Cargando…

Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin

Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn's disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic...

Descripción completa

Detalles Bibliográficos
Autores principales: Fatahi, Azam, Rahimmanesh, Ilnaz, Mirian, Mina, Rohani, Fattah, Boshtam, Maryam, Gheibi, Azam, Shariati, Laleh, Khanahmad, Hossein, Kouhpayeh, Shirin
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/PMC6040166/
https://www.ncbi.nlm.nih.gov/pubmed/30065768
http://dx.doi.org/10.4103/1735-5362.235162
_version_ 1783338809634586624
author Fatahi, Azam
Rahimmanesh, Ilnaz
Mirian, Mina
Rohani, Fattah
Boshtam, Maryam
Gheibi, Azam
Shariati, Laleh
Khanahmad, Hossein
Kouhpayeh, Shirin
author_facet Fatahi, Azam
Rahimmanesh, Ilnaz
Mirian, Mina
Rohani, Fattah
Boshtam, Maryam
Gheibi, Azam
Shariati, Laleh
Khanahmad, Hossein
Kouhpayeh, Shirin
author_sort Fatahi, Azam
collection PubMed
description Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn's disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic evolution of ligands by exponential enrichment) method requires positive and negative expressing α4 integrin cell lines. For a better isolation, we intended to construct a negative cell line lacking of specific ligand binding site of α4 integrin. Escherichia coli strain top 10 was used for truncated integrin subunit α4 (TITGA-4) expression vector. Human embryonic kidney (HEK)-293T cell was transfected with linearized TITGA-4 plasmid and subsequently screened for stable truncated TITGA-4 expressing cells. Chromosomal DNA of truncated TITGA-4-transfected cells was extracted and the presence of truncated TITGA-4 gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of truncated TITGA-4 on HEK-293T cells was also analysed by real-time PCR and flow cytometry. Real-time PCR and flow cytometric analysis showed significant difference of truncated TITGA-4 expression between untransfected HEK-293T cells compared to transfected cells. The results suggest that we have successfully constructed the truncated integrin α4 expressing HEK-293T cell, which will facilitate further research into the production of antibody, nanobody, and aptamer against α4 integrin.
format Online
Article
Text
id pubmed-6040166
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Medknow Publications & Media Pvt Ltd
record_format MEDLINE/PubMed
spelling pubmed-60401662018-08-01 Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin Fatahi, Azam Rahimmanesh, Ilnaz Mirian, Mina Rohani, Fattah Boshtam, Maryam Gheibi, Azam Shariati, Laleh Khanahmad, Hossein Kouhpayeh, Shirin Res Pharm Sci Original Article Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn's disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic evolution of ligands by exponential enrichment) method requires positive and negative expressing α4 integrin cell lines. For a better isolation, we intended to construct a negative cell line lacking of specific ligand binding site of α4 integrin. Escherichia coli strain top 10 was used for truncated integrin subunit α4 (TITGA-4) expression vector. Human embryonic kidney (HEK)-293T cell was transfected with linearized TITGA-4 plasmid and subsequently screened for stable truncated TITGA-4 expressing cells. Chromosomal DNA of truncated TITGA-4-transfected cells was extracted and the presence of truncated TITGA-4 gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of truncated TITGA-4 on HEK-293T cells was also analysed by real-time PCR and flow cytometry. Real-time PCR and flow cytometric analysis showed significant difference of truncated TITGA-4 expression between untransfected HEK-293T cells compared to transfected cells. The results suggest that we have successfully constructed the truncated integrin α4 expressing HEK-293T cell, which will facilitate further research into the production of antibody, nanobody, and aptamer against α4 integrin. Medknow Publications & Media Pvt Ltd 2018-08 /pmc/articles/PMC6040166/ /pubmed/30065768 http://dx.doi.org/10.4103/1735-5362.235162 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
Fatahi, Azam
Rahimmanesh, Ilnaz
Mirian, Mina
Rohani, Fattah
Boshtam, Maryam
Gheibi, Azam
Shariati, Laleh
Khanahmad, Hossein
Kouhpayeh, Shirin
Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin
title Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin
title_full Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin
title_fullStr Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin
title_full_unstemmed Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin
title_short Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin
title_sort construction and characterization of human embryonic kidney-(hek)-293t cell overexpressing truncated α4 integrin
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040166/
https://www.ncbi.nlm.nih.gov/pubmed/30065768
http://dx.doi.org/10.4103/1735-5362.235162
work_keys_str_mv AT fatahiazam constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin
AT rahimmaneshilnaz constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin
AT mirianmina constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin
AT rohanifattah constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin
AT boshtammaryam constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin
AT gheibiazam constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin
AT shariatilaleh constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin
AT khanahmadhossein constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin
AT kouhpayehshirin constructionandcharacterizationofhumanembryonickidneyhek293tcelloverexpressingtruncateda4integrin