Computational protocol: Genetic variability of the activity of bidirectional promoters: a pilot study in bovine muscle

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Protocol publication

[…] Each putative bidirectional promoter region was PCR amplified from bovine genomic DNA using a pair of primers. The genomic DNA sequences were retrieved from the UCSC Genome Database. Sequence repeats were masked using RepeatMasker and Primer3 was then used to design primer pairs to amplify each putative bidirectional promoter region. PCR primers were synthesized by Eurofins MWG Operon. Restriction enzyme cutting sites for EcoRI (GAATTC) and BamHI (GGATCC) were artificially added into the PCR primers to facilitate directional cloning. Primer sequences are presented in . Polymerase chain reactions were performed in 50 μl using 120 ng genomic DNA, 1 × PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.3 μM of each primer and 1U AccuPrime GC-rich Taq DNA polymerase (Invitrogen). The following cycling protocol was used: 95 °C for 15 min, followed by 35 cycles of 95 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min, and a final extension step at 72 °C for 10 min. To check the quality of the amplification, 5 μl of PCR products were then analysed by gel electrophoresis with a 1% agarose gel. The PCR products were purified using the Qiagen MinElute Gel Extraction kit (Qiagen), digested with EcoRI and BamHI (New England Biolabs), purified with Qiagen Reaction Cleanup kit, quantified and then ligated using T4 DNA ligase (New England Biolabs) to the pBiP0 vector at a 3:1 ratio. The vector was previously digested with EcoRI/BamHI and dephosphorylated with alkaline phosphatase (New England Biolabs). The ligation products were transformed into Escherichia coli DH5α competent cells (Invitrogen). Ten clones were then amplified and plasmids were purified with the Pureyield Plasmid Miniprep System DNA purification kit (Promega). Positive clones were identified by digesting plasmid DNA with EcoRI and BamHI. Digestion products were then visualized after gel electrophoresis on a 1% agarose gel. One clone carrying the right plasmid construct was then amplified and plasmids were purified with the Macherey-Nagel Midi Endotoxin-free plasmid DNA purification kit. The plasmids were then sequenced bidirectionally using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and the primers used for the PCR amplification. After purification on Sephadex G50 superfine column (GE Healthcare), the sequencing reaction products were analyzed using 3130 Genetic Analyzer sequencer (Applied Biosystems). One clone containing for each cloning orientation the right plasmid construct was then chosen.Murine C2C12 myoblastic cells (ATCC CRL-1772) were grown in Dulbecco’s Modified Eagle’s Medium with Glutamax-I (4.5 g/l glucose, Invitrogen) supplemented with 1% penicillin/streptomycin and 20% heat-inactivated fetal bovine serum. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in air. C2C12 (∼25,000 cells/well) were seeded into 24-well plates 1 day before transfection. Transfections were performed on 40% confluent C2C12 cells with TurboFect transfection reagent (Fermentas) using 2 μl of reagent per μg of DNA plasmid, during 4 h according to the manufacturer’s protocol. The cells were transfected with 0.8 μg of the vectors carrying unidirectional pCMV1/pCMV2 promoters or putative bidirectional promoters or with pBI-CMV2, pBiP-DsRed, pBiP0 control vectors. All transient transfection experiments were done in triplicate and repeated three times. Thirty-six hours after transfection cells were washed two times with PBS 1×, and then viewed with an Axio Observer Z1 microscope (Zeiss) and images were acquired using a CoolSNAP HQ2 camera (Photometrics) driven by the Axiovision imaging system software. Analyses of micrographies were performed using the AxioVision 4.7.2 software (Zeiss). Filter sets 38HE [excitation BP470/40(HE) and emission BP525/50(HE)] and 43HE [excitation BP545/30(HE) and emission BP620/60(HE)] were used to visualize AcGFP1 and DsRed monomer signals, respectively. Each reporter gene assay was performed in triplicate and with three independent transfection experiments. […]

Pipeline specifications

Software tools RepeatMasker, Primer3
Application qPCR
Organisms Bos taurus