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Current Protocols in Molecular Biology

F William Studier
Inducible T7 expression systems are capable of producing a wide range of proteins in E. coli. Improvements over common practice include: (1) preventing unintended induction by establishing and maintaining expression strains in non-inducing growth media composed entirely of purified components instead of complex growth media that may variably induce target proteins on approach to saturation; and (2) expressing many target proteins in parallel by convenient and productive auto-induction in BL21(DE3) and other suitable hosts, instead of IPTG induction...
July 17, 2018: Current Protocols in Molecular Biology
Cheryl Isaac Murphy, Helen Piwnica-Worms, Stefan Grünwald, William G Romanow, Nicole Francis, Hua-Ying Fan, Sharon Marr
This article describes how to analyze protein expression in cells infected with recombinant baculovirus on a small scale for optimizing protein production, how to maximize and scale up recombinant protein production, and how to purify recombinant proteins. © 2018 by John Wiley & Sons, Inc.
July 2018: Current Protocols in Molecular Biology
Xin Liu, Yuannyu Zhang, Yong Chen, Mushan Li, Zhen Shao, Michael Q Zhang, Jian Xu
Cis-regulatory elements (CREs) play a pivotal role in spatiotemporal control of tissue-specific gene expression, yet the molecular composition of the vast majority of CREs in native chromatin remains unknown. In this article, we describe the clustered regularly interspaced short palindromic repeats (CRISPR) affinity purification in situ of regulatory elements (CAPTURE) approach to simultaneously identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single guide RNAs (sgRNAs), this approach allows for high-resolution and locus-specific isolation of protein complexes and long-range chromatin looping associated with single copy CREs in mammalian cells...
July 2018: Current Protocols in Molecular Biology
Taichi Umeyama, Takashi Ito
The genome exerts its functions through interactions with proteins. Hence, comprehensive identification of protein-occupied sites by genomic footprinting is critical to an in-depth understanding of genome functions. This unit describes the protocol of dimethyl sulfate-sequencing (DMS-seq). DMS is an alkylating reagent that methylates guanine and adenine in double-stranded DNA. DMS added to the culture medium readily enters the cell and methylates its DNA throughout the genome except for the regions bound by proteins, thereby obviating the need for nuclear isolation in genomic footprinting...
July 2018: Current Protocols in Molecular Biology
Ying Xie, Xiao Han, Yansong Miao
The expression and purification of recombinant proteins using bacterial vectors is a mature and preferred system to obtain folded and stable proteins. However, functional post-translational protein modifications, such as glycosylation or phosphorylation, can only be achieved using eukaryotic expression systems. In addition, insolubility is another challenge when using proteins expressed in Escherichia coli, such as certain intrinsically disordered proteins, which are more prone to aggregation than folded proteins...
July 2018: Current Protocols in Molecular Biology
Heng-Chang Chen, Eduard Zorita, Guillaume J Filion
The latent HIV reservoir is the main barrier to curing AIDS, because infected cells escape the immune system and antiretroviral therapies. Developing new treatment strategies requires technologies to trace latent proviruses. Here, we describe a genome-wide technique called Barcoded HIV Ensembles (B-HIVE) to measure HIV expression at the single provirus level. The principle of B-HIVE is to tag the genome of HIV with DNA barcodes to trace viral transcripts produced by single proviruses in an infected cell population...
April 2018: Current Protocols in Molecular Biology
Barton E Slatko, Andrew F Gardner, Frederick M Ausubel
High throughput DNA sequencing methodology (next generation sequencing; NGS) has rapidly evolved over the past 15 years and new methods are continually being commercialized. As the technology develops, so do increases in the number of corresponding applications for basic and applied science. The purpose of this review is to provide a compendium of NGS methodologies and associated applications. Each brief discussion is followed by web links to the manufacturer and/or web-based visualizations. Keyword searches, such as with Google, may also provide helpful internet links and information...
April 2018: Current Protocols in Molecular Biology
Thale Kristin Olsen, Ninib Baryawno
During the last decade, high-throughput sequencing methods have revolutionized the entire field of biology. The opportunity to study entire transcriptomes in great detail using RNA sequencing (RNA-seq) has fueled many important discoveries and is now a routine method in biomedical research. However, RNA-seq is typically performed in "bulk," and the data represent an average of gene expression patterns across thousands to millions of cells; this might obscure biologically relevant differences between cells...
April 2018: Current Protocols in Molecular Biology
Arun Sharma, Michael Mücke, Christine E Seidman
We describe a method to transform blood lymphocytes into human-induced pluripotent stem cells by delivering four transcription factors with a non-integrative virus. Using human peripheral blood mononuclear cells (PBMCs) as the source cell type for hiPSC reprogramming is advantageous since blood samples are rapidly and safely obtained from nearly-all subjects. Reprogramming factors needed to make hiPSCs are introduced by infecting the PBMCs with non-integrating Sendai virus vectors. Reprogrammed cells can subsequently be quickly expanded for downstream use...
April 2018: Current Protocols in Molecular Biology
Daniel M DeLaughter
Understanding the transcriptional heterogeneity that occurs on the level of a single cell is critical to understanding the gene-regulatory mechanisms underlying development and disease. Population-level whole-transcriptome profiling approaches average gene expression across thousands to millions of cells and are unable to delineate the transcriptional signature of individual cells. Considerable biological heterogeneity between individual cells arises from differences in cell lineage, environment, or response to stimulus...
