Comparative genomics reveals a functional thyroid-specific element in the far upstream region of the PAX8 gene
- Roberto Nitsch†1,
- Valeria Di Dato†2,
- Alessandra di Gennaro3,
- Tiziana de Cristofaro1,
- Serena Abbondante1,
- Mario De Felice2, 3,
- Mariastella Zannini1Email author and
- Roberto Di Lauro2Email author
© Nitsch et al; licensee BioMed Central Ltd. 2010
Received: 17 November 2009
Accepted: 14 May 2010
Published: 14 May 2010
The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the Pax8 gene.
We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human Pax8 gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the Pax8 transcription start site. The in silico data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind in vitro and in vivo the Pax8 far upstream element, and is capable to activate transcription from it.
Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the Pax8 gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control Pax8 transcription during thyroid differentiation and are relevant to further studies on Pax8 as a candidate gene for thyroid dysgenesis.
The thyroid gland is a very important organ for the development of vertebrates as it synthesizes hormones that are essential for growth, development and survival such as tetraiodothyronine (thyroxine or T4) and triodothyronine (T3). Thyroglobulin (Tg), thyroperoxidase (TPO), sodium/iodide symporter (NIS), and TSH receptor (TSHr) are genes necessary for the synthesis of such hormones which takes place in the fully differentiated thyroid cell, called the thyrocite [1, 2]. Indeed, some of these genes mark a differentiated thyroid cell; in particular, thyroglobulin and thyroperoxidase are genes exclusively expressed in thyroid cells. The promoters of these two genes have been extensively studied and three transcription factors, namely TTF-1 (also named Titf1/Nkx2-1), Foxe1 and Pax8, have been demonstrated to be involved in the activation of these genes [3, 4]. During development and in the adult life, these factors are also present in other tissues, but the three of them are co-expressed only in the thyroid. It has been shown that their expression is required for the early stages of thyroid morphogenesis and is crucial for normal thyroid function. Indeed, for all its life a thyroid cell will be hallmarked by the simultaneous presence of TTF-1, Foxe1 and Pax8. Interestingly, these thyroid-enriched transcription factors are likely linked in a regulatory network such that each of them can be involved in the initiation or maintenance of the others .
During the past years, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that Pax8 plays a key role in thyroid development and differentiation . The first evidence of a role for Pax8 in the fully developed thyroid gland was provided by Mansouri et al.  by the generation of a Pax8 knockout mouse. Interestingly, Pax8+/- mice had no phenotype, while homozygous Pax8-/- mice showed growth retardation and died within 2-3 weeks. The cause of the death of the mutated animals was hypothyroidism, and the administration of thyroxine to Pax8-/- mice allowed the animals to survive. In fact, these mice did not display any apparent defects in Pax8 territories of expression except for the thyroid gland that appeared smaller and no follicles were detectable, demonstrating that Pax8 is necessary for the survival of follicular thyroid cells. Furthermore, it was shown that in the thyroid anlage of Pax8-/- mice the expression of Foxe1 is strongly down-regulated . These observations demonstrated that Pax8 not only is required for the survival of thyroid precursor cells, but also holds a specific upper role in the genetic regulatory cascade, which controls thyroid development and its functional differentiation.
Indeed, the reintroduction in vitro of an exogenous Pax8 in the PCPy transformed thyroid cell line, in which Pax8 is absent as well as all the differentiation markers, was sufficient to re-activate transcription of the endogenous genes encoding thyroglobulin, thyroperoxidase and sodium/iodide symporter . Furthermore, it has been reported that Pax8, together and synergistically with another thyroid-enriched transcription factor TTF-1, is able to activate transcription from the thyroglobulin gene promoter .
Although the function as well as the downstream targets of Pax8 are well studied, very little is known about its transcriptional regulation. Actually, an exhaustive knowledge of such a regulation is essential for a comprehensive view of thyroid gland development and differentiation pathways. Understanding those pathways is important not only to develop new treatments for thyroid gland diseases but also to add new dowels in the understanding of gene deregulation-dependent syndromes.
