384
Related Techniques
type cells. Extended culture in selection medium will result in the development of cells resistant to G418.
Results
Maisonhaute and Echalier (1986) selected stably transformed Drosophila
cells using this method. Jarvis et al. (1990) also used a neomycin-resistance gene as the selectable marker gene, and isolated stably transformed
Spodoptera frugiperda cells by culturing transfected cells in culture medium containing G418 at a concentration of 1 mg/ml.
Subprotocol 6
Selection of Transfected Cells with Puromycin
The pac (puromycin acetyltransferase) gene is a selectable marker gene,
and this gene has been used as a dominant selectable marker gene in
mammalian cells. The introduction of this gene confers the resistance to
puromycin on transfected cells.
Materials
Equipment - The equipment is the same as that given for subprotocol 5
Antibiotics - Puromycin
Procedure
Vector 1. Construct a vector containing the pac gene as a selectable marker gene.
construction
I transfection
Results
McLachlin and Miller (1997) selected Spodoptera frugiperda cells
cotransfected with pac and a non-selectable apoptosis inhibiting gene
from a baculovirus in medium containing puromycin at a concentration
of 2.0 f,lg/ml.
Subprotocol 7
Selection of Transfected Cells with Hygromycin B
Hygromycin B inhibits protein synthesis in both prokaryotes and eukaryotes by interfering with translocation and causing mistranslation.
The hph gene from Escherichia coli is a gene coding a hygromycin
phosphotransferase.
Related Techniques
type cells. Extended culture in selection medium will result in the development of cells resistant to G418.
Results
Maisonhaute and Echalier (1986) selected stably transformed Drosophila
cells using this method. Jarvis et al. (1990) also used a neomycin-resistance gene as the selectable marker gene, and isolated stably transformed
Spodoptera frugiperda cells by culturing transfected cells in culture medium containing G418 at a concentration of 1 mg/ml.
Subprotocol 6
Selection of Transfected Cells with Puromycin
The pac (puromycin acetyltransferase) gene is a selectable marker gene,
and this gene has been used as a dominant selectable marker gene in
mammalian cells. The introduction of this gene confers the resistance to
puromycin on transfected cells.
Materials
Equipment - The equipment is the same as that given for subprotocol 5
Antibiotics - Puromycin
Procedure
Vector 1. Construct a vector containing the pac gene as a selectable marker gene.
construction
I transfection
Results
McLachlin and Miller (1997) selected Spodoptera frugiperda cells
cotransfected with pac and a non-selectable apoptosis inhibiting gene
from a baculovirus in medium containing puromycin at a concentration
of 2.0 f,lg/ml.
Subprotocol 7
Selection of Transfected Cells with Hygromycin B
Hygromycin B inhibits protein synthesis in both prokaryotes and eukaryotes by interfering with translocation and causing mistranslation.
The hph gene from Escherichia coli is a gene coding a hygromycin
phosphotransferase.