Other Considerations and Alternative Protocols

PEG is a key reagent in this protocol. PEG with a molecular weight of 1000 to 6000 can be used in cell fusion. Exposure of cells to 1 mL of 50% PEG 1500 solution for 2 min works well for most types of cells but the molecular weight, concentration, and treatment length of the polymer should be established to suit individual cell types. Some protocols use phytohemagglutinin to enhance cell fusion efficiency. In our experience, however, no significant enhancement of phytohemagglutinin in fusion efficiency was observed. In some protocols for microcell fusion, microcells are purified by filtration through 3- to 5-|m polycarbonate filters to remove whole cells and nuclei. This step is tedious and not necessary if proper double selection is applied. After selection applied to fusion culture, medium with proper selection is usually changed every 2 to 4 d, but in our protocol, medium changes are not required for the DT40 suspension cells. For the maintenance of hybrids, chemicals used for selection can be reduced to 30-50% of initial concentration for hybrid isolation that is enough to induce expression of selectable markers in most cases. In regard to FEG-medi-ated nuclear transfer from embryonic or somatic cells into enucleated oocytes, particular protocols should be considered (6,10).

4. Notes

1. Choice of cells for fusion and selectable markers: both donor and recipient cells must be exponentially growing before being harvested for fusion. In principle, cells between either different species or the same species can be fused with PEG, leading to the generation of interspecific or intraspecific hybrids. However, the fusibility varies depending on cell types. For example, it is much easier to fuse the mouse LA9 cells than it is the mouse E14 embryonic stem cells and the chicken DT40 cells. When fusion is performed between adherent and suspension cells, it is relatively easy to isolate hybrids of particular cell types if no efficient double positive selection is available. However, double positive selection usually is required to isolate hybrids if both donors and recipients in a fusion are suspension or adherent cells. Double selection requires donor and recipient cells to carry a different selectable marker respectively. A number of dominant markers are available for positive selection in vertebrate cells. The widely used markers include the Hyg gene, the neomycin resistance gene (neo), the bleomycin resistance gene (ble), the blasticidin resistance gene (bsd), the puromycin N-acetyl transferase gene (pac), and the histidinol dehydrogenase gene (hisD). These markers, driven by a suitable promoter, such as the cytomegalovirus promoter, can be introduced into donor or recipient cells by transfection or viral infection. However, these markers with a given promoter have to be tested in individual cell lines for function before being used in cell fusion. In addition, counter selection may be useful if cell fusion is used to transfer a single or a limited number of chromosomes between cells. Examples of selectable markers that can be used for counter selection are the HPRT gene and the TK gene. Cells with the HPRT gene are killed when grown in medium containing 6-thioguanine and cells with the TK gene are killed when grown in medium containing bromodeoxyuridine.

2. Whole cell fusion and microcell fusion. Whole cell fusion is simple and can be used in many areas involving production of somatic cell hybrids and nuclear transfer. Microcell fusion is particularly useful for transfer of a single or a limited number of chromosomes between cell lines. Microcells are prepared from micronucleated donor cells induced by colcemid. The colcemid concentration and treatment length are important factors for microcell preparation that vary with cell types. Treatment with 0.05 to 0.10 ^g/mL colcemid for 10 to 48 h can be tried for most of vertebrate cell lines.

3. Characterization of hybrids: the characterization of hybrids may involve analysis of cell morphology, karyotypes, genome structure, gene expression, and gene function. Analysis of mitotic and structural stability of heterologous chromosomes can be extended to several months in the presence or absence of selection. Choice of techniques for hybrid characterisation is determined by particular purposes of a given cell fusion experiment.

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