Therefore, the aim of this work was to shed light on the transcriptional regulation of Pax8 gene expression. The identification of distant acting gene regulatory sequences that direct precise spatial and temporal patterns of expression has been limited, despite their established roles in development, phenotypic diversity and human disease. Comparative genomic-based approaches have proved to be useful in identifying gene regulatory sequences, primarily on a gene-by-gene basis. These studies involved sequence comparisons of human (or other vertebrate) genomic intervals to orthologous regions from organisms separated by varying evolutionary distances, ranging from primates to fish .
We decided to use the bioinformatics approach to search for putative enhancer elements located in the 5'-flanking region of Pax8. We utilized a common interspecies sequence comparison and we analyzed 275 kb of orthologous regions of the human and mouse Pax8 gene. The rationale for using cross-species sequence comparisons to identify biologically active regions of a genome is based on the observation that sequences with important functions are frequently conserved among phylogenetically distant species, distinguishing them from non-functional surrounding sequences. We searched for non-coding genomic sequences (CNS), conserved between human and mouse, located in the 5'-flanking region of the Pax8 chromosomic locus, and we identified one sequence with a high thyroid-specific transcriptional activity. We demonstrated that this sequence is the first discovered enhancer of the Pax8 gene.
Genomic comparison and identification of conserved non-coding sequences
91 CNS fitting the above-cited criteria were identified. We discarded 11 of them because of their overlap with coding sequences inside the Pax8 gene (exons and UTR) and the remaining 80 were defined as non-coding conserved elements. We decided to focus our attention on 32 segments in the 5'-flanking region of the Pax8 gene extending for 116 kb upstream the start of transcription and located more than 30 kb away from any known gene.
Interestingly, the CNS87 manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but it showed no activity in HeLa cells (Figure 2B). Similar results were obtained if the CNS87 was inserted in a reporter vector containing the minimal promoter of the thymidine-kinase gene (TK-pGL3basic). We conclude that the CNS87 is an enhancer with thyroid-specific transcriptional activity.
The thyroid-specific transcription factor TTF-1 binds to the CNS87
To demonstrate the ability of TTF-1 to interact with the CNS87 also in vivo, we performed chromatin immunoprecipitation (ChIP) experiments on PC Cl3 cells. The crosslinked chromatin was immunoprecipitated using the antibody against TTF-1. As control, to rule out unspecific background of the ChIP assay, we performed one reaction using an unrelated antibody. The enrichment of the endogenous CNS87 region was monitored by PCR amplification using specific primers. Indeed, we demonstrate that, in agreement with the in vitro data, TTF-1 antibody is able to immunoprecipitate the chromatin containing the CNS87 element (Figure 4C). This result confirms the in vitro binding data presented in this paper, and clearly demonstrate that the thyroid-enriched transcription factor TTF-1 is able to bind in vivo the Pax8 far upstream element CNS87.
FT1 and FT6 are key regions for the transcriptional activity of the CNS87
TTF-1 activates transcription from the CNS87 and is involved in Pax8 expression in thyroid cells
Since TTF-1 was the only confirmed binding factor on the CNS87, and to better understand its role in the transcriptional activity of this element, we decided to perform transactivation experiments in a heterologous recipient. Hence, we transfected HeLa cells with the wild-type CNS87 vector alone or in co-transfection with an expression vector encoding TTF-1. The results showed in Figure 5C demonstrated that the co-transfection of the transcription factor TTF-1 is capable to strongly activate transcription from the CNS87, and in particular the fold activity of luciferase expression obtained in co-transfection experiments clearly resembles the levels obtained transfecting the CNS87 in the physiological PC Cl3 cell recipient.
All together these data confirm that TTF-1 is responsible for the full expression of the Pax8 gene and fit perfectly with our previous data in which we demonstrated the transcriptional activity of TTF-1 on the Pax8 far upstream enhancer CNS87.
CNS87 is a distant regulatory element that controls Pax8 expression
Comparative analysis of non-coding sequences between evolutionary correlated species is one of the new tools of the genomic era widely used when researchers want to look for regulatory mechanisms that are conserved throughout evolution. Such an approach in fact leads to the identification of highly conserved sequences that may be functionally relevant and to the discovery of new transcription factors binding sites and mechanisms that were to be known yet. Inter-genomic comparisons indeed, are rapidly evolving for investigations of regulatory regions involved in promoter activity [15–19].
