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Past Issue in 2019
Volume: 9, Issue: 4
Cell Biology
Determination of Cellular Uptake and Endocytic Pathways
Efficiency of drug and gene delivery via nonviral vehicles is contingent on proper cellular uptake and intracellular release. Further, various cargos, such as nucleases for gene editing or inhibitors for endosomal receptors, require transport to specific compartments of the cell. Hence, characterization of cellular uptake and endocytic pathways is crucial for the optimization of any nanoparticle-mediated intracellular delivery system. Previous work on endocytic pathways looks at the effect of various pathway inhibitors on the uptake efficiency of nanoparticles carrying fluorescently-labeled cargo. While this helps attribute particle uptake to specific pathways like caveolae-mediated or clathrin-mediated endocytosis, this does not provide a holistic picture of the delivery process. Here, we provide a general protocol that combines systematic studies of inhibitor effects on efficiency with quantification of particle-induced cell membrane permeability. By applying this methodology to a nucleic acid delivery system, for example a helical polypeptide-based nanoparticle for plasmid and guide RNA delivery, we gain understanding of the endocytic mechanisms and cell uptake for intelligent design of intracellular delivery.
On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates
Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial approaches have fused fluorescent proteins such as green fluorescent protein (GFP) to the viral protein-of-interest. In contrast, self-labeling enzyme tags such as the commercial SNAP-tag, a modified version of human O6-alkylguanine-DNA-alkyltransferase, covalently link synthetic ligands, which users can add on demand. The first two protocols presented here build on previously published protocols for fluorescent labeling in pulse-chase and quench-pulse-chase experiments; the combination of fluorescent labeling with advanced light microscopy visualizes the dynamic turnover of the SNAP-tagged viral protein in intact mammalian cells. A third protocol also outlines how to inspect cellular lysates microscopically for detergent-resistant assemblies of the labeled viral protein. These protocols showcase the flexibility of the SNAP-based labeling system for tracking a viral protein-of-interest in live cells, intact fixed cells, and cell lysates. Moreover, the protocols employ recently developed commercial microscopes (e.g., Airyscan microscopy) that balance resolution, speed, phototoxicity, photobleaching, and ease-of-use.
Immunology
Induced Germinal Center B Cell Culture System
The germinal center (GC) is the site where B cells undergo clonal expansion, affinity-based selection, and differentiation into memory B cells or plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells in vivo has been limited and no in vitro system has been available that resembles B cell reaction in GC. Here we describe the protocol for a culture system named “induced GC B (iGB) culture system” which can induce massive expansion of B cells that exhibit GC B cell-like phenotype, and thus it mimics the GC reaction. This protocol can be useful to elucidate the molecular mechanisms of GC B cell differentiation.
DNA Immunization Using in vivo Electroporation for Generating Monoclonal Antibodies Against Mouse IL-9R
Membrane proteins such as cytokine receptors and G protein-coupled receptors can be drug targets. Recently, we have generated specific monoclonal antibodies (mAbs) against the mouse IL-9 receptor (IL-9R) and found that IL-9R on memory B cells have critical roles in T-dependent immune response. So far, most antibodies against cell surface proteins have been generated by immunization of animals with recombinant proteins produced in Escherichia coli (E. coli) or peptides derived from the protein. However, such antibodies often fail to recognize native proteins on cell surfaces because these antigens lack posttranslational modification and natural protein conformations. To circumvent such problems, we have developed a mouse immunization method, the DNA-immunization utilizing hyaluronidase and E. coli GroEL. Herein, we report an application of the original mouse immunization method in rats to generate anti-mouse IL-9R mAbs which could react with the native form of mouse IL-9R on cell surfaces. Thus, we suggest that the DNA-immunization method is feasible for generating monoclonal antibodies against cell surface proteins in rats.
