Table of contents

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The fetal mouse metatarsal bone explant as a model of angiogenesis pp1459 - 1473

Weihua Song, Chee Wai Fhu, Koon Hwee Ang, Cheng Hao Liu, Nurul Azizah Binte Johari, Daniel Lio, Sabu Abraham, Wanjin Hong, Stephen E Moss, John Greenwood & Xiaomeng Wang


This protocol describes how to set up an assay investigating vessel outgrowth from mouse fetal metatarsals. This assay is an ex vivo assay to investigate sprouting angiogenesis.

Efficient genetic engineering of human intestinal organoids using electroporation pp1474 - 1485

Masayuki Fujii, Mami Matano, Kosaku Nanki & Toshiro Sato


This protocol describes how to grow untransformed human colonic organoids and deliver genes of interest into the organoids via the piggyBac transposon or gene editing using the CRISPR-Cas9 system.

Live imaging of Tribolium castaneum embryonic development using light-sheet–based fluorescence microscopy pp1486 - 1507

Frederic Strobl, Alexander Schmitz & Ernst H K Stelzer


The Stelzer lab describes how to live-image Tribolium embryos using light-sheet-based fluorescence microscopy (LSFM). Imaging can proceed for up to 120 h, allowing the entire embryonic development of this important model insect to be recorded.

Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers pp1508 - 1524

Yifan Lv, Rong Hu, Guizhi Zhu, Xiaobing Zhang, Lei Mei, Qiaoling Liu, Liping Qiu, Cuichen Wu & Weihong Tan


This protocol describes how to prepare multifunctional DNA nanoflowers. Rolling-circle amplification of a designed template containing drug binding, cell targeting, and fluorescent dye binding sites creates structures of controllable size for drug delivery.

Studying tumor growth in Drosophila using the tissue allograft method pp1525 - 1534

Fabrizio Rossi & Cayetano Gonzalez


The Gonzalez laboratory provides its allograft protocol for studying tumorigenesis in Drosophila. Transplanting tissue from donor larvae to adult hosts allows its tumorigenic potential to be determined.

Whole-mount immunolocalization to study female meiosis in Arabidopsis pp1535 - 1542

Rocio Escobar-Guzmán, Daniel Rodríguez-Leal, Jean-Philippe Vielle-Calzada & Arnaud Ronceret


The study of meiosis in plants is considered to be gender-biased owing to the easy accessibility of male meiocytes. This protocol describes how to prepare and image female Arabidopsis meiocytes to investigate protein localization during meiosis.

MicroRNA-based conversion of human fibroblasts into striatal medium spiny neurons pp1543 - 1555

Michelle Richner, Matheus B Victor, Yangjian Liu, Daniel Abernathy & Andrew S Yoo


In this protocol, neuronal miRNAs and transcription factors are used to directly convert human fibroblasts to striatal medium spiny neurons, a neuronal subtype important in motor control and the main cell type affected in Huntington's disease.

Genomic variant annotation and prioritization with ANNOVAR and wANNOVAR pp1556 - 1566

Hui Yang & Kai Wang


This protocol describes how to annotate genomic variants using either the ANNOVAR software or the web-based wANNOVAR tool.

Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry pp1567 - 1593

Holger Franken, Toby Mathieson, Dorothee Childs, Gavain M A Sweetman, Thilo Werner, Ina Tögel, Carola Doce, Stephan Gade, Marcus Bantscheff, Gerard Drewes, Friedrich B M Reinhard, Wolfgang Huber & Mikhail M Savitski


Unbiased proteome-level discovery of intracellular drug targets can be achieved by plotting melting curves of proteins from untreated and drug-treated cells. Multiplexed quantitative mass spectrometry using TMT10 reagents makes this possible.

Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation pp1594 - 1611

Daniel Rösner, Tatjana Schneider, Daniel Schneider, Martin Scheffner & Andreas Marx


This protocol uses CuAAC click chemistry along with two different methods for expansion of the genetic code to assemble protein-protein conjugates for studying the post-translational modification of proteins by ubiquitin (ubiquitylation).

Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting pp1612 - 1624

Ling Liu, Tom H Cheung, Gregory W Charville & Thomas A Rando


In this protocol, limb muscles are physically and enzymatically dissociated to maximally release resident mononucleated cells. Pure populations of either quiescent or activated muscle stem cells are then isolated by flow cytometry.

Directed evolution of artificial enzymes (XNAzymes) from diverse repertoires of synthetic genetic polymers pp1625 - 1642

Alexander I Taylor & Philipp Holliger


The authors describe methods for the directed evolution of artificial endonuclease and ligase enzymes by X-SELEX, from diverse repertoires of synthetic genetic polymers (XNAzymes). The protocol has been applied to four different XNA chemistries and three different reactions, and it is, in principle, applicable to many more.