ACT-LABS gun system Instruction Manual download pdf (Page 36)

Section 7

Optimization of Gene Gun Parameters

7.1 Overview

The flexibility of the particle delivery system allows fine-tuning of experimental param-

eters; 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 biologi-

cal 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 iden-

tify 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 dis-

tribution 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 plas-

mid 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 bom-

bardment 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 exper-

iments.

For example, Figure 20 shows a test of discharge pressure on transient gene expression,

in mouse skin transfected in vivo as described in Section 6.3. Optimum pressure was deter-

mined 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 epithe-

lial layer rather than the relatively acellular underlying dermal tissue.

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ACT-LABS gun system Instruction Manual Page 36

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