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

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.

Keywords: antibiotics; cell culture; exosomes (vesicles); extracellular vesicle; recombinant protein expression; selectable markers; tetraspanin; transgenic.

Figure 1

Transgene expression profiles of HEK293 cell lines arising from two-gene and bicistronic selections.A, line diagram showing the NeoR and 3xNLS-tdTomato-2a-BsdR transgenes of the plasmid pJM825. B and C, flow cytometry scatter plots of HEK293 cells that had been transfected with pJM825 and then selected in (B) G418 or (C) blasticidin for 4 weeks. Numbers of cells are shown on the y-axis while relative fluorescent brightness (arbitrary units (a.u.)) is shown on the x-axis (log scale). R3 shows the experimentally determined background fluorescence of HEK293 control cells, whereas R4 denotes red fluorescence above background. D–I, fluorescence micrographs of DAPI-stained (D–F) HEK293/pJM825/G418-resistant cells and (G–I) HEK293/pJM825/blasticidin-resistant cells, showing (D, G) 3xNLS-tdTomato, (E, H) DAPI, and (F, I) merged images. Bar: 100 μm. These experiments were performed in triplicate.

Figure 2

Effect of selectable marker on linked expression of 3xNLS-tdTomato. A, line diagram of transgenes encoding 3xNLS-tdTomato, the viral p2a peptide, and the NeoR, BsdR, HygR, PuroR, and BleoR selectable markers (not to scale). Scatter plots of flow cytometric analyses of (B) HEK293 cells or (C–G) HEK293 cells transfected with plasmids encoding the above transgenes and selected for 4 weeks in media containing (C) G418, (D) blasticidin, (E) hygromycin, (F) puromycin, or (G) zeocin. Numbers of cells are shown on the y-axis while relative fluorescent brightness (arbitrary units (a.u.)) is shown on the x-axis (log scale). R7 shows the experimentally determined background fluorescence of HEK293 cells, whereas R8 denotes red fluorescence due to 3xNLS-tdTomato expression. These experiments were performed twice.

Figure 3

Effect of selectable marker on linked expression of CD81-mNG.A, line diagram of transgenes encoding CD81mNG, the viral p2a peptide, and the NeoR, BsdR, HygR, PuroR, and BleoR selectable markers (not to scale). Scatter plots of flow cytometric analyses of (B) HEK293 cells or (C–G) HEK293 cells transfected with plasmids encoding the above transgenes and selected for 4 weeks in media containing (C) G418, (D) blasticidin, (E) hygromycin, (F) puromycin, or (G) zeocin. Numbers of cells are shown on the y-axis while relative fluorescent brightness (arbitrary units (a.u.)) is shown on the x-axis (log scale). R3 shows the experimentally determined background fluorescence of HEK293 cells, whereas R4 denotes green fluorescence due to CD81mNG expression. These experiments were performed twice.

Figure 4

Fluorescence micrographs of HEK293 cells transfected with CD81mNG-expressing transgenes. HEK293 cells transfected with the five transgenes described in Figure 3A were selected for 4 weeks in (A–C) G418, (D–F) blasticidin, (G–I) hygromycin, (J–L) puromycin, or (M–O) zeocin, respectfully. Each of these five cell lines were then grown overnight on sterile cover glasses, fixed, stained with DAPI. Images show (A, D, G, J, M) mNeonGreen fluorescence, (B, E, H, K, N) DAPI fluorescence, and (C, F, I, L, O) the merge of the two. Bar: 100 μm. These experiments were performed in triplicate.

