Homology searches were carried out using BLAST (Basic Local Alignment Search Tool): http://www.ncbi.nlm.nih.gov. The search for putative transcription factor binding site was carried our using the Lasergene Sequence Analysis Software (DNASTART, Inc, Wisconsin, USA). The CpG island was searched using the CpG Island Explorer Program at http://www.bioinfo.hku.hk/cpgieintro.html.
Generation of mTert-EGFP construct
Three constructs were generated using the available sequence of 4471 bp of the promoter of mouse mTert gene (NCBI Gene Banck; accession number AF 121949). Three fragments of the mTert promoter of 1086, 1846, and 4471 bp, downstream of the first ATG of the open reading frame of mTert gene, were amplified by PCR and ligated to pEGFP (Clontech Laboratories, Inc., Palo Alto, CA, USA) to promote the expression of EGFP. Orientation and sequence of each insert was checked by sequencing. The constructs were named 1 k-, 2 k-, and 5 k-mTert-EGFP to respectively indicate the construct with 1086, 1846 or 4471 bp of the mTert promoter. The plasmids were amplified and linearized before being used for electroporation of ES cell.
Culture, transformation and differentiation of ES Cells
Undifferentiated mouse ES cells (R1 from A. Nagy lab. and MAR1 a B6DF1 origin established by our group) were maintained on mitomycin-C treated (Sigma-Aldrich corporation St. Louis, MO, USA) mouse embryonic fibroblast (MEF) cells in 0.1% vol/vol gelatine coated tissue plates in Dulbecco's modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 20% FBS (PAA Laboratories Cölbe Germany), 2 mM glutamine, 1 mM MEM nonessential amino acids solution, 1 mM β-mercaptoethanol, leukaemia Inhibitory Factor (LIF) 1000 U/ml and an antibiotic mixture containing 100 U/ml penicillin and 100 μg/ml Streptomycin (all from Sigma-Aldrich).
Constructs 1 k-, 2 k- y 5 k-mTert-GFP were transfected by electroporation in mouse stem cell lines R1 and MAR1 as follow: ten micrograms of the linealized mTert-EGFP construct were electroporated into 3 × 106 cells using a Multiporator (Eppendorf, Hamburg, Germany) and a pulse of 300 V, 500 μs. Cells were allowed to recover for 24 h before G418 was added to a final concentration of 150 μg /ml. Cellular clones were selected by geneticine resistance over 7 days. At least ten clones of every construct were taken up for further analysis. Clones were disaggregated by trypsinization and allowed to attach on 96 well cell culture cluster, maintained for 2 days in LIF (Leukemia Inhibitory factor)-supplemented media until colonies were formed. Then LIF was removed from the culture media to allow the differentiation into embryoid bodies (EBs) and different cellular types.
For the promoter assays, transient transfections of the three transgenic constructs were performed into R1 ES cells by electroporation as indicated above, and the GFP activity of each construct was determined by flow cytometry using FACSCalibur System and CellQuest software. Cells transformed with an EGFP plasmid without promoter were used as controls for each transfection. Three independent experiments, each performed in duplicates, were realized. The GFP mean fluorescence intensity was expressed as relative intensity per positive cell with respect to that of the promoterless construct, pEGFP.
Generation and screening of transgenic mice
Transgenic mice (C57BL6xCBAF1) generation and identification was carried out as previously described . Briefly, transgene constructs (1 k-, 2 k, and 5 k-mTert- EGFP) were released from the vector by restriction endonuclease digestion and purified for embryo microinjection. Transgenic founders were backcrossed to C57BL6xCBA mice to obtain homozygotic lines. PCR-positive founder animals were confirmed by dot blot analysis and both the integrity and the transgene transmission were analysed during two generations. All the animals were maintained in an animal facility using procedures and protocols that are approved by our Institutional Animal Care and Use Committee.
Analysis of GFP mRNA expression in different tissues of transgenic mice by RT-PCR
Poly (A+) RNA was extracted from liver, spleen, kidney, heart, testis, ovary, and brain of transgenic mice at different ages using the Ultraspect™ RNA Isolation System following the manufacturer's instructions (Biotecx Lab. Inc., Houston, Texas, USA). The precipitated mRNA was dissolved in DEPC-treated water and then treated with 1 U of RQ DNAse I (Promega) at 37°C for 20 min to ensure that the only source of DNA in the polymerase chain reaction (PCR) was cDNA from cellular RNA. Finally, the RNA was extracted with phenol purification and ethanol precipitation, reconstituted in 50 μl of DEPC-treated water and stored at -70°C until the RT-PCR.
