374
Related Techniques
Subprotocol 4
Method for the Selection of Hybrid Cells: Use of Auxotrophic
Cells
In mammalian cell culture, auxotrophic cell lines (mutant cell lines that
are unable to proliferate in a chemically defined medium deprived of
some specific metabolite but can be rescued by acquisition of the corresponding wild allele) are often used as parent cell lines of hybrid cells.
The HGPRT- (hypoxanthine-guanine-phosphoribosyl-transferase deficient) and TK- (thymidine kinase deficient) cell lines are examples of
auxotrophic cell lines. If these auxotrophic cell lines are used as parent
cell lines of hybrid cells, the resulting hybrid cells can be selected by
cultivation in a selection medium, HAT. However, it is extremely difficult
to obtain auxotrophic cell lines for invertebrate cell cultures. So far only
an APRT- (adenosine-phosphoribosyl-transferase deficient) cell line
from Drosophila has been shown to be used in a manner similar to auxotrophic mammalian cells (Wyss, 1979). Another difficulty with this process in invertebrate cell culture is the lack of chemically defined media
for invertebrate cells. Most invertebrate cell culture media contain complex components, and this does not permit the starvation of cells for a
single given nutrient.
Wyss (1979) has devised a thymidine-adenine-methotrexate (TAM)selection system for Drosophila cell lines. The following is his method.
Materials
Equipment - Centrifuge and centrifuge tube
- Pasteur pipettes
- Culture flasks
Solutions - ZH 1 % medium (see Appendix 1)
- TAM-selection medium (ZH 1% medium containing 10- 4 M thymidine, 10- 4 M adenine, and 10- 6 M methotrexate)
- Conditioned medium
Cells - Drosophila cell clone resistant to 6-methylpurine and diamino purine
(this clone is an adenine salvage-deficient mutant, and can proliferate
in ZH 1 % medium, but it is sensitive to TAM-selection medium)
- Wild-type cell lines (resistant to TAM, and unable to proliferate in
medium ZH 1 %)
Procedure
Mixing 1. Mix the suspension in culture medium of the adenine salvage-deficient clone cells and wild-type cells.
2. Centrifuge the mixed cell suspension at 600 g for 10 min.
Related Techniques
Subprotocol 4
Method for the Selection of Hybrid Cells: Use of Auxotrophic
Cells
In mammalian cell culture, auxotrophic cell lines (mutant cell lines that
are unable to proliferate in a chemically defined medium deprived of
some specific metabolite but can be rescued by acquisition of the corresponding wild allele) are often used as parent cell lines of hybrid cells.
The HGPRT- (hypoxanthine-guanine-phosphoribosyl-transferase deficient) and TK- (thymidine kinase deficient) cell lines are examples of
auxotrophic cell lines. If these auxotrophic cell lines are used as parent
cell lines of hybrid cells, the resulting hybrid cells can be selected by
cultivation in a selection medium, HAT. However, it is extremely difficult
to obtain auxotrophic cell lines for invertebrate cell cultures. So far only
an APRT- (adenosine-phosphoribosyl-transferase deficient) cell line
from Drosophila has been shown to be used in a manner similar to auxotrophic mammalian cells (Wyss, 1979). Another difficulty with this process in invertebrate cell culture is the lack of chemically defined media
for invertebrate cells. Most invertebrate cell culture media contain complex components, and this does not permit the starvation of cells for a
single given nutrient.
Wyss (1979) has devised a thymidine-adenine-methotrexate (TAM)selection system for Drosophila cell lines. The following is his method.
Materials
Equipment - Centrifuge and centrifuge tube
- Pasteur pipettes
- Culture flasks
Solutions - ZH 1 % medium (see Appendix 1)
- TAM-selection medium (ZH 1% medium containing 10- 4 M thymidine, 10- 4 M adenine, and 10- 6 M methotrexate)
- Conditioned medium
Cells - Drosophila cell clone resistant to 6-methylpurine and diamino purine
(this clone is an adenine salvage-deficient mutant, and can proliferate
in ZH 1 % medium, but it is sensitive to TAM-selection medium)
- Wild-type cell lines (resistant to TAM, and unable to proliferate in
medium ZH 1 %)
Procedure
Mixing 1. Mix the suspension in culture medium of the adenine salvage-deficient clone cells and wild-type cells.
2. Centrifuge the mixed cell suspension at 600 g for 10 min.