Viral-Mediated Gene Transfer to Study the Behavioral Correlates of CREB Function in the Nucleus Accumbens of Rats - Drugs of Abuse: Neurological Reviews and Protocols - page 324

simplex virus (HSV) vectors to study the molecular biology of cocaine addic-
tion. Included are time-effi cient steps to ensure that the viral vector increases
expression of the target gene, that it is not toxic in the brain, and that it causes
meaningful changes in behavior. The steps in engineering and constructing a
viral vector are beyond the scope of this chapter; we assume that the reader has
obtained the vectors that will be used, although we make reference to virtually
all of the steps involved in performing a viral vector study and we provide
citations for those procedures that are not covered in depth. As a template,
we describe here some experiments that we conducted to examine how drug-
induced activation of the transcription factor cAMP response element binding
protein (CREB) in the basal forebrain (nucleus accumbens) [NAc] affects
behavior (1) .

2. Materials
2.1. Viral Packaging
For an overview of the packaging procedure, see Carlezon et al. (2) .

1. Laminar fl ow hood and 37°C humidifi ed incubator with 10% CO 2 atmosphere.

2. Tissue culture microscope and hemocytometer.

3. 60-mm, 100-mm, and 150-mm tissue culture dishes.

4. 5 dl 1.2 helper virus derived from HSV-1 strain KOS by deletion of most of the
IE2 (or ICP27 ) gene.

5. 2-2 cell line derived from the African green monkey VERO kidney epithelium
cell line by transfection with a plasmid expressing the IE2 gene.

6. 0.05% Trypsin-0.02% EDTA (Sigma T3924).

7. Dulbecco's minimum essential medium (DMEM): 1% penicillin-streptomycin
and 4 m M glutamine (50 glutamine in water can be stored at -20°C) with either

5% or 10% fetal bovine serum (FBS; Hyclone) as indicated.
8. Dulbecco's phosphate-buffered salt solution (D-PBS): both Ca 2+ , Mg 2+ -
free D-PBS (Fisher MT-21-031-CV) and D-PBS + Ca 2+ , Mg 2+ (Fisher
MT-21-30-CV).
9. LipofectAMINE (Life Technologies).
10. OptiMEM (Life Technologies).
11. Cell lifters.
12. Dry ice-ethanol bath, 37°C water bath.
13. 15- and 50-mL conical tubes, polypropylene, with plug seals.
14. 50-mL conical tubes, polystyrene.
15. Cup-type sonicator.
16. 10%, 30%, and 60% sucrose solutions in D-PBS with Ca 2+ , Mg 2+ .
17. Beckman Ultra-Clear 25

×

89 (SW41)-mm tubes.
18. Ultracentrifuge, Beckman SW28 and SW41 (or SW40) rotors.
19. 3-mL syringes and 18-gauge needles.
20. Screw-capped vials for virus storage.

× 89-mm (SW28), and 14 ×