Cell culture, growth media and cultivation
Dihydrofolate-reductase deficient CHO cells (ATCC, Rockville, MD, USA) were cultured in Dulbecco's modified eagle's medium (DMEM) and Ham's F-12 medium mixed 1:1, supplemented with 4 mM L-glutamine, 0.1% (w/v) pluronic, 0.25% (w/v) soya-peptone, 13.60 mg/l of hypoxanthine and 3.88 mg/l of thymidine, at 37°C under 7% CO2. The cells were routinely passaged twice a week (1:6) at a starting density of 1 × 105 cells/ml in a spinner flask and used for transient transfection assays at the amount of 4 × 106 cells per sample.
Screening of a CHO genomic lambda-phage library
A CHO-K1 genomic library in the Lambda FIX II vector (Stratagene Inc, La Jolla, Calif) was screened with a probe specific for the Chinese hamster S100a6 gene. cDNA was synthesized with SuperScript® III Reverse transcriptase (Invitrogen) in a volume of 20 μl and 250 ng of CHO total RNA at 42°C for 1 h. 2 μl of the reaction product were further amplified by PCR at 98°C for 30 s and 34 cycles of 98°C for 10 s, 60°C for 30 s and 72°C for 15 s followed by a final extension at 72°C for 5 min using Phusion DNA polymerase (Finnzymes) and primers S100B (5'-ATGGCATGCCCCCTGGATCAG-3') and S100F (5'-CATTGTAGATCAAAGCCAAGG-3'), respectively. 100 pg of the amplified RT-PCR product were labeled with DIG-dUTP in a final volume of 50 μl using PCR DIG Probe Synthesis Kit, according to the DIG application manual (Roche). All cloning procedures were performed as described by Sambrook and Russell 2001 [41], restriction enzymes and other modifying enzymes were purchased from New England Biolabs (NEB, USA) and used according to the manufacturer's recommendations.
Plaque lifts
For one dish (145 mm) that was used for screening and plaque hybridization, fresh Escherichia coli XL1-Blue MRA (P2) (600 μl of OD600 = 0.5 cells diluted with 10 mM MgSO4) were infected with 10 μl of 50,000 plaque-forming units for 20 minutes at 37°C. Then 7 ml of NZY top agar at ~50°C were added, mixed and spread onto a prewarmed (37°C) NZY agar plate. The plaques became visible after 8 hours incubation at 37°C. After plaque formation, the culture dishes were stored at least 2 hours at 4°C, blotted on nylon membrane (Roche), denatured and neutralized. The step from preparing the plating cultures and performing the plaque lifts were done according to the instruction manual for Lambda FIX II Library (Stratagene).
Hybridization and detection
Before hybridization, the DNA was fixed to the nylon membrane by baking at 80°C for 2 hours. Pre-hybridization (39°C for 1 hour) and hybridization (39°C for overnight) were performed in the same solution, standard buffer (5x SSC, 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS and 1% blocking reagent). 10 ml buffer containing 10 μl of DIG-labeled probe were used for hybridization for one nylon membrane. Membranes were washed twice with low stringency wash buffer (2x SSC, 0.1% SDS) at room temperature followed by high stringency wash buffer (0.5x SSC, 0.1% SDS) at 65°C. After washing and blocking, the antibody solution (1:10,000 diluted Anti-Digoxigenin-AP (Roche) in blocking solution) was added to the membrane. Membranes were washed, equilibrated, covered with CDP-Star (NEB) and exposed to Lumi-Imager (Roche) to detect with a chemiluminescent assay. All steps after performing the plaque lifts were done according to the DIG application manual (Roche). A square area of agar corresponding to the location of a single positive clone was picked from the plate and confirmed by a second and third screening step. Purification of the lambda DNA was done with a Lambda kit (Qiagen Inc). The DNA was used in PCR reactions with T3 (5'-AATTAACCCTCACTAAAGG-3') and T7 (5'-TAATACGACTCACTATAGGG-3') and S100a6 gene specific primers A-F for veryfing the presence and location of the gene.
(primer name, sequence (5'-3'), strand/location).
A CTCCTTTGGCTCTTCGCTGTC sense/exon 1
B ATGGCATGCCCCCTGGATCAG sense/exon 2
C CCTTCTTGTGGCCATCTTCC sense/exon 2
D CTGAGATTGCAAGGCTGATGG sense/exon 3
E GCCAATGGTGAGCTCCTTCTG reverse/exon 2
F CATTGTAGATCAAAGCCAAGG reverse/exon 3
The lambda DNA was sequenced by GATC Biotech (Germany).
