Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 225

Vector preparations are considered pure if only three bands that
correspond to the three VP proteins are found with Coomassie rev-
elation. Additional bands or smears are contaminant proteins ( see
Note 16 ). If needed, a more sensitive silver staining can be per-
formed on the gel to reveal less abundant protein contaminants.

3.3 AAV6 Vector
Injection and Analysis
of Transgene
Expression

1. Material preparation . Connect a 10 l Microliter Hamilton

syringe with a 15 cm polyethylene tubing. Remove the metallic
needle tip of a 30 G insulin syringe using forceps and connect
it to the end of the polyethylene tubing. Fill the device with
ultrapure water using a 5 ml syringe connected to a 25 G nee-
dle. Make sure there are no leaks or bubbles. Aspirate 0.5

μ

3.3.1 Intramuscular
Injection

μ l of

2. Animal handling and vector injection . Newborn mice (P0-P5,
depending on the application) are anesthetized with a mix of
medetomidine (0.67 mg/ml) and midazolam (1.7 mg/ml).
Inject 4

air before sucking up to 9.5 μ l of the virus preparation.

l of the mix intraperitoneally. Anesthetized pups
should be kept on a heating mattress. Intramuscular injections
require two experimenters. One should hold the mouse and
expose muscles to be injected while another one injects the
required amount of virus directly through the skin ( see Note 17 ).
Animal handling for injection in individual muscles is
depicted in Fig. 2 .

μ

(a) Triceps surae
Expose the triceps surae muscle by pulling the hindlimb
and applying a slight pressure on the anterior tibia. Slowly
inject up to 2 l of virus preparation, while closely moni-

toring the muscle, which should get slightly white from
the increase in pressure and subsequent reduction in blood
fl ow. The injection of 2

μ

μ l virus should last around 5 s.

(b) Tibialis anterior
Slightly pull the hindlimb and gently press on the internal-
posterior side of the tibia to expose the tibialis anterior
muscle. Injection volume should not exceed 1 μ l of virus.

(c) Biceps and Triceps
Pull the forelimb to stretch the skin and expose the biceps
anteriorly and the triceps posteriorly. Slowly inject up 1 μ l

of virus in the biceps and 1.5 μ l of virus in the triceps.

(d) Masseter
The needle should penetrate the muscle with a slight angle
at mid-jaw level in direction of the ear. Slowly inject 2

μ l

of virus.