Figure 5

Immunoblot analysis of HEK293 cells expressing CD81mNG. HEK293 cells transfected with the five transgenes described in Figure 3A were selected for 4 weeks in G418, blasticidin, hygromycin, puromycin, or zeocin, respectfully. A, immunoblot analysis of cell lysates was probed using antibodies specific for (upper panel) the p2a tag (which is fused to the C-terminus of CD81mNG) and (lower panel) actin. MW markers are, from top, 250 kDa, 150 kDa, 100 kDa, 75 kDa (pink), 50 kDa, 37 kDa, 25 kDa (pink), 20 kDa, and 15 kDa. B, bar graphs show (upper graph) anti-2a signal intensity and (lower graph) the ratio of anti-2a immunoblot signals/actin immunoblot signals from three independent trials. Averages (bar height), standard error of the mean (error bars), and statistical significance (∗p ≤ 0.05; ∗∗∗p ≤ 0.0005; ∗∗∗∗p ≤ 0.00005) were calculated using Prism software and Welch's unequal variances t test. The raw data used in these analyses is also accessible (supporting information).

Figure 6

Size distribution profiles of exosomes released by transgenic 293F cells. Exosomes were collected from the tissue culture supernatants of (A, B) 293F/pC-CD81mNG-2a-PuroR and (C, D) 293F/pC-CD81mNG-2a-BleoR cell lines and assayed by nanoparticle tracking analysis. A and B, scatter plots of exosome concentration and size for (A) all 293F/pC-CD81mNG-2a-PuroR-derived exosomes and (B) green fluorescent 293F/pC-CD81mNG-2a-PuroR-derived exosomes. C and D, scatter plots of exosome concentration and size for (C) all 293F/pC-CD81mNG-2a-BleoR-derived exosomes and (D) green fluorescent 293F/pC-CD81mNG-2a-BleoR-derived exosomes. These experiments were performed once.

Figure 7

Effect of transcriptional control elements and mode of transgene delivery on CD81mNG expression.A and B, line diagrams of plasmid, Sleeping Beauty transposon, EBV-based episome, and lentiviral vectors carrying the (A) CMV-CD81mNG-2a-Puro and (B) SFFV LTR-CD81mNG-2a-Puro transgenes. C–J, HEK293 cells were transfected or transduced with each of these vectors, selected in puromycin, grown in selective media for 4 weeks, and assayed for mNeonGreen fluorescence by flow cytometry. Numbers of cells are shown on the y-axis while relative fluorescent brightness (arbitrary units (a.u.)) is shown on the x-axis (log scale). K–P, HEK293 cells were transfected with the six plasmid vectors shown (A, B), grown for 2 days in normal media and assayed for mNeonGreen fluorescence by flow cytometry. Numbers of cells are shown on the y-axis while relative fluorescent brightness (arbitrary units (a.u.)) is shown on the x-axis (log scale). R7 shows the experimentally determined background fluorescence of HEK293 control cells, whereas R8 denotes green fluorescence above background. These experiments were performed twice.

Figure 8

Fluorescence micrographs of COS7 cell lines carrying CD81mNG-expressing transgenes. COS7 cells transfected with the five transgenes described in Figure 3A were selected for 4 weeks in (A–C) G418, (D–F) blasticidin, (G–I) hygromycin, (J–L) puromycin, or (M–O) zeocin, respectfully. Each of these five cell lines were then grown overnight on sterile cover glasses, fixed, stained with DAPI. Images show (A, D, G, J, M) mNeonGreen fluorescence, (B, E, H, K, N) DAPI fluorescence, and (C, F, I, L, O) the merge of the two. Bar: 100 μm. These images were selected from three technical replicates of the experiment.

Figure 9

Line diagrams of nonreplicating and Sleeping Beauty expression vectors. The top two lines show the DNA sequence of the polylinkers common to all p2a-containing and all pl-designated vectors. The linear plasmid maps depict the relative positions of major design elements of the circular plasmids we created, with the pC plasmids showing nonreplicating vectors with the CMV transcriptional control sequences, the pS plasmids showing the nonreplicating vectors with the SFFV LTR, the pITRSB-C plasmids showing the Sleeping Beauty vectors with the CMV transcriptional control elements, and the pITRSB-S showing the Sleeping Beauty vectors with the SFFV LTR.