Chapter 41
Gene Technology
Attempts have been made to use cultured insect cells for the production
of foreign proteins by stably transforming the cultured cells. For this
purpose, usually the target gene and a gene that facilitates the selection
of transformed cells such as antibiotics resistant gene are introduced at
the same time. There are various methods to transfect cultured cells.
Subprotocol 1
DNA-Calcium Phosphate Coprecipitation Method
Materials
- Plastic tissue culture flasks
- Pasteur pipettes
- Test tubes
PROTOCOL
Equipment
- Sterile 250 mM calcium chloride solution (3.7 g CaCI 2 ·2H20 per 100 ml Solutions
distilled water)
- Sterile 2x HEPES-buffered saline (1.6 g NaCI, 0.027 g Na 2 HP0 4 ·2H 2 0,
and 1.2 g HEPES (Sigma) per 100 ml distilled water; pH adjusted to
7.08 with 1 N NaOH)
- Culture medium
- Selecting medium
- 250 mM CaCl 2 solution
- TE (10 mM Tris-HCI and 1 mM EDTA)
- Plasmid DNA containing genes to be inserted into host genome DNA DNA
- DNA-calcium phosphate coprecipitate (prepared 1 h before transfection)
- Mix the transfecting DNA (10-39 Ilg plasmid DNA/flask) dissolved
in TE and 250 mM CaCl 2 solution
- Add the DNA-calcium solution drop wise to an equal volume of2x
HEPES solution
- Incorporate air bubbles into the HEPES solution from the tip of a
pipette while the DNA-calcium solution is being added
- After that, continue coprecipitation for 30-45 min at room temperature without agitation