Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 188

polyQ pathogenesis. The possibility of designing and integrating
small interfering RNAs (siRNAs) into expression vectors makes
this therapeutic approach particularly attractive. Several groups,
including ours, have recently demonstrated the potential of LVs
for gene silencing in the rodent model of HD [ 44 - 47 ].

2 Materials

2.1 Cloning
of Transfer Plasmids

Sterile water
Easy cloning kits:

pENTR/D-TOPO or pCR2.1 cloning kit (Invitrogen)
pENTR4 cloning kit (Invitrogen)
LR clonase enzyme mix (Invitrogen)
Agarose (Invitrogen)
Purifi cation columns (Macherey-Nagel)
Restriction enzymes and associated buffers (Invitrogen or
New England Biolabs)
Antarctic phosphatase/Calf intestinal phosphatase/Shrimp alka-
line phosphatase (New England Biolabs)
T4 DNA polymerase (Invitrogen)
T4 DNA ligase (Invitrogen)
PCR: Pfx , PfuUltra II , or Taq kit/Deoxynucleotides (dNTPs) kit
(Invitrogen)
Antibiotics: ampicillin (75 μ g/ml)/kanamycin (50 μ g/ml)/spec-

tinomycin (100 μ g/ml) (Sigma)

Tris-EDTA (Sigma)
Luria Broth medium/agar (Invitrogen)
SOC medium, provided with competent cells (Invitrogen)
Petri dishes for microbiological cultures (CML)
Competent cells: DH10B, TOP10 (Invitrogen)
Incubator

2.2 293T Cells
and LV Production

Culture
Dulbecco's Modifi ed Eagle Medium (DMEM) (Invitrogen)
5 % Trypsin (Invitrogen)
Penicillin (10,000 units/ml)/streptomycin (10,000 μ g/ml)

(Invitrogen)
Phosphate-buffered saline (PBS) (Invitrogen)
Fetal bovine serum (FBS) for cell culture (Sigma)
Cell culture equipment (CO 2 incubator, laminar fl ow hood)