Generation of promoter constructs
5'- truncation constructs: according to the sequence information of the lambda DNA, several 5' deletion constructs were generated by PCR with the use of reverse primer (-1)F GATGATAAGCTTAATTGACCACTGGGCTAGAAG together with 1 of 11 sense primers as listed in below:
(primer name, sequence 5'-3'):
(-1505)B GATGATGGTACCGCATGCTGGCTGGGCTGGG
(-1343)B GATGATGGTACCTGAGACAGGGTTTTATATAGCC
(-1294)B GATGATGGTACCGGATCAACCTACTGAGCTATAT
(-1206)B GATGATGGTACCCACAAGTATTTACACTGAGATTC
(-910)B GATGATGGTACCATGCCGGAGTCACGAGTCAC
(-763)B GATGATGGTACCAGAGGCGTGGAAAACTGAGG
(-683)B GATGATGGTACCACTCCTTGGGCGGGCCTC
(-663)B GATGATGGTACCGGATGCTAGCCGCTATAAGG
(-534)B GATGATGGTACCAGGTCGGCTCCTGGGCTGG
(-322)B GATGATGGTACCTAGGGCGGCTCCCCGAGT
(-256)B GATGATGGTACCTCGCAGTGTGTGGTCCTGTC
The resulting constructs were designated Sps1 to Sps11, starting with the full-length construct derived with primer (-1505)B to the smallest obtained with primer (-256)B.
3'- truncation constructs: Constructs were generated by PCR with the use of sense primer (-910)B GATGATGGTACCATGCCGGAGTCACGAGTCAC together with one of the following reverse primers (name, sequence 5'-3'):
S-HINDIII(-115)-F GATGATAAGCTTTCAGGGATGTAAGAACGGAAGC
S-HINDIII(-231)-F GATGATAAGCTTCTTCCTGACAGGACCACACAC
S-HINDIII(-344)-F GATGATAAGCTTGCTTGCCTGGCACAACCAAGC
S-HINDIII(-461)-F GATGATAAGCTTTAGACCACCCGCGGAACCCG
S-HINDIII(-579)-F GATGATAAGCTTGGCAGGTAGACAGCGAAGAGC
S-HINDIII(-651)-F GATGATAAGCTTGCGGCTAGCATCCGGGAGG
The resulting constructs were designated from dS13 to dS17 according to the series of primers from (-115)F to (-651)F.
5' and 3' double truncation constructs: fragments P3 and P4 were amplified by PCR using primers: -1425B (5'-GATGATGGTACCGGAAGAAGCGAGTTAGACAG-3') and -1167B (5'-GATGATGGTACCTACGGTGTGAAGCAGCAGTG-3') together with the reverse primer -450F (5'-GATGATAAGCTTAGACCCCAGTGTAGACCACC-3').
deletion construct: a 64 bp GC-rich fragment located at positions -722 to -658 of the S100a6 promoter (5'CTAGCCTCAGGCGCCGGGTGGGGCTCGGGGCGGGCCGGCACTCCTTGGGCGGGCCTCCCGGATG-3'), containing two putative SP1 binding sites, was removed from construct dS16 by NheI digestion. 4 μg of construct dS16 DNA were digested with 20 U of NheI, purified by a GFX kit and subsequently religated with T4 DNA ligase, yielding construct 16-∆SP1
reverse-orientated, minimal and double promoter constructs: To generate the minimal, reverse and double promoter constructs primers listed below were used (primer name, sequence 5'-3'):
HINDIII(-910)B GATGATAAGCTTATGCCGGAGTCACGAGTCAC
KPNI(-579)F GATGATGGTACCGGCAGGTAGACAGCGAAGAGC
KPNI(-800)B GATGATGGTACCCCTCATGCCACTCCCAATCC
HINDIII(-800)B GATGATAAGCTTCCTCATGCCACTCCCAATCC
XHOI(-791) B GATGATCTCGAGACTCCCAATCCGGGACAGTC
XHOI(-589) F GATGATCTCGAGCAGCGAAGAGCCAAAGGAGTG
A reverse-oriented construct of dS16 (16-rev) was generated by cloning of the fragment amplified with the use of primers HindIII(-910)B and KpnI(-579)F into the pGL3-Basic vector. The minimal promoter construct, dS18, was generated by removing a 110 bp upstream region from dS16, using primers KpnI(-800)B and (-579)F for PCR amplification. Construct 18-rev was generated by PCR with primers HindIII(-800)B and KpnI(-579)F, followed by cloning into the vector in reverse orientation. Construct 18double, consisting of two tandem repeats of the core promoter sequence (dS18), was generated by amplifying two copies of the core sequence, one with KpnI(-800)B and XhoI(-589)F and the second with XhoI(-791)B and (-579)F. The products of these PCRs were cut with corresponding enzymes and subsequently ligated together.
