Targeted Gene Therapy for Ischemic Stroke - Gene Delivery and Therapy for Neurological Disorders - page 196

1,261-bp long, located between EcoN I and BamH I sites. To give
more rooms for cloning genes of interest, this fragment was deleted
from the vector and replaced with a 200-bp polyadenylation signal
between Bcl I and BamH I sites of SV40 large T antigen (SV40
plasmid, New England Biolabs Inc., Beverly, MA). The resulting
plasmid is known as H9LacZ (Fig. 1b ). The lacZ gene in the
H9LacZ can be replaced with other genes. For example, we gener-
ated a hypoxia-inducible VEGF expression vector by replacing lacZ
gene in H9LacZ plasmid with human VEGF 165 cDNA (Fig. 1b ).
The expression cassette in H9LacZ plasmid can be released from
the vector by Sma I and Sal I digestion and cloned to pAAV-LacZ
(Stratagene, La Jolla, CA). The resulting AAV vectors can be pack-
aged in different capsids of different AAV serotypes ( see Note 2 ).

3.2 pMCAO Model

1. This protocol is approved by the Animal Care Committee of
the University of California, San Francisco.
2. Anesthetize adult mouse using 5 % isofl urane inhalation, main-
taining at 1 ~ 1.5 % isofl urane during the whole procedure.
3. Place the mouse at a posture on a heating pad to maintain the
head and body temperature around 37 ± 0.5 °C throughout
the entire surgical procedure.
4. Make a vertical skin incision long enough to gain access to the
zygomatic arch between the left external auditory canal and
orbit.
5. Defl ect the temporal muscle anteriorly.
6. Remove the middle part of the left zygomatic arch without
damaging the mandibular nerve.
7. Retract muscles both downward and anteriorly.
8. Make a 4-ram craniectomy just anterior and superior to the
foramen ovale using a microdrill with 0.35- ~ 0.42-mm diam-
eter ultrafi ne burrs (Fig. 2a ).
9. Peel the bone off using blunted no. 1 forceps.

Fig. 2 Schematic illustrations of craniectomy position and middle cerebral artery.
( a ) Schematic illustration shows the position of the craniotomy after removal of
the skin and muscle. ( b ) Schematic drawing of MCA and ligation site: the left
MCA is exposed and occluded. In order to make the infarct at the same location
and comparable size among the animals, the MCA should be occluded at the
same site in all the experimental animals