Nucleotides 3853 - 4363 of the EREG mRNA (accession number: NM_001432.3) were amplified from human gDNA by PCR and inserted into pMIR-RNL-TK reporter plasmid (Ambion, Kaufungen, Germany). Mutagenesis of the predicted target site seed sequences of reporter constructs were performed by site directed mutagenesis. The miRNA expression plasmids were generated by PCR amplification of nucleotides 91,350,658 - 91,351,156 of chromosome 13 (+) for miR-19a-3P and nucleotides 91,350,960 - 91,351,560 of chromosome 13 (+) for miR-19b-3P from human gDNA. Subsequently, the DNA fragments were inserted into the pSG5 vector (Agilent technologies, Ratingen, Germany). Expression plasmid for miR-20b is described elsewhere (8 (link)). The oligonucleotide sequences used for molecular cloning and site directed mutagenesis are shown in Supplementary Table 3
Psg5 vector
Manufactured by Agilent Technologies
Sourced in Germany
The PSG5 vector is a high-performance signal generator that provides a versatile solution for a wide range of applications. It offers stable and precise signal generation capabilities, making it a reliable tool for various testing and measurement needs.
Automatically generated - may contain errors
5 protocols using psg5 vector
1
Cloning of miRNA Expression Constructs
2
Overexpression of MECR in HeLa Cells
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All cell cultures were maintained at 37°C and 5% CO2. HeLa cells were plated in 10-cm cell culture dishes (CellTreat) at a density of 1.5 x 105 cells/mL in DMEM + 10% FBS (Mediatech, Gibco). After 24 h, MECR OX was achieved by transfecting cells with the pSG5 vector with cloned-in MECR under SV40 promoter control. Control cells received an equal amount of empty pSG5 vector (Agilent Technologies, Inc). HeLa cells were transfected using FuGENE transfection reagent (Promega). After an additional 24 h, cells were harvested by trypsinization and centrifugation. MECR OX was confirmed using real-time quantitative RT-PCR. Cell viability was unaltered in the MECR OX cells as determined by cell count and trypan blue uptake.
3
HCMV UL54 Promoter Analysis
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The pUL54-luciferase indicator plasmids pUL54-0.4, bearing the entire HCMV UL54 promoter, and pUL54-0.15, containing UL54 promoter sequence from -150 to +15 relative to the transcription start site, were previously reported (Gariano et al., 2012) . The pSGIE72 (IE1 expression construct), the luciferase indicator construct phTS-243/+30 [that contains a portion of the promoter of the human thymidylate synthase gene (-243 and +30 relative to the AUG start codon)] were previously described (Gribaudo et al., 2002) . pSG5 vector was purchased from Stratagene/Agilent Technologies and the pRL-TK vector expressing Renilla luciferase was from Promega.
4
Mimic miRNA Overexpression Plasmids
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The miRNA expression plasmids were generated by PCR amplification of nucleotides 91,350,335–91,350,756 of chromosome 13 for miR-17, 91,350,951–91,351,273 of chromosome 13 for miR-20a, 134,169,622–134,170,000 of X chromosome for miR-20b, 134,169,722–134,170,440 of X chromosome for miR-106a and nucleotides 100,093,902–100,094,174 of chromosome 7 for miR-106b from human genomic DNA and by insertion into the pSG5 vector (Agilent technologies, Ratingen, Germany). Overexpression of the corresponding miRNAs after transfection in HEK293T and LNCaP cells was verified by qRT-PCR (S1 Fig ). The nucleotides 2005–3089 of the CCND1 mRNA (accession number: NM_053056.2) containing a part of the corresponding 3’UTR were amplified via PCR using specific primers from human genomic DNA and inserted into pMIR-RNL-TK reporter vector which is described elsewhere [20 (link)]. The mutagenesis of the predicted target site seed sequences of pMIR-RNL-TK reporter constructs were performed with QuickChange Site Directed Mutagenesis Kit (Stratagene, Heidelberg, Germany), following the instructions of the manufacturer's manual. The primer sequences used for cloning and site directed mutagenesis are shown in S1 Table .
5
In Vitro Transcription and Translation of RFX Proteins
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Human RFX1 and RFX3 cDNA subcloned respectively into EcoRI and EcoRI-BamHI sites of pSG5 vector (Agilent Technologies) were transcribed and translated in vitro using a T7 TNT coupled reticulocyte lysate system (Promega) and used in EMSA with radiolabeled D2 or X-box oligonucleotides.
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