Introduction

Cell fusion has become of major scientific interest with the establishment of monoclonal antibodies, derived from fused antibody-producing B-lymphocytes with myeloma cells (1). Apart from the derivation of clonal antibodies, cell fusion has been utilized in a variety of applications, including transfer of cellular contents from one cell to its fusion partner, as recently demonstrated by the fusion of cardiomyocytes with CHO cells (2). Interestingly, fusion was also identified as a physiological process that occurs under different circumstances in vivo. It has been observed that bone marrow–derived cells are capable of fusing with a variety of cell types, including cardiomyocytes, hepatocytes, and Purkinje neurons (3-5). Detailed analysis of these processes revealed that the fusion of bone marrow cells with the different cell types is enhanced by tissue damage or inflammation (5-7). Formation of heterokaryons by in vivo fusion of bone marrow–derived cells with Purkinje neurons was followed by a reprogramming of the bone marrow–derived nucleus into a state that corresponds to that of Purkinje neurons in terms of chromatin structure and gene expression profile (8).

In the last 10 years, the fusion of embryonic stem (ES) cells with somatic cells in vitro has been intensively studied owing to the ability of ES cells to reprogram somatic nuclei to an ES cell–like state (9-12). In 2005, Melton and colleagues demonstrated that fusion of human ES cells with human fibroblasts results in the reprogramming of the somatic nucleus, recovering a gene expression profile similar to that found in human ES cells alone (13).

The selection of fused cells requires marker genes to detect which cells have undergone fusion as well as to isolate these cells from non-fused cells. Usually this is accomplished by introducing two different selectable markers in each of the fusion partners (e.g., neomycin and hygromycin resistance) (13). After cell fusion, application of both selection agents enables purification of fused cells. This technique allows selection for tetraploid cells as a result of the fusion event. Here we describe a novel selection methodology that not only allows tracking of fusion events between cells, but also enables selection of fused cells independently from the formation of polyploid derivatives.

Material and methods

RFPpig indicator plasmid construction

HcRed-pA was isolated from pHcRed1.1 (Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France) by BamHI and AflII digest (New England BioLabs, Frankfurt, Germany). The HcRed fragment was inserted between the first loxP site and the Westphal stop of pRFPGFP (Thomas Wunderlich, Institute for Genetics, University of Cologne; unpublished data) replacing DsRed. The GFP was excised by EcoRV digestion and puromycin-acetyltransferase-IRES-GFP was inserted as described (14).

Cell lines, culture conditions, and transfection

HEK 293 cells (provided by Toni Schneider, University of Cologne, Cologne, Germany) were cultured using Iscove's Medium (IMDM) containing 100 µM β-mercaptoethanol (Sigma-Aldrich, Munich, Germany), 1% non-essential amino acids (Invitrogen Life Technologies, Karlsruhe, Germany), 1% penicillin/streptomycin solution (Invitrogen), and 17% FCS (Invitrogen). We generated stably transfected cell lines by transfection with linearized RFPpig vector and linearized Cre-ER (ER, estrogen receptor) expression vector. Transfections were performed by electroporation using a Gene Pulser (Bio-Rad, Munich, Germany) (260 V; 500 µF; 0.4-cm gap). Cell selection was started 48 h after transfection with 500 µg/mL geneticin (PAA, Pasching, Austria) for RFPpig or 200 µg/mL hygromycin (Invitrogen) for Cre-ER. Clones were picked after 2 weeks of selection. Cre-ER transgenic clones were assessed for Cre expression by staining with anti-Cre antibody.

Immunocytochemistry and cytosol staining by CMAC

For Cre staining, cells were fixed in ice-cold 99% methanol for 15 min. Blocking was performed with 5% BSA in PBS pH 7.4 for 1 h. Anti-Cre (polyclonal, Novagen, distributed by Merck KgaA, Darmstadt, Germany) was diluted 1:500 in 1% BSA and incubation was done overnight at 4°C. Secondary detection was performed with anti-rabbit Alexa Fluor 647 (Invitrogen) for 90 min at room temperature.

For 7-amino-4-chloromethylcoumarin (CMAC, Molecular Probes) labeling, cells were washed twice with PBS and incubated with 10 µM CMAC in cell culture medium without FCS for 60 min. Cells were washed and incubated with IMDM supplemented with 17% FCS for 45 min to bind residual dye. Thereafter, cells were trypsinized and used for fusion immediately. Pictures were taken using a Zeiss Axiovert 200 equipped with an Apotom (Zeiss, Jena, Germany) device to facilitate optical sectioning. Images were processed with Axiovision 4.5 (Zeiss) and Corel Graphic Suite 11.