Horse genomic DNA was obtained from Zyagen (San Diego, USA). Equine chemokine receptors CXCRA, CXCR2, CCR2, CCR5, C5aR1, and the predicted Duffy antigen receptor (DARC) were amplified from equine genomic DNA by PCR using PfuTurbo DNA polymerase (Stratagene). Primers and accession numbers are listed in Supplementary Table 6. Exons encoding DARC were assembled using overlap extension PCR. All coding sequences were cloned into the pIRESpuro3 vector (Clontech) according to methods described elsewhere12 (link). The human Ga16 cDNA (pCISG16 plasmid) was kindly provided by Melvin I. Simon47 (link). The Ga16 gene was recloned in between the BstBI and EcoRV sites of the pIREShyg3 vector (Clontech) using the following primers: 5′-AACTATTTCGAAGCCGCCACCATGGCCCGCTCGCTGACCTG-3′ and 5′-ATCGAGGATATCTCACAGCAGGTTGATCTCGTC-3′.
Koop G., Vrieling M., Storisteanu D.M., Lok L.S., Monie T., van Wigcheren G., Raisen C., Ba X., Gleadall N., Hadjirin N., Timmerman A.J., Wagenaar J.A., Klunder H.M., Fitzgerald J.R., Zadoks R., Paterson G.K., Torres C., Waller A.S., Loeffler A., Loncaric I., Hoet A.E., Bergström K., De Martino L., Pomba C., de Lencastre H., Ben Slama K., Gharsa H., Richardson E.J., Chilvers E.R., de Haas C., van Kessel K., van Strijp J.A., Harrison E.M, & Holmes M.A. (2017). Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus. Scientific Reports, 7, 40660.
Antigen receptor C5ar1 Ccr5 Cdna Chemokine receptors Coding sequences Dna polymerase Equine Gene Genomic Human Plasmid Primers Vector
Corresponding Organization :
Other organizations :
Utrecht University, University Medical Center Utrecht, Papworth Hospital, University of Cambridge, MRC Human Nutrition Research, Medical Research Council, Roslin Institute, University of Edinburgh, Moredun Research Institute, Universidad de La Rioja, Animal Health Trust, Royal Veterinary College, University of Veterinary Medicine Vienna, The Ohio State University, University of Naples Federico II, University of Lisbon, Universidade Nova de Lisboa, Tunis University, University of Birmingham, Addenbrooke's Hospital
PfuTurbo DNA polymerase (Stratagene) used for PCR amplification of equine chemokine receptors CXCRA, CXCR2, CCR2, CCR5, C5aR1, and the predicted Duffy antigen receptor (DARC) from equine genomic DNA
Primers used for amplification of equine chemokine receptors and DARC
dependent variables
Expression of equine chemokine receptors CXCRA, CXCR2, CCR2, CCR5, C5aR1, and the predicted Duffy antigen receptor (DARC)
Cloning of all coding sequences into the pIRESpuro3 vector
control variables
Equine genomic DNA obtained from Zyagen (San Diego, USA)
PIRESpuro3 vector (Clontech) used for cloning all coding sequences
Human Ga16 cDNA (pCISG16 plasmid) provided by Melvin I. Simon
Ga16 gene recloned into the pIREShyg3 vector (Clontech) using specified primers
positive controls
Human Ga16 cDNA (pCISG16 plasmid) provided by Melvin I. Simon
negative controls
No negative controls were explicitly mentioned.
Annotations
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