Computational protocol: Development of a common carp (Cyprinus carpio) pregnane X receptor (cPXR) transactivation reporter assay and its activation by azole fungicides and pharmaceutical chemicals

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Protocol publication

[…] Total RNA was isolated from frozen carp liver using Tri-reagent () following the manufacturer's instructions. Following DNase treatment with RQ1 DNase (Promega, Southampton, UK), cDNA was synthesised from 1 μg total RNA using random hexamers and MMLV reverse transcriptase (Promega), according to the manufacturer's instructions. This cDNA was used as template for the polymerase chain reaction (PCR) amplification of a partial cPXR sequence using the degenerate oligonucleotides 5′-TYTTCAGRMGKGCSATGAAR-3′ and 5′-CCHGGVYGRTCTGGDGARAA-3′ designed in conserved regions from aligned PXR sequences in other, closely related species (zebrafish, grass carp, fathead minnow and rainbow trout). The partial cPXR sequence was amplified using GoTaq DNA polymerase (Promega) and the following PCR conditions: 96 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and elongation at 72 °C for 3 min. A 865 bp product was obtained, purified using NucleoSpin Extract II columns (Macherey-Nagel, Dϋren, Germany) according to the manufacturer's instructions and sequenced (Eurofins Genomics, Ebersberg, Germany). BLAST searches (National Centre of Biotechnology Information ()) of the obtained sequences confirmed similarity with known PXR sequences.The full cPXR sequence was obtained by rapid amplification of cDNA ends (RACE) using the SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) and gene specific primers Cc_GSP1: 5′-ACT ATG AAA GCT GGA GGA TGG GGA CGA G-3′ (antisense), Cc_GSP2: 5′-CTC ACT GCA CAT CAC AAG ACC TTC GAC A-3′ (sense) and Cc_GSP3: 5′-CCG CAA CCA GGA AAT AGT AGC ACT CAC C-3′ (sense) according to the manufacturer's instructions. Both 5′ and 3′ RACE products were purified as described previously, sequenced, and characterised using BLAST and Clustal W (, ). A neighbour-joining phylogenetic tree was constructed in MEGA7 () using a 1000 replicate bootstrap analysis. [...] Data are presented throughout as mean ± standard error of the mean (SEM). All transfections were performed in triplicate and repeated three times on cells with different passage numbers. Concentration–response data using a four-parametric curve fitting and EC50 (for agonists) were analysed using GraphPad Prism (Graph Pad Software Inc.). Chemical responses were normalised against their relevant respective controls. Statistical analyses were carried out using SigmaPlot® software (Systat Software, Inc.) and p < 0.05 was considered statistically significant. […]

Pipeline specifications

Software tools Clustal W, MEGA, SigmaPlot
Applications Miscellaneous, Phylogenetics
Organisms Cyprinus carpio, Danio rerio, Homo sapiens
Chemicals Azoles, Clotrimazole, Rifampin