BMC Cancer 2006, 6:66 http://www.biomedcentral.com/1471-2407/6/66 Helper, came from Stratagene (La Jolla, California, USA). The RevTet-On system, including the plasmids of pRev- TRE and pRevTet-On, was supplied by Clontech (Palo, Alto, CA). The construction of pAAV/TRE/HSVtk/Tet-On pRevTRE/HSVtk was constructed as previously described [22]. In brief, Tet-On gene fragment was amplified by PCR technique with primers Tet1/Tet2 (Tet1 5 ' -CTC TAT CGA TGG ATC CGG CCA TTA GCC AT-3'; Tet2 5 ' -CTC TGT TAA CTA CCC ACC GTA CTC GTC-3'; Cla I and Hpa I restriction sites are shown in boldface) from the plasmid pRevTet-On. The PCR product was recovered from agarose gel and digested with Cla I and Hpa I. The recovered DNA fragment was inserted into pRevTRE/HSVtk to produce the recombinant plasmid pRevTRE/HSVtk/Tet-On. The fusion DNA fragment of HSVtk and Tet-On was cut with Xho I and Cla I and inserted into pAAV-MCS. The recom- binant plasmid pAAV/TRE/HSVtk/Tet-On was constructed followed by partial digestion of Not I and Cla I, filling the 3' end and self-ligation. Preparation, purification and titre detection of rAAV Packaging cells 293 were transfected by the method of modified calcium phosphate co-precipitation with pAAV/ TRE/HSVtk/Tet-On, pAAV-RC and pAAV-Helper when the cell monolayers were 50–60% confluent. The cells were collected after 72 days of transfection, and resuspended in cell lysis buffer (10 mM Tris-HCl, pH 8.5, 150 mM NaCl). The cells were frozen and thawed 3 times in liquid nitro- gen and 37°C respectively. The lysate was centrifuged at 4,000 g for 10 min and the supernatant was collected. Solid NaCl and PEG-8000 were added in supernatant to reach concentrations of 1 M and 10% (W/V) respectively, and the mixture agitated to dissolve these components whilst being incubated on ice for 1 h. After 9,000 g centrif- ugation for 15 min, the pellet was mixed with 1.38 g/ml of CsCl2 and centrifuged at 500,000 g for 24 h at 4°C to isolate the AAV-2/TRE/HSVtk/Tet-On viral particles. Titre detection was performed by dot blot hybridization for purified rAAV. The physical titre of AAV-2/TRE/HSVtk/Tet- On virus was 2.38 × 1011 viral particle/ml. rAAV infections and MTT assay MCF-7 cells were cultured at 2 × 103 cells per well in 96- well plates. The experimental cultures were divided into 3 groups, to which 2 × 107, 2 × 108 and 2 × 109 vp of rAAV- 2/TRE/HSVtk/Tet-On were added per well, at 37°C for 8 h. The ratio of viral particles and cells was as 104 vp/cell, 105 vp/cell, and 106 vp/cell. Twenty-four hours later, 1.0 µg/ ml Dox and 3.0µg/ml GCV (Sigma, St. Louis, MO) were added to each well of group 1, 3.0 µg/ml GCV was added to each well of group 2, and 1.0 µg/ml Dox was added to each well of group 3. Cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphe nyltetrazolium bromide (MTT, Sigma) assay after another 72 h incuba- tion. The cells were incubated with 0.5% MTT for 4 h. The medium was then removed and 100 µl of 0.04 M dime- thyl sulfoxide solution added, followed by incubation at 37°C for a further 4 h. The absorbance of the reaction product in solution at 570 nm was measured. The data were expressed as mean ± S.D and used to draw growth curves. To calculate the percentage of living cells, we divided the cell number in the experimental wells by the cell number in the control wells (no Dox or no GCV). The growth curves were protracted by a manual count method. After 48 h culture, viable cells were counted by the trypan blue dye exclusion method. All experiments were per- formed 4 times independently. Detection of HSVtk gene expression Expression of HSVtk gene in the infected MCF-7 cells and tumor tissues of BALB/c nude mice was analyzed by RT- PCR. 0.1 g of fresh tumor tissue with 1 ml of pre-cooled TRIzol reagent (Invitrogen, La Jolla, CA) was homoge- nized and total RNA was extracted according to instruc- tion. RT-PCR was performed with nested primers TK3/TK4 (TK3: 5'-TGG AGC AGA AAA TGC CCA CG-3' TK4: 5'-TGC TGC CCA TAA GGT ATC GC-3') (a DNA fragment of 420 bp). For internal control, β-actin (sense: 5'-TGA CGG TCA GGT CAT CAC TAT CGG CAA TGA-3' and antisense: 5'- TTG ATC TTC ATG GTG (AGC) TAG GAG CGA GGG CA- 3') were co-amplified with HSVtk (a DNA fragment of 260 bp). Before reverse transcription (RT), residual genomic DNA was removed by treatment with RQ DNase (Promega, Madison, WI). Total RNA (1.5 µg) was used to synthesize cDNA in 20 µl of reaction mixture by standard protocol with oligo(dT)15 as primer, and AMV reverse transcriptase (Promega). PCR reactions were carried out with 5 µl of RT products in 50 µl of reaction system using Taq DNA polymerase. After an initial denaturation 94°C for 5 min, 30 cycles of amplification were performed under following condition: 94°C for 30 seconds, 56°C for 45 seconds, and 72°C for 40 seconds. PCR products were run on 1.5 % agarose gel (containing 0.5 mg of ethidium bromide per liter). Establishment of human breast cancer models in BALB/c nude mice and GCV treatment Female BALB/c nude mice, 4–5 week-old and weighing 20.0 ± 2.0 grams, came from the Laboratory Animal Center of the Academy of Medical Sciences of China (Shanghai, China). The mice were housed in vinyl cages equipped with air filter lids, which were kept in laminar airflow hoods and maintained under pathogen-limiting conditions. All mice were cared for according to institu- tional guidelines, and all food, water, caging, and bedding were sterilized before use. All procedures were approved by the National Animal Care and Use Committee in China and the Laboratory Animal Center of the Academy Page 3 of 8 (page number not for citation purposes)