Retroviral Delivery of ECM Genes to Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 193

2. Doxycycline hydrochloride (Sigma # D9891): Dissolve in 1 mg/mL in H 2 O, filter

sterilize (0.22-22 mm filter) and store at 4 ° C in dark (stable within 1 mo). Small

aliquots can be frozen at -20 ° C.

2.6. Measurement of Infection Using Reporter Gene (LacZ)
1. Reagents for -gal assay: Staining kit is available from Strategene, La Jolla, CA

(#200284). You can also make the solution by using the following chemicals for
5 mL of staining solution: 0.008 g potassium ferricyanide, 0.01 g potassium fer-
rocyanide, 1.5 mL 2 M MgCl 2 , 75

β

µ L X-gal (40 mg/mL in DMF stock solution).

2. Reagents for fixation of cells: PBS containing 1% glutaraldehide (Sigma #G-5882).

2.7. Cloning and Plasmid Preparation
1. Cloning: All standard reagents for cloning, restriction enzymes, T4 ligase, alka-

line phosphatase, Klenow DNA polymerase.

2. E. coli competent cells: DH5 (Gibco, Gaithersburg, MD, #18258-012),

Epicurian Coli Æ SURE Æ competent cells (Stratagene #200152).

α

3. Plasmid preparation: L-broth, ampicillin, gel extraction kit (Quagen, Chatsworth,

CA, #28704), Mini Prep kit (Quagen #27104), Maxi Prep kit (Quagen #12162),
CHROMASPIN+TE-1000 Column (Clontech #K1324-1, 2).

3. Methods
3.1. Preparation of Recombinant Retroviral Vector
1. Choose one of the desirable retroviral vectors ( see Subheading 2. ). For a large-

scale preparation of plasmid, transform a plasmid into a suitable E. coli strain
(e.g., DH5 or SURE™) in accordance with manuals. Perform a large-scale DNA

preparation using Maxi Prep Kit or CHROMASPIN+TE-1000 column. Also, it is
possible to isolate and purify DNA by CsCl gradient centrifugation (Sambrook,
et al., 1989).

α

2. Digest the retroviral vector with suitable restriction enzymes and check the

digestion of the vector on agarose gel. If a blunt-ligation is required, both 5' and
3' ends of the vector can be filled up by addition of 1 U of the Klenow large
fragment of DNA polymerase and dNTPs at 50 M . Incubate a reaction mixture

for 15 min at room temperature and inactivate the enzyme at 75

µ

C for 10 min. To
prevent self-ligation of the vector, incubate the digested retroviral vector with the
calf intestine (CIP) or the shrimp (SAP) alkaline phosphatase and purify DNA
using phenol/chloroform/isoamylalcohol (24/23/1) solution. Isolate the insert
from an agarose gel according to manufacturer's specification (Quiagen Gel
Extraction kit).

°

3. Isolate an insert using similar procedures described above. It is important that the

5' and 3' overhang sequences of the insert and the retroviral vector are compatible
for ligation. If available restriction sites are not compatible, a blunt ligation can
be carried out after filling the overhangs of both insert and vector.