NDLI: Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B.

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Using the SERV Applet to Detect Tandem Repeats in DNA Sequences and to Predict Their Variability.

Immunohistochemistry of embryo sections.

Immunohistochemistry of whole-mount mouse embryos.

Infrared-guided laser stimulation of neurons in brain slices.

Preparation and loading of gelatin nanoparticles.

Intracellular trafficking studies using gold-encapsulated gelatin nanoparticles.

Cell Transfection and Analysis Using DNA-Loaded Gelatin Nanoparticles.

Preparation of biodegradable nanoparticles and their use in transfection.

Long-term, high-resolution imaging in the neocortex in vivo.

Development of Mammalian Cell Lines with lac Operator-Tagged Chromosomes.

Whole-Genome Amplification by Degenerate Oligonucleotide Primed PCR (DOP-PCR).

GenomePlex Whole-Genome Amplification.

Whole-Genome Amplification by Improved Primer Extension Preamplification PCR (I-PEP-PCR).

Whole-Genome Amplification by Adaptor-Ligation PCR of Randomly Sheared Genomic DNA (PRSG).

Whole-Genome Amplification by Single-Cell Comparative Genomic Hybridization PCR (SCOMP).

Using in ovo electroporation to transfect cells in avian somites.

Transfecting neural crest cells in avian embryos using in ovo electroporation.

Transfecting avian motor neurons and their axons using in ovo electroporation.

Tracking receptors by imaging single molecules.

Protein nanospheres for gene delivery.

The genus antirrhinum (snapdragon): a flowering plant model for evolution and development.

Planarians: a versatile and powerful model system for molecular studies of regeneration, adult stem cell regulation, aging, and behavior.

Pristionchus pacificus: A Genetic Model System for the Study of Evolutionary Developmental Biology and the Evolution of Complex Life-History Traits.

The American Wandering Spider Cupiennius salei.

Opossum (Monodelphis domestica): A Marsupial Development Model.

High-Throughput Cloning of Open Reading Frames (ORFs): Assembling Large Sets of Expression Constructs.

Cultivating antirrhinum.

Propagating antirrhinum.

Establishing and maintaining a colony of planarians.

Gene knockdown in planarians using RNA interference.

Live Imaging of Planarian Membrane Potential Using DiBAC4(3).

Isolation of pristionchus nematodes from beetles.

Assessment of the olfactory response to chemicals or bacteria in pristionchus nematodes.

Isolation of RNA and DNA from biofilm samples obtained by laser capture microdissection microscopy.

qRT-PCR of Microbial Biofilms.

Collection and fixation of spider embryos.

Whole-mount in situ hybridization of spider embryos.

Detection of Cell Death in Spider Embryos Using TUNEL.

Gene Silencing via Embryonic RNAi in Spider Embryos.

Detection of Cell Proliferation in Spider Embryos Using BrdU Labeling.

Dissecting spider embryos for light microscopy.

Basic Maintenance and Breeding of the Opossum Monodelphis domestica.

Harvesting monodelphis embryos.

Monodelphis whole-embryo culture.

Whole-mount in situ hybridization in monodelphis embryos.

Tomato (Solanum lycopersicum): A Model Fruit-Bearing Crop.

Comb jellies (ctenophora): a model for Basal metazoan evolution and development.

Astyanax mexicanus, the Blind Mexican Cave Fish: A Model for Studies in Development and Morphology.

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 1: Coating Glass Slides with Amino Silane.

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 2: Preparing Bacterial Cultures in a 96-Well Format.

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 3: Isolating DNA Plasmids in a 96-Well Plate Format.

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 4: DNA Biotinylation, Precipitation, and Arraying of Samples.

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 5: Expressing Proteins on NAPPA Slides.

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 6: Detecting Proteins on NAPPA Slides.

Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 7: Detecting DNA on NAPPA Slides.

Isolation, culture, and differentiation of progenitor cells from the central nervous system.

Preparing recombinant gonad organ cultures.

Sex chromatin staining in amnion cells.

How to grow tomatoes.

Crossing Tomato Plants

Grafting tomato plants.

Tomato transformation.

Mnemiopsis leidyi Spawning and Embryo Collection.

Ctenophore whole-mount antibody staining.

Ctenophore whole-mount in situ hybridization.

Ctenophore tissue preparation and extraction of DNA.

Ctenophore tissue preparation and extraction of RNA.

Determining the Sex of Adult Astyanax mexicanus.

Breeding Astyanax mexicanus through Natural Spawning.

In Vitro Fertilization of Astyanax mexicanus.

Handling Astyanax mexicanus Eggs and Fry.

The Demosponge Amphimedon queenslandica: Reconstructing the Ancestral Metazoan Genome and Deciphering the Origin of Animal Multicellularity.

Dictyostelium discoideum: The Social Ameba.

