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siRNA/shRNA gene knockdown

Introduction RNA interference (RNAi) is the process by which expression of a target gene is effectively silenced or knocked down by the selective inactivation of its corresponding mRNA by double-stranded RNA (dsRNA). RNAi is activated by dsRNA species delivered to the cytoplasm of cells. The slicing mechanisms can either lead to the degradation of a target mRNA, as induced by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs). Both siRNA and shRNA can be used for gene knockdown, but the mechanisms are different. In cells, most of the siRNAs are accumulated in the cytompasm, upon introduction to the cell, the long dsRNAs form a complex with Dicer. The cleaved products are then incorporated into the RISC, which is composed of Argonaute-2 (Ago-2), Dicer, and TAR-RNA-binding protein (TRBP). The RNA duplex is separated, and one strand is removed from the complex. Fig.1 Mechanism of RNAi induced gene silencing. shRNAs are synthesized in the nucleus of transfected/transduced cells and form hairpin structures that consist of a stem region of paired antisense and sense strands connected by unpaired nucleoti...

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Construction And Packaging of Adenovirus Vectors

Introduction The adenovirus genome is a linear, 36-Kb double-stranded DNA (dsDNA) molecule containing multiple, heavily spliced transcripts. At either end of the genome are inverted terminal repeats (ITRs). Genes are divided into early (E1-E4) and late (L1-L5) transcripts. As a cell expression vectors, adenoviral vectors have a number of distinct advantages: its genome is relatively easy to manipulate by recombinant DNA techniques, and the adenovirus vectors are relatively stable, can grow to high titers, and can transduce a variety of cell types in cell culture in vivo. Vectors can be designed that are either relocation competent or replication defective, and in the latter case, they are highly efficient at delivering and expressing genes in mammalian cells without killing the cells. Procedures: 1. Cloning a Gene of Interest into Shuttle Vectors. 2. Transfer the expression cassette into the Adenovirus Vector. 3. Producing first generation, Low-titer Virus Stock. 4. Virus Amplification and Concentration. 5. Titration. Why Leading Biology? Working with us, you will get stability, and it means...

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Construction And Packaging of Lentiviral Vectors

Introduction Lentiviruses are a subset of retroviruses and are derived from the human immunodeficiency virus (HIV-1) with most of the viral genes removed, only contain the Long terminal repeats (LTRs) and the packaging sequence (Psi-sequence), thus they have the ability to transduce both dividing and non-dividing cells without significant immune responses. They also enable long-term transgene expression by integrating stably into the host genome. To obtain a lentiviral vector, a target gene is cloned into a vector sequence which is flanked by LTRs and the Psi-sequence. The LTRs are necessary to integrate the therapeutic gene into the genome of the target cell, and the Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the target gene in virions. Procedures: 1. Construction of a lentivirus overexpressing plasmid vector. 2. Interference of lentivirus in the construction of plasmid vector. 3. Packaging and concentrated purification of lentiviral vectors. Why Leading Biology? Working with us, you will get stability, and it means a reliable partner to help streamline your R&...

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Recombinant Plasmid Construction and Gene Targeting

Recombinant Plasmid Construction Recombinant Plasmids are the plasmid molecules which contain the target DNA fragment, and the process of introducing genes into a vector to form a new DNA molecule which can be replicated in a host cell is called Recombinant Plasmid Construction (Recombinant Cloning), the word “Recombinant” means that two different strands of DNA that would not normally occur together are combined and the cloning means creating multiple copies of genetically identical organisms. There are five main steps in recombinant plasmid construction: 1. Target DNA extraction and PCR amplification. 2. PCR product purification. 3. Using restriction enzymes cut both plasmids and PCR products. 4. Connect the two loose pieces of DNA together using DNA ligase, form the modified plasmid. 5. Transform the modified plasmid into bacteria. Gene Targeting Gene targeting is the process of disrupting or mutating a specific genetic locus in embryonic stem (ES) cells, usually with the intention of making knock-out or knock-in mice. This method can be used for many proposes, including delete or inse...

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Cell Functional Analysis

Introduction Cell functional analysis is an important technology in studying the physiologic control of cell function, the assays could reflect bioenergetics, metabolism, cell signaling and biosynthetic/secretory function. The cell functional analysis including: 1. Assays of the molecules and chemicals that would affect the cell function, including lactate production, glucose production and uptake, oxygen consumption, NADH, CO2 production, reductive state of cytochrome C, ROS production, mitochondrial membrane potential, ATP content, calcium and potassium metabolism. 2. Determination of protein kinase A and C activates. 3. Cell morphological and functional characterization. Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get stability, and it means a reliable partner to help streamline your R&D process. Working with us, you will get the guaranteed service to accommodate your requirements. ...

