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

Abstract

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.

GPI-anchored glutathione S-transferase as marker allows affinity sorting of transfection-positive cells

“…To obtain efficient membrane translocation, we tested six membrane anchoring modules for their ability to translocate the GST fusion to the cell surface. Three modules are GPI-anchored protein signal sequences from DAF, BY55, and CEAM7, and the other three are transmembrane domains (TMDs) from ITAV, ITA5, and ITB3 as used previously ( Yang et al, 2022 ).…”

“…The recently reported positive cell sorting system utilizes GPI-anchored Twin-Strep-Tag as the affinity tag and allows the positive cells to be enriched with Strep-Tactin magnetic beads Frontiers in Molecular Biosciences frontiersin.org (Yang et al, 2022). It works efficiently in gene function studies, but however, the usage of Strep-Tactin magnetic beads still remains a big cost.…”

“…Alternatively, we also optimized the cell sorting procedure by separating the bead/cell complexes through free settling instead of using the magnetic stand, as described previously ( Yang et al, 2022 ). Briefly, D-PBS containing 0.1% BSA was used to prepare GSH magnetic beads and cell suspension, and the binding reaction was performed with a 1.5 ml volume in the1.5 ml tube.…”

“…The transfected K-562 cells with or without cell sorting and the negative control cells transfected with empty plasmids pcDNA3.1-GST DAF or pcDNA3.1-GST CEAM7 were counted and seeded into 96-well plates in triplicates at a density of 1,000 per well. Cell viability and proliferation were determined using the CCK-8 kit (MA0218, Meilun) at 0, 24, 48, 72, and 96 h post seeding, as described previously ( Yang et al, 2022 ). Briefly, 10 μL of CCK-8 reagent was added to the 100 μL cell suspension and incubated for 3 h at 37°C.…”

“…Recently, we developed a gene transfer-positive cell sorting system using membrane anchoring Twin-Strep-Tag ( Yang et al, 2022 ). The system allows efficient enrichment of gene transfection-positive cells and can boost gene functional studies, including gene overexpression, gene knockdown and gene editing experiments et al However, the cell sorting system utilizes Strep-Tactin magnetic beads to bind and separate positive cells, which still has the space to reduce cost.…”