Microbiology
I Plate-based Assay for Studying How Fungal Volatile Compounds (VCs) Affect Plant Growth and Development and the Identification of VCs via SPME-GC-MS
Biogenic volatile compounds (VCs) mediate various types of crucial intra- and inter-species interactions in plants, animals, and microorganisms owing to their ability to travel through air, liquid, and porous soils. To study how VCs produced by Verticillium dahliae, a soilborne fungal pathogen, affect plant growth and development, we slightly modified a method previously used to study the effect of bacterial VCs on plant growth. The method involves culturing microbial cells and plants in I plate to allow only VC-mediated interaction. The modified protocol is simple to set up and produces reproducible results, facilitating studies on this poorly explored form of plant-fungal interactions. We also optimized conditions for extracting and identifying fungal VCs using solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS).
Analysis of Early Phase HIV-1 Replication and Integration Events by Using Real-time PCR
Upon entry into a host cell, the HIV-1 virus undergoes a series of critical early replication events including reverse transcription, nuclear import, and integration of its cDNA into the host genome. Molecular assays used to detect and analyze changes in HIV-1 early phase replication events are valuable tools in developing potential antiretroviral drugs, as well as studying the pathogenesis of HIV. Described here are the molecular assays utilized to detect and quantify HIV-1 early, intermediate, and late reverse transcription (RT) products. In addition to this, protocols for quantifying HIV-1 2-LTR circle DNA and proviral DNA after integration are also included. In these protocols, the optimized TaqMan Real-time PCR reagent is used to increase assay sensitivity and reproducibility. Furthermore, a nested PCR is applied to HIV-1 integration quantification with increased accuracy.
Molecular Biology
Preparing Single-cell DNA Library Using Nextera for Detection of CNV
Single-cell DNA sequencing is a powerful tool to evaluate the state of heterogeneity of heterogeneous tissues like cancer in a quantitative manner that bulk sequencing can never achieve. DOP-PCR (Degenerate Oligonucleotide-Primed Polymerase Chain Reaction), MDA (Multiple Displacement Amplification), MALBAC (Multiple Annealing and Looping-Based Amplification Cycles), LIANTI (Linear Amplification via Transposon Insertion) and TnBC (Transposon Barcoded) have been the primary choices to prepare single-cell libraries. TnBC library prep method is a simple and versatile methodology, to detect copy number variations or to obtain the absolute copy numbers of genes per cell.
Rapid Multiplexed Reduced Representation Bisulfite Sequencing Library Prep (rRRBS)
DNA methylation is a common mechanism of epigenetic regulation involved in transcriptional modulation and genome stability. With the evolution of next-generation sequencing technologies, establishing quantitative genome-wide DNA methylation profiles is becoming routine in many laboratories. However, many of these approaches take several days to accomplish and use subjective PCR methods to amplify sequencing libraries, which can induce amplification bias. Here we propose a rapid Reduced Representation Bisulfite Sequencing (rRRBS) protocol to minimize PCR amplification bias and reduce total time of multiplexed library construction. In this modified approach, the precise quantification of the final library amplification step is accomplished and monitored by qPCR, instead of using standard PCR and gel electrophoresis, to determine the appropriate number of cycles to perform. The main advantages of this rRRBS method are: i) Reduced amount of amplification enzyme used for library prep, ii) Reduced number of PCR cycles resulting in less PCR amplification bias, and iii) Preparation of quality multiplexed rRRBS libraries in only ~2 days.
Neuroscience
Assessing Olfaction Using Ultrasonic Vocalization Recordings in Mouse Pups with a Sono-olfactometer
Olfaction is the first sensory modality to develop during fetal life in mammals, and plays a key role in the various behaviors of neonates such as feeding and social interaction. Odorant cues (i.e., mother or predator scents) can trigger potentiation or inhibition of ultrasonic vocalizations (USV) emitted by pups following their isolation. Here, we report how USV are inhibited by olfactory cues using a sono-olfactometer that has been designed to quantify precisely olfaction in pups congenitally infected by cytomegalovirus. This olfactory-driven behavioral test assesses the USV emitted in presence of unfamiliar odorants such as citral scent or adult male mouse scent. We measure the number of USV emitted as an index of odorant detection during the three periods of the 5-min isolation time of the pup into the sono-olfactometer: first period without any odorant, second period with odorant exposure and last period with exhaust odorant. This protocol can be easily used to reveal olfactory deficits in pups with altered olfactory system due to toxic lesions or infectious diseases.
