Choice of selectable marker affects recombinant protein expression in cells and exosomes

Chenxu Guo,Francis K Fordjour,Shang Jui Tsai,James C Morrell,Stephen J Gould

doi:10.1016/j.jbc.2021.100838

Chenxu Guo, Francis K Fordjour + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100838

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Abstract

Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.

Highlights

Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines
The cells were placed into complete media containing either 400 ug/ml G418 to select for G418resistant cell lines or 20 ug/ml blasticidin to select for blasticidin-resistant cell lines
Assaying thousands of cells from these polyclonal cell lines revealed that ~50% of cells in the G418-resistant line cells lacked detectable levels of 3xNLS-tdTomato expression. This is not surprising, given that the 3xNLS-tdTomato gene represents a sizeable proportion of the transfected plasmid and will be disrupted in a significant proportion of G418resistant cell lines due to the random nature of plasmid linearization that occurs during transgene insertion into host chromosomes [4, 5]

Summary

Introduction

Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. These site-directed insertional strategies represent a significant technological advance, several are limited to specific, previously engineered recipient cell lines, all require the isolation, expansion, and characterization of numerous SCCs, and none are designed for eliciting the very highest levels of transgene expression While these and other studies have led to significant improvements and advanced the field of mammalian cell transgenesis, none have systematically interrogated the relative effectiveness of the available dominant selectable markers. The five dominant selectable markers in widespread are the NeoR, BsdR, HygR, PuroR, and BleoR genes, which confer resistance to the selective antibiotics G418/geneticin, blasticidin, hygromycin B, puromycin, and zeocin, respectively [3, 26,27,28,29] It is unclear whether the choice of selectable marker has any predictable effects on the outcome of mammalian cell transgenesis experiments. Extracellular vesicles (sEVs) of ~30–150 nm in diameter that are released by all human cell types, can transmit signals and molecules to other cells in a pathway of intercellular vesicle traffic, and are of increasing use as delivery vehicles for vaccines and therapeutics [30]

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