Pax8, a thyroid-enriched transcription factor, plays a key role in thyroid development and differentiation . Despite its essential function, the transcriptional regulation of the Pax8 gene remains poorly characterized. In this paper, we describe the identification of the first transcriptional regulatory element of the PAX8/Pax8 gene. We achieved our results by coupling modern computational studies with experimental EMSA analysis and promoter/reporter assays.
We performed comparative studies on human and mouse genomic sequences flanking the Pax8 gene looking for conserved sequences to explore the possibility that such conserved non-coding sequences (CNS) may be instrumental in guiding the thyroid-specific expression of Pax8. By globally aligning a large fragment of the human and mouse Pax8 genomic locus, we found about 80 CNS that fitted our search criteria. However, we decided to focus our attention only on 32 of those sequences located in the 5'-flanking region of the Pax8 gene extending for about 110 kb upstream the start of transcription. Using promoter/reporter assays, we identified one conserved and functional sequence with a typical enhancer behavior specifically acting in thyroid cells and we named this sequence CNS87 (conserved non-coding sequence 87). In particular, this conserved sequence is a far upstream element, conserved between human and mouse, that turned out to be a target of the action of another thyroid-enriched transcription factor, i.e. TTF-1/NKX2.1.
Using several mutant mouse lines, Parlato et al. have established that in thyroid cell precursors the transcription factor TTF-1 and Pax8 are linked in a complex network of reciprocal regulatory interaction . Our data strongly support this scenario indicating that TTF-1, through the CNS87 element, is involved in the regulation of Pax8 gene expression in the environment of the thyroid cell. In fact, the CNS87 has all the features typical of an enhancer since it can stimulates the transcription of a reporter gene in a very strong and tissue-specific manner. A peculiar feature of the CNS87 element is its location about 90 kb upstream from the 5'UTR of the mouse/human Pax8 gene. It is noteworthy that a sequence located so far from a gene could be related with its expression. However, many examples exist in the literature on regulatory elements located very far from both the 5'end and the 3'end or even inside other genes. The most representative example came from Sonic Hedgehog gene (Shh), where its regulatory element is located 1 Mb upstream of Shh, embedded in a gene that resides in a cluster of unrelated genes [20–23]. In addition, many model of action have been developed for long distance acting regulatory elements. Such models imply the existence of factors that have the ability to bring and stimulate genes in active compartment of the chromatin. Therefore regulatory elements get closer to the proximal regulatory region of a gene allowing the start of the transcription .
The notion that the CNS87 could indeed serve as a regulatory element of Pax8 gene expression was further reinforced by the experimental observation with the BAC transfection assays. Those data clearly confirm that the CNS87 fragment is required for the full expression of the Pax8 gene.
The bioinformatics predictions of the transcription factors binding sites in this CNS element further highlight the physical experimental data. The TRANSFAC program predicted two binding sites for the thyroid-enriched transcription factor TTF-1 within footprints 1 and 6. Indeed, when the sequences corresponding to the FT-1 and FT-6 are mutated the CNS87 activity is severely reduced respect to that of the wild-type fragment confirming that TTF-1 is leading the activity of this element.
Taken together, our data suggest that the transcription factor TTF-1 participates to Pax8 gene expression directly binding to its 5'-flanking region and activating transcription from the CNS87 regulatory element that we have identified and characterized. In conclusion, this paper puts a milestone in the understanding of the regulation of the expression of the transcription factor Pax8 and will contribute to the establishment of a fine intergenic network between thyroid enriched transcription factors and their roles during development and adult life.
Mouse and human DNA sequences were obtained from the University California Santa Cruz Genome Browser (mouse genome, release Feb 2007; human genome, release June 2007). From this database, a region was selected spanning 275 kb of the entire Pax8 genomic locus in Homo sapiens and Mus musculus. The first known gene at the 3' and the first known gene at 5' of the Pax8 gene were taken as border limits of the genomic fragment selected. The genomic fragment was first chosen from the mouse genome. Then, the corresponding genomic fragment from the human genome containing the same size of DNA at the 5' and 3' flanking sides of the PAX8 gene (77.653 bp at the 3' and 140.982 at the 5') was taken. Comparative genomic analysis was carried out using the PipMaker http://bio.cse.psu.edu/pipmaker 51 and VISTA http://www.gsd.lbl.gov/vista/ 50 programs. The conserved sequences identified in this way were analyzed and compared with the TRANSFAC 4.0 database using Mat Inspector vertebrate matrix database to search for potential transcription factor binding sites. Sum of both minimized false positive and false negative errors were considered to ensure proper interpretation of the survey results.
