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Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells

OBJECTIVE(S): Checkpoint blocking is considered a revolutionary method in cancer treatment. This method eliminates cancer cells by maintaining the sensitivity of immune cells. Today, cell therapy through checkpoint blocking is known as the most efficient method of cancer treatment. The programmed ce...

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Autores principales: Alambeladi, SeyedAli, Hosseiny, SeyedEbrahim, Jafarinia, Mojtaba, Dianatpour, Mehdi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Mashhad University of Medical Sciences 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894638/
https://www.ncbi.nlm.nih.gov/pubmed/33643569
http://dx.doi.org/10.22038/ijbms.2020.48566.11146
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author Alambeladi, SeyedAli
Hosseiny, SeyedEbrahim
Jafarinia, Mojtaba
Dianatpour, Mehdi
author_facet Alambeladi, SeyedAli
Hosseiny, SeyedEbrahim
Jafarinia, Mojtaba
Dianatpour, Mehdi
author_sort Alambeladi, SeyedAli
collection PubMed
description OBJECTIVE(S): Checkpoint blocking is considered a revolutionary method in cancer treatment. This method eliminates cancer cells by maintaining the sensitivity of immune cells. Today, cell therapy through checkpoint blocking is known as the most efficient method of cancer treatment. The programmed cell death protein-1(PD-1), as an immune check protein, has a vital role in weakening the immune responses by reducing the number of stimulated T cells. In normal situations, a decline in the immune responses can cause induced tolerance and prevent autoimmune diseases. MATERIALS AND METHODS: In this study, to reduce the induction of tolerance due to PDL-1 binding to PD-1, the PD-1 gene was destroyed in PBMCs by the means of CRISPR-Cas9 and dual-transfection of two plasmids containing the Cas 9 gene and two different sgRNAs specific to two region of PD-1 gene in order to produce a deletion mutation. Six different sgRNA were designed and cloned in PX-458 plasmid vector, and PBMCs were transfected using lipofectamine 2000 and electroporation. Indels were evaluated by gel electrophoresis and Sanger sequencing. RESULTS: We showed the PD-1 gene in PBMCs was knocked out successfully by CRISPR-Cas9 and dual-transfection of two sgRNAs. The minimum interval between the two sgRNAs was 448 nucleotides. CONCLUSION: The results of this research demonstrated that the use of dual-transfection of CRISPR-Cas9 sgRNA is a suitable method to knock out the PD-1 gene and prevention of inducing tolerance in PBMCs.
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spelling pubmed-78946382021-02-26 Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells Alambeladi, SeyedAli Hosseiny, SeyedEbrahim Jafarinia, Mojtaba Dianatpour, Mehdi Iran J Basic Med Sci Original Article OBJECTIVE(S): Checkpoint blocking is considered a revolutionary method in cancer treatment. This method eliminates cancer cells by maintaining the sensitivity of immune cells. Today, cell therapy through checkpoint blocking is known as the most efficient method of cancer treatment. The programmed cell death protein-1(PD-1), as an immune check protein, has a vital role in weakening the immune responses by reducing the number of stimulated T cells. In normal situations, a decline in the immune responses can cause induced tolerance and prevent autoimmune diseases. MATERIALS AND METHODS: In this study, to reduce the induction of tolerance due to PDL-1 binding to PD-1, the PD-1 gene was destroyed in PBMCs by the means of CRISPR-Cas9 and dual-transfection of two plasmids containing the Cas 9 gene and two different sgRNAs specific to two region of PD-1 gene in order to produce a deletion mutation. Six different sgRNA were designed and cloned in PX-458 plasmid vector, and PBMCs were transfected using lipofectamine 2000 and electroporation. Indels were evaluated by gel electrophoresis and Sanger sequencing. RESULTS: We showed the PD-1 gene in PBMCs was knocked out successfully by CRISPR-Cas9 and dual-transfection of two sgRNAs. The minimum interval between the two sgRNAs was 448 nucleotides. CONCLUSION: The results of this research demonstrated that the use of dual-transfection of CRISPR-Cas9 sgRNA is a suitable method to knock out the PD-1 gene and prevention of inducing tolerance in PBMCs. Mashhad University of Medical Sciences 2021-01 /pmc/articles/PMC7894638/ /pubmed/33643569 http://dx.doi.org/10.22038/ijbms.2020.48566.11146 Text en This is an Open Access article distributed under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Alambeladi, SeyedAli
Hosseiny, SeyedEbrahim
Jafarinia, Mojtaba
Dianatpour, Mehdi
Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells
title Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells
title_full Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells
title_fullStr Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells
title_full_unstemmed Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells
title_short Use of dual-transfection for programmed death cell protein 1 disruption mediated by CRISPR-Cas9 in human peripheral blood mononuclear cells
title_sort use of dual-transfection for programmed death cell protein 1 disruption mediated by crispr-cas9 in human peripheral blood mononuclear cells
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894638/
https://www.ncbi.nlm.nih.gov/pubmed/33643569
http://dx.doi.org/10.22038/ijbms.2020.48566.11146
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