Enhancer Analysis of the a1(II) and a2(XI) Collagen Genes in Transfected Chondrocytes and Transgenic Mice - Methods in Molecular Biology: Extracellular Matrix Protocols - page 179

enhancer activity. Further, some cell types (e.g., ameloblasts and odontoblasts)
are difficult to culture in vitro.
The generation of transgenic mice provides a powerful molecular tool for
identification of tissue- and developmental stage-specific enhancers of genes.
This method allows the analysis of promoter and enhancer activity in all
tissues at various developmental stages. A drawback of this approach is that it
is a time-consuming and expensive process. However, the problem may be
overcome by analyzing generation 0 (G0) embryos instead of establishing
transgenic mouse lines.

2. Materials
2.1. CAT Reporter Gene Assay
in Transfected Chick Chondrocytes
1. Ham's F-12 medium.

2. Fetal bovine serum (FBS).

3. Collagenase B (0.4% w/v): prepare by dissolving 100 mg collagenase B

(Boehringer Mannheim, Mannheim, Germany) in 25 mL Hank's balanced salt
solution (HBSS). Sterilize by passing solution through a 22- µ m filter apparatus.

4. Sterile instruments for dissection of sterna (small scissors, forceps, and scalpel).

5. Sterilized Nintex membranes (Tetko, New York, NY) and Swinex filters

(Millipore, Bedford, MA).

6. Sterile stirring bar and magnetic stir plate.

7. HBBSS: 0.14 g CaCl 2 2H 2 O, 0.40 g KCl, 0.06 g KH 2 PO 4 , 0.098 g MgSO 4

(anhyd.), 8.0 g NaCl, 0.048 g Na 2 HPO 4 , 1.00 g D-glucose made up to 900 ml;
adjust pH with 2 M NaOH to 7.4, adjust final volume to 1 L. Sterilize by auto-
claving ( see Note 1 ).

8. Complete growth medium: Ham's F-12 medium, 10% FBS, 100 U per mL peni-

cillin, and 50 µ g/mL streptomycin.

9. pDAS1BB5 containing the CAT reporter gene under the control of the promoter

and intron enhancer of the α 1(II) collagen gene (5) .

10. For LipofectAMINE™ (Life Technologies, Bethesda, MO) transfections, pre-

pare the following solutions in sterile 15 mL tubes:
Solution A: Dilute 5 µ g DNA in 300 µ L of serum-free medium for each transfection.

Solution B: Dilute 10 µ L of LipofectAMINE reagent (Life Technology) in to

300 µ L of serum-free medium for each transfection.

2.2. CAT Assay
1. 5 m M chloramphenicol: dissolve 16 mg of chloramphenicol in 10 mL of ethanol,

store at -20 ° C.

2. CAT scraping buffer: 0.04 M Tris-HCl pH 7.4, 1 m M EDTA, and 0.15 M NaCl.

3. CAT extraction buffer: 0.25 M Tris-HCl pH 7.8.

4. [ 3 H] Acetyl CoA (200 mCi/mmol, New England Nuclear, Boston, MA).

5. Water-immiscible scintillation fluid (Econofluor, New England Nuclear).