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丁香实验推荐阅读
Strategies to Insulate Lentiviral Vector-Expressed Transgenes

Lentiviruses are capable of infecting many cells irrespective of their cycling status, stably inserting DNA copies of the viral RNA genomes into host chromosomes. This property has led to the development of lentiviral vectors for high-efficiency gene transfer to a wide variety of cell typ ...

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Flexibility in Cell Targeting by Pseudotyping Lentiviral Vectors

Lentiviral vectors have become an important research tool and have just entered into clinical trials. As wild-type lentiviruses engage specific receptors that have limited tropism, most investigators have replaced the endogenous envelope glycoprotein with an alternative en ...

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New Protocol for Lentiviral Vector Mass Production

Multiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugat ...

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Lentiviral Vector-Mediated Gene Transfer and RNA Silencing Technology in Neuronal Dysfunctions

Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animal models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. L ...

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Lentiviral Vector Transduction of Fetal Mesenchymal Stem Cells

Human fetal mesenchymal stem cells (hfMSC) demonstrate extensive expansion and differentiation capacities and are hence being studied for use in stem cell therapeutics, including gene delivery. With advanced prenatal diagnosis, fetal gene therapy represents an additional av ...

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The RNA Silencing Technology Applied by Lentiviral Vectors in Oncology

Since the discovery of RNA interference (RNAi) in Caenorhabditis elegans in 1998, this mechanism has been found to be conserved in a wide variety of species, including insects, plants, and mammals. In mammals, small (or short) interfering RNA (siRNA) or short hairpin RNA (shRNA) can be expressed by u ...

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Lentiviral Vector-Mediated Expression of pre-miRNAs and AntagomiRs

Micro (mi)RNAs are highly conserved small regulatory RNAs, which regulate gene expression by hybridization to specific binding sites in the 3′untranslated region (UTR) of many mRNAs. Upon miRNA-guided recruitment of a multiprotein complex, target mRNAs are either degraded or their t ...

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Lentiviral Vector Engineering for Anti-HIV RNAi Gene Therapy

RNA interference or RNAi-based gene therapy for the treatment of HIV-1 infection has recently emerged as a highly effective antiviral approach. The lentiviral vector system is a good candidate for the expression of antiviral short hairpin RNAs (shRNA) in HIV-susceptible cells. However, ...

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Lentivirus-Expressed siRNA Vectors Against Alzheimer Disease

Amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease, and the accumulation of APP products ultimately leads to the familiar histopathological and clinical manifestations associated with this most common form of dementia. A protein that has b ...

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Immunoelectron Microscopy of Chemically Fixed Developing Plant Embryos

The hydrophobic plant cell wall, large acidic central vacuole, diverse secondary compounds, intercellular airspaces, and rigid starch granules present obstacles to ultrastructure preservation and specimen sectioning. We describe modifications of fixation and embeddi ...

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Monitoring Rapid Endocytosis in the Electron Microscope via Photoconversion of Vesicles Fluorescently Labeled with FM1-43

Cells communicate via endo- and exocytosis with their environment and neighboring cells. At synapses of the nervous system, fast exocytosis is coupled to fast endocytosis, which forms the basis for neurotransmitter release. The introduction of the unique fluorescent FM dyes allowed t ...

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Horseradish Peroxidase as a Reporter Gene and as a Cell-Organelle-Specific Marker in Correlative Light-Electron Microscopy

A modern electron microscopic approach to the investigation of the structural organization of proteins and subcellular structures demands the use of molecular genetic techniques. The successful implementation of genetic techniques is closely tied to a reporter gene such as the gr ...

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Immunogold Labelling for Scanning Electron Microscopy

Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo or real 3D at magnifications of anything from ...

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Localization of rDNA at Nucleolar Structural Components by Immunoelectron Microscopy

Fluorescence in situ hybridization (FISH) shows that DNA encoding ribosomal RNA cistron (rDNA) is localized to small speckles scattered in the nucleolus and the nucleolus-associated chromatin (NAC). This technique cannot however precisely locate rDNA in the nucleolar ultrastr ...

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Freeze-Etch Electron Tomography for the Plasma Membrane Interface

To visualize the basal or apical cytoplasmic surface just beneath the plasma membrane, we developed two different methods (“unroof” and “rip-off”). The immunoreplica technique for “unroof” and “rip-off” sample preparation that will be presented in this chapter can determine the distri ...

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Serial Section Immunoelectron Microscopy of Algal Cells

Electron microscopy when combined with immunogold labeling provides a 2D image of intracellular protein distribution. Cells are however 3D structures. We describe a method of serial section immunogold electron microscopy that allows a 3D cellular image to be reconstructed from a ser ...

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Isolation, Identification, and Validation of Microtubule-Associated Proteins from Drosophila Embryos

The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert ...

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How to Measure Microtubule Dynamics

Microtubules are one of the most spectacular features in the cell: long, fairly rigid tubules that provide physical strength while at the same time serving as tracks of the intracellular transport network. In addition, they are the main constituents of the cell division machinery, and guide ax ...

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Laser Ablation of the Microtubule Cytoskeleton: Setting Up and Working with an Ablation System

Laser ablation is a powerful tool that can be used to study a variety of biological mechanisms. Microscopes with high optical performances are nowadays available, and lasers that could be used to perform ablations have become accessible to every laboratory. Setting up a laser ablation system ...

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Investigating Microtubule Dynamic Instability Using Microtubule-Targeting Agents

This chapter presents protocols not only used to investigate the effect of microtubule-targeting agents on microtubule dynamic instability parameters, but also their impact on loading +TIPs at microtubule plus ends. These agents can be considered either as drugs to analyze their pha ...

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