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FRET Analysis

Introduction Fluorescence resonance energy transfer (FRET) is a distance-dependent physical process by which energy is transferred nonradiative from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole-dipole coupling. The theory supporting energy transfer is based on the concept of treating an excited fluorophore as an oscillating dipole that can undergo an energy exchange with a second dipole having a similar resonance frequency. Unlike radiative mechanisms, resonance energy transfer can yield a significant amount of structural information concerning the donor-acceptor pair. FRET can be an accurate measurement of molecular proximity at angstrom distances and highly efficient if the donor and acceptor are positioned within the Förster radius. FRET now is widely used in cell biology, among all the applications, there are five general FRET approaches have proven particularly useful: 1. Sensitized Emission - Two-channel imaging using an algorithm that corrects for excitation and emission crosstalk 2. Acceptor Photobleaching - Also known as donor d...

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Vector Construction, transfection&co-localization analysis

Vector Construction Plasmid vector construction is an essential step for molecular microbiology. Manipulation of the fungal genome to express genes to activate secondary metabolite production often requires creation of plasmid constructs in a reiterative fashion. Plasmids were recognized as a convenient means to package foreign DNA for molecular cloning strategies allowing for mass production of certain genes and encoding proteins in a desired host. Thus, gene cloning and vector construction are routine procedures in genomic studies. Fig. 1 Construction of genomic vectors for gene expression Procedures: 1. Target gene determination and primer design. 2. PCR amplification. 3. DNA fragments mixed with TAR-cloning vector. 4. Introduce the vector into E. Coli Transfection Transfection is the method that introducing naked or purified nucleic acids into eukaryotic cells, and the cells that have incorporated the foreign DNA are called transfectants. There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on physical treatment (electroporation, cell squeezing, nanoparticle...

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Agarose Cell Migration Assay

Introduction The Under-Agarose assay is a useful method for observing the response of a cell population to one or more chemoattractant sources. The behavior of individual migrating cells can be studied by modifying the assay for video microscopy. The assay works well with freshly-isolated human neutrophils. Monocytes can also be observed to migrate but require more time. The procedure makes use of a tissue culture dish filled with an agarose mixture. Chemoattractant diffuses from wells in the agarose to form a gradient. Cells in nearby wells can be monitored as they migrate in the direction of the chemoattractant source. Procedures: 1. Preparation of agarose filled plates. 2. Cut the wells. 3. Performing the assay. 4. Fixing and Staining. 5. Detection and Analyzing. Why Leading Biology? Working with us, you will get stability, and it means a reliable partner to help streamline your R&D process. Working with us, you will get the guaranteed service to accommodate your requirements. · Vigorous quality control system to ensure the requir...

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Cell Crawling Slides Preparation

Introduction Cell crawling is of primary importance for fundamental biological processes such as embryonic development, wound healing, inflammation, and cancer metastasis. Motion of cells along extracellular substrates of matrices requires a fairly small force which applied to the cell to move it against the friction between the cell surface and the surrounding liquid, since there are no external fields or factors that would generate such forces, the force must be produced by the cell itself. We could prepare slides to buy using cell crawling, the procedure is like below: 1. Put sterilized coverslips into the cell culture wells 2. Add gelatin-coating solution and incubate the slides with the cells. 3. Remove the solution, and air dry the coverslips. Why Leading Biology? At Leading Biology, we custom protein purification design for every single protein to ensure the production and recovery rate as high as possible. Working with us, you will get stability, and it means a reliable partner to help streamline your R&D process. Working with us, you will get the gua...

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PARP Assays

Introduction The Poly (ADP-ribose) Polymerases (PARPs) are an emerging family of enzymes that detects and signals DNA damage to repair mechanisms. It’s activated in response to single-strand DNA breaks and subsequently attaches to regions of damaged DNA. PARP then catalyzes the synthesis of poly (ADP-ribose) (PAR) chains on itself and adjacent nuclear proteins. PAR chains also serve as a signal for other DNA repair enzymes. After the DNA is repaired, the PAR chains are degraded by PAR glycohydrolase (PARG). However, the PAR generates ADP-ribose (ADPr) modifications onto target proteins using NAD+ as a substrate. Extensive DNA damage can lead to the depletion of intracellular NAD+ and energy depletion-induced necrosis. PARP Universal Colorimetric Assay is one of the methods which could be used to analyze the PARP. This method measures PARP in cells and tissues by detecting the incorporation of biotinylated poly (ADP-ribose) (PAR) onto histone proteins in a 96-strip well microplate format, it’s useful for testing whether DNA is damaged, if damaged DNA is from non-apoptotic cells, or the effectiveness of PARP inhibitors. ...

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