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A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag

Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluores...

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Autores principales: Yang, Lele, Cui, Lifang, Ma, Shumin, Zuo, Qingqing, Huang, Qilai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9244562/
https://www.ncbi.nlm.nih.gov/pubmed/35782508
http://dx.doi.org/10.3389/fbioe.2022.930966
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author Yang, Lele
Cui, Lifang
Ma, Shumin
Zuo, Qingqing
Huang, Qilai
author_facet Yang, Lele
Cui, Lifang
Ma, Shumin
Zuo, Qingqing
Huang, Qilai
author_sort Yang, Lele
collection PubMed
description Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells.
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spelling pubmed-92445622022-07-01 A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag Yang, Lele Cui, Lifang Ma, Shumin Zuo, Qingqing Huang, Qilai Front Bioeng Biotechnol Bioengineering and Biotechnology Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positive cells, and the TST tag at the N terminal of EGFP allows efficient affinity sorting of the positive cells using Strep-Tactin magnetic beads. The cell sorting system enables efficient isolation of gene transfer-positive cells in a simple, convenient, and fast manner. Cell sorting on transfected K-562 cells resulted in a final positive cell percentage of up to 95.0% with a positive cell enrichment fold of 5.8 times. The applications in gene overexpression experiments could dramatically increase the gene overexpression fold from 10 times to 58 times, and in shRNA gene knockdown experiments, cell sorting increased the gene knockdown efficiency from 12% to 53%. In addition, cell sorting in CRISPR/Cas9 genome editing experiments allowed more significant gene modification, with an editing percentage increasing from 20% to 79%. The gene transfer-positive cell sorting system holds great potential for all gene transfer studies, especially on those hard-to-transfect cells. Frontiers Media S.A. 2022-06-16 /pmc/articles/PMC9244562/ /pubmed/35782508 http://dx.doi.org/10.3389/fbioe.2022.930966 Text en Copyright © 2022 Yang, Cui, Ma, Zuo and Huang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Yang, Lele
Cui, Lifang
Ma, Shumin
Zuo, Qingqing
Huang, Qilai
A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag
title A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag
title_full A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag
title_fullStr A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag
title_full_unstemmed A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag
title_short A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag
title_sort gene transfer-positive cell sorting system utilizing membrane-anchoring affinity tag
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9244562/
https://www.ncbi.nlm.nih.gov/pubmed/35782508
http://dx.doi.org/10.3389/fbioe.2022.930966
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