If Using Transient Producer Cells

1. Purified retroviral vector DNA: A number of retroviral vectors are available. Appropriate vectors should be chosen according to the needs of the individual investigator. These vectors differ in the promoter driving the gene of interest, and the presence or absence of drug resistance genes. Similarly, different reporter genes are available, such as lacZ and alkaline phosphatase. The use of a lacZ reporter gene is detailed here.

2. Ecotropic envelope-expressing packaging cell line: Bosc 23 (American Type Culture Collection, Accession Number CCRL 11270). For culture of these cells, the same reagents as detailed in Subheading 2.1.1., items 2-4 are needed.

3. Transfection reagents:

a. 25 mM Chloroquine stock solution in PBS-CMF (see Note 2). Filter through a 0.2-|m disposable sterile filter unit and store at -20°C.

b. 2X N,N-bis[2-Hydroxyethyl]-2-aminoethanesulfonic acid (BES): 50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4. The final pH should be 7.0 ± 0.05. Filter through a 0.2-|im disposable sterile filter unit and store at 4°C.

c. 2 M CaCl2: Filter through a 0.2-|im disposable sterile filter unit and store at -20°C.

d. 8 mg/mL polybrene stock solution in PBS. Filter through a 0.2-|im disposable sterile filter unit and store at -20°C.

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