We have generated ‘green’ and ‘red’ BeWo cell lines: the green one constitutively expresses the green fluorescent protein (GFP) attached to histone H2B (Kimura & Cook, 2001), and the red one the red fluorescent protein tagged to the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (Mit-DsRed; Bevis & Glick, 2002). Our quantitative assay for cell fusion (syncytialisation) utilizes the fluorescence-activated cell sorter (FACS) to detect ‘cells’ that are both green and red.

Exponentially growing BeWo cells (gift from S. Greenwood) were transfected with vectors encoding either H2B-GFP or Mit-DsRed, and stable autofluorescent clones selected. Fluorescence microscopy revealed that one line had green fluorescent nuclei, the other red fluorescent mitochondria. The fluorescent proteins had no effect on the rates of DNA, RNA or total protein synthesis (monitored using the rate of incorporation of radiolabelled thymidine, uridine or leucine into acid-insoluble material) or on the secretion of the peptide hCG (measured by immunoassay).

When these two cell lines were mixed and cultured in forskolin, cells with green nuclei and red mitochondria appeared. Such green and red (fused) cells were readily detected amongst green or red (unfused) cells by FACS. Using this novel assay, growth in 100 mM forskolin for 48 h increased BeWo syncytialisation approximately 5-fold over cells incubated in the absence of forskolin.

We will use this specific and rapid non-destructive assay to quantify the rate of syncytialisation and to study factors affecting the process.