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 180

2.3. Luciferase Reporter Gene Assay
in Transfected RCS Cells Using FuGene
1.

FuGene 6 transfection reagent kit (Boehringer Mannheim).

2. Dulbecco's modified Eagle's medium high glucose without pyruvate, (Life Tech-

nologies, Inc).

3. RSC cells (4) .

4. Penicillin (50 U/mL), streptomycin (50 g/mL), 10% heat-inactivated FBS

(Hyclone, Laboratories, Inc., Logan, UT) for RCS cells and 10% heat-inacti-
vated calf serum (BALB/3T3 cells) (Hyclone, Laboratories, Inc.).

µ

5. Dual-Luciferase™ Reporter Assay System (Promega, Madison, WI).

6. The 742Luc/Int reporter gene construct derived from the pGL3 -Basic (Promega)

containing the firefly luciferase reporter gene. Control pRL family vectors (e.g.,
pRLSV40 , pRL-CMV , Promega) for renilla luciferase as an internal control.

7. Luminometer (e.g., Model MLX from DYNEX, Chantilly, VA).

2.3. Transgenic Mice
1. The most commonly used reporter gene in transgenic mice is the bacterial lacZ

( -galactosidase) gene. The basic reporter gene vector is commercially available

(e.g., pNASS

β

, Clontech, Palo Alto, AC). The 742LacZInt construct contains the
lacZ reporter gene under the control of the promoter and enhancer of the

β

α 2(XI)

collagen gene (6 , 8) . ( See Note 2 .)

2. LB broth with ampicillin (100 g/mL), Plasmid preparation kit (e.g., Maxi Kit,

Qiagen, Chatsworth, CA), QIAquick Gel Extraction Kit (Qiagen).

µ

3. Fertilized ova isolated from female donors that are produced through timed mat-

ing with fertile males. Recipient females are made by timed mating with vasecto-
mized males.

4. PBS (pH 7.4)

5. 4% paraformaldehyde, Dissolve 4 g paraformaldehyde in 100 mL PBS (pH 7.4)

at 80 ˚C under stirring in a chemical hood. Prepare fresh.

6. Washing solution: 0.1 M phosphate buffer (pH 7.4), 2 m M MgCl 2 , 0.01% sodium

deoxycholate, 0.02% Nonidet P-40.

7. Staining solution: 0.1 M phosphate buffer (pH 7.4), 2 m M MgCl 2 , 0.01% sodium

deoxycholate, 0.02% Nonidet P-40, 5 m M potassium ferricyanide, 5 m M potas-
sium ferrocyanide, 20 m M Tris (pH 7.4).

8. X-gal stock, 25 mg/mL in dimethylformamide (store in the glass tube at -20 C

protected from light). Just before use, dilute X-gal stock to give a final concen-
tration of 1 mg/mL.

°

3. Methods
3.1. CAT Reporter Gene Assay
in Transfected Chick Chondrocytes
Although there are a few established cell lines that exhibit a chondrogenic
phenotype, primary chondrocytes derived from the sterna of embryonic chick-