380
Related Techniques
Procedure
Pretreatment 1. Seed cells at a density of 2 x 10 7 cells/plastic tissue culture flask with
2-3 ml culture medium 1 day before transfection.
2. Add an equal volume of sterile distilled water to the culture medium
2 h before transfection.
3. Two hours after the addition of the distilled water, replace the medium with 5 ml fresh medium containing serum.
Endocytosis 1. Take 0.5 ml of the coprecipitate suspension after gentle pipetting, and
pour it into the 5 ml of medium of the culture to be transfected.
2. Leave the coprecipitate to sediment onto and then penetrate into the
cells for 18-24 h at 25°C.
3. Replace the medium with 2-3 ml fresh growth medium, and culture
for 2-3 days before using the selective medium.
Results
When the DNA-calcium solution is added to the HEPES solution, the
mixture will progressively become opalescent. This coprecipitation technique was first developed for mammalian cells (Graham and Van der Eb,
1973), and was adapted for insect cells by Bourouis and Jarry (1983), Di
Nocera and Dawid (1983), and Sinclair et al. (1983). This technique has
been used routinely in most transformation experiments with Drosophila cells. Using this method, McLachlin and Miller (1997) transfected
the Spodopterafrugiperda cell line IPLB-Sf21 with an expression vector
that co expresses two genes, namely pac (which confers resistance to puromycin) and an apoptosis-inhibiting gene from a baculovirus, and obtained transformants stably expressing both genes.
I Subprotocol 2
Polybrene-DMSO Method
The following procedure is based on the method of Fallon (1986).
Materials
Equipment - Plastic tissue culture flasks (25 ml; Falcon)
- Pasteur pipettes
- Test tubes
Solutions - 0.1% Polybrene (Sigma) aqueous solution
- DMSO
DNA - Plasmid DNA containing genes to be inserted into host genome DNA
Related Techniques
Procedure
Pretreatment 1. Seed cells at a density of 2 x 10 7 cells/plastic tissue culture flask with
2-3 ml culture medium 1 day before transfection.
2. Add an equal volume of sterile distilled water to the culture medium
2 h before transfection.
3. Two hours after the addition of the distilled water, replace the medium with 5 ml fresh medium containing serum.
Endocytosis 1. Take 0.5 ml of the coprecipitate suspension after gentle pipetting, and
pour it into the 5 ml of medium of the culture to be transfected.
2. Leave the coprecipitate to sediment onto and then penetrate into the
cells for 18-24 h at 25°C.
3. Replace the medium with 2-3 ml fresh growth medium, and culture
for 2-3 days before using the selective medium.
Results
When the DNA-calcium solution is added to the HEPES solution, the
mixture will progressively become opalescent. This coprecipitation technique was first developed for mammalian cells (Graham and Van der Eb,
1973), and was adapted for insect cells by Bourouis and Jarry (1983), Di
Nocera and Dawid (1983), and Sinclair et al. (1983). This technique has
been used routinely in most transformation experiments with Drosophila cells. Using this method, McLachlin and Miller (1997) transfected
the Spodopterafrugiperda cell line IPLB-Sf21 with an expression vector
that co expresses two genes, namely pac (which confers resistance to puromycin) and an apoptosis-inhibiting gene from a baculovirus, and obtained transformants stably expressing both genes.
I Subprotocol 2
Polybrene-DMSO Method
The following procedure is based on the method of Fallon (1986).
Materials
Equipment - Plastic tissue culture flasks (25 ml; Falcon)
- Pasteur pipettes
- Test tubes
Solutions - 0.1% Polybrene (Sigma) aqueous solution
- DMSO
DNA - Plasmid DNA containing genes to be inserted into host genome DNA