The Two-Spotted Cricket Gryllus bimaculatus: An Emerging Model for Developmental and Regeneration Studies.

The Dogfish Scyliorhinus canicula: A Reference in Jawed Vertebrates.

Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions: General Guidelines.

Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.

Gene Silencing by Injection and Electroporation of dsRNA in Avian Embryos.

Isolation of amphimedon developmental material.

Whole-mount in situ hybridization in amphimedon.

Analysis of cell movement in amphimedon embryos by injection of fluorescent tracers.

Genotyping individual amphimedon embryos, larvae, and adults.

Growth and maintenance of dictyostelium cells.

Multicellular Development of Dictyostelium

Making permanent stocks of dictyostelium.

Transformation of dictyostelium with plasmid DNA by calcium phosphate precipitation.

Transformation of dictyostelium with plasmid DNA by electroporation.

Selection of dictyostelium transformants.

Extraction of DNA from dictyostelium.

Extraction of RNA from dictyostelium.

Cultures of cerebellar granule neurons.

Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 1: Coexpression of Query Protein on NAPPA Slides.

Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 2: Detection of Query Proteins on NAPPA Slides.

Collection, cryopreservation, storage, and revitalization of transgenic mouse embryos.

Chicken erythrocyte histone octamer preparation.

Salt gradient dialysis reconstitution of nucleosomes.

Drosophila RNA Interference (RNAi) Using a Gal-4 Inducible Transgene Vector.

PCR Mutagenesis by Overlap Extension and Gene SOE.

Preparation of Double-Stranded RNA for Drosophila RNA Interference (RNAi).

Collection of Drosophila Embryos for RNA Interference (RNAi).

Injection of dsRNA into Drosophila Embryos for RNA Interference (RNAi).

Mounting live cells attached to coverslips for microscopy.

Labeling Acetylcholine Receptors in Live Cells Using Rhodamine {alpha}-Bungarotoxin for Imaging.

Labeling the components of the plasma membrane with fluorescent dyes for imaging.

Labeling the Golgi Apparatus with BODIPY-FL-Ceramide (C5-DMB-ceramide) for Imaging.

Calculation of Spot Reliability Evaluation Scores (SRED) for DNA Microarray Data.

Calculation of absolute expression values for DNA microarray data.

In vitro histone methyltransferase assay.

Stable Isotope Labeling of Mammals (SILAM).

Fixation, embedding, and sectioning of plant tissues.

Preparation and hydrolysis of digoxygenin-labeled probes for in situ hybridization of plant tissues.

Nonradioactive in situ hybridization of RNA probes to sections of plant tissues.

Whole-mount in situ hybridization of RNA probes to plant tissues.

Immunohistochemistry on sections of plant tissues using enzyme-coupled avidin-biotin complex.

Immunohistochemistry on sections of plant tissues using alkaline-phosphatase-coupled secondary antibody.

Whole-mount fluorescence immunocytochemistry on Xenopus embryos.

Media for mounting fixed cells on microscope slides.

Streamlined gene assembly PCR.

Cell-Penetrating Peptide-Mediated Delivery of Peptide Nucleic Acid (PNA) Oligomers.

Labeling lysosomes in live cells with fluorescent dyes for imaging.

Vital stain for plant cytoplasm.

Preparation of Arabidopsis tissue sections of fixed material.

Phenotypic analysis of Arabidopsis mutants: quantitative analysis of root growth.

Computer-based analysis of the optokinetic response in zebrafish larvae.

Phenotypic analysis of Arabidopsis mutants: hypocotyl length.

DNA Immunoprecipitation (DIP) for the Determination of DNA-Binding Specificity.

Electroretinogram (ERG) Measurements in Larval Zebrafish.

Methylated CpG Island Amplification and Microarray (MCAM) for High-Throughput Analysis of DNA Methylation.

Photoactivation cell labeling for cell tracing in avian development.

Culture of HEK293-EBNA1 Cells for Production of Recombinant Proteins.

Transfection of HEK293-EBNA1 Cells in Suspension with Linear PEI for Production of Recombinant Proteins.

Transfection of Adherent HEK293-EBNA1 Cells in a Six-Well Plate with Branched PEI for Production of Recombinant Proteins.

Transfection of HEK293-EBNA1 Cells in Suspension with 293fectin for Production of Recombinant Proteins.

Purification of his-tagged proteins using fractogel-cobalt.

Strategies for overcoming PCR inhibition.

Genetic Analysis of Arabidopsis Mutants

Sectioning of brain tissues.

Choosing a method for phylogenetic prediction.

Perfusion of brain tissues with fixative.

Dissection of rat brains.

Retroviral vector production by transient transfection.

Generation of stable vector-producing cells for retroviral vectors.

Transduction of cell lines by retroviral vectors.

Transduction of primary hematopoietic cells by retroviral vectors.

