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  • Genome Editing: Which Should I Choose, TALEN or CRISPR?
    Introduction Genomic editing - the ability to make specific changes to targeted genomic sites - is critical for biological and medical researchers (Bogdanove and Voytas, 2011; van der Voystet et al., 2013). Two genomic editing techniques have recently emerged using bacterial systems for plant pathogenesis or adaptive immunity: TALEN (transcriptionactivator-like nuclease) and CRISPR (clustering, periodic intervals, and short Parindro repeating separately). TALEN and CRISPR both use endoenzymes to initiate double-stranded fractures (DSBs) in almost any genome target sequence and are used in many applications, including gene knock-off, genetically modified knock-on, gene markers, and gene defect correction. While both technologies are popular, the decision to choose one technology over another is not always clear, and Gene Copoeia customers occasionally ask us for advice. In this technical note, we compare the advantages and disadvantages of TALEN and CRISPR in order to provide customers with sufficient information to choose which technology to use to order reagents from us. Genome editing Genome editing starts with efficient DSB generation in the target DNA (Figu...
  • TALEN - Transcription activator-like effector nuclease
    TALENs have recently swooped down and snatched the genome editing monopoly from ZFNs. TALENs works almost the same way as ZFNs. TALENs is a fusion of transcriptionactivator-like (TAL) proteins with FokI nucleases. The TAL protein consists of 33-34 amino acid repeat patterns, with two variable positions, and has a strong ability to identify specific nucleotides. Specific cutting of the genome can be achieved by assembling these TLL arrays and incorporating them into FokI nucleases. When two TALENs are combined and meet, the FokI domain induces a double-stranded break, which can cause the gene to become inactive or can be used to insert the DNA of interest. Tallun is more specific than ZFN, and the monomer has no cross-reaction problems that plague ZFN researchers. This simplicity facilitates automation of THE TALEN structure. On the downside, because each nucleotide requires a TAL, TALEN is larger and more difficult to deliver than ZFN. Transcriptionactivator-like nuclease technology utilizes artificially restricted enzymes produced by integrating the TAL effectr DNA binding domain into the DNA cleavage domain. Restrictive enzymes are enzymes that cut DNA strand...
  • Transgenic Plants Construction
    1. Introduction Transgenic plants are plants that have had their genomes modified through genetic engineering techniques either by the addition of a foreign gene or removal of a certain detrimental gene. A foreign gene inserted into a plant can be of a different species or even kingdom. The first transgenic plant was developed through the insertion of nptII bacterial antibiotic resistance gene into tobacco. Since then, with the rapid development in plant molecular biology and genetic engineering technology, a wide variety of transgenic plants with important agronomic traits such as pest resistance and drought tolerance have been developed, ranging from dicots to monocots that are amenable to genetic modifications. The main purpose in the production of transgenic plants is to produce crops, which have ideal traits, quality, and high yield. Besides being beneficial to the agriculture sector, the plants are found to be able to act as the factory for pharmaceutical protein production. 2. Application of transgenic plants 2.1. Resistance to biotic or abiotic stresses Biotic stresses occur naturally as a result of stress exerted from other living...
  • CRISPR-CAS Technology
    Q: What are genome editing and CRISPR-Cas9?  Genome editing (also known as gene editing) is a set of techniques that enable scientists to alter the DNA of an organism. These techniques allow genetic material to be added, removed, or altered at specific locations in the genome. Several genome editing methods have been developed. The most recent one is called CRISPR-Cas9, which is short aggregation regular interval short backtracking repeat and CRISPR-related protein 9. The CRISPR-Cas9 system has caused a stir in the scientific community because it is faster, cheaper, more accurate and more efficient than other existing genome editing methods.   CRISPR-Cas9 is adapted from a genome editing system that naturally exists in bacteria. Bacteria capture DNA fragments from invading viruses and use them to create fragments of DNA called CRISPR arrays. THE CRISPR array allows bacteria to "remember" viruses (or closely related viruses). If the virus attacks again, bacteria produce RNA fragments from the CRISPR array to target the virus's DNA. Bacteria then use Cas9 or similar enzymes to isolate DNA, which disables the virus.   Th...
