * Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, USA. * Rob Knight * Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. * Rob Knight * Department of Biology, San Diego State University, San Diego, California, USA. * Scott T Kelley Contributions D.K. designed the algorithm and software, and performed computational experiments; D.K., R.K. and S.T.K. wrote the manuscript; J.K., E.S.C., J.Z., M.C.M., R.G.C. and F.D.B. contributed to writing the manuscript; J.K. and M.C.M. contributed to algorithm design; J.K. processed the data after sequencing; E.S.C. collected the data; R.G.C. and F.D.B. organized and supervised the data collection; R.G.C., F.D.B., R.K. and S.T.K. supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Scott T Kelley Author Details * Dan Knights Search for this author in: * NPG journals * PubMed * Google Scholar * Justin Kuczynski Search for this author in: * NPG journals * PubMed * Google Scholar * Emily S Charlson Search for this author in: * NPG journals * PubMed * Google Scholar * Jesse Zaneveld Search for this author in: * NPG journals * PubMed * Google Scholar * Michael C Mozer Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald G Collman Search for this author in: * NPG journals * PubMed * Google Scholar * Frederic D Bushman Search for this author in: * NPG journals * PubMed * Google Scholar * Rob Knight Search for this author in: * NPG journals * PubMed * Google Scholar * Scott T Kelley Contact Scott T Kelley Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–7 and Supplementary Tables 1–2 Additional data - Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection
- Nat Methods 8(9):765-770 (2011)
Nature Methods | Article Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection * Vikram Pattanayak1 * Cherie L Ramirez2, 3 * J Keith Joung2, 3 * David R Liu1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:765–770Year published:(2011)DOI:doi:10.1038/nmeth.1670Received27 April 2011Accepted20 July 2011Published online07 August 2011 Abstract * Abstract * 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 Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224–induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design. View full text Subject terms: * Chemical Biology * Genetics * Synthetic Biology Figures at a glance * Figure 1: In vitro selection for ZFN-mediated cleavage. Preselection library members are concatemers (represented by arrows) of identical ZFN target sites lacking 5′ phosphates (P). L, left half-site; R, right half-site, S, spacer; and L′, S′ and R′ are sequences complementary to L, S and R, respectively. ZFN cleavage reveals a 5′ phosphate, which is required for ligation of sequencing adapters (red and blue). The only sequences that can be amplified by PCR using primers complementary to the red and blue adapters are sequences that have been cleaved twice and have adapters on both ends. DNA cleaved at adjacent sites is purified via gel electrophoresis and sequenced. A computational screening step after sequencing ensures that the filled-in spacer sequences (S and S′) are complementary and therefore from the same molecule. * Figure 2: DNA cleavage sequence specificity profiles for CCR5-224 and VF2468 ZFNs. (,) Heat maps show specificity scores compiled from all sequences identified in selections for cleavage with 2 nM CCR5-224 () or 1 nM VF2468 (). The target DNA sequence is shown below each half-site. Black boxes indicate target base pairs. Specificity scores were calculated by dividing the change in frequency of the base pair at each position in the postselection DNA pool compared to the preselection pool by the maximal possible change in frequency from preselection library to postselection library of the base pair at each position. Blue and red boxes indicate enrichment for and against a base pair at a given position, respectively, as indicated by the color scale. Specificity scores of 1 and −1 correspond to absolute preference for or against a given base pair, respectively. * Figure 3: Evidence for a compensation model of ZFN target site recognition. (,) Heat maps show the changes in specificity score upon mutation at the black-boxed positions in selections with 2 nM CCR5-224 () or 1 nM VF2468 (). Each row corresponds to a different mutant position (explained graphically in Supplementary Fig. 8). Sites are listed in their genomic orientation; the (+) half-site of CCR5-224 and the (+) half-site of VF2468 are therefore listed as reverse complements of the sequences found in Figure 2. * Figure 4: ZFNs can cleave a large fraction of target sites with three or fewer mutations in vitro. (,) Percentages of sequences with one, two or three mutations that are enriched for in vitro cleavage (enrichment factor >1) by CCR5-224 ZFN () and VF2468 ZFN (). Enrichment factors were calculated for each sequence identified in the selection by dividing the observed frequency of that sequence in the postselection library by the frequency of that sequence in the preselection library. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Chemistry and Chemical Biology, and Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA. * Vikram Pattanayak & * David R Liu * Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. * Cherie L Ramirez & * J Keith Joung * Department of Pathology and Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA. * Cherie L Ramirez & * J Keith Joung Contributions V.P. and C.L.R. performed the experiments, designed the research, analyzed the data and wrote the manuscript. J.K.J. and D.R.L. designed the research, analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David R Liu Author Details * Vikram Pattanayak Search for this author in: * NPG journals * PubMed * Google Scholar * Cherie L Ramirez Search for this author in: * NPG journals * PubMed * Google Scholar * J Keith Joung Search for this author in: * NPG journals * PubMed * Google Scholar * David R Liu Contact David R Liu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5427K) Supplementary Figures 1–15, Supplementary Tables 1–7, Supplementary Notes 1–2, Supplementary Protocols 1–9, Supplementary Data Additional data - A photoswitchable orange-to-far-red fluorescent protein, PSmOrange
- Nat Methods 8(9):771-777 (2011)
Nature Methods | Article A photoswitchable orange-to-far-red fluorescent protein, PSmOrange * Oksana M Subach1 * George H Patterson2 * Li-Min Ting3 * Yarong Wang1 * John S Condeelis1 * Vladislav V Verkhusha1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MethodsVolume: 8,Pages:771–777Year published:(2011)DOI:doi:10.1038/nmeth.1664Received07 December 2010Accepted29 June 2011Published online31 July 2011 Abstract * Abstract * 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 We report a photoswitchable monomeric Orange (PSmOrange) protein that is initially orange (excitation, 548 nm; emission, 565 nm) but becomes far-red (excitation, 636 nm; emission, 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to our knowledge, the most far-red excitation peak of all GFP-like fluorescent proteins, provides diffraction-limited and super-resolution imaging in the far-red light range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluo! rescence of photoconverted PSmOrange results from a new chromophore containing N-acylimine with a co-planar carbon-oxygen double bond. View full text Subject terms: * Imaging * Microscopy * Chemical Biology * Molecular Engineering Figures at a glance * Figure 1: Characterization of purified PSmOrange in vitro. () Absorbance and emission spectra of PSmOrange before and after photoswitching with a 489-nm LED array. () PSmOrange far-red brightness and photostability in the presenence of indicated amounts of K3Fe(CN)6 concentrations. () Photoswitching half-times (t0.5) for PSmOrange at indicated K3Fe(CN)6 concentrations. Half-time at 0.25 mM oxidant is shown by dotted line. () Formation of the far-red form over time for purified PSmOrange (in 0.25 mM K3Fe(CN)6) and for cytoplasmic PSmOrange inside the indicated mammalian cells. The half-time for the purified protein is indicated by dotted line. (–) Photoswitching kinetics for orange () and far-red () forms of the indicated proteins. () PSmOrange initial photoswitching rate at indicated power values of the photoswitching 480/40 nm light. (,) Photobleaching half-times for the orange () and far-red () forms of the indicated fluorescent proteins at indicated power densities. Inset, magnification of data for 1,130 mW cm−2 power. The po! wer densities were estimated at the sample. The photobleaching data (,) were normalized to the spectral output of the lamp, transmission profile of the filter and dichroic mirror and absorbance spectra of the proteins. Error bars, s.d.; n = 10 (,) and n = 5 (–). * Figure 2: Imaging of PSmOrange in mammalian cells. (,) Micrographs of HeLa cells expressing NLS-PSCFP2 in the nucleus