The RT reaction was carried out following the manufacturer's instructions of Epicentre (Tech. Corp., Madison, Wisconsin). Five micrograms of poly(A+)RNA were dissolved in water, heat-denatured (65°C, 2 min) and reverse-transcribed at 37°C for 60 min in a final volume of 25 μl containing 0.5 mM each dNTP, 0.2 μM oligo (dT), MMLV-RT (0.5 μl), RNAsin (0.2 μl), 1× MMLV-RT buffer and 8 mM DTT. After reverse transcription, PCR was performed adding 5 μl aliquot of each sample to the PCR mix containing the specific forward primers GFP-1 (5'-TGA ACC GCA TCG AGC TGA AGG G-3') and GFP-2 (5'-TCC AGC AGG ACC ATG TGA TCG C-3'), which specifically amplified a 340 bp GFP DNA. For mice Tert amplification, primers and length of the PCR product were mTert-F1 (5'-ACT TCA ACC GCA AGA CCG ACA GG-3') and mTert-R2 (5'-GGG TGG CCA TCA GTC CAG GAT GG-3') (451 bp). Amplification was carried out in a total volume of 25 μl (1× of PCR mix containing 1 u Taq polymerase, 2.5 μl 10× buffer, both from Promega, 100 μM each dNTP, 0.1 μM each primer and 2.5 mM MgCl2) Samples were loaded directly from ice into the heating block at 92°C to minimize the time required to reach denaturation temperatures. For semi-quantification of the mRNA, the amount of cDNA in all of the samples was equalized. In order to determine the lowest PCR cycle number that gave a reliable detectable product, a linear range of amplification was previously set. Amplification was done with an initial step of 92°C (2 min), followed by 23 cycles for β-actine and GFP, and 26 for mTert of 92°C (30 sec), 59°C (30 sec) and 72°C (30 sec). The final cycle extension was at 72°C (10 min). PCR products were resolved in 1.5% TBE agarose gels, followed by staining with ethidium bromide, and visualized using UV light. The relative abundance of each PCR product was determined by quantitative analysis of digital photographs of gels using the plug-in of Image Processing Tool Kit 5.0 (Reindeer Games Inc., Asheville, NC, USA). Negative control experiments in the absence of template RNA were performed. Generation of the expected fragments was strictly dependent on the presence of RNA in the RT reaction, as demonstrated with β-actine amplification, as a non-competitive internal standard control of the RT-PCR. To minimize variability, duplicate runs were performed for each mRNA amplified and the data were averaged.
Ten- to twenty-micrometer cryosections were blocked with 5% goat serum in TBST (Tris 0.1 M, NaCl 150 mM, pH 7.5, Triton 0.1% vol/vol blocking solution), then incubated for 1 hour at room temperature (r.t.) with anti-GFP rabbit polyclonal antibodies (1:600. Molecular P robes, Eugene, OR. USA) diluted in TBST 5% goat serum. Sections then were washed three times with TBS (Tris 0.1 M, NaCl 150 mM, pH 7.5) and incubated with highly cross-adsorbed Alexa 488 anti-rabbit secondary antibodies (1:500. Molecular Probes) for 1 hour at r.t. Finally, sections were counterstained with Hoechst 33342 (Molecular Probes), and mounted with Aqua Polymount mounting media (Poly-Labo).
Analysis of transgene expression in pluripotent stem cell isolated from testis of transgenic mice
ES-like colonies and germinal stem cells were obtained from neonatal and adult testis cells which were collected from a newborn transgenic 1 k-mTert- GFP mouse line of C57BL6/CBA F1 background (0–3 days old) and from 21–28 days old mice. Testis cell culture was performed according to the previously published protocol  with slight modifications. Briefly, testis cells were digested with 1 mg/ml collagenase (type IV, Sigma-Aldrich corporation St. Louis, MO, USA) for 10 min at 37°C, followed by 0.05% vol/vol trypsin/0,1 mM EDTA digestion for 3 min at 37°C. Testis cells were allocated to a tissue culture plate. 2.5 hours later, floating cells were recovered and passed to secondary culture plates on mitomycin-C treated (Sigma-Aldrich) mouse embryonic fibroblast (MEF) cell in 0.1% vol/vol gelatine coated tissue plates in Dulbecco's modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 20% FBS (PAA Laboratories Cölbe Germany), 2 mM glutamine, 1 mM MEM nonessential amino acids solution, 1 mM β-mercaptoethanol, leukaemia Inhibitory Factor (LIF) 1000 U/ml and an antibiotic mixture containing 100 U/ml penicillin and 100 μg/ml Streptomycin 20 ng/ml mouse epidermal growth factor, 10 ng/ml human basic fibroblast growth factor, 10 ng/ml recombinant rat glial cell line-derived neurotrophic factor (GDNF) (all from Sigma-Aldrich).
Analysis of transgene expression in neuroesferes obtained from E14 foetus brain of transgenic mice
Fore brain of E14 foetus of 1 k-, 2 k, and 5 k-mTert-EGFP transgenic mice were dissected and cut in small pieces. The tissue was suspended in HBSS (Sigma) with DNAsa 0.005% wt/vol (Sigma) and Papain 20 U/ml (Roche). Digestion was carried out at 37°C with gentle agitation every five minutes during 15 minutes. Digested tissue was centrifuged and pellet was suspended in DNAsa HBSS and mechanically dissociated with decreasing fire polished Pasteur pipettes. Cells were centrifuged twice with DNAsa HBSS and pellet was suspended in DMDM:F12, N2 supplements, AANE, Hepes 10 mM, Glucose 6 g/l, Albumax 0.1% vol/vol counted and seed at 400.000 cells/ml in the same medium plus 20 ng/ml of FGF (AbCys) and 20 ng/ml of EGF (AbCys). Neurospheres were mechanically triturated and diluted 1/5 weekly.