A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds

Elisa Michelini, Luca Cevenini, Laura Mezzanotte, Piia Leskinen, Marko Virta, Matti Karp, Aldo Roda

Tutkimustuotos: ArtikkelijulkaisuArtikkeliTieteellinenvertaisarvioitu

Kuvaus

We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control of the androgen response element (ARE). In the presence of androgens, activated hAR binds to the ARE sequences and activates luciferase expression. After addition of D-luciferin, luciferase activity measurements can be performed, and the BL signal is proportional to the androgenic activity of the sample. Cytotoxic effects of the sample are monitored by the use of a control yeast strain that allows BL signal correction according to cell viability. After overnight culture of the recombinant strain, the assay can be accomplished in a 96-well microplate format in 1 working day with a detection limit of 0.05 nM for testosterone and intra-and interassay variability of 14% and 23%, respectively.
Alkuperäiskielienglanti
LehtiNature Protocols
Vuosikerta3
Numero12
Sivut1895 - 1902
ISSN1754-2189
DOI - pysyväislinkit
TilaJulkaistu - 2008
OKM-julkaisutyyppiA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä, vertaisarvioitu

Tieteenalat

  • 1182 Biokemia, solu- ja molekyylibiologia

Lainaa tätä

Michelini, E., Cevenini, L., Mezzanotte, L., Leskinen, P., Virta, M., Karp, M., & Roda, A. (2008). A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds. Nature Protocols, 3(12), 1895 - 1902. https://doi.org/10.1038/nprot.2008.189
Michelini, Elisa ; Cevenini, Luca ; Mezzanotte, Laura ; Leskinen, Piia ; Virta, Marko ; Karp, Matti ; Roda, Aldo. / A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds. Julkaisussa: Nature Protocols. 2008 ; Vuosikerta 3, Nro 12. Sivut 1895 - 1902.
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title = "A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds",
abstract = "We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control of the androgen response element (ARE). In the presence of androgens, activated hAR binds to the ARE sequences and activates luciferase expression. After addition of D-luciferin, luciferase activity measurements can be performed, and the BL signal is proportional to the androgenic activity of the sample. Cytotoxic effects of the sample are monitored by the use of a control yeast strain that allows BL signal correction according to cell viability. After overnight culture of the recombinant strain, the assay can be accomplished in a 96-well microplate format in 1 working day with a detection limit of 0.05 nM for testosterone and intra-and interassay variability of 14{\%} and 23{\%}, respectively.",
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Michelini, E, Cevenini, L, Mezzanotte, L, Leskinen, P, Virta, M, Karp, M & Roda, A 2008, 'A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds', Nature Protocols, Vuosikerta 3, Nro 12, Sivut 1895 - 1902. https://doi.org/10.1038/nprot.2008.189

A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds. / Michelini, Elisa; Cevenini, Luca; Mezzanotte, Laura; Leskinen, Piia; Virta, Marko; Karp, Matti; Roda, Aldo.

julkaisussa: Nature Protocols, Vuosikerta 3, Nro 12, 2008, s. 1895 - 1902.

Tutkimustuotos: ArtikkelijulkaisuArtikkeliTieteellinenvertaisarvioitu

TY - JOUR

T1 - A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds

AU - Michelini, Elisa

AU - Cevenini, Luca

AU - Mezzanotte, Laura

AU - Leskinen, Piia

AU - Virta, Marko

AU - Karp, Matti

AU - Roda, Aldo

PY - 2008

Y1 - 2008

N2 - We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control of the androgen response element (ARE). In the presence of androgens, activated hAR binds to the ARE sequences and activates luciferase expression. After addition of D-luciferin, luciferase activity measurements can be performed, and the BL signal is proportional to the androgenic activity of the sample. Cytotoxic effects of the sample are monitored by the use of a control yeast strain that allows BL signal correction according to cell viability. After overnight culture of the recombinant strain, the assay can be accomplished in a 96-well microplate format in 1 working day with a detection limit of 0.05 nM for testosterone and intra-and interassay variability of 14% and 23%, respectively.

AB - We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control of the androgen response element (ARE). In the presence of androgens, activated hAR binds to the ARE sequences and activates luciferase expression. After addition of D-luciferin, luciferase activity measurements can be performed, and the BL signal is proportional to the androgenic activity of the sample. Cytotoxic effects of the sample are monitored by the use of a control yeast strain that allows BL signal correction according to cell viability. After overnight culture of the recombinant strain, the assay can be accomplished in a 96-well microplate format in 1 working day with a detection limit of 0.05 nM for testosterone and intra-and interassay variability of 14% and 23%, respectively.

KW - 1182 Biochemistry, cell and molecular biology

U2 - 10.1038/nprot.2008.189

DO - 10.1038/nprot.2008.189

M3 - Article

VL - 3

SP - 1895

EP - 1902

JO - Nature Protocols

JF - Nature Protocols

SN - 1754-2189

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ER -