PCR Amplification and subcloning of the Conserved Sequences
32 CNS positioned at the 5' of the Pax8 transcription start site or immediately downstream of it were selected for experimental test. These fragments were amplified by PCR using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and the PCR products were separated on a 2% agarose gel electrophoresis stained with ethidium bromide.
The conserved DNA sequences amplified by PCR were cloned in the pGL3basic vector (Promega) containing an E1b TATA box upstream the Luciferase reporter gene (pGL3-LUC), or into pGL3-TK vector containing the thymidine-kinase promoter .
The constructs CNS87-FT1m, CNS87-FT6m and CNS87-FT1-6m containing the mutated sequences of the CNS87 were generated by recombinant PCR.
Preparation of Cell Nuclear Extract and DNaseI Footprinting Assay
Total cell extracts were prepared from PC Cl3 cells grown in one hundred plates (diameter, 150 mm) as previously described . The footprinting probes were generated by digestion of the p87-E1b-LUC plasmid. The plasmid was linearized with the restriction enzyme XhoI, dephosphorylated with calf intestinal alkaline phosphatase (Roche Diagnostics) and extracted from agarose gel using the Qiaquick gel extraction Kit (Qiagen). 1.24 μg of linearized and purified plasmid DNA were end-labelled with γ-ATP-32P using T4 DNA polynucleotide kinase (BioLabs). The reverse strand probe was prepared in a similar way using as restriction enzyme Mlu1. The end-labelled probes were released from the plasmid with a second digestion and purified on a 5% acrylamide gel. 7 kcpm of the eluted probes were digested for 1 min at 20°C with 1:5000 and 1:2000 serial dilutions of a 3 mg/ml DNaseI (Roche Diagnostics) stock solution. The eluted probes were incubated with 30 μg of PC Cl3 nuclear extract and digested for 1 min at 20°C with 1:100, 1:50 and 1:25 serial dilutions of a 3 mg/ml DNaseI (Roche) stock solution.
Cells and Transient Transfection Assay
PC Cl3 and FRTL-5 cells were grown in Coon's modified F-12 medium (Euroclone) supplemented with 5% v/v calf serum and a six-hormone mixture (6H), as described .
HeLa cells were grown in Dulbecco's modified Eagle's medium (Euroclone) supplemented with 10% v/v fetal calf serum (Hyclone). For transient transfection experiments, cells were plated at 3 × 105 cells/60-mm tissue culture dish 5 to 8 h prior to transfection, whereas PC Cl3 cells were plated at a density of 5 × 105 cells/60-mm tissue culture dish 18 h prior to transfection. Transfections were carried out with the FuGENE6 reagent (Roche Diagnostics) according to the manufacturer's directions. The DNA/FuGENE ratio was 1:2 in all the experiments. The plasmid pRL-TK (Promega) was used as internal control in the transfection assays. Cells extracts were prepared 48 h after transfection to determine the levels of the firefly and renilla luciferase with the Dual Luciferase Reporter Assay System (Promega). Transfection experiments were done in duplicate and repeated at least three times.
Protein extracts and Gel Mobility Shift Assay
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in a buffer containing 10 mM Hepes pH 7.9, 400 mM NaCl, 0.1 mM EGTA pH 7.8, 5% v/v glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF).
TTF-1 purified protein (bTTF1) was produced as previously described . For EMSA assays, double-stranded oligonucleotides were labeled with γ-32P ATP and T4 polynucleotide kinase (New England BioLabs) and used as probes. The binding reactions were carried out in a buffer containing 10 mM HEPES (pH 7.9), 10% glycerol, 0.1 mM EDTA, 8 mM MgCl2, 1 mM dithiothreitol, 0.15 μg/ml of poly (dI-dC) for 30 min at room temperature. DNA-protein complexes were resolved on a 6% nondenaturing polyacrylamide gel and visualized by autoradiography.
