Horikawa et al. 10.1073/pnas.0508964102. |
Fig. 4. hTERT and mTERT expression in transgenic mice. Spleen lymphocytes from wild-type (mTERT+/+), mTERT–/–, mTERT+/–hTERT+, and mTERT–/–hTERT+ mice were stimulated with Con A (5 mg/ml), LPS (15 mg/ml), and rIL2 (150 IU/ml) for 48 h. The RNA samples were examined by conventional RT-PCR for mTERT mRNA, hTERT mRNA, and mouse actin mRNA (control), as described in Materials and Methods.
Fig. 5. RT-PCR comparison of mTERT and hTERT mRNA expression in each organ. The PCR primer sequences are perfectly conserved between mTERT and hTERT: 5'-CAT GGA GAA CAA GCT GTT TGC-3' in exon 9; and 5'-CAG GGA AGT TCA CCA CTG TC-3' in exon 11. The 182-bp PCR products are from both mTERT and hTERT (Undigested). The NcoI endonuclease cleaves mTERT-derived product into 122-bp and 60-bp fragments, but does not cleave hTERT-derived product. The HinfI endonuclease cleaves hTERT-derived product into 156-bp and 26-bp fragments but does not cleave mTERT-derived product. (a) Different ratios of mTERT and hTERT cDNA were used as the templates. Lane 1, 1.0 fg of mTERT; lane 2, 0.9 fg of mTERT, and 0.1 fg of hTERT; lane 3, 0.5 fg of mTERT and 0.5 fg of hTERT; lane 4, 0.1 fg of mTERT and 0.9 fg of hTERT; and lane 5, 1.0 fg of hTERT. The result suggests that this RT-PCR assay amplifies mTERT and hTERT at similar efficiencies. (b) RNA samples [11 organs and a mouse-embryonic fibroblast (MEF)] from mTERT+/–hTERT+ transgenic mice (line BAC-C10) were reverse-transcribed and examined for the relative abundance of mTERT and hTERT mRNA. Mouse GAPDH was a loading control. The results are consistent with the data shown in Fig. 2 a and b.
Fig. 6. Sp1/Sp3 complex binds to the nonconserved GC-box within the hTERT promoter in electrophoretic mobility-shift assay. The 45-bp probe corresponding to –38 to +7 of the hTERT promoter (wt), but not its GC-box-mutated version (mut), showed a stronger band shift when both Sp1 and Sp3 were included in the reaction (arrow) than when either Sp1 or Sp3 alone was included (arrowheads). This binding was specifically abrogated by preincubation with either anti-Sp1 or anti-Sp3 antibody but not anti-b-galactosidase antibody. The asterisk indicates a nonspecific band shift observed with both wt and mut probes.
Supporting Materials and Methods
Cells.
A normal human fibroblast strain NHF was derived from foreskin and is described in ref. 1. WI-38 human fibroblasts and WI-38/VA13 cells (SV40-immortalized, telomerase-negative derivative of WI-38) were from Coriell Cell Repository (Camden, NJ). For preparation of mouse embryonic fibroblasts (MEF), mTERT+/–hTERT+ mouse embryos were harvested at embryonic day 12.5, dissociated by incubation in 0.25% trypsin/EDTA, and cultured in RPMI medium 1640 containing 10% FBS. RCC23 (2), TE85 (3), and CMV-Mj-HEL-1 (3) have been described, as indicated. The other human cell lines used were obtained from American Type Culture Collection.Sources of Human Tissue RNA Samples.