April 2018: Current Protocols in Molecular Biology
Sizun Jiang, Liang Wei Wang, Michael J Walsh, Stephen J Trudeau, Catherine Gerdt, Bo Zhao, Benjamin E Gewurz
Epstein-Barr virus (EBV) efficiently transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs), which are extensively used in human genetic, immunological and virological studies. LCLs provide unlimited sources of DNA for genetic investigation, but can be difficult to manipulate, for instance because low retroviral or lentiviral transduction frequencies hinder experiments that require co-expression of multiple components. This unit details Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 engineering for robust LCL genome editing...
January 16, 2018: Current Protocols in Molecular Biology
Huntington Potter, Richard Heller
Electroporation-the use of high-voltage electric shocks to introduce DNA into cells-can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination...
January 16, 2018: Current Protocols in Molecular Biology
Federica Piccioni, Scott T Younger, David E Root
Pooled cell-based screens of mammalian genetic perturbations enable systematic large-scale, even genome-scale, evaluation of gene function. Pooled screens introduce genetic perturbations into a cell population through viral transduction such that each cell integrates into its DNA a single or small number of library perturbations with barcodes identifying the perturbations. One then selects and physically isolates the subset of cells that exhibit the phenotype of interest. Sequencing the barcodes in the hit cells reveals which genes favored or inhibited the hit phenotype...
January 16, 2018: Current Protocols in Molecular Biology
Chad J Creighton
The vast amounts of genomic data now deposited in public repositories represent rich resources for cancer researchers. Large-scale genomics initiatives such as The Cancer Genome Atlas have made available data from multiple molecular profiling platforms (e.g., somatic mutation, RNA and protein expression, and DNA methylation) for the same set of over 10,000 human tumors. There has been much collective effort toward providing user-friendly software tools for biologists lacking computational skills to ask questions of large-scale genomic datasets...
January 16, 2018: Current Protocols in Molecular Biology
Yaligara Veeranagouda, Delphine Debono-Lagneaux, Hamida Fournet, Gilbert Thill, Michel Didier
The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells)...
January 16, 2018: Current Protocols in Molecular Biology
Wei Wang, Bingning Dong, Feng Yang
The RCAS (replication-competent avian sarcoma leukosis virus long-terminal repeat with splice acceptor)-TVA (tumor virus A) gene delivery system has been successfully used in modeling human cancers. Based on this, we have recently developed a novel RCI-Oncogene (RCAS-Cre-IRES-Oncogene) gene delivery system that can be used to efficiently manipulate gene expression in spontaneous tumors in vivo. We used this system for tumor gene knockout (TuKO) and demonstrated a crucial role of FGFR1 in driving mammary tumor metastasis...
January 16, 2018: Current Protocols in Molecular Biology
Liang Wei Wang, Stephen J Trudeau, Chong Wang, Catherine Gerdt, Sizun Jiang, Bo Zhao, Benjamin E Gewurz
Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus...
January 16, 2018: Current Protocols in Molecular Biology
Andreas Mayer, L Stirling Churchman
In eukaryotic cells, RNAs at various maturation and processing levels are distributed across cellular compartments. The standard approach to determine transcript abundance and identity in vivo is RNA sequencing (RNA-seq). RNA-seq relies on RNA isolation from whole-cell lysates and thus mainly captures fully processed, stable, and more abundant cytoplasmic RNAs over nascent, unstable, and nuclear RNAs. Here, we provide a step-by-step protocol for subcellular RNA-seq (subRNA-seq). subRNA-seq allows the quantitative measurement of RNA polymerase II-generated RNAs from the chromatin, nucleoplasm, and cytoplasm of mammalian cells...
October 2, 2017: Current Protocols in Molecular Biology
April Cook, Jakub Mieczkowski, Michael Y Tolstorukov
This unit describes a method for determining the accessibility of chromatinized DNA and nucleosome occupancy in the same assay. Enzymatic digestion of chromatin using micrococcal nuclease (MNase) is optimized for liberation, retrieval, and characterization of DNA fragments from chromatin. MNase digestion is performed in a titration series, and the DNA fragments are isolated and sequenced for each individual digest independently. These sequenced fragments are then collectively analyzed in a novel bioinformatics pipeline to produce a metric describing MNase accessibility of chromatin (MACC) and nucleosome occupancy...
October 2, 2017: Current Protocols in Molecular Biology
Nathan J VanDusen, Yuxuan Guo, Weiliang Gu, William T Pu
In vivo loss-of-function studies are currently limited by the need for appropriate conditional knockout alleles. CRISPR/Cas9 is a powerful tool commonly used to induce loss-of-function mutations in vitro. However, CRISPR components have been difficult to deploy in vivo. To address this problem, we developed the CASAAV (CRISPR/Cas9/AAV-based somatic mutagenesis) platform, in which recombinant adeno-associated virus (AAV) is used to deliver tandem guide RNAs and Cre recombinase to Cre-dependent Cas9-P2A-GFP mice...
October 2, 2017: Current Protocols in Molecular Biology
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