amplicons were as follows: amplicon A: 59-GATGAATGTCAG- GATGCTCTAAACAT-39 and 59-CCACCATGCTGACG- TTTGTG-39; amplicon B: 59-CAGCAGCACATTTCACTCAT- TAGAG-39 and 59-TCACTTGATCCCACCGATGTC-39; am- plicon C: 59-GAGCTGCTGGAGGCAGTAGAG-39 and 59- GGAGTCAGGCTGTTCAAGACAA-39; amplicon D: 59- TGGCAACCTGTGCTCAGTCT-39 and 59-GGCATGTG- CTGCACAGTTATG-39. The primer sequences for the NBPF1 amplicon were 59-GCGAGGCTGCCCGAGCTTCT-39 and 59- GACTTCGCGTAACTTCCCATTCA-39. All measurements were performed in triplicates and the mean Cp-value was used in the calculations. Statistical analysis of the groups with NBPF1 loss and those without loss was performed with the Wilcoxon rank sum test. Cell culture and transfection The colon cancer cell line DLD1Tr21 Tet-On was obtained from Van de Wetering et al. [21]. This Tet-On system activates transcription of the gene of interest in the presence of doxycycline (Dox). The cells were cultured in RPMI with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml strepto- mycin. The cDNA for NBPF1-IRES-GFP, fused to an amino- terminal flag tag, was cloned in the pcDNA4/TO vector (Invitrogen). This construct was stably transfected in DLD1Tr21 cells using Lipofectamine reagent (Invitrogen) according to the manufacturer’s instructions. Fluorescence activated cell sorting allowed rapid isolation of NBPF1-positive cells, and further subcloning of this population yielded a clone that was more than 90% positive for NBPF1 expression upon induction with Dox (2 mg/ml, Sigma, Steinheim, Germany) for 48 h. As the transfected vector encodes a flag-tagged NBPF1 fusion protein, single colonies were tested for induction of NBPF1 expression by immunofluorescence staining with the anti-flag antibody (Sigma). Colony formation in soft agar Soft agar assays were performed with 6,500 DLDTr21 cells, either stably transfected with a GFP-expressing vector or with the NBPF1-IRES-GFP-expressing vector, both in the presence and absence of Dox. The cells were cultured in six-well dishes in a two-layer soft agar system, with 0.7% (w/v) Bacto agar (Difco, Detroit, MI, USA) on the bottom and 0.35% (w/v) Bacto agar on the top. Liquid medium was placed on top of the agar. During incubation, this medium was refreshed every 2 days to provide a continuous source of Dox. An additional top-agar overlay was given after one week. Experiments were performed in duplicates and the mean value was used for further analysis. Plates were incubated at 37uC with a humidified atmosphere of 5% CO2 for 2 weeks and colonies were counted using an inverted microscope. Pictures are taken with a Leica microscope (0.86magnification) or with an Olympus microscope (46mag- nification). Ethics The reported procedures using human samples were fully approved by the Ethics Committee at Ghent University and written informed consent was obtained from each patient since 1983. For patients whose DNA was obtained before 1983, only verbal consent was obtained. Indeed, the Ethics Committee advised that oral consent was sufficient for those patients, and that written consent should not be sought because most of these patients had died, and it was considered more ethical not to raise the matter with the family members again. Neuroblastoma Breakpoint Results Identification of rearranged genomic fragments encompassing the constitutional t(1;17) translocation breakpoint Karyotypic analysis of the lymphocytes of a neuroblastoma patient revealed a de novo, constitutional, apparently balanced translocation between chromosomes 1p36.2 and 17q11.2 [7]. Karyotyping of the primary tumor was unsuccessful, mostly due to necrosis. Due to the highly repetitive structure of the chromosome 1p36 region, breakpoint cloning proved to be extremely difficult. For this reason, we constructed a PAC/BAC contig of 1.5 Mbp covering the 17q breakpoint, and by screening a chromosome-17 specific cosmid library we identified three cosmids overlapping the breakpoint [18]. To facilitate the identification and cloning of the t(1;17) translocation breakpoints, genomic DNA from the somatic cell hybrids 32-7A and 32-2F53VIII, the Chinese hamster ovary cell line A/3, and normal human placenta were used for Southern blot analysis. The somatic cell hybrids 32-7A and 32- 2F53VIII contain, respectively, the der(1) and the der(17) chromosomes of this neuroblastoma patient [7]. Based on the sequence of the cosmid contig spanning the chromosome 17 translocation breakpoint (GenBank Acc Nu AF148647), nine DNA probes free of repetitive sequences were selected (Figure 1A). These probes were used for Southern blot analysis to search for abnormal, patient-specific hybridization bands. Probes 6 and 9 revealed additional bands in the DNA of cell lines 32-2F53VIII and 32-7A, respectively, but not in any of the other DNAs analyzed, indicating that these fragments span the chromosome 17 translocation breakpoint (Figure 1B). Both hybrid cell lines also contain a copy of the normal chromosome 17, resulting in the appearance of a ‘normal’ band in these cell lines. Cloning of the constitutional t(1;17) translocation breakpoints in somatic cell hybrids 32-2F53VIII and 32-7A We used GenomeWalker PCR to clone the breakpoints of the der(17) and der(1) chromosomes, present in the somatic cell hybrids 32-2F53VIII and 32-7A, respectively. For 32-2F53VIII, we used gene-specific primers (GSP1 and GSP2) localized in the probe #6 sequence, together with an adaptor-specific primer (AP1). For 32-7A we used GSP3 and GSP4, localized in or nearby the probe #9 sequence, together with AP1 and AP2, respectively. Cloning from 32-2F53VIII. Primer pair GSP1 and AP1 was used in a first round of amplification. Specific products of 1,457 bp and 3,575 bp were amplified from, respectively, the 32-2F53VIII/StuI and 32- 2F53VIII/PvuII GenomeWalker libraries (Figure 1C). With the GSP2 and AP1 primer pair, products of 1,029 bp and 3,459 bp were amplified from, respectively, the 32-2F53VIII/DraI and 32- 2F53VIII/EcoRV GenomeWalker libraries. All four 32- 2F53VIII-specific products were cloned, and complete sequencing showed overlapping novel sequences derived from chromosome 1 in all clones. In total, we sequenced 3,743 bp flanking the breakpoint region on the der(17) chromosome, of which 3,134 nucleotides were derived from chromosome 1 (GenBank Acc Nu AF379607; partly shown in Figure 2A). Cloning from 32-7A. Using primer pair GSP3 and AP1, specific products of 1,649 bp and 1,888 bp were amplified from the 32-7A/DraI and 32- 7A/EcoRV GenomeWalker libraries, respectively (Figure 1D). PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2207