Engineered designer nucleases can be used to efficiently modify genomic sequence in a wide variety of model organisms and cell types. Zinc finger nucleases (ZFNs), consisting of an engineered zinc finger array fused to a non-specific cleavage domain, have been extensively used to modify a broad range of endogenous genes. Protocols for engineering ZFNs targeted to specific gene sequences of interest using the context-dependent assembly (CoDA) method are described in this unit.

Current Protocols in Molecular BiologyVolume 96, Issue 1 p. 19.0.1-19.0.3 Introduction Informatics for Molecular Biologists Frederick Ausubel, Frederick AusubelSearch for more papers by this authorNicholas C. Dracopoli, Nicholas C. DracopoliSearch for more papers by this authorMary Wildermuth, Mary WildermuthSearch for more papers by this author Frederick Ausubel, Frederick AusubelSearch for more papers by this authorNicholas C. Dracopoli, Nicholas C. DracopoliSearch for more papers by this authorMary Wildermuth, Mary WildermuthSearch for more papers by this author First published: 15 October 2011 https://doi.org/10.1002/0471142727.mb1900s96Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. View the latest in Molecular Biology RelatedInformation

This unit presents the protocol for the NanoString nCounter Gene Expression Assay, a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction with high sensitivity and linearity across a broad range of expression levels. The methodology serves to bridge the gap between genome-wide (microarrays) and targeted (real-time quantitative PCR) expression profiling. The nCounter assay is based on direct digital detection of mRNA molecules of interest using target-specific, color-coded probe pairs. It does not require the conversion of mRNA to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR. Each target gene of interest is detected using a pair of reporter and capture probes carrying 35- to 50-base target-specific sequences. In addition, each reporter probe carries a unique color code at the 5' end that enables the molecular barcoding of the genes of interest, while the capture probes all carry a biotin label at the 3' end that provides a molecular handle for attachment of target genes to facilitate downstream digital detection. After solution-phase hybridization between target mRNA and reporter-capture probe pairs, excess probes are removed and the probe/target complexes are aligned and immobilized in the nCounter cartridge, which is then placed in a digital analyzer for image acquisition and data processing. Hundreds of thousands of color codes designating mRNA targets of interest are directly imaged on the surface of the cartridge. The expression level of a gene is measured by counting the number of times the color-coded barcode for that gene is detected, and the barcode counts are then tabulated.

Current Protocols in Molecular BiologyVolume 92, Issue 1 p. 14.0.1-14.0.3 Introduction In Situ Hybridization and Immunohistochemistry J.G. Seidman, J.G. SeidmanSearch for more papers by this author J.G. Seidman, J.G. SeidmanSearch for more papers by this author First published: 15 October 2010 https://doi.org/10.1002/0471142727.mb1400s92Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. View the latest in Molecular Biology RelatedInformation

Along with genes and proteins, metabolites play important roles in sustaining life. Their functions include "primary" functions in metabolism and energy storage, as well as "secondary" functions in cell-to-cell signaling, metal acquisition, and virulence. There remains much to be learned about the in vivo roles of metabolites. Approaches that accelerate measurement of metabolite levels directly from cells and tissues should increase our understanding of the diverse roles of metabolites and potentially lead to discovery of novel metabolites and metabolic pathways. Metabolomics is an important comparative tool to study global metabolite levels in samples under various conditions. In this unit, the steps needed to perform a mass spectrometry (MS)--based untargeted metabolomics experiment using bacterial supernatants are detailed. In contrast to a targeted metabolomics experiment, which measures ions from known metabolites, an untargeted metabolomics experiment registers all ions within a certain mass range, including ions belonging to structurally novel metabolites. The protocols in this unit describe the conditions necessary for analyzing hydrophobic metabolites and provide an example of how to structurally characterize a novel metabolite.