Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering

Abstract

Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as "exosome painting," we show here that CP05 binds equally well to CD63^-/- cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.

Keywords: CD63; G418; antibiotic; blasticidin; extracellular vesicle; hygromycin; puromycin; selectable marker; transgenic; zeocin.

Figure 1

Line diagrams of AR gene test vectors. Fifteen distinct Sleeping Beauty transposon–containing vectors were created, each carrying a single transposon–carried gene in which the CMV enhancer/promoter was positioned to drive the expression of a bicistronic ORF encoding (i) mCherry, (ii) a viral 2a peptide, and (iii) an AR protein. These AR proteins included untagged ER50 degron-tagged or ecDHFR degron-tagged forms of the five AR proteins BsdR, BleoR, PuroR, HygR, and NeoR (19). Following transfection of these vectors into 293F cells, expression of the Sleeping Beauty transposase SB100X is driven from the Rous sarcoma virus long-terminal repeat, leading to mobilization of all sequences including and between the inverted tandem repeats, from the vector and into one or more sites in the host cell genome, leading subsequently to expression of mCherry in a manner dependent upon integration site and integrant copy number, both of which can vary dramatically within the population of transgenic cells. AR, antibiotic resistance; CMV, cytomegalovirus; ecDHFR, Escherichia coli dihydrofolate reductase; ER50, estrogen receptor 50.

Figure 2

Degron-tagging BsdR genes results in approximately fivefold higher expression of the linked recombinant protein mCherry. Flow cytometry measurements of mCherry expression levels (fluorescence brightness, arbitrary units) in polyclonal cell lines that were generated following transfection with transposons carrying (A) the untagged BsdR ORF, (B) the ER50BsdR ORF, or (C) the ecDHFRBsdR ORF. Data are from three technical replicates of each cell line. Approximately 20,000 cells were assayed in each replicate, gray shows the background fluorescence of 293F cells, and the median, mean, and CV from each replicate are shown to the right. Light purple shows mCherry expression in the BsdR cell line data, medium purple shows the ER50BsdR cell line data, and dark purple shows the ecDHFRBsdR cell line data. Data from 293F cells are shown in the box at the bottom right of the figure. ecDHFR, Escherichia coli dihydrofolate reductase; ER50, estrogen receptor 50.

Figure 3

ER50BleoR selects for twofold higher levels of linked mCherry expression. Flow cytometry histograms of mCherry expression levels (fluorescence brightness, arbitrary units) in the polyclonal cell lines selected using transposons carrying (A) the untagged BleoR ORF and (B) the ER50BleoR ORF. Data are shown for three technical replicates of each cell line, involving ∼20,000 independent cell fluorescence measurements for each replicate, with gray showing the background fluorescence of 293F cells, and the median, mean, and CV from each replicate are shown to the right. Light chartreuse shows the BleoR cell line data, whereas dark chartreuse shows the ER50BleoR cell line data. Data from 293F cells are shown in the box at the bottom right of the figure. ER50, estrogen receptor 50.

Figure 4

Degron-tagging PuroR increases linked mCherry expression by 70%. Flow cytometry measurements of mCherry expression levels (fluorescence brightness, arbitrary units) in polyclonal cell lines selected via the (A) untagged PuroR ORF, (B) ER50BleoR ORF, and (C) ecDHFRPuroR ORF. Data are shown for three technical replicates of each cell line, involving ∼20,000 independent cell fluorescence measurements for each replicate, with gray showing the background fluorescence of 293F cells, and the median, mean, and CV from each replicate are shown to the right. Light coral shows the PuroR-selected cell line data, medium coral shows the ER50PuroR-selected cell line data, and dark coral shows the ecDHFRPuroR-selected cell line data. Data from 293F cells are shown in the box at the bottom right of the figure. ecDHFR, Escherichia coli dihydrofolate reductase; ER50, estrogen receptor 50.

Figure 5

Degron tagging has only minimal effects on HygR-selected transgene expression. Flow cytometry measurements of mCherry expression levels (fluorescence brightness, arbitrary units) in the polyclonal cell lines generated following transfection with transposons carrying transgenes expressing the (A) untagged HygR ORF, (B) ER50HygR ORF, and (C) ecDHFRHygR ORF. Data are shown for three technical replicates of each cell line, involving ∼20,000 independent cell fluorescence measurements for each replicate, with gray showing the background fluorescence of 293F cells, and the median, mean, and CV from each replicate are shown to the right. Light cyan shows the HygR-selected cell line data, medium cyan shows the ER50HygR-selected cell line data, dark cyan shows the ecDHFRHygR-selected cell line data. Data from 293F cells are shown in the box at the bottom right of the figure. ecDHFR, Escherichia coli dihydrofolate reductase; ER50, estrogen receptor 50.

