Retroviral Delivery of ECM Genes to Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 192

high-titer virus in comparison to a self-inactivating or a Tc-regulatable vector.
Once the retroviral vector is chosen, viral transduction can proceed by trans-
fection of the retroviral vector into packaging cells, virus production, and
infection of target cells ( see Subheading 3. ).

2. Materials
2.1. Retroviral Vectors
Retroviral vectors listed below are available from Clontech (Palo Alto, CA).
1. Constitutive retroviral vector: pLXSN (#K1060-B), pLNCX (#K1060-C).

2. Tc-regulatable vector: RevTet -On vector (#K1659-1), RevTet -Off vector

(#K1640-1).

3. Self-inactivating vector: pSIR (#6063-1).

2.2. Packaging Cells
1. 293-based packaging cells: Φ NX -eco (ATTC), Φ NX -ampho (ATCC).

2. NIH3T3-based packaging cells: PA317 (ATCC #f-13677, 9078-CRL), PT67

(Clontech #K1060-D).

2.3. Tissue Culture, Transfection, and Selection
1.

Tissue culture. All standard tissue culture reagents can be obtained from Gibco-BRL
(Bethesda, MD). Dulbecco modified Eagle medium (DMEM), fetal bovine serum
(FBS), glutamine, streptomycin, penicillin, trypsin, PBS.

2. Transfection of packaging cells: CalPhos Maximizer™ Transfection kit

(Clontech #K2050-1), ProFection mammalian Transfection system - calcium
Phosphate (Promega, Madison, WI #E 12). Also, you can make transfection
reagents as follows. Dissolve the mixture listed below in 80 mL of H 2 O and adjust
the pH exactly to 7.0-7.05 using 5 M NaOH or HCl ( see Note 2 ). Adjust the
volume up to 100 mL. Sterilize the 2xHBS solution through 0.22- m filter. All

reagents should be at room temperature prior to use. The following chemicals are
used to make 100 mL of 2XHBS solution: 1.6 g NaCl, 0.2 g KCl, 1 g dextrose,
0.027 g HEPES acid, 0.074 g Na 2 HPO 4 ×

µ

2H 2 O.

3. Selection of mammalian cells: Hygromycin B (Sigma, St. Louis, MO #H-3274),

Puromycin (Clontech #8052-1, -2).

2.4. Infection of Target Cells
Culture medium for target cells, 0.45 m Millipore filters, 5- or 10-mL

syringes, Polybrene (Hexadimethrine Bromide) Sigma #H9268.

µ

2.5. Regulation of Gene Expression
1.

Tetracycline hydrochloride (Sigma #T3383): Dissolve in 1 mg/mL in 70% etha-
nol, filter sterilize (0.22-Millipore filter), and store at -20 ° C (stable within 2 mo).