Methods in enzymology
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Methods in enzymology · Jan 2014
The iCRISPR platform for rapid genome editing in human pluripotent stem cells.
Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. ⋯ We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.
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Methods in enzymology · Jan 2014
Methods to monitor ROS production by fluorescence microscopy and fluorometry.
Mitochondria are considered one of the main sources of reactive oxygen species (ROS). The overgeneration of ROS can evoke an intracellular state of oxidative stress, leading to permanent cell damage. ⋯ Here, we describe the use of chemical probes for the rapid detection of ROS in intact and permeabilized adherent cells by fluorescence microscopy and fluorometry. Moreover, after discussing the limitations described in the literature for the fluorescent probes presented herein, we recommend methods to assess the production of specific ROS in various fields of investigation, including the study of oncometabolism.
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The rapid advancement of genome-editing techniques holds much promise for the field of human gene therapy. From bacteria to model organisms and human cells, genome editing tools such as zinc-finger nucleases (ZNFs), TALENs, and CRISPR/Cas9 have been successfully used to manipulate the respective genomes with unprecedented precision. With regard to human gene therapy, it is of great interest to test the feasibility of genome editing in primary human hematopoietic cells that could potentially be used to treat a variety of human genetic disorders such as hemoglobinopathies, primary immunodeficiencies, and cancer. ⋯ By using two guide RNAs directed at a single locus, we achieve highly efficient and predictable deletions that ablate gene function. The use of a Cas9-2A-GFP fusion protein allows FACS-based enrichment of the transfected cells. The ease of designing, constructing, and testing guide RNAs makes this dual guide strategy an attractive approach for the efficient deletion of clinically relevant genes in primary human hematopoietic stem and effector cells and enables the use of CRISPR/Cas9 for gene therapy.
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Methods in enzymology · Jan 2014
3-D adipocyte differentiation and peri-adipocyte collagen turnover.
Peri-adipocyte extracellular matrix (ECM) remodeling is a key biological process observed during adipose tissue development and expansion. The genetic loss of a pericellular collagenase, MMP14 (also known as MT1-MMP), renders mice lipodystrophic with the accumulation of undigested collagen fibers in adipose tissues. ⋯ Studying adipogenesis in 3-D may serve as an effective experimental approach to bridge gaps in our understanding of in vivo adipocyte biology. Moreover, by assessing the content of collagen family members and their rate of degradation in adipose tissues, we should be able to better define the role of dynamic ECM remodeling in the pathogenesis of obesity and diabetes.
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Methods in enzymology · Jan 2012
Generation of dual-variable-domain immunoglobulin molecules for dual-specific targeting.
Bispecific antibodies may be used to improve clinical efficacy by targeting two disease mechanisms for the treatment of complex human diseases in a single agent. Bispecific antibodies also hold promise for certain therapeutic applications difficult to achieve by single-targeting monospecific antibodies, such as immune (T cell or NK) cell retargeting, site-specific targeting, enabling therapeutics to cross the blood-brain barrier, and unique receptor modulation. Although the history of bispecific antibody research is almost as long as hybridoma technology, it is not until recent that bispecific antibodies have made substantial breakthrough, thanks to promising clinical trial results of a few bispecific antibodies and the development of new formats which largely ease manufacturing and physicochemical property challenges encountered by early bispecific antibody formats. ⋯ An optimized DVD-Ig™ molecule has many desirable properties of a mAb, such as good expression in mammalian cells, easy purification to homogeneity using standard approaches, displaying good drug-like biophysical and pharmacokinetic properties, and amenability to large-scale manufacturing. Several DVD-Ig molecules have demonstrated favorable pharmacokinetic properties and efficacy in preclinical animal models. Here, we provide an example of construction and preliminary characterization of a DVD-Ig™ molecule and discuss the general approach used in optimization.