The antibodies, αTTF-1  and αtubulin (sc5286, Santa Cruz), used in the supershift experiments were incubated with the protein extract for 20 min before adding the probe.
Oligonucleotides were derived from the protected sequences in the footprinting assays on the CNS87 and were as follows: FT-1: CGCACAAGAGCCCTTCTCAAGGGAT; FT-2: CTGGCTAAAGCCCAACGACACAGGT; FT-3: AACACTTGGGTGATCTACGTGAAGC; FT-4: GGGGGCAGGTTGGACAAAAGCCCCA; FT-5: CCTCAACAGCTTCTGACCTTCCTCT; FT-6: GAGAACGTTTATAAGTGTCTGGCTG.
RNA Extraction, cDNA Synthesis, and Real time-PCR
Total RNA was prepared using TRIZOL Reagent (Invitrogen) according to the manufacturer's directions. Total RNA (1 μg) was retrotranscribed using the iScript cDNA Synthesis kit (Bio-Rad). Real-time PCR analysis was performed using an iCycler-iQ real-time detection system and SYBR green chemistry (Bio-Rad, Hercules, CA). Reactions were carried out in duplicate in four independent experiments. The specific primers sets used for this analysis were for amplification of β-actin (Fwd: GGCAATGAGCGGTTCCGATG; Rev: ATGGTGGTGCCACCAGACAG), of Pax8 (Fwd: CAGCTATGCCTCTTCCGCTATT; Rev: TGTGGCTGTAGGCATTGCC), for TTF-1 (Fwd: AGGACACCATGCGGAACAGC; Rev: GGCCGCCCATGCCGCTCATA) and thyroglobulin (Fwd: TGTGGAATCTAATGCCAAGAACTG; Rev: TCCCTGAGAGCTTTTGGAATG).
For each gene, values are means ± SD of four independent experiments, normalized by the expression of β-actin, and expressed as a percentage of the value measured in parental FRTL-5 cells.
The cross-linking solution, containing 1% formaldehyde, was added directly to cell culture media. The fixation proceeded for 10 min and was stopped by the addition of glycine to a final concentration of 125 mM. PC Cl3 cells were rinsed twice with cold PBS plus 1 mM PMSF, and then scraped. Cells were collected by centrifugation at 800 × g for 5 min at 4 C. Cells were swelled in cold cell lysis buffer containing 5 mM piperazine-N, N'-bis(2-ethanesulfonic acid) (pH 8.0), 85 mM KCl, 0.5% Nonidet P-40, 1 mM PMSF, and inhibitors cocktail (Sigma) and incubated on ice for 10 min. Nuclei were spun down by microcentrifugation at 2000 × g for 5 min at 4 C, resuspended in nuclear lysis buffer containing 50 mM Tris-HCl (pH 8), 10 mM EDTA, 0.8% sodium dodecyl sulfate (SDS), 1 mM PMSF and inhibitors cocktail (Sigma), and then incubated on ice for 10 min. Samples were broken by sonication into chromatin fragments of an average length of 500/1000 bp and then microcentrifuged at 16,000 × g. The sonicated cell supernatant was diluted 8-fold in ChIP Dilution Buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), and 167 mM NaCl, and precleared by adding Salmon Sperm DNA/Protein A Agarose (Upstate Biotechnology, Inc., Lake Placid, NY) for 30 min at 4 C. Precleared chromatin from 1 × 106 cells was incubated with 1 μg of affinity-purified rabbit polyclonal antibody, αTTF-1  and an unrelated one, rotated at 4 C for 16 h. Immunoprecipitates were washed five times with RIPA buffer containing 10 mM Tris-HCl (pH 8), 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 140 mM NaCl, and 1 mM PMSF; twice with LiCl buffer containing 0.25 M LiCl, 1% Nonidet P-40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), and then three times with TE (10 mM Tris-HCl, pH 8; 1 mM EDTA). Before the first wash, the supernatant from the reaction lacking primary antibody was saved as total input of chromatin and was processed with the eluted immunoprecipitates beginning at the cross-link reversal step. Immunoprecipitates were eluted by adding 1% SDS, 0.1 M NaHCO3 and reverse cross-linked by addition of NaCl to a final concentration of 200 mM and by heating at 65 C for 16 h. Recovered material was treated with proteinase K, extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated. The pellets were resuspended in 30 μl of TE and analyzed by PCR using specific primers for the CNS87. The input sample was resuspended in 30 μl of TE and diluted 1:10 before PCR.