Figure 6

Degron tagging has only minimal effects on NeoR-selected transgene expression. Flow cytometry measurements of mCherry expression levels (fluorescence brightness, arbitrary units) in the polyclonal cell lines selected using transposons carrying (A) the untagged NeoR ORF, (B) the ER50NeoR ORF, and (C) the ecDHFRNeoR ORF. Data are shown for three technical replicates of each cell line, involving ∼20,000 independent cell fluorescence measurements for each replicate, with gray showing the background fluorescence of 293F cells, and median, mean, and CV from each replicate are shown to the right. Light lavender shows the average data from the NeoR-selected cell line, medium lavender shows the average data from the ER50NeoR-selected cell line, and dark lavender shows the average data from the ecDHFRNeoR-selected cell line. Data from 293F cells are shown in the box at the bottom right of the figure. ecDHFR, Escherichia coli dihydrofolate reductase; ER50, estrogen receptor 50.

Figure 7

ER50BleoR selects for higher expression and improved exosome engineering.A, line diagrams of Sleeping Beauty transposon vectors YA22 and YA24, which drive the expression of CD63/Y235A linked to the BleoR and ER50BleoR antibiotic resistance proteins, respectively. It should be noted that these bicistronic ORFs were expressed from the spleen focus-forming virus (SFFV) long terminal repeat (LTR), which appears to drive slightly higher transgene expression from integrated transgenes than the CMV enhancer/promoter elements (19). B–D, immunofluorescence micrographs showing anti-CD63 fluorescent antibody staining of (B) 293F cells, (C) the zeocin-resistant 293F-derived cell line YA22, and (D) the zeocin-resistant 293F-derived cell line YA24. Top panels are brightfield images, and bottom panels are anti-CD63 immunofluorescent images collected at the same exposure time for all three samples. The bar represents 150 μm. E, immunoblots of cell lysates interrogated using (upper panel) a monoclonal antibody specific for CD63 and (lower panel) an antibody-specific for HSP90. In an effort to accurately convey the difference in CD63 expression levels between 293F, F/YA22, and F/YA24 cells, we present an overexposed image of the immunoblot in this figure, though we used a nonsaturated exposure for subsequent quantification. F, immunoblot of equal proportions of exosomes collected from the same triplicate cultures as in (E), demonstrating that high-level expression of CD63/Y235A results in elevated levels of exosome-associated CD63 proteins. G, bar graph showing the amount of CD63 in cell and exosome lysates, with bar height denoting the average, error bars representing the standard error of the mean, asterisks denoting p value significance (∗∗<0.005, ∗∗∗<0.0005, and ∗∗∗∗<0.00005), and individual data points shown as points. Differences between the F/YA22 and F/YA24 samples were 2.1× for cell-associated CD63 and 3.5-fold for exosome-associated CD63. Numerical values were obtained by quantification of nonsaturated exposures of each immunoblot. CMV, cytomegalovirus; ER50, estrogen receptor 50; HSP90, heat shock protein 90.

Figure 8

CP05-Cy5 displays similar binding to 293F, F/CD63^−/−, and F/YA24 cells.A, CD63 genomic DNA sequence in the vicinity of the Cas9/gRNA target site. Shaded sequence corresponds to the 3′ end of exon 5, whereas the unshaded sequence corresponds to the 5′ end of intron 5. Underlined sequence denotes the gRNA target site. B, DNA sequence of alleles 1 and 2 in the F/CD63^−/− cell line, resulting from Cas9/gRNA-mediated gene editing. C, CD63 mRNA abundance in 293F and F/CD63^−/− cells, as determined by qRT–PCR. D, flow cytometry histograms of (purple) F/CD63^−/− cells, (red) 293F cells, and (green) F/YA24 cells, each stained with the same FITC-labeled anti-CD63 monoclonal antibody. E and F, flow cytometry measurements of (purple) F/CD63^−/− cells, (red) 293F cells, and (green) F/YA24 cells stained with the CP05-Cy5 peptide (D) at a concentration of 0.34 μM of CP05-Cy5 peptide and (E) at a concentration of 3.4 μM of CP05-Cy5 peptide. gRNA, guide RNA; qRT–PCR, quantitative PCR.

Figure 9

Schematic representation of how choice of AR gene affects transgene expression.Gray bar represents the range of transgene expression within the entire population of transgenic cells in a transfected cell population, prior to addition of a selective antibiotic. Black, blue, and orange bars represent the range of transgene expression in polyclonal antibiotic-resistant cell lines selecting using AR proteins that have high, moderate, or low activity/stability, respectively. Black, blue, and orange arrows denote the threshold of transgene expression below which no cell can survive. Hatched bars represent the population of transgenic cells that will perish after the addition of selective antibiotic. AR, antibiotic resistance.