RNA samples included in the Human Total RNA Master Panel II (BD Biosciences CLONTECH) were as follows: liver, 27-year-old male Asian; spleen, pooled from 14 male/female Caucasians, ages 30-66; testis, pooled from 19 Caucasians, ages 17-61; skeletal muscle, pooled from 2 male/female Caucasians, ages 43 and 46; kidney, 32-year-old male Asian; heart, 25-year-old male Asian; thymus, pooled from 9 male/female Caucasians, ages 15-25; lung, 27-year-old male Asian; brain, 28-year-old male Asian; bone marrow, pooled from 7 male/female Caucasians, ages 18-54; and uterus, pooled from 10 Caucasians, ages 15-77. Another total RNA sample from human livers (pooled from 23 male/female) was a kind gift from Dr. Snorri Thorgeirsson (National Cancer Institute).Primers and Probes for Real-Time Quantitative RT-PCR.
The specific sets of primers and probe were as follows: for hTERT, TaqMan predeveloped assay reagents for human TERT (#4319447F, Applied Biosystems), which amplify hTERT exons 3 to 4 (nucleotides 1,790 to 1,881 in GenBank NM_003219), and for mTERT, 5'-CGT AAG AGT GTG TGG AGC AAG C-3' (primer in exon 3), 5'-CCA GGT GTC CTG GTG ATG C-3' (primer in exon 4) and 5'-AGG CAA CAC CTT GAG AGA GTG CGG CTA C-3' (probe). As the endogenous control gene, hGAPDH (#4326317E, Applied Biosystems) was used for human tissues and mGAPDH (Assay ID Mm99999915_g1, Applied Biosystems) was used for tissues from mTERT+/–hTERT+ mice.Standard Curves for Quantitative Analysis of Gene-Expression Data.
A serial dilution of testis RNA samples from human or mTERT+/–hTERT+ mice (1.563 ng to 50 ng per reaction) was used to obtain the following standard curves [where y means Ct value (cycle threshold) and x means log ng of starting RNA]: y = –4.0361x + 37.68 (R2 = 0.9844) for mTERT in mTERT+/–hTERT+ mice; y = –3.5086x + 34.563 (R2 = 0.988) for hTERT in mTERT+/–hTERT+ mice; y = –3.545x + 34.773 (R2 = 0.9851) for hTERT in human; y = –3.6095x + 25.07 (R2 = 0.99) for mGAPDH; and y = –3.9473x + 27.409 (R2 = 0.9921) for hGAPDH.Electrophoretic Mobility-Shift Assay.
The double-stranded DNA probe corresponding to nucleotides –38 to +7 of the hTERT promoter (TCC GCG GCC CCG CCC TCT CCT CGC GGC GCG AGT TTC AGG CAG CGC) and its mutant version (TCC GCG GCC CAT ACC TCT CCT CGC GGC GCG AGT TTC AGG CAG CGC, mutated sequences underlined) were synthesized and 5'-labeled with fluorescein at Invitrogen. The recombinant human Sp1 and Sp3 proteins were from Alexis Biochemicals (San Diego, CA). The anti-Sp1 and anti-Sp3 antibodies were from Santa Cruz Biotechnology, catalog numbers sc59-X and sc-644X. The anti-b-galactosidase antibody was used as a control antibody. The protein-DNA binding reactions were performed in buffer containing 10 mM Hepes (pH 7.9), 0.1 mM EDTA, 8.5% glycerol, 170 mM KCl, 1 mM ZnSO4, 37.5 mg/ml poly(dI-dC), 10 mM DTT, and 1 mg/ml BSA, separated in a 5% polyacrylamide gel, and detected by using the Typhoon system (Molecular Dynamics). For supershift experiments, protein samples were preincubated with anti-Sp1, anti-Sp3, or anti-b-galactosidase antibody.1. Horikawa, I., Parker, E. S., Solomon, G. G. & Barrett, J. C. (2001) J. Cell. Biochem. 82, 415–421.
2. Horikawa, I., Oshimura, M. & Barrett, J. C. (1998) Mol. Carcinog. 22, 65–72.
3. Hensler, P. J., Annab, L. A., Barrett, J. C. & Pereira-Smith, O. M. (1994) Mol. Cell. Biol. 14, 2291–2297.