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    Lecture Reporter Gene-Luciferase Assays 2024

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    kinjal010902

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    Lecture Reporter Gene-Luciferase Assays 2024

    Uploaded by

    kinjal010902
    2 views8 pages

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    Lecture Reporter gene-Luciferase assays 2024

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    2 views8 pages

    Lecture Reporter Gene-Luciferase Assays 2024

    Uploaded by

    kinjal010902

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 8

    Luciferase reporter gene assays

    Reporter genes:
    • Gene that is attached to the regulatory sequence (e.g. promoter) of another gene of interest.
    • Indicators to study gene expression and cellular events coupled to gene expression.
    • Typically, a reporter gene is cloned with a DNA sequence of interest into an expression
    vector that is then transferred into cells. Following this transfer, the cells are assayed for
    the presence of the reporter by directly measuring the reporter protein itself (e.g. GFP) or
    the enzymatic activity of the reporter protein (e.g. luciferase activity).
    • A good reporter gene can be identified easily and measured quantitatively when it is
    expressed (in the organism or cells of interest).

    Wikipedia
    Bioluminescence:

    • Production and emission


    of light by living organisms
    • encompasses a number of different chemistries evolved for light production and is based
    on the interaction of the enzyme, luciferase, with a luminescent substrate, luciferin.
    • Bioluminescence is found in many phyla including coelenterates, echinoderms, mushrooms
    and insects. Only a few of these chemistries have been characterized in detail e.g. luciferases
    from firefly (Photinus pyralis) and Renilla (Renilla reniformis, a sea pansy)

    Luciferin + O2 + ATP Oxyluciferin + CO2 + AMP + PPi + Light


    Luciferase
    Allard et al, Cell Notes 2008, Promega
    Study of transcription factor binding/activity

    IL-6

    STAT3

    rPAP1 promoter

    Secreted
    luciferase

    Cell
    supernatant

    !!
    Adapted from http://photobiology.info/Ohmiya.html
    Vector used in transfection

    pNL2.3
    pNL2.3 /rPAP1

    Empty vector carrying Same vector carrying NanoLuc luciferase


    NanoLuc luciferase (blue) (blue) under the control of the rPAP1
    promoter (red)

    rPAP1 promoter: STAT3-responsive rat pancreatitis-associated protein 1 promoter


    Promega
    Transfection
    • process of artificially introducing nucleic acids (DNA or RNA) into cells, by ways
    other than viral infection
    • the introduced nucleic acid may exist in the cells transiently, such that it is only
    expressed shortly and does not replicate, or it may be stable, integrating into the host
    genome, replicating when the host genome replicates
    • Challenge to introduce negatively charged DNA or RNA molecules into cells with negatively
    charged membrane

    Chemical methods neutralize the


    negative charge of DNA, facilitating its
    uptake

    Lipofection Lipid-based reagents can also coat


    DNA while forming micelles.
    Electroporation makes the membrane
    more permeable transiently, allowing
    DNA to enter the cell.
    https://www.promega.lu/resources/guides/cell-biology/transfection/
    Lipofection – Lipofectamine LTX

    • Principle: association between nucleic acids with cationic lipids and resulting molecular
    complexes, known as lipoplexes, are taken up by the cells.
    • Lipid-based reagents generally composed of synthetic cationic lipids
    • Lipid mixtures form liposomes or micelles with an overall positive charge at physiological pH
    able to form complexes (lipoplexes) with negatively charged nucleic acids through
    electrostatic interactions.
    • Association of the lipid-based transfection reagent with nucleic acids results in tight
    compaction and protection of the nucleic acids and cationic complexes are mainly
    internalised by endocytosis

    https://www.thermofisher.com/lu/en/home/life-science/cell-culture/transfection/transfection-reagents/lipofectamine-ltx-plus-reagent.html

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