  • Microorganisms Gene Modification Services
    Current Use of Genetically Modified Organisms Agricultural plants are one of the most commonly cited examples of genetically modified organisms (GMOs). Some of the benefits of genetic engineering in agriculture are to increase crop yields, reduce food or drug production costs, reduce the need for pesticides, improve nutritional content and food quality, fight pests and diseases, enhance food security, and health benefits for the benefit of the world's growing population. Progress has been made in the development of crops that mature faster and are resistant to aluminum, boron, salt, drought, frost and other environmental stressfactors so that plants can grow under conditions that would not otherwise grow (table 1, Takeda - Matsuoka, 2008). Other applications include the production of non-protein (bioplastic) or non-industrial (decorative plant) products. Some animals have also been genetically engineered to increase yields and reduce vulnerability. For example, salmon are designed to grow more (Figure 1) and mature faster (Table 1), and cattle have increased resistance to mad cow disease (U.S. Department of Energy, 2007).  Potential GMO Applications ...
  • HE Stain
    Hematoxylin and eosin stain (HE stain) is one of the widely used stains in histology. This stain produces colors different tissue structures, which would otherwise be transparent, so that you can get a detailed view of the tissue. As its name suggests, H&E stain makes use of a combination of two dyes: haematoxylin and eosin. Haematoxylin can be considered as a basic dye (general formula for basic dyes is:dye+ Cl-) which is dark blue or violet, it binds to basophilic substances (such DNA/RNA - which are acidic and negatively charged); and Eosin is a red or pink stain that is Acidic / Negative. It binds to acidophilic substances such as positively charged amino acid side chains (e.g. lysine, arginine).
  • Fluorescent Protein Transfection
    The Fluorescent Protein contained vectors can be used as transfection markers and to label living cells. They do not contain an MCS. In these vectors, the fluorescent protein is constitutively expressed and can be detected by fluorescence microscopy or flow cytometry, providing direct visual evidence of transfection/cotransfection or the ability to monitor cells after transplantation experiments in vivo. Transfected cells can also be used in assays that require fluorescent labeling of whole cells. The most widely used Fluorescent protein is Green Fluorescent Protein (GFP), and Red Fluorescent Protein (RFP).  GFP and RFP are both versatile biological markers for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression in vivo, GFP can be excited by the 488 nm laser line and is optimally detected at 510 nm, while RFP can be excited by the 488 nm or 532 nm laser line and is optimally detected at 588 nm.
  • Immunohistochemistry Test
    Immunohistochemistry (IHC) is a technique used to determine the presence and level of specific cellular proteins. IHC measures protein expression using specially labeled antibodies that can bind to the proteins of interest. The antibody is mixed with the cellular components of the tumor. After a set amount of time, the mixture is rinsed and only those antibodies attached to their protein targets will remain. The presence of the antibodies can be detected by viewing the sample under a microscope because areas containing bound antibodies will appear a different color than areas lacking antibodies. Samples with more protein will bind more antibody and therefore appear darker. This allows the test to reveal not only whether a protein is present but also the relative amount of the protein. Test results are based on the strength of the staining and the percent of cells stained. Procedures: 1. Tissue Fixation 2. Antigen Retrieval 3. Staining/Add primary antibody 4. Add secondary antibody 5. Add chromogen 6. Counterstain 7. Detection
  • Immunofluorescence Technique
    Immunofluorescence (IF) or cell imaging techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyanate (FITC) to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. IF allows researchers to evaluate whether cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, immunofluorescence also allows researchers to determine which subcellular compartments are expressing the antigen. IF can be used on cultured cell lines, tissue sections, or individual cells. There are two different immunofluorescence assays: indirect immunofluorescence assay and direct immunofluorescence assay. For indirect immunofluorescence assay, the protocol mainly include tissue or cell treparation, tissue or cell fixation, serum blocking, primary antibody incubation, marked second antibody incubation, staining, result judgment and imaging. For direct immunofluorescence assay, there are only marked primary antibody been incubated without second ant...
  • In Situ Hybridization
    In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand which allows for precise localization of a specific segment of nucleic acid within a histologic section. The underlying basis of ISH is that nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached. Visualization of the reporter molecule allows to localize DNA or RNA sequences in a heterogeneous cell populations including tissue samples and environmental samples, Riboprobes also allow to localize and assess degree of gene expression.  There are four types of ISH probes: Double-stranded DNA (dsDNA) probes, Single-stranded DNA (ssDNA) probes, RNA probes (riboprobes), and Synthetic oligonucleotides (PNA, LNA). The probes can be labeled both by radioactive isotopes (e.g. 32P, 35S, 3H) and non-radioactive labels (e.g. biotin, digoxigenin, fluorescent dye (FISH)). FISH (Fluorescence In Situ Hybridization) is a method which based on the in situ hybridization techniques and consist in ...
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