All fragments were cloned into pGL3-Basic at KpnI-HindIII sites and sequenced.
The PCR reactions for the constructs were performed in a reaction volume of 25 μl containing 1 ng of lambda DNA as template, 0.2 mM of each dNTPs, 0.4 μM of each primer and 1 unit of KOD Hifi DNA polymerase (Novagen) at the following cycling conditions: 95°C 2 min, 30 cycles 98°C 15 s, 58°C 5s and 72°C 20s, final extension at 72°C 5 min. Amplifications for constructs P3 and P4 were performed in 50 μl containing the same amount of template DNA and dNTPs as above, 0.5 μM of each primer and 1 unit of Phusion DNA polymerase (Finnzymes). PCR cycles were: 98°C 2 min, 30 cycles of 98°C 10 s, 60°C 15 s and 72°C 45 s and a final extension at 72°C for 7 min.
Transient transfection assay
The constructs were co-transfected along with the Renilla luciferase reporter vector pRL-SV40 into CHO dhfr- by using Amaxa's Necleofector (Lonza). 4 × 106 cells were transferred to a 15 ml tube and centrifuged at 1000 rpm 10 min and resuspended with the nucleofection mixture containing 10 μg of plasmid DNA, 1 μg of pRL-SV40 in 100 μl of NucleofectorTM solution and subjected to electroporation. Subsequently, the cells were transferred into a pre-warmed culture medium in 6-well plates and incubated at 33°C and 37°C. The luciferase activities were measured on a Biotek Synergy 2 luminometer (Gen5 software) by using the Dual-Luciferase Reporter Assay System (Promega) preferentially after 24 hours. To normalize for transfection efficiency, the promoter activity of each construct was expressed as the ratio of firefly luciferase activity relative to Renilla luciferase activity. pGL3-Basic and pGL3-Promoter were used as negative and positive controls, respectively.
Quantitative real-time (qRT) PCR
Total RNA was extracted from 4 × 106 cells transfected with the promoter constructs, using the TRIZOL reagent, following the instructions for RNA isolation (Invitrogen TM, Life technologies). The yield and purity of the isolated RNA were determined by measuring the absorbance at 230, 260 and 280 nm using an ND-1000 spectrophotometer (NanoDrop Technologies, DE). The Eukaryote total RNA Nano assay together with Agilent's 2100 bioanalyzer were used for quality control of the isolated RNA according to the RNA 6000 Nano LabChip kit (5065-4476) standard protocol. First-strand cDNA synthesis of 1.5 μg total RNA was performed with the SuperScript III Reverse Transcriptase kit (Invitrogen). The synthesized cDNA served as template for quantitative real-time PCR reactions (1 cycle at 95°C, 2 min; 40 cycles at 95°C, 15 s; 60°C, 60 s) with the Rotor-Gene Q Real-Time PCR cycler (Quiagen). GoTaq qPCR Master Mix (Promega) was used in combination with the primers listed below and the corresponding cDNA template. For relative quantification of the mRNA levels of the samples the Rotor gene 6000 software and the 2-ΔΔC T comparative method [42] were used.
Luc135_se ACATATCGAGGTGGACATCAC
Luc199_as TAGCTTCTGCCAACCGAACG
n-Actb-back TTGACTCAGGATTTAAAAACTGG
n-Actb-for TGCTCCAACCAACTGCTGTCG
Measurement of mRNA stability
The mRNA stability of the Luciferase reporter gene normalized to the β-actin reference gene was determined via quantitative real-time PCR after blocking of new RNA synthesis by addition of actinomycin D at a final concentration of 5 μg/ml. Following actinomycin D treatment, 4 × 106 cells were harvested at designated time points (0 h, 2 h, 4 h, and 6 h) and total RNA was prepared and reverse transcribed into cDNA as described in section "Quantitative real-time (qRT) PCR". The fold change in Luc mRNA level is indicated as percentage mRNA decay. The mRNA level at time point zero (before treatment with actinomycin D) was considered as 100%.