CRISPR-Cas9-mediated genome editing in apple and grapevine.

abstract：:Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraint...

journal_title：Nature protocols

authors： Sharan SK,Thomason LC,Kuznetsov SG,Court DL

更新日期：2009-01-01 00:00:00

abstract：:Stabilized α-helices and nonpeptidic helix mimetics have emerged as powerful molecular scaffolds for the discovery of protein-protein interaction inhibitors. Protein-protein interactions often involve large contact areas, which are often difficult for small molecules to target with high specificity. The hypothesis beh...

journal_title：Nature protocols

authors： Patgiri A,Menzenski MZ,Mahon AB,Arora PS

更新日期：2010-11-01 00:00:00

abstract：:The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and ...

journal_title：Nature protocols

authors： Palmisano G,Lendal SE,Engholm-Keller K,Leth-Larsen R,Parker BL,Larsen MR

更新日期：2010-12-01 00:00:00

abstract：:Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein o...

journal_title：Nature protocols

authors： Asara JM,Zhang X,Zheng B,Maroney LA,Christofk HR,Wu N,Cantley LC

更新日期：2006-01-01 00:00:00

abstract：:Neurons in cortical sensory regions receive modality-specific information through synapses that are located on their dendrites. Recently, the use of two-photon microscopy combined with whole-cell recordings has helped to identify visually evoked dendritic calcium signals in mouse visual cortical neurons in vivo. The c...

journal_title：Nature protocols

authors： Jia H,Rochefort NL,Chen X,Konnerth A

更新日期：2011-01-01 00:00:00

abstract：:The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for ...

journal_title：Nature protocols

authors： Beloqui A,Brayden DJ,Artursson P,Préat V,des Rieux A

更新日期：2017-07-01 00:00:00

abstract：:Understanding chemical reactivity through the use of state-of-the-art computational techniques enables chemists to both predict reactivity and rationally design novel reactions. This protocol aims to provide chemists with the tools to implement a powerful and robust method for analyzing and understanding any chemical ...

journal_title：Nature protocols

authors： Vermeeren P,van der Lubbe SCC,Fonseca Guerra C,Bickelhaupt FM,Hamlin TA

更新日期：2020-02-01 00:00:00

abstract：:Desorption electrospray ionization (DESI) allows the direct analysis of ordinary objects or pre-processed samples under ambient conditions. Among other applications, DESI is used to identify and record spatial distributions of lipids and drug molecules in biological tissue sections. This technique does not require sam...

journal_title：Nature protocols

authors： Wiseman JM,Ifa DR,Venter A,Cooks RG

更新日期：2008-01-01 00:00:00

abstract：:Here we describe a protocol for the production of frozen competent yeast cells that can be transformed with high efficiency using the lithium acetate/single-stranded carrier DNA/PEG method. This protocol allows the production of highly competent yeast cells that can be frozen and used at a later date and is especially...

journal_title：Nature protocols

authors： Gietz RD,Schiestl RH

更新日期：2007-01-01 00:00:00

abstract：:To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC an...

journal_title：Nature protocols

authors： Booth MJ,Ost TW,Beraldi D,Bell NM,Branco MR,Reik W,Balasubramanian S

更新日期：2013-10-01 00:00:00

abstract：:This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. The characterization consists of the following steps: (i) loading heat-activated dormant spores into a temperature-controlled micros...

journal_title：Nature protocols

authors： Kong L,Zhang P,Wang G,Yu J,Setlow P,Li YQ

更新日期：2011-05-01 00:00:00

abstract：:An important area of proteomics involves the need for quantification, whether relative or absolute. Many methods now exist for relative quantification, but to support biomarker proteomics and systems biology, absolute quantification rather than relative quantification is required. Absolute quantification usually invol...

journal_title：Nature protocols

authors： Pratt JM,Simpson DM,Doherty MK,Rivers J,Gaskell SJ,Beynon RJ

更新日期：2006-01-01 00:00:00

abstract：:Adult stem cell-based organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting organoid cultures. Patient-deriv...

journal_title：Nature protocols

authors： Driehuis E,Kretzschmar K,Clevers H

更新日期：2020-10-01 00:00:00

abstract：:Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms. Here, I review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster o...

journal_title：Nature protocols

authors： Kim JS

更新日期：2016-09-01 00:00:00

abstract：:Gene tagging with fluorescent proteins is essential for investigations of the dynamic properties of cellular proteins. CRISPR-Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and allows functionality and physiological expression of the fusion protein. ...

journal_title：Nature protocols

authors： Koch B,Nijmeijer B,Kueblbeck M,Cai Y,Walther N,Ellenberg J

更新日期：2018-06-01 00:00:00

abstract：:Determining enantiomeric excess (e.e.) in chiral compounds is key to development of chiral catalyst auxiliaries and chiral drugs. Here we describe a sensitive and robust fluorescence-based assay for determining e.e. in mixtures of enantiomers of 1,2- and 1,3-diols, chiral amines, amino alcohols, and amino-acid esters....

