**4.2.3 Transfection, dual-luciferase assay and representation of HR repair activity**

	- a. Add 4 l each of HR13 and HR24 DNA fragments together with 0.25 g of internal control plasmid (pCMV-Luc) in 50 μl of Opti-MEM (Invitrogen) medium, mix gently.
	- b. Mix 1 l LipofectamineTM 2000 gently in 50 μl of Opti-MEM medium and incubate for 5 minutes at room temperature.

CMV IE promoter and 5'-part of *Renilla* luciferase gene, the later ones result in 3'-part of *Renilla* luciferase gene and the poly-A signal. These two DNA fragments contain a 222-bp

Fig. 2. Schematic representation of substrate preparation for HCR assay of homologous

(C) Homologous recombination of the two PCR fragments results in expression of luciferase. (D) An alternative way to produce DNA fragments for HR by using restriction enzymes.

2. Prepare DNA-LipofectamineTM 2000 (Invitrogen) complexes for each sample as follows: a. Add 4 l each of HR13 and HR24 DNA fragments together with 0.25 g of internal control plasmid (pCMV-Luc) in 50 μl of Opti-MEM (Invitrogen) medium, mix gently. b. Mix 1 l LipofectamineTM 2000 gently in 50 μl of Opti-MEM medium and incubate

(B) Agarose gel electrophoresis of the PCR products, which serve as HR substrates.

**4.2.3 Transfection, dual-luciferase assay and representation of HR repair activity**  1. The HEp-2 cells (6104) are seeded in 24-well plates 24 h prior to transfection (the

appropriate cell numbers for seeding are dependent on cell types).

(A) Location of PCR primers on the pRL-CMV reporter plasmid.

for 5 minutes at room temperature.

recombination (HR) repair.

overlapping region for recombination (Fig. 2D).

5. Determine the HR activity by comparing the firefly luciferase-calibrated *Renilla* luciferase activities between the environmental toxicants- or anticancer drugs-treated cells and vehicle-treated control cells. For examples, the treatment of anticancer drug camptothecin (CPT, a topoisomerase I inhibitor) can potentiate HR repair activity but the areca nut extracts (ANE) repress HR repair in HEp-2 cells (Fig. 3).

Fig. 3. The use of HCR assays in evaluating the effects of camptothecin and areca nut extracts on homologous recombination repair.

(A) Using the method illustrated in Fig. 2D, the *Renilla* luciferase activity (reflecting the HCR activity in Y-axis) can only be detected in the presence of both two DNA fragments (N+P, lane 3) but not in cells transfected with only one fragment (NheI or PstI, lanes 1 and 2). Treatment of camptothecin (CPT) stimulates HR repair efficiency in the cells (lane 4). (B) Dose-dependent repression of HR repair activity by areca nut extracts (ANE).
