40 Cell Fusion
375
3. Remove the supernatant.
4. Stir up the cell pellet with a Pasteur pipette without adding any solution.
1. Add 0.3 ml of 50% PEG solution.
2. After 30 s, add 20 ml serum-free medium to the treated cells.
3. Centrifuge the cells at 600 g for 10 min.
Fusion
1. Suspend the cells in TAM-selection medium and transfer the suspen- Selection
sion into a culture flask.
2. Maintain the growing cells (hybrids) further in TAM-selection medium.
Results
Wyss (1979) obtained hybrids between an auxotrophic mutant cell line
and a wild-type cell line using the method outlined above.
Comments
Another method for the selection of hybrid cells is to rescue metabolically blocked cells by the introduction of a wild-type gene. Miltenburger
et al. (1985) used an actinomycin D- and puromycin-treated Cydia
pomonella cell line, which lacked proliferating ability, and obtained proliferating hybrid cells by fusion with non-cultured wild-type embryonic
cells (ref. chapter 4, subprotocol 5: Hybird cell technique for obtaining
growing cells).
Subprotocol 5
Method for the Selection of Hybrid Cells: Use of ts Mutants
If ts mutants sensitive to high temperature can be obtained by the deletion of different genes, a hybrid produced from these mutants may tolerate high temperature because of the compensation by deficient genes for
one another. Nakajima and Miyake (1978) obtained ts mutants of Drosophila melanogaster, and used them as a parent cell line. Hybrid cells were
selected by culturing PEG-treated cells at 30°C, a non-permissive temperature. The following is the method of Nakajima and Miyake (1978)
for obtaining ts mutants.
Materials
- CO 2 incubator
- Glass dishes (60 x 15 mm)
- Pasteur pipettes
- Culture plates (Microtest II; Falcon, Becton-Dickinson)
Equipment
375
3. Remove the supernatant.
4. Stir up the cell pellet with a Pasteur pipette without adding any solution.
1. Add 0.3 ml of 50% PEG solution.
2. After 30 s, add 20 ml serum-free medium to the treated cells.
3. Centrifuge the cells at 600 g for 10 min.
Fusion
1. Suspend the cells in TAM-selection medium and transfer the suspen- Selection
sion into a culture flask.
2. Maintain the growing cells (hybrids) further in TAM-selection medium.
Results
Wyss (1979) obtained hybrids between an auxotrophic mutant cell line
and a wild-type cell line using the method outlined above.
Comments
Another method for the selection of hybrid cells is to rescue metabolically blocked cells by the introduction of a wild-type gene. Miltenburger
et al. (1985) used an actinomycin D- and puromycin-treated Cydia
pomonella cell line, which lacked proliferating ability, and obtained proliferating hybrid cells by fusion with non-cultured wild-type embryonic
cells (ref. chapter 4, subprotocol 5: Hybird cell technique for obtaining
growing cells).
Subprotocol 5
Method for the Selection of Hybrid Cells: Use of ts Mutants
If ts mutants sensitive to high temperature can be obtained by the deletion of different genes, a hybrid produced from these mutants may tolerate high temperature because of the compensation by deficient genes for
one another. Nakajima and Miyake (1978) obtained ts mutants of Drosophila melanogaster, and used them as a parent cell line. Hybrid cells were
selected by culturing PEG-treated cells at 30°C, a non-permissive temperature. The following is the method of Nakajima and Miyake (1978)
for obtaining ts mutants.
Materials
- CO 2 incubator
- Glass dishes (60 x 15 mm)
- Pasteur pipettes
- Culture plates (Microtest II; Falcon, Becton-Dickinson)
Equipment