A new luciferase-based quantitative assay for the evaluation of human trophoblast fusion

Reproductive Sciences
A VargasB Barbeau

Abstract

The syncytiotrophoblast is a multinuclear cell layer maintained through fusion events with cytotrophoblasts and plays a key role in the properties of the placenta. Monitoring fusion in this cell layer is important in studies aimed at understanding its function. We herein propose a new fusion assay based on the transactivating potential of the human immunodeficiency virus type 1 (HIV-1) Tat protein on its promoter present in the long terminal repeat (LTR) region. We used 2 BeWo cell populations, one stably transfected with the HIV-1 LTR positioned upstream of the luciferase gene and the other stably transfected with a Tat expression vector. Both stable cell lines were responsive to Tat-mediated LTR transactivation and demonstrated normal fusion and human chorionic gonadotropin (hCG) secretion upon stimulation. When both BeWo cell lines were cocultured, forskolin-mediated induction of fusion led to an increase in luciferase activity, which was sensitive to anti-syncytin 1 and -2 antibodies and syncytin 2 small interfering RNAs (siRNAs). Similar results were obtained in primary trophoblasts. Our results highlight the effectiveness and accuracy of this new quantification assay for trophoblast fusion.

References

Jan 1, 1991·American Journal of Obstetrics and Gynecology·R N TaylorS A Chen
Feb 1, 1990·Experimental Cell Research·B WiceA L Schwartz
Apr 5, 2003·Biochimica Et Biophysica Acta·Yoshiki KudoI L Sargent
May 2, 2003·Molecular and Cellular Biology·Jean-Louis FrendoFrançois Mallet
Jan 13, 2004·Human Reproduction Update·A MalassinéD Evain-Brion
Jun 21, 2005·Virology·Marie-Eve ParéBenoit Barbeau
Feb 25, 2006·Micron : the International Research and Review Journal for Microscopy·Berthold HuppertzMaria Kokozidou
Jun 6, 2009·Methods in Molecular Biology·Catherine MounierJulie Lafond
Jul 21, 2009·Journal of Molecular Biology·Amandine VargasBenoit Barbeau

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Methods Mentioned

BETA
flow cytometry
transfection
enzyme-linked immunosorbent assay
protein assay
PCR
confocal microscopy
Fluorescence Activated Cell Sorting

Software Mentioned

StatGraphics

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