Bio-Rad PDS-1000 / He™ and Hepta™ Systems - Manual (Page 39)

more appropriate to use an extractive assay procedure in which expression level can be
measured, for example, as a function of enzyme activity per unit of total protein. Several
excellent markers (and associated assay procedures) are available for this type of study. These
include luciferase and ß-glucuronidase measured by a fluorometric process, and neomycin
phosphotransferase, for which a commercial ELISA assay has recently been marketed.
Yeast/Bacteria/Microbes
Cells which grow in suspension should be grown to late log to early stationary phase in
media under the appropriate conditions. Pellet the cells and resuspend in sterile water. Estimate
the cell density by determining the absorbence (for yeast, 1 O.D.
600
is approximately 2.8 x 10 9
cells/ml; for E. coli, 1 O.D.
600
is approximately 1.0 x 10 9 cells/ml). Spread 1 x 10 8 yeast of
2 x 10 9 E.coli
on 100 mm Petri plates containing the appropriate media. In addition to the
necessary supplements, the media for growing yeast should contain 0.75 M mannitol and
0.75 M sorbitol, while media for growing bacteria should contain 0.75 M sorbitol. The plates
should be allowed to air dry briefly, but used within 1 or 2 hours. Transformation efficiency
decreases if the plates are allowed to sit even for several hours.
Mammalian Cells
Cells in culture
Both primary and established animal cell lines are transformable using particle bom-
bardment. Because optimum bombardment conditions are usually found when mammalian
cells are placed at the top level of the bombardment chamber, the cells can be inoculated into
35 mm tissue culture plates. Only if cells are to be bombarded at Target Level 2 or farther
should larger plates be used.
Cells which grow as attached cultures should be inoculated into tissue culture plates one
day prior to bombardment so that they are in log phase and 50–80% confluent at the time of
transformation. Cells which grow as suspension cultures should be inoculated from log phase
cultures onto plates which have been asceptically coated by adsorption with Cell-Tak
(Collaborative Biomedical Products, Bedford, MA) according to the manufacturer’s instruc-
tions. The cells should be allowed to attach for 1 or 2 hours and should be approximately
75% confluent at the time of bombardment.
Immediately prior to bombardment, tissue culture media should be aspirated so that the
cells are directly exposed to air. Cells to be bombarded are placed in the bombardment cham-
ber centered under the stopping screen, the chamber door closed, the chamber evacuated to
the proper vacuum, and the cells bombarded. After bombardment, add fresh tissue culture
media to the cells and incubate for the appropriate time.
Organ cultures
Animal organs may be transformed by particle bombardment 1 hour to several days after
preparation. Tissues should be approximately 400 µm thick sections and no more than 1 cm
square. The tissue may be grown in cell culture inserts containing a permeable membrane. The
tissue section (in the cell culture insert) is placed in a Petri plate containing 1% agar, then
transferred to the bombardment chamber. After bombardment, the insert containing the tissue
can be transferred back to the growth chamber.
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