Volume 10 Issue 10, October 2015

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.

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.

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.

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.

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.

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.

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

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.

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).

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.

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.