BIO RAD Helios Gene Gun System, Инструкция по эксплуатации

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Section 7 Optimization of Gene Gun Parameters
38 | Helios Gene Gun System
Another variable parameter, DNA loading rate (DLR), is determined by varying the concentration of DNA
precipitated on to the gold particles. Table 3 shows DNA dosage results for transfection of CHO cells
using the method described in In Vitro Delivery to Suspension Cultures, in Section 6. Secretion of murine
granulocyte macrophage colony stimulating factor (mGM-CSF) was greater at higher DNA dosages. Similar
results have been found for several in vivo and in vitro systems, with maximal expression observed for
1–5 µg DNA/mg gold particles. At dosages above ~5 µg/mg, the DNA and gold can form a single clump
which is unsuitable for cartridge preparation.
Table 3. Effect of DNA concentration on mGM-CSF expression in CHO cells.
*
DNA loading rate
**
Expression,
***
ng/106 cells
2.5 515
0.25 239
0.025 25
0.0025 0.9
* CHO cells were transfected as suspension cultures using the Accell Gene Gun.
** Irrelevant plasmid DNA was added to each preparation to bring the total amount of
DNA precipitated constant.
*** mGM-CSF in the media was assayed 24 hours postbombardment by ELISA as
described by Mahvi et al. (1996).
Parameters for In Vitro Delivery
The following conditions have been found to be effective for transformation of mammalian cell lines.
However, each laboratory should determine the optimum parameters for their particular application.
(See previous section.)
Table 4. Suggested starting conditions for in vivo transformation of tissue
culture cells using the Helios Gene Gun.
Parameter Conditions
Helium pressure 50–200 psi
PVP 0–0.015 mg/ml
MLQ 0.125–0.5 mg/tube
Microcarriers 1.0 or 1.6 µ gold
DLR 0.5–2.5 µg DNA/mg gold (0.06–1.25 µg DNA/cartridge)