Plant Science
Top Starch Plating Method for the Efficient Cultivation of Unicellular Red Alga Cyanidioschyzon merolae
The unicellular red alga Cyanidioschyzon merolae has been used as a model photosynthetic eukaryote for various basic and applied studies, and several of these molecular genetics techniques have been reported. However, there are still improvements to be made concerning the plating method. The conventional plating method often generates diffuse colonies and single colonies cannot be easily isolated. To overcome these problems, we established a novel plating method for C. merolae, making use of melted cornstarch as the use of top agar plating in bacterial genetics. This method improved the formation of defined colonies in at least 4-fold higher efficiency than the conventional method, and made the handling procedure much easier than the previous method.
Quantification of Serotonin in Rice and Insect Pest and its Functional Analysis in Insects Using Artificial Diet Feeding
Rice is one of the world’s most important crops, but its production suffers from insect pests. Rice brown planthopper (BPH; Nilaparvata lugens Stål) and striped stem borer (SSB, Chilo suppressalis Walker) are the two most serious pests in rice production. We reported that serotonin is an essential mediator in the interaction between rice and insect. Here, we established a method for extraction and determination of serotonin in rice and BPH.
Stem Cell
Isolation of Neural Stem Cells from the Embryonic Mouse Hippocampus for in vitro Growth or Engraftment into a Host Tissue
For both stem cell research and treatment of the central nervous system disorders, neural stem/progenitor cells (NSPCs) represent an important breakthrough tool. In the expanded stem cell-based therapy use, NSPCs not only provide a powerful cell source for neural cell replacement but a useful model for developmental biology research. Despite numerous approaches were described for isolation of NSPCs from either fetal or adult brain, the main issue remains in extending cell survival following isolation. Here we provide a simple and affordable protocol for making viable NSPCs from the fetal mouse hippocampi, which are capable of maintaining the high viability in a 2D monolayer cell culture or generating 3D neuro-spheroids of cell aggregates. Further, we describe the detailed method for engraftment of embryonic NSPCs onto a host hippocampal tissue for promoting multilinear cell differentiation and maturation within endogenous environment. Our experimental data demonstrate that embryonic NSPCs isolated using this approach show the high viability (above 88%). Within a host tissue, these cells were capable of differentiating to the main neural subpopulations (principal neurons, oligodendrocytes, astroglia). Finally, NSPC-derived neurons demonstrated matured functional properties (electrophysiological activity), becoming functionally integrated into the host hippocampal circuits within a couple of weeks after engraftment.
Isolation of Multipotent Mesenchymal Stem Cells from Human Extraocular Muscle Tissue
Mesenchymal stem cells (MSCs) have attracted significant attention as potential therapeutic cells to treat various diseases ranging from tissue injuries, graft versus host disease, degenerative diseases and cancer. Since the initial discovery of MSCs in the bone marrow cells, MSCs have been successfully isolated from various adult and neo-natal tissues, albeit the procedures are often coupled with difficulties in harvesting tissue and produce low yield of cells, requiring extensive expansion in vitro. Here, we explored extra-ocular muscle tissues obtained from patients as a novel source of MSCs which express characteristic cell surface markers of MSCs and show multilineage differentiation potential with high proliferation capacity.
Cell Microencapsulation and Cryopreservation with Low Molecular Weight Hyaluronan and Dimethyl Sulfoxide
Cryopreservation is commonly used for the storage of cells, tissues, organs or 3D cell-based products using ultra-low temperatures, which involves the immersion in liquid nitrogen for their long-term preservation. The cryopreservation of several microencapsulated cells is usually performed by the slow freezing with the dimethyl sulfoxide (DMSO) as a cryoprotectant agent (CPA). In this study, we cryopreserved several microencapsulated cells with the natural, non-toxic low molecular-weight hyaluronan (LMW-HA) at 5% and DMSO 10% solution assessing cell viability and metabolic activity after thawing. The cryopreservation of microencapsulated D1 mesenchymal stem cells (D1MSC) and murine myoblast cells (C2C12) with the LMW-HA 5% presented similar outcomes after thawing compared to the DMSO solution, showing the low molecular weight hyaluronan as a natural, non-toxic CPA that can be used preventing the DMSO related adverse effects after the implantation of the cryopreserved cell-based products.