with cytoplasmic PSmOrange () and vimentin-PSmOrange (). Photoswitching of PSCFP2 and PSmOrange was performed with 390/40 nm and 540/20 nm light, respectively. () Micrographs show dynamics of PSmOrange-tubulin expressed together with NLS-mCherry in live HeLa cells. Photoswitching of PSmOrange was performed with 480/40 nm light for 60 s. The zoomed area is marked as a green box in the first row and is shown in all subsequent rows. The area of the PSmOrange photoconversion is indicated as a white box. Filter set information is available in Online Methods. Scale bars, 10 μm (–). () TIRF microscopy and a PALM images of EGFR-PSmOrange in fixed COS-7 cells. Scale bars, 5 μm. The histograms show distributions of photons and localization uncertainties. The mean number of photons is 337, and the mean molecular localization uncertainty (sigma) is 45 nm. Data are from 576,290 molecules collected from 5 cells. * Figure 3: Imaging of PSmOrange in vivo. () Whole body images of a mouse injected intramuscularly with 106 cells expressing mKate2 (right flank) or photoconverted PSmOrange (left flank). Images on the right are copies of images on the left but with fluorescence signals shown in green (605 nm excitation channel) and red (640 nm excitation channel) pseudocolors. () Total radiant efficiency corresponding to data in . Total radiant efficiency of mKate2 was set as 100% in 605/30 nm excitation channel, and total radiant efficiency of PSmOrange was set as 100% in 640/30 nm excitation channel. Error bars, s.d. (n = 3). () Whole body images at the indicated excitation and emission wavelengths before and after photoconversion of PSmOrange in mammary tumor xenograft in a mouse. () Total radiant efficiency corresponding to data in . Maximal total radiant efficiency in each channel was normalized to 100%. Error bars, s.d. (n = 4). In – the first and second numbers in 535 nm/580 nm, 605 nm/660 nm and 640 nm/680 nm indicate exc! itation and emission wavelengths, respectively. Scale bars (black, top of images in ,), 1 cm. * Figure 4: Mass spectrometry analysis of the PSmOrange chromophore. () SDS-PAGE analysis of PSmOrange samples before and after photoswitching. (,) The chromophore-bearing peptides and structures of chromophores for PSmOrange before () and after () photoswitching. Calculated (first number) and observed (second number) m/z ratios for the orange-form peptide were: y3, 381.21 and 381.21; y4, 468.25 and 468.29; y6, 696.38 and 696.32; y8, 914.45 and 914.35; b2, 185.09 and 185.13; b3, 298.18 and 298.18; b4, 445.24 and 445.26; b7, 744.38 and 744.31; b8, 831.41 and 831.42; b9, 959.50 and 959.47; b10, 1,030.54 and 1,030.45; and for the far-red–form peptide: y3, 381.21 and 381.22; y4, 468.25 and 468.26; b4, 413.17 and 413.16; b5, 541.27 and 541.23; b6, 612.30 and 612.28. () Proposed scheme for the PSmOrange photoconversion. Asterisk indicates position 2 of the GFP-like chromophore core. Ox, oxidant molecule; OxH, reduced oxidant molecule; and hv, irradiation with blue-green light. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA. * Oksana M Subach, * Yarong Wang, * John S Condeelis & * Vladislav V Verkhusha * Biophotonics Section, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA. * George H Patterson * Department of Immunology and Microbiology, and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA. * Li-Min Ting Contributions O.M.S. developed the protein and characterized it in vitro. O.M.S. and G.H.P. characterized the protein in mammalian cells. O.M.S. and L.-M.T. characterized the protein in mouse models. O.M.S., Y.W. and J.S.C. performed tumor experiments. V.V.V. designed the project and, together with O.M.S., planned and discussed the project, and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Vladislav V Verkhusha Author Details * Oksana M Subach Search for this author in: * NPG journals * PubMed * Google Scholar * George H Patterson Search for this author in: * NPG journals * PubMed * Google Scholar * Li-Min Ting Search for this author in: * NPG journals * PubMed * Google Scholar * Yarong Wang Search for this author in: * NPG journals * PubMed * Google Scholar * John S Condeelis Search for this author in: * NPG journals * PubMed * Google Scholar * Vladislav V Verkhusha Contact Vladislav V Verkhusha Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–11 and Supplementary Tables 1–2 Additional data
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