Gene Therapy Approaches Using Reproducible and Fully Penetrant Lentivirus-Mediated Endogenous Glioma Models - Gene Delivery and Therapy for Neurological Disorders - page 351

6. Start treatment with ganciclovir (GCV) 24 h after delivery of
Ad vectors. Administer twice daily 7 mg/100 l rat by intra-

peritoneal injection to each rat that received Ad-TK treatment.
GCV treatment should be administered for 7-10 days.
7. Monitor the animals daily, and euthanize them according to
the guidelines of the Institutional Animal Care and Use
Committee at your institution at the fi rst signs of moribund
behavior.

ʼ

4 Notes

1. This protocol uses the Clontech Lenti-X system (Clonetech.
com).
2. Virus titers will generally be the highest at 48 h after the start
of transfection. Caution: discarded medium contains infectious
lentivirus.
3. The fi lter used should be made of cellulose acetate or polysul-
fonic (low protein binding), instead of nitrocellulose.
Nitrocellulose binds proteins present in the membrane of len-
tivirus and destroys the virus.
4. Lentiviral titers can drop as much as two- to fourfold with each
freeze-thaw cycle.

4.1 Lentiviral Vector
Production

4.2 Glioma Models:
Intracranial Lentiviral
Injection in Rats

5. To generate reproducible tumor models, it is critical that the rat
is immobilized properly in the stereotactic frame. Correct place-
ment of the ear bars is challenging but is crucial for proper
alignment of the skull. The ear bars must be inserted slowly and
carefully into the ear canals of the rat without causing injury.

6. The operator should ensure that the dorsoventral plane of the
cranium is as fl at as possible. This can be adjusted by changing
the vertical position of the incisor bar.
7. Mounting of the rat onto the frame should be performed with
extreme care so that the position of the rat does not cause any
respiratory issues. The breathing rate of the rat should be
monitored during the entire procedure.
8. The stereotactic frame used is adequate for adult rats of 250 g
body weight. Fitting larger animals into the frame may be dif-
fi cult, and the coordinates will also be inaccurate. The operator
is advised to refer to a rodent brain atlas for fi nding the coor-
dinates of the injection site of interest [ 25 ].
9. The brain is well vascularized, and the operator is likely to
encounter blood vessels during the drill step. We recom-
mended drilling slowly and in the vicinity around the blood