TY - JOUR
T1 - Choice of selectable marker affects recombinant protein expression in cells and exosomes
AU - Guo, Chenxu
AU - Fordjour, Francis K.
AU - Tsai, Shang Jui
AU - Morrell, James C.
AU - Gould, Stephen J.
N1 - Funding Information:
Acknowledgments—We thank members of the Gould lab and the Department of Biological Chemistry for their suggestions during the course of this study and particularly Dr Michael Caterina. We thank Zhaolin Chen and Yiyan Lin for expert technical assistance, Hao Zhang for flow cytometric analysis, and Nazar Filonov and Nadia Filonov for assistance with NTA analysis. This work was supported, in part, by the National Institutes of Health (grant number 5U19CA179563). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2021 THE AUTHORS.
PY - 2021/7/1
Y1 - 2021/7/1
N2 - 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-tocell 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-tocell 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.
AB - 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-tocell 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-tocell 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.
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U2 - 10.1016/j.jbc.2021.100838
DO - 10.1016/j.jbc.2021.100838
M3 - Article
C2 - 34051235
AN - SCOPUS:85109449876
SN - 0021-9258
VL - 297
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
M1 - 100838
ER -