Both injectors are equipped with a nozzle (rectangular for
Isa10 and circular for Isa40) comprising five orifices.
4. These systems deliver fixed volumes of 100 μl per shot. The
void volume corresponding to the space between the reservoirs
and nozzles is of about 0.8 ml in Isa10 and 1.5 ml in Isa40.
3.1.1 Animal
Experiments in Mice
1. Mice are anaesthetized by i.p. injection of a solution containing
8 mg/ml ketamine (Imalge `ne 500, Rho ˆne-Me ´rieux) and
1.6 mg/ml xylazine (Rompun 2%, Bayer) in a total volume of
0.2 ml of saline.
2. Intramuscular injections: 50 μl of DNA are injected into the
quadriceps muscle by using a Micro-Fine insulin syringe
equipped with a 29 G1/2 needle.
3. Intradermal injections: 100 μl of DNA is injected into five spots
of dorsal skin by using the Isa10 injector. The site of injection
located all along the spinal cord is shaved and swabbed with
ethanol prior to the injection. For serum antibodies titration,
mice are bled by retroorbital sinus puncture, the bleedings
could clot, centrifuged and the sera were stored at À20
C
until the assay (see Note 1).
3.1.2 Animal
Experiments in Non-human
Primates
1. Monkeys are anaesthetized by intramuscular administration in
the thigh of 50 mg of ketamine (0.5 ml of Imalgene 1000,
Rho ˆne-Me ´rieux).
2. Intramuscular injections: 0.5 ml of DNA in 0.9% NaCl is
injected into the quadriceps muscle by using a 3 ml syringe
equipped with a 26 G3/8 needle (see Note 2).
3. Intradermal injections: 100 μl of DNA in 0.9% NaCl is injected
into five spots of thigh skin by using the Isa40 injector. The
injection site is shaved and swabbed with ethanol prior to the
injection (see Note 3).
3.2 Particle
Bombardment of Skin
1. For extracellular delivery, the following inert microparticles
8.5 Æ 0.4, 11 Æ 1.5, 20.5 Æ 1.2, and 52.6 Æ 2.0 μm polystyrene spheres (mean radius Æ standard deviation); 2.6 Æ 0.4,
10 Æ 0.7, and 20 Æ 1.4 μm glass spheres; and stainless-steel
beads are manually sieved to bands of radii 10–19, 19–27, and
27–38 μm.
2. The micro-particles used for intracellular studies are gold with
radii of 0.89 Æ 0.58, 1.12 Æ 0.56, and 1.52 Æ 0.58 μm and a
measured density of between 16.8 and 18.2 g/cm
3 . Gold payloads were either 0.5, 0.75, or 1.5 mg.
3. DNA vaccines to be injected are coated on the particles by
incubating with spermidine for 2 h prior to accelerating them
using commercially available gene guns (see Note 4).
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Akshay Datey et al.
Isa10 and circular for Isa40) comprising five orifices.
4. These systems deliver fixed volumes of 100 μl per shot. The
void volume corresponding to the space between the reservoirs
and nozzles is of about 0.8 ml in Isa10 and 1.5 ml in Isa40.
3.1.1 Animal
Experiments in Mice
1. Mice are anaesthetized by i.p. injection of a solution containing
8 mg/ml ketamine (Imalge `ne 500, Rho ˆne-Me ´rieux) and
1.6 mg/ml xylazine (Rompun 2%, Bayer) in a total volume of
0.2 ml of saline.
2. Intramuscular injections: 50 μl of DNA are injected into the
quadriceps muscle by using a Micro-Fine insulin syringe
equipped with a 29 G1/2 needle.
3. Intradermal injections: 100 μl of DNA is injected into five spots
of dorsal skin by using the Isa10 injector. The site of injection
located all along the spinal cord is shaved and swabbed with
ethanol prior to the injection. For serum antibodies titration,
mice are bled by retroorbital sinus puncture, the bleedings
could clot, centrifuged and the sera were stored at À20
C
until the assay (see Note 1).
3.1.2 Animal
Experiments in Non-human
Primates
1. Monkeys are anaesthetized by intramuscular administration in
the thigh of 50 mg of ketamine (0.5 ml of Imalgene 1000,
Rho ˆne-Me ´rieux).
2. Intramuscular injections: 0.5 ml of DNA in 0.9% NaCl is
injected into the quadriceps muscle by using a 3 ml syringe
equipped with a 26 G3/8 needle (see Note 2).
3. Intradermal injections: 100 μl of DNA in 0.9% NaCl is injected
into five spots of thigh skin by using the Isa40 injector. The
injection site is shaved and swabbed with ethanol prior to the
injection (see Note 3).
3.2 Particle
Bombardment of Skin
1. For extracellular delivery, the following inert microparticles
8.5 Æ 0.4, 11 Æ 1.5, 20.5 Æ 1.2, and 52.6 Æ 2.0 μm polystyrene spheres (mean radius Æ standard deviation); 2.6 Æ 0.4,
10 Æ 0.7, and 20 Æ 1.4 μm glass spheres; and stainless-steel
beads are manually sieved to bands of radii 10–19, 19–27, and
27–38 μm.
2. The micro-particles used for intracellular studies are gold with
radii of 0.89 Æ 0.58, 1.12 Æ 0.56, and 1.52 Æ 0.58 μm and a
measured density of between 16.8 and 18.2 g/cm
3 . Gold payloads were either 0.5, 0.75, or 1.5 mg.
3. DNA vaccines to be injected are coated on the particles by
incubating with spermidine for 2 h prior to accelerating them
using commercially available gene guns (see Note 4).
442
Akshay Datey et al.