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

Lele Yang1, Lifang Cui1, Shumin Ma1

  • 1Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Science, Shandong University, Qingdao, China.

Frontiers in Bioengineering and Biotechnology
|July 5, 2022
PubMed
Summary
This summary is machine-generated.

This study introduces an affinity cell sorting system for efficient gene transfer. The system enhances gene delivery in challenging cells, significantly improving gene overexpression, knockdown, and genome editing efficiencies.

Keywords:
affinity cell sortingco-transfectiongene deliverygene function studytwin-strep-tag (TST)

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Gene delivery efficiency is a critical bottleneck in gene research and therapy, particularly for hard-to-transfect cells.
  • Existing methods often lack the efficiency and specificity required for isolating gene-modified cells.

Purpose of the Study:

  • To develop and validate an affinity cell sorting system for the efficient enrichment of gene transfer-positive cells.
  • To demonstrate the system's utility in improving outcomes for various gene manipulation techniques.

Main Methods:

  • Development of a cell surface expression system combining enhanced green fluorescent protein (EGFP) with a Twin-Strep-Tag (TST) and a GPI membrane anchor.
  • Utilizing Strep-Tactin magnetic beads for affinity-based sorting of EGFP-TST expressing cells.
  • Application of the system in K-562 cells for gene overexpression, shRNA knockdown, and CRISPR/Cas9 genome editing.

Main Results:

  • Achieved up to 95.0% positive cell purity with a 5.8-fold enrichment in transfected K-562 cells.
  • Significantly enhanced gene overexpression (10x to 58x), shRNA gene knockdown (12% to 53%), and CRISPR/Cas9 editing (20% to 79%).
  • Demonstrated a simple, convenient, and fast method for isolating gene transfer-positive cells.

Conclusions:

  • The developed affinity cell sorting system provides a powerful tool for enriching gene transfer-positive cells, especially in challenging cell types.
  • This technology has broad applications and significant potential to advance gene transfer studies, gene therapy, and genome engineering.
  • The system offers a substantial improvement in the efficiency of various gene manipulation applications.