41 Gene Technology
383
1. Suspend the cell pellet in serum-free culture medium so as to make a Electroporation
cell density of between 10 7 and 10 8 cells/ml.
2. Transfer the cell suspension to a cuvette electrode.
3. Add plasmid DNA and leave for 5 min.
4. Apply an electric pulse of 440 V/cm field strength, 1,200 fiF capacitance, and < 1 s pulse duration.
s. Leave the cells for 10 min to recover from the electric shock.
6. Wash the cells out from the cuvette with 9.2 ml complete medium in a
100 mm plate.
Results
Working with the Drosophila Kcl67 cell line, Cherbas et al. (1994) reported that, with 20 fig DNA/plate, the expression in transient assays and
the frequency of stable transformants were comparable with those obtained by the calcium phosphate-DNA coprecipitation method.
Subprotocol 5
Selection of Transfected Cells with G418
In order to select transfected cells efficiently, a dominant selective marker
generally derived from bacterial plasmids, which confers a high level of
resistance against a specific antibiotic, is used. This method requires no
special selection medium, but uses any standard culture medium by adding a relatively high concentration of the antibiotic. G418 is an antibiotic
belonging to the same group of drugs as kanamycin and neomycin. The
bacterial resistance to this group of antibiotics relies on the gene neo.
Materials
- Cell culture system
- Plasmid-constructing system
- G418
Procedure
Equipment
Antibiotics
1. Use a vector carrying the gene and a non-selectable target gene (for Vector
the construction of the vector, refer to gene technology guides).
construction and
2. Cotransfect cells with the hybrid vector.
transfection
1. After cotransfection of cells with the neo selectable gene and a non- Selection
selectable gene, culture the cells in medium containing G418 sulfate at
a concentration of 1 to several mg/ml. Insect cells are rather insensitive to G418, but the concentration of G418 cited here can kill wild-
383
1. Suspend the cell pellet in serum-free culture medium so as to make a Electroporation
cell density of between 10 7 and 10 8 cells/ml.
2. Transfer the cell suspension to a cuvette electrode.
3. Add plasmid DNA and leave for 5 min.
4. Apply an electric pulse of 440 V/cm field strength, 1,200 fiF capacitance, and < 1 s pulse duration.
s. Leave the cells for 10 min to recover from the electric shock.
6. Wash the cells out from the cuvette with 9.2 ml complete medium in a
100 mm plate.
Results
Working with the Drosophila Kcl67 cell line, Cherbas et al. (1994) reported that, with 20 fig DNA/plate, the expression in transient assays and
the frequency of stable transformants were comparable with those obtained by the calcium phosphate-DNA coprecipitation method.
Subprotocol 5
Selection of Transfected Cells with G418
In order to select transfected cells efficiently, a dominant selective marker
generally derived from bacterial plasmids, which confers a high level of
resistance against a specific antibiotic, is used. This method requires no
special selection medium, but uses any standard culture medium by adding a relatively high concentration of the antibiotic. G418 is an antibiotic
belonging to the same group of drugs as kanamycin and neomycin. The
bacterial resistance to this group of antibiotics relies on the gene neo.
Materials
- Cell culture system
- Plasmid-constructing system
- G418
Procedure
Equipment
Antibiotics
1. Use a vector carrying the gene and a non-selectable target gene (for Vector
the construction of the vector, refer to gene technology guides).
construction and
2. Cotransfect cells with the hybrid vector.
transfection
1. After cotransfection of cells with the neo selectable gene and a non- Selection
selectable gene, culture the cells in medium containing G418 sulfate at
a concentration of 1 to several mg/ml. Insect cells are rather insensitive to G418, but the concentration of G418 cited here can kill wild-