Gene Therapy in Spinal Muscular Atrophy (SMA) Models Using Intracerebroventricular Injection into Neonatal Mice - Gene Delivery and Therapy for Neurological Disorders - page 315

1. Injection stock solutions can be viral vector, plasmid DNA,
oligonucleotides, drugs, etc. In case of using viral vector, mix
a desired titer of viral vector (5-7

3.1.2 Preparation
of the Injection Stock
Solution

l per ventricle multiplied by
the number of the animals to be injected) with 1 % fi ltered
food coloring dye [ 23 , 96 ]. Addition of the dye is extremely
helpful for the visualization of the injection site to verify a suc-
cessful injection.
2. In case of using plasmid DNA for injection (in vivo transfec-
tion), the DNA solution will be 4

ʼ

ʼ l in total containing D -(+)-

glucose 20 % (w/v) (1 l),

and 2.5 kDa linear PEI homopolymer ( Polysciences , Inc .)
(150 mM) (1

ʼ l), plasmid DNA ( ≈ 5 ʼ g/ ʼ l) (2 ʼ

l) [ 82 , 83 ].
3. In case of using synthetic oligonucleotides, dissolve the oligo-
nucleotides to the desired concentration in ddH 2 O, multiplied
by the number of the animals to be injected, and mix with
food coloring dye [ 44 , 80 ].

ʼ

1. The tip of the glass micropipette (needle) is approximately
5 mm long (Fig. 6b ). Break the tip of the needle with a spatula
(Fig. 6c ) to adjust the length for the penetration of 2-2.5 mm
into the skull (Fig. 6d ).
2. The needle should be attached to the narrow end of the tub-
ing system (purchased from a hardware store and prepared
manually) with the syringe attached to the other end (Fig. 7a ).
3. Place the needle diagonally inside the microcentrifuge tube
containing the injection solution, and load the injection solu-
tion into the needle by slowly pulling the plunger of the
syringe (Fig. 7b ). After a suffi cient volume (approximately
5-7

3.1.3 Loading
the Injection Solution into
the Glass Micropipette

l) is loaded into the needle (Fig. 7c ), immediately stop
pulling the plunger; otherwise, the solution will be taken up
into the tubing. If the solution is taken into the tubing, wash
the tubing and start with a new needle.
4. After the loading is complete, detach the syringe from the tub-
ing system; pull the plunger up further and then reattach it to
the tubing system (Fig. 7d ).

ʼ

1. The P1 or 2 neonates are immobilized via cryoanesthesia for
1-2 min.
2. For injection, place the syringe in the injecting hand between
the index and middle fi ngers with the plunger touching the
palm of the hand under the thumb, and hold the needle
between the thumb and index fi ngers (Fig. 8a ). The length of
the tubing that connects the needle to the syringe should be
adjusted for each person's hand to allow fl exible handling of
the needle and the syringe.

3.1.4 Injection