BIO RAD Helios Gene Gun System, Инструкция по эксплуатации

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Section 7 Optimization of Gene Gun Parameters
36 | Helios Gene Gun System
Section 7
Optimization of Gene Gun Parameters
Overview
The flexibility of the particle delivery system allows fine-tuning of experimental parameters; however, it is
necessary for each laboratory to determine the optimal parameters for their particular instrument and in vivo
or cell culture system. Any quantitative assay may be used to determine the optimum combination of critical
parameters for the particular biological system under investigation. Important parameters to evaluate include
the helium pressure, the PVP concentration, the microcarrier loading quantities (MLQ), and the DNA loading
ratio (DLR). It should be noted that absolute transgene expression levels are only a part of the processes
leading to immune or other biological responses, thus, each researcher must identify those parameters that
result in the appropriate level, location, and duration of transgene expression following particle-mediated
delivery. The representative results shown below reveal a commonly observed and useful trait of the Helios
Gene Gun: a broad, bell shaped distribution of effective delivery parameters.
Scientists are advised to optimize bombardment parameters for their particular gene gun and biological
system. The experimental approach which appears to be most effective in determining the optimum
bombardment conditions is the following.
Expt 1: Optimize helium pressure: Coat plasmid onto gold particles at 2 µg plasmid/mg gold. Prepare tubes
with 0.05 mg/ml PVP and 500 µg gold/shot (1 µg plasmid/shot). Bombard the target tissue at 50,
100, 200, and 400 psi helium to determine the optimum helium pressure.
Expt 2: Optimize PVP concentration: Use gold particles coated with 2 µg plasmid/mg gold as in Experiment
1. Prepare tubes with 0, 0.05, and 0.1 mg/ml PVP. Bombard the target tissue using the optimum
helium pressure determined in Experiment 1.
Expt 3: Optimize MLQ: Coat plasmid onto gold particles at 2 µg, 4 µg, and 8 µg plasmid/mg gold; prepare
tubes with each DNA/gold sample at 1 µg plasmid/shot (this is 500 µg gold/shot, 250 µg gold/shot,
and 125 µg gold/shot, respectively), and containing the optimum amount of PVP determined in
Experiment 2. Bombard the target tissue at the optimum helium pressure determined in Experiment
1 or 2.
Expt 4: Optimize DLR: The lowest amount of DNA that has been found to give a detectable level of gene
expression is 1 ng of plasmid, but this is dependent on the vector, the target, and the assay system.
For most experimental systems, bombardment with 1 µg of plasmid produces near maximal
expression. Increasing the amount of plasmid per bombardment does not result in a proportional
increase in gene expression. However, bombardment with higher amounts of DNA may be
important when using several expression vectors. To verify the optimum amount of plasmid per
bombardment precipitate different amounts of plasmid onto gold particles – depending on how
extensive a study desired, this may be as simple as a single two-fold dilution to multiple dilutions
using between 1 ng and 5 µg of plasmid per bombardment; the other bombardment parameters
should be as determined in the previous experiments.
For example, Figure 20 shows a test of discharge pressure on transient gene expression, in mouse skin
transfected in vivo as described in In Vivo Delivery to Epidermis, in Section 6. Optimum pressure was
determined to occur at 200 psi. This pressure resulted in adequate penetration of the particles without
excessive tissue damage, and deposition of the majority of the particles in the epithelial layer rather than the
relatively acellular underlying dermal tissue.