Scanning Electron Microscopy (SEM) of Plant Tissues.

Labeling the nucleus with fluorescent dyes for imaging.

Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (ENU).

Desorption Electrospray Ionization (DESI) Analysis of Intact Proteins/Oligopeptides.

Desorption Electrospray Ionization (DESI) Analysis of Tryptic Digests/Peptides.

In Situ Desorption Electrospray Ionization (DESI) Analysis of Tissue Sections.

In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).

Retroviral vectors for gene transfer.

Maximum parsimony method for phylogenetic prediction.

Distance methods for phylogenetic prediction.

The maximum likelihood approach for phylogenetic prediction.

Desorption Electrospray Ionization: Proteomics Studies by a Method That Bridges ESI and MALDI.

Making solutions from dry chemicals.

Making solutions from hydrated compounds.

Making and diluting stock solutions.

Alkaline Phosphatase Treatment of Phosphopeptides: In-Solution Dephosphorylation prior to MALDI-MS Analysis.

Alkaline Phosphatase Treatment of Phosphopeptides: In-Solution Dephosphorylation after MALDI-MS Analysis.

Alkaline Phosphatase Treatment of Phosphopeptides: On-Probe Dephosphorylation after MALDI-MS Analysis.

Karyotyping mouse cells.

Dissection of larval salivary glands and polytene chromosome preparation.

Mapping protein distributions on polytene chromosomes by immunostaining.

Phenotypic analysis of Arabidopsis mutants: gibberellin/abscisic acid/paclobutrazol hormone response.

Hematoxylin and eosin staining of tissue and cell sections.

Cutting sections of paraffin-embedded tissues.

Preparation of slides and coverslips for microscopy.

Paraffin embedding tissue samples for sectioning.

Decalcifying tissues for paraffin embedding.

Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): I. CIP Treatment of Samples and Tailing Reaction with Terminal Transferase.

Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): II. Second-Strand Synthesis and In Vitro Transcription.

Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): III. Sample Purification.

Fixation and permeabilization of cells and tissues.

Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): Overview.

A test of the markov model of evolution in proteins.

Comparison of the PAM and BLOSUM Amino Acid Substitution Matrices.

Studies of varying alignment algorithm, amino Acid scoring matrix, and gap penalties.

Reduction of contamination of epithelial cultures by fibroblasts.

In-gel s-pyridylethylation of gel-resolved proteins: whole gel method.

In-gel s-pyridylethylation of gel-resolved proteins: individual spot method.

Phenotypic analysis of Arabidopsis mutants: auxin hormone response.

Phenotypic analysis of Arabidopsis mutants: ethylene hormone response.

Phenotypic analysis of Arabidopsis mutants: brassinosteroid hormone response.

Isolation and Whole-Cell Patch Clamping of Arabidopsis Guard Cell Protoplasts

Immunogold Staining of Epoxy Resin Sections for Transmission Electron Microscopy (TEM).

Immunogold Staining of London Resin (LR) White Sections for Transmission Electron Microscopy (TEM).

Immunogold Staining Following Freeze Substitution and Low Temperature Embedding after Chemical Fixation or after Cryoimmobilization for Transmission Electron Microscopy (TEM).

Immunogold Staining of Ultrathin Thawed Cryosections for Transmission Electron Microscopy (TEM).

High-resolution fluorescent in situ hybridization of Drosophila embryos and tissues.

Using PAM Matrices in Sequence Alignments.

Using BLOSUM in Sequence Alignments.

Using gaps and gap penalties to optimize pairwise sequence alignments.

Copy Number Variation Detection via High-Density SNP Genotyping.

Ultrastructural immunochemistry.

Isolation and culture of mouse keratinocytes.

Identification of differential genes by suppression subtractive hybridization: I. Preparation of subtracted cDNA or genomic DNA library.

Identification of Differential Genes by Suppression Subtractive Hybridization: II. Subtractive Hybridization.

Identification of Differential Genes by Suppression Subtractive Hybridization: III. PCR Amplification of Differentially Presented DNAs.

Identification of Differential Genes by Suppression Subtractive Hybridization: IV. Mirror Orientation Selection (MOS).

Identification of Differential Genes by Suppression Subtractive Hybridization: V. PCR-Based DNA Dot Blot.

Identification of Differential Genes by Suppression Subtractive Hybridization: VI. Differential Hybridization with Tester and Driver DNA Probes.

Phenotypic analysis of Arabidopsis mutants: flowering time.

Browsing HapMap Data Using the Genome Browser.

Generating HapMap Data Text Reports Using the Genome Browser.

Manipulating HapMap Data Using HaploView.

Retrieving HapMap Data Using HapMart.

Retrieving HapMap Data via Bulk Download.

In Vitro Screening for Regulated Transcription Factors with Differential Display of DNA-Binding Proteins (DDDP).

