41 Gene Technology
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Materials
- The equipment is the same as that given for subprotocol 5
- Hygromycin B
Procedure
1. Select transfected cells by culturing in medium containing between
200 fIg/ml and 1 mg/ml hygromycin B.
Results
Van der Straten et al. (1988) used this system to select transformed Drosophila cells.
Subprotocol 8
Selection of Transfected Cells with a-Amanitin
u-Amanitin is a toxin obtained from a mushroom, and it specifically inhibits RNA polymerase II. A gene responsible for resistance to u-amanitin has been isolated from the Drosophila mutant C4, which is itself resistant to the drug. This gene can be used as a selectable marker when a
non-selectable gene is used for transfection (Echalier, 1997).
Materials
- The equipment is the same as that given for subprotocol 5
- u-Amanitin
Procedure
1. Select transfected cells by culturing cells in medium containing uamanitin at a concentration of 5 fIg/ml (u-amanitin is first dissolved
to a concentration of 1 mg/ml in 100% ethanol, and is then dispersed
in culture medium).
Results
Transfected cells from Drosophila melanogaster were selected using this
method.
Equipment
Antibiotics
Equipment
Chemicals