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Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study w...

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Autores principales: Boshtam, Maryam, Asgary, Seddigheh, Rahimmanesh, Ilnaz, Kouhpayeh, Shirin, Naderi, Jamal, Hejazi, Zahra, Mohammad-Dezashibi, Hoda, Pieper, Ina Laura, Khanahmad, Hossein
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Medknow Publications & Media Pvt Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082034/
https://www.ncbi.nlm.nih.gov/pubmed/30271445
http://dx.doi.org/10.4103/1735-5362.236836
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author Boshtam, Maryam
Asgary, Seddigheh
Rahimmanesh, Ilnaz
Kouhpayeh, Shirin
Naderi, Jamal
Hejazi, Zahra
Mohammad-Dezashibi, Hoda
Pieper, Ina Laura
Khanahmad, Hossein
author_facet Boshtam, Maryam
Asgary, Seddigheh
Rahimmanesh, Ilnaz
Kouhpayeh, Shirin
Naderi, Jamal
Hejazi, Zahra
Mohammad-Dezashibi, Hoda
Pieper, Ina Laura
Khanahmad, Hossein
author_sort Boshtam, Maryam
collection PubMed
description Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F’ was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface.
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spelling pubmed-60820342018-10-01 Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface Boshtam, Maryam Asgary, Seddigheh Rahimmanesh, Ilnaz Kouhpayeh, Shirin Naderi, Jamal Hejazi, Zahra Mohammad-Dezashibi, Hoda Pieper, Ina Laura Khanahmad, Hossein Res Pharm Sci Original Article Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F’ was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface. Medknow Publications & Media Pvt Ltd 2018-10 /pmc/articles/PMC6082034/ /pubmed/30271445 http://dx.doi.org/10.4103/1735-5362.236836 Text en Copyright: © 2018 Research in Pharmaceutical Sciences http://creativecommons.org/licenses/by-nc-sa/4.0 This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
spellingShingle Original Article
Boshtam, Maryam
Asgary, Seddigheh
Rahimmanesh, Ilnaz
Kouhpayeh, Shirin
Naderi, Jamal
Hejazi, Zahra
Mohammad-Dezashibi, Hoda
Pieper, Ina Laura
Khanahmad, Hossein
Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface
title Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface
title_full Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface
title_fullStr Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface
title_full_unstemmed Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface
title_short Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface
title_sort display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293t cell surface
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082034/
https://www.ncbi.nlm.nih.gov/pubmed/30271445
http://dx.doi.org/10.4103/1735-5362.236836
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