Generation of Driver and Reporter Constructs for the GAL4 Expression System in Drosophila.

High-throughput paraffin section in situ hybridization and dual immunohistochemistry on mouse tissues.

Identification of differential genes by suppression subtractive hybridization: an overview.

The GAL4 System: A Versatile Toolkit for Gene Expression in Drosophila.

Nuclear stains.

Anionic counterstains.

Inoculation and passaging of Mammalian monolayer cell cultures.

Quantifying Protein by Bicinchoninic Acid

The PAS Reaction for Staining Cell Walls.

Phenotypic analysis of Arabidopsis mutants: trypan blue stain for fungi, oomycetes, and dead plant cells.

Cryosectioning tissues.

Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version A.

Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B.

Phage-display methodology for the study of protein-protein interactions: stage 1, generating libraries.

Phage-display methodology for the study of protein-protein interactions: stage 2, panning.

Phage-display methodology for the study of protein-protein interactions: stage 3, binding analysis.

Analyzing DNA Replication I: Labeling Animals, Tissues, and Cells with Bromodeoxyuridine (BrdU).

Analyzing DNA Replication II: Fixation and Processing of Tissues and Cells Labeled with Bromodeoxyuridine (BrdU).

Analyzing DNA Replication III: Antibody Labeling of Incorporated Bromodeoxyuridine (BrdU) in Tissues and Cells.

In vitro assays for the extracellular matrix protein-regulated extravasation process.

Whole mount RNA in situ hybridization on zebrafish embryos: probe synthesis.

Whole mount RNA in situ hybridization on zebrafish embryos: hybridization.

Whole mount RNA in situ hybridization on zebrafish embryos: mounting.

Isolation of centrosomes from cultured Mammalian cells.

Imaging Calcium Dynamics Using Targeted Recombinant Aequorins

Phage-display methodology for the study of protein-protein interactions: overview.

Antibody staining of Drosophila tissues--helpful hints.

Preparation of baskets for Drosophila egg collections, treatments, and incubations.

Estimation of Free Thiols and Disulfide Bonds Using Ellman's Reagent.

Subbing slides.

The nissl stain: a stain for cell bodies in brain sections.

Analyzing glucose phosphate isomerase isozymes in chimeric mouse tissues by electrophoresis.

Clearing plant tissue for observation of vascular strands.

Phenotypic analysis of Arabidopsis mutants: heavy metal stress.

Streamlined protocol for antibody staining of large numbers of samples in Drosophila.

Human lymphocyte culture and chromosome analysis.

Noninvasive human nuclear transfer with embryonic stem cells.

Preparation of mouse embryonic fibroblast feeder cells for human embryonic stem cell culture.

Culturing human embryonic stem cells with mouse embryonic fibroblast feeder cells.

Preparation of human foreskin fibroblasts for human embryonic stem cell culture.

Culturing human embryonic stem cells in feeder-free conditions.

Method for single-cell sorting and expansion of genetically modified human embryonic stem cells.

DNA methylation analysis of human imprinted Loci by bisulfite genomic sequencing.

Metaphase spreads and spectral karyotyping of human embryonic stem cells.

Human germ cell lineage differentiation from embryonic stem cells.

Coimmunoprecipitation (co-IP) of Nuclear Proteins and Chromatin Immunoprecipitation (ChIP) from Arabidopsis.

Year: 2007 Volume: 2007

Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Tiscornia, Gustavo Singer, Oded Verma, Inder M.
Abstract	INTRODUCTIONThis protocol describes the use of lentiviral vectors to deliver small interfering RNA (siRNA)-mediated silencing cassettes. The combination of these two technologies allows for the development of a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo. It combines the specificity of RNA interference with the versatility of lentiviral vectors to stably transduce a wide range of cell types. In this method, a small hairpin (shRNA) is cloned initially into an entry vector (pENTR/U6) immediately downstream from an hU6 promoter. The silencing cassette is flanked by recombination sites from bacteriophage λ (attL1 and attL2). Once an effective shRNA is obtained, it can be transferred to the destination vector. The destination vector is a lentiviral vector carrying a marker (green fluorescent protein [GFP] or a selection marker) with a destination cassette cloned upstream of the marker (attR1 and attR2 flanking a ccdB toxic gene). Thus, the silencing cassette can be transferred from the entry vector to the destination vector in a simple Gateway LR cloning reaction. The positioning of the silencing cassette upstream of the marker expression cassette avoids down-regulation of the marker.
File Format	HTM / HTML
ISSN	19403402
Journal	Cold Spring Harbor Protocols
Volume Number	2008
e-ISSN	15596095
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2008-08-01
Publisher Place	United States
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Discipline Clinical Laboratory Techniques
Content Type	Text
Resource Type	Article
Subject	Biochemistry, Genetics and Molecular Biology

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