journal_title：Nature protocols

authors： Shcherbakova EG,James TD,Anzenbacher P Jr

更新日期：2020-07-01 00:00:00

abstract：:The scototaxis (dark/light preference) protocol is a behavioral model for fish that is being validated to assess the antianxiety effects of pharmacological agents and the behavioral effects of toxic substances, and to investigate the (epi)genetic bases of anxiety-related behavior. Briefly, a fish is placed in a centra...

journal_title：Nature protocols

authors： Maximino C,Marques de Brito T,Dias CA,Gouveia A Jr,Morato S

更新日期：2010-02-01 00:00:00

abstract：:We describe the use of a microfabricated cell culture substrate, consisting of a uniform array of closely spaced, vertical, elastomeric microposts, to study the effects of substrate rigidity on cell function. Elastomeric micropost substrates are micromolded from silicon masters comprised of microposts of different hei...

journal_title：Nature protocols

authors： Yang MT,Fu J,Wang YK,Desai RA,Chen CS

更新日期：2011-02-01 00:00:00

abstract：:Methods for installing natural and unnatural amino acids and their modifications into proteins in a benign and precise manner are highly sought-after in protein science. Here we describe a protocol for 'post-translational mutagenesis' that enables the programmed installation of protein side chains through the use of r...

journal_title：Nature protocols

authors： Wright TH,Davis BG

更新日期：2017-10-01 00:00:00

abstract：:Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have b...

journal_title：Nature protocols

authors： Tulkens J,De Wever O,Hendrix A

更新日期：2020-01-01 00:00:00

abstract：:The analysis of large microbiome data sets holds great promise for the delineation of the biological and metabolic functioning of living organisms and their role in the environment. In the midst of this genomic puzzle, viruses, especially those that infect microbial communities, represent a major reservoir of genetic ...

journal_title：Nature protocols

authors： Paez-Espino D,Pavlopoulos GA,Ivanova NN,Kyrpides NC

更新日期：2017-08-01 00:00:00

abstract：:The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confo...

journal_title：Nature protocols

authors： Hatta K,Tsujii H,Omura T

更新日期：2006-01-01 00:00:00

abstract：:Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of es...

journal_title：Nature protocols

authors： Mariappan I,Maddileti S,Savy S,Tiwari S,Gaddipati S,Fatima A,Sangwan VS,Balasubramanian D,Vemuganti GK

更新日期：2010-08-01 00:00:00

abstract：:Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the s...

journal_title：Nature protocols

authors： Mederacke I,Dapito DH,Affò S,Uchinami H,Schwabe RF

更新日期：2015-02-01 00:00:00

abstract：:Live-imaging technology has markedly advanced in the field of neural injury and axon degeneration; however, studies are still predominantly performed in in vitro settings such as cultured neuronal cells or in model organisms such as Caenorhabditis elegans in which axons lack glial wrappings. We recently developed a ne...

journal_title：Nature protocols

authors： Fang Y,Soares L,Bonini NM

更新日期：2013-04-01 00:00:00

abstract：:The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunode...

journal_title：Nature protocols

authors： Lin Z,Chiang NY,Chai N,Seshasayee D,Lee WP,Balazs M,Nakamura G,Swem LR

更新日期：2014-07-01 00:00:00

abstract：:Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol i...

journal_title：Nature protocols

authors： Huang J,Doria-Rose NA,Longo NS,Laub L,Lin CL,Turk E,Kang BH,Migueles SA,Bailer RT,Mascola JR,Connors M

更新日期：2013-10-01 00:00:00

abstract：:Here, we describe a simple micromolding method to construct three-dimensional arrays of organotypic epithelial tissue structures that approximate in vivo histology. An elastomeric stamp containing an array of posts of defined geometry and spacing is used to mold microscale cavities into the surface of type I collagen ...

journal_title：Nature protocols

authors： Nelson CM,Inman JL,Bissell MJ

更新日期：2008-01-01 00:00:00

abstract：:With the advent of massively parallel sequencing, considerable work has gone into adapting chromosome conformation capture (3C) techniques to study chromosomal architecture at a genome-wide scale. We recently demonstrated that the inactive murine X chromosome adopts a bipartite structure using a novel 3C protocol, ter...

journal_title：Nature protocols

authors： Ramani V,Cusanovich DA,Hause RJ,Ma W,Qiu R,Deng X,Blau CA,Disteche CM,Noble WS,Shendure J,Duan Z

更新日期：2016-11-01 00:00:00

abstract：:During biosynthesis on the ribosome, an elongating nascent polypeptide chain can begin to fold, in a process that is central to all living systems. Detailed structural studies of co-translational protein folding are now beginning to emerge; such studies were previously limited, at least in part, by the inherently dyna...

journal_title：Nature protocols

authors： Cassaignau AM,Launay HM,Karyadi ME,Wang X,Waudby CA,Deckert A,Robertson AL,Christodoulou J,Cabrita LD

更新日期：2016-08-01 00:00:00