370
Related Techniques
4. Suspend the mixed cells in a mixture of equal parts of MEM and Grace's
medium (EPM) containing UV-inactivated HVJ. The hemagglutinating titer of the virus should be predetermined.
5. Keep the mixture in a 4°C water bath on an automatic shaker, and
shake at low speed for 20 min.
6. Then, shake for 60 min with the same speed at 37°C.
7. Centrifuge the cells at 250 g for 5 min.
8. Suspend the cells in fresh EPM and culture at 37°C.
Results
The Ae cells cannot survive at either 37°C, or in MEM. In contrast, HeLa
cells do not grow at 26° or 32°C, but can grow slowly in EPM at 37°C.
Therefore, culture in EPM at 37°C can roughly select hybrid cells, which
can grow under these conditions. Using this technique, Zepp et aI. (1971)
obtained a hybrid of He La and Ae cells. The formation of hybrids was
dearly shown by karyotype analysis. Heterokaryon formations were consistently obtained when the amount of virus used contained 1,600,3,200,
or 4,800 hemagglutinating units. At the latter two doses, the incidence of
heterokaryon formation was 25%.
Comments
In the report of Zepp et aI., the insect cell line used was reported to be a
mosquito cell line. However, it was proven by Greene et aI. (1972) that
the cell line used was actually the moth (Antheraea eucalypti) cell line
Ae.
In addition to some animal viruses, such as HVJ, some invertebrate
and plant viruses are known to cause cell fusion. Hsu (1978) reported
that the fusion of cultured cells from leafhoppers (Aceratagallia
sanguinolenta or Dalbulus elimatus) was induced by inoculation by potato yellow dwarf virus (rhabdovirus).
Subprotocol 2
Cell Fusion with Polyethyleneglycol
In later cell fusion studies, chemical fusogens were often used. Concanavalin A, wheat germ hemagglutinin, and polyethylene glycol (PEG) are some
of the chemicals used as fusogens. Of these, PEG has been used most
frequently.
Materials
Equipment - Centrifuge and centrifuge tubes
- Glass rod
- Culture vessels
Related Techniques
4. Suspend the mixed cells in a mixture of equal parts of MEM and Grace's
medium (EPM) containing UV-inactivated HVJ. The hemagglutinating titer of the virus should be predetermined.
5. Keep the mixture in a 4°C water bath on an automatic shaker, and
shake at low speed for 20 min.
6. Then, shake for 60 min with the same speed at 37°C.
7. Centrifuge the cells at 250 g for 5 min.
8. Suspend the cells in fresh EPM and culture at 37°C.
Results
The Ae cells cannot survive at either 37°C, or in MEM. In contrast, HeLa
cells do not grow at 26° or 32°C, but can grow slowly in EPM at 37°C.
Therefore, culture in EPM at 37°C can roughly select hybrid cells, which
can grow under these conditions. Using this technique, Zepp et aI. (1971)
obtained a hybrid of He La and Ae cells. The formation of hybrids was
dearly shown by karyotype analysis. Heterokaryon formations were consistently obtained when the amount of virus used contained 1,600,3,200,
or 4,800 hemagglutinating units. At the latter two doses, the incidence of
heterokaryon formation was 25%.
Comments
In the report of Zepp et aI., the insect cell line used was reported to be a
mosquito cell line. However, it was proven by Greene et aI. (1972) that
the cell line used was actually the moth (Antheraea eucalypti) cell line
Ae.
In addition to some animal viruses, such as HVJ, some invertebrate
and plant viruses are known to cause cell fusion. Hsu (1978) reported
that the fusion of cultured cells from leafhoppers (Aceratagallia
sanguinolenta or Dalbulus elimatus) was induced by inoculation by potato yellow dwarf virus (rhabdovirus).
Subprotocol 2
Cell Fusion with Polyethyleneglycol
In later cell fusion studies, chemical fusogens were often used. Concanavalin A, wheat germ hemagglutinin, and polyethylene glycol (PEG) are some
of the chemicals used as fusogens. Of these, PEG has been used most
frequently.
Materials
Equipment - Centrifuge and centrifuge tubes
- Glass rod
- Culture vessels