For the construction of SV40p TV, the bovine growth hormone polyadenylation site [BGH poly(A)] derived from pcDNA3.1 (Life Technologies, Carlsbad, CA, USA) was first inserted at the EcoRV site of plasmid 5A (a gift from Dr. Ben H. Park), a platform vector to construct targeting vectors carrying a NeoR gene expressed by an SV40 early promoter (17). The resulting plasmid containing BGH poly(A) in the forward direction was cleaved with NheI and XmnI to remove the backbone of the plasmid. The recovered fragment was then blunt-ended and ligated in the forward direction to a blunt-ended SpeI-EcoRV fragment from ATG-less TV containing a backbone and both of the homology arms.
For 3′ EGFP TV, a fragment between two XbaI sites in ATG-less TV (i.e., the EGFP coding region without an authentic start codon) was replaced with a PCR-amplified 3′ partial EGFP coding region (606 bp).
To produce the 5′ EGFP reporter vector, the 5′ portion of EGFP (500 bp) with a cytomegalovirus promoter was recovered as a SpeI fragment derived from pEGFPx2 (a gift from Dr. Ben H. Park) and inserted into pSEPT in the forward direction. To introduce a 3′ homology arm, the resulting plasmid was cleaved with XhoI and then ligated to the internal portion of SV40 large T excised from the HygR-5′ EGFP reporter vector. After transfection of the resulting plasmid into cells, stable transfectants were infected with an adenovirus encoding Cre, and a subclone undergoing Cre-loxP recombination was isolated from each stable clone; the H/R ratio was then measured by infection with ATG-less TV. Throughout the construction of all plasmids described above, fragments amplified by PCR were confirmed by DNA sequencing after cloning into plasmids. The sequences of the plasmids have been deposited in GenBank (accession noumbers: HygR-5′ EGFP reporter, JQ394982; ATG-less TV, JQ394983; SV40p TV, JQ394984; 3′ EGFP TV, JQ394985; and 5′ EGFP reporter, JQ394986).Transfection and infection
Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Wako Chemicals, Osaka, Japan) supplemented with 5% fetal bovine serum. Transfection of plasmids into cells was performed using TransIT-LT1 (Mirus Bio, Madison, WI, USA) as per the manufacturer's instructions. Adeno-associated viral (AAV) particles (serotype 2) were produced by transfection of 293T cells in 75 cm2 flasks with 7.5 µg AAV-based targeting plasmids and the AAV Helper-Free system (Agilent Technologies). The copy numbers of the AAV particles were determined by real-time PCR performed with StepOnePlus (Life Technologies), using the solutions of the AAV-based targeting plasmids as controls. The copy numbers of the AAV-based targeting plasmids in the control samples were calculated using their molecular weights and concentrations measured by NanoDrop (NanoDrop Technologies, Wilmington, DE, USA).
Throughout the study, cells were infected with AAV particles as previously described (7) at a multiplicity of infection of 1 × 104. Selection with G418 (Life Technologies) was carried out at concentrations of 0.4 and 1 mg/mL for the HCT116 and DLD-1 cell lines, respectively. In addition, 0.2 and 0.25 mg/mL hygromycin (Wako Chemicals) were used for the selection of HCT116 and DLD-1 cells, respectively. For the assay of the H/R ratio of the targeting vectors, reporter cell clones were infected with AAV-based targeting vectors in 75-cm2 flasks, selected with G418 for 1–2 weeks, trypsinized, suspended in phosphate-buffered saline (PBS), and analyzed with a FACSCanto II flow cytometer (BD Biosciences). Cell sorting was performed with a FACSVantage SEM instrument (BD Biosciences).Polymerase chain reaction
PCR was performed using Platinum Taq (Life Technologies) or Pwo DNA Polymerase (Roche, Basel, Switzerland) and a GeneAmp 9700 thermal cycler (Life Technologies). Genomic DNA (gDNA) for PCR templates was extracted using PureLink Genomic DNA Mini kit (Life Technologies) according to the manufacturer's instructions. To verify homologous integration of the targeting vector into the genome, PCR was performed using the following oligonucleotide primers: F1, 5′-TGTGTAGAAGTACTCGCCGA-3′; R1, 5′-TCCAGCAGGACCAT-GTGATC-3′; F2, 5′-ACCATCTTCTT-CA AGGACGA-3 ′; and R2, 5′-AGTACTCACCCCAACAGCTG-3′.Southern blot analysis
For Southern blot analyses, gDNA was extracted using standard phenol/chloroform extraction and ethanol precipitation. Five micrograms gDNA were digested with the appropriate restriction enzymes, fractionated on 0.7% agarose gels, and blotted onto Hybond-N+ membrane (GE Healthcare, Piscataway, NJ, USA). Probes were labeled using AlkPhos Direct Labeling and Detection System with CDP-Star kit (GE Healthcare) and used for hybridization.