Mouse BAC clone bMQ-241M1 was obtained for the bMQ mouse BAC library made at the Wellcome Trust Sanger Institute constructed from the 129SvEv/AB2.2 mouse strain. Engineered versions were produced via recombinogenic targeting in Escherichia coli by the method of Lee et al. , also know as recombineering. Briefly, a luciferase expression cassette with a neo resistance cassette was inserted in the exon 2 of Pax8 into the ATG start codon. To obtain this construct luciferase cDNA form PGL3 was cloned upstream PGK-EM7-Neo cassette from pl452.
The recombination cassette was constructed by subcloning a 50-bp 5' recombination arm overlapping part of pax8 intron 1 and part of exon 2 upstream ATG start codon and a 50-bp 3'arm recombination arm containing part of exon 2 and intron 2 downstream ATG start codon such that the recombination arms flanked the Luc-PGK-EM7-Neo cassette. The forward strand sequences of the 50-bp homology arms were as follows, for the 5'arm: TGCGTAGGAAAGCTGCGAGTGTCCCTCAGTCTGTGAGCGACTCCCCGGCG; for the 3' arm: ATGCCTCACAACTCGATAGATCCGGTAAGGACCGCGGAGGGGCCAGGAC.
The final cassette with recombination arms was digested from the vector, gel purified and then recombined with pax8 BACs as described previously. Successful recombinants BAC Luc-Neo were selected by plating the electroporated cells on LB plates containing 20 μg/ml Kanamycin, 20 μg/ml chloroamphenicol. Correct modified BACs were verified by PCR analysis. For BAC 87- (lacking 87 region) the BAC luc-Neo was modified by insertion of Amp-resistance cassette into the 87 sequence. Amp-resistance cassette was amplified from pBS vector using primer 5' flanked by recombination arm overlapping the sequence upstream 87 region and using 3' primer flanked by recombination arm overlapping sequence downstream 87 region.
The forward strand sequences of the 50-bp Homology arms were as follows, for the 5'arm: AAAGAGAGGCAAAGAAAGCTAGGGGTCTGCAGTCTCCAAACCTGCAGGGCTGGCTAAAG; for the 3'arm: TTTCTGAATCTAAATCCAAAACTTTACCCTCTTCTGATTGGTAATGAGTC.
The PCR product was purified and recombined with BAC Luc-Neo. Recombinants were selected by plating the electroporated cells on LB plates containing 20 μg/ml Kanamycin, 20 μg/ml chloroamphenicol and 50 μg/ml ampicillin and the correct modified BACs were verified by PCR analysis.
BAC DNA for transfection was prepared with Qiagen Large-Construct kit.
Stable transfection were carried out with PEI22 (MBI Fermentas, St.Leon-Rot, Germany) as described previously . PC Cl3 cells were seeded in 6-well dishes (2 × 105) the day before transfection. For each construct 1 μg of BAC DNA and 2 μg of BAC DNA were transfected with an amount of PEI giving an N/P ratio of 7.5. The medium was replaced on day 3 with fresh medium containing 150 μg/ml G418. Stably transfected cell pools were grown to confluence. Cells were then washed twice with phosphate-buffered saline and incubate for 30 min at room temperature with passive lysis buffer (Promega) with vigorous shaking. Firefly luciferase activity was determined using "Luciferase assay System" (Promega). Luciferase activity was then normalized with protein concentration of cleared lysates.
We are grateful to M. Dentice for sharing with us useful reagents. We would also like to thank the Service of Molecular Biology (SBM) of the Stazione Zoologica A. Dohrn of Naples and F. D'Agnello of the IEOS-CNR for their technical assistance.
This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) to MZ and RDL and by grants from the Italian Ministry of Education, University and Research (MIUR-PRIN 2007) to MZ and RDL.
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