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  • 1
    ISSN: 0884-3996
    Keywords: Bioluminescence ; dehydrogenase ; nucleic acid ; hybridization ; amplification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase.On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample.In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0884-3996
    Keywords: Stable transfection ; firefly luciferase ; nuclear receptors ; membrane-bound receptors ; MCF-7 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in Intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time.Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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