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. 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. The creation of transgenic mammalian cell lines was pioneered in the 1980s by Berg and colleagues (1Mulligan R.C. Berg P. Expression of a bacterial gene in mammalian cells.Science. 1980; 209: 1422-1427Crossref PubMed Scopus (440) Google Scholar, 2Mulligan R.C. Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase.Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2072-2076Crossref PubMed Scopus (634) Google Scholar, 3Southern P.J. Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar, 4Canaani D. Berg P. Regulated expression of human interferon beta 1 gene after transduction into cultured mouse and rabbit cells.Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5166-5170Crossref PubMed Scopus (49) Google Scholar). In general, this process involves transfecting or transducing cells with a recombinant DNA vector that carries the gene of interest and a selectable marker gene and then selecting for transgene-expressing cells using an appropriate antibiotic (3Southern P.J. Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar, 4Canaani D. Berg P. Regulated expression of human interferon beta 1 gene after transduction into cultured mouse and rabbit cells.Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5166-5170Crossref PubMed Scopus (49) Google Scholar). This approach has been widely employed, has led to the creation of many useful transgenic cell lines, and is still in use today. However, many of the antibiotic-resistant cell clones generated by this approach express low or undetectable levels of the linked transgene (5Hunter M. Yuan P. Vavilala D. Fox M. Optimization of protein expression in mammalian cells.Curr. Protoc. Protein Sci. 2019; 95: e77Crossref PubMed Scopus (52) Google Scholar). As a result, experiments that require high-level expression of the transgene of interest often require the isolation, expansion, and screening of dozens (or more) single-cell clones (SSCs) before one can obtain a cell line that displays the desired level of transgene expression. Many researchers have devoted significant time, effort, and resources to improving the outcomes of mammalian cell transgenesis experiments. Much of this effort has been directed at improved vector design, resulting in the identification of many cis-acting features that affect transgene expression, including transcriptional regulatory regions (6Humphries R.K. Ley T. Turner P. Moulton A.D. Nienhuis A.W. Differences in human alpha-, beta- and delta-globin gene expression in monkey kidney cells.Cell. 1982; 30: 173-183Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 7Gillies S.D. Morrison S.L. Oi V.T. Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene.Cell. 1983; 33: 717-728Abstract Full Text PDF PubMed Scopus (713) Google Scholar, 8Joyner A. Yamamoto Y. Bernstein A. Retrovirus long terminal repeats activate expression of coding sequences for the herpes simplex virus thymidine kinase gene.Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1573-1577Crossref PubMed Scopus (14) Google Scholar, 9Boshart M. Weber F. Jahn G. Dorsch-Hasler K. 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PubMed Scopus Google or DNA (e.g., of a and in human cells.Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, E. B. A. A. K. J. E. D. of a gene in Genet. 41: PubMed Scopus Google or M. J. E. T. T. Mol. Biol. PubMed Scopus Google Scholar, J. T. to clinical PubMed Scopus Google can also as virus of and the 1 gene transgene expression gene B. of virus in mammalian 1985; PubMed Scopus Google Scholar). have also that transgene into sites of the cell that are with transgene expression, as S. J. J. J. a for PubMed Scopus Google Scholar), DNA B. P.J. J. of in the human Mol. Biol. PubMed Scopus Google Scholar), and F. and of for Full Text Full Text PDF PubMed Scopus Google Scholar). these a significant are to cell lines, require the isolation, expansion, and of numerous and are for the very highest levels of transgene expression. these and have led to significant and the of mammalian cell have the of the dominant selectable markers. The dominant selectable markers in are the and BleoR which resistance to the antibiotics blasticidin, and (3Southern P.J. Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar, J. F. A. and expression of a puromycin gene in and Escherichia 1985; 33: PubMed Scopus Google Scholar, of a bacterial resistance gene by transcriptional and and by DNA 1983; PubMed Scopus Google Scholar, P. A. M. G. resistance as a dominant selectable marker in Cell Mol. Genet. PubMed Scopus Google Scholar, M. T. of the gene and use as a selectable marker for and Genet. PubMed Scopus Google Scholar). is the choice of selectable marker has on the of mammalian cell transgenesis experiments. This that the choice of selectable marker and the process of antibiotic selection has a significant on the expression of linked recombinant many of the experiments in this the of selectable marker on expression of a recombinant protein, that results have to the of are of in that are by human cell can signals and to cells in a of and are of use as for and 2019; PubMed Scopus (583) Google Scholar). HEK293 cell lines are used for and cell biological and are an cell for biological and (5Hunter M. Yuan P. Vavilala D. Fox M. Optimization of protein expression in mammalian cells.Curr. Protoc. Protein Sci. 2019; 95: e77Crossref PubMed Scopus (52) Google Scholar, HEK293 in cell and and PubMed Scopus Google Scholar). the results of a mammalian cell transgenesis D. Berg P. Regulated expression of human interferon beta 1 gene after transduction into cultured mouse and rabbit cells.Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5166-5170Crossref PubMed Scopus (49) Google Scholar, M. Yuan P. Vavilala D. Fox M. Optimization of protein expression in mammalian cells.Curr. Protoc. Protein Sci. 2019; 95: e77Crossref PubMed Scopus (52) Google Scholar), we HEK293 cells with which carries functional is to express the NeoR gene the SV40 early and the is to express a a of the protein and PubMed Scopus Google that carries of a D. A to Full Text PDF PubMed Scopus Google at the A.L. of in the of Biol. PubMed Scopus Google Scholar), and the selectable marker M. T. of the gene and use as a selectable marker for and Genet. PubMed Scopus Google Scholar). after the cells were into G418 to for cell lines or to for cell lines. the cells were and by to the of expression in each cell and of cells these cell lines that of cells in the line cells levels of expression. This is that the gene a of the and in a significant of cell lines to the of that transgene into D. Berg P. Regulated expression of human interferon beta 1 gene after transduction into cultured mouse and rabbit cells.Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5166-5170Crossref PubMed Scopus (49) Google Scholar, M. Yuan P. Vavilala D. Fox M. Optimization of protein expression in mammalian cells.Curr. Protoc. Protein Sci. 2019; 95: e77Crossref PubMed Scopus (52) Google Scholar). In contrast, the cell line a of cells, consistent with the that this transgene a between expression and expression. However, this cell line also displayed a cell-to-cell variability in expression, that resistance to BsdR no to level of transgene expression. These were also experiments These results the of dominant selectable markers cell lines with low and levels of recombinant protein expression. this we a of expression vectors to expression of a gene of a upstream of a that by the NeoR (3Southern P.J. Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google BsdR M. T. of the gene and use as a selectable marker for and Genet. PubMed Scopus Google Scholar), of a bacterial resistance gene by transcriptional and and by DNA 1983; PubMed Scopus Google Scholar), J. F. A. and expression of a puromycin gene in and Escherichia 1985; 33: PubMed Scopus Google Scholar), or BleoR P. A. M. G. resistance as a dominant selectable marker in Cell Mol. Genet. PubMed Scopus Google markers HEK293 cells were with each of these for a in and then for in blasticidin, hygromycin, or respectively. each were then to cell lines, which were then for expression by The and cell lines displayed the lowest and of cell-to-cell in with a of and with a In contrast, the and cell lines displayed higher and levels of expression and the cell line displayed the highest and expression of data for cell lines in a these to recombinant we of vectors in which the coding region was with that of R. S. R. S. the of an a of Cell Biol. PubMed Scopus Google Scholar), an protein that is in W. of on the of and on by human Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), to green protein A. Y. P.J. M. J. A green protein PubMed Scopus Google Scholar), the of this protein by HEK293 cells were with these and the antibiotic-resistant clones were to cell lines. These lines were then by to the levels of in of cells each The and cell lines displayed the lowest and expression of linked recombinant protein, with levels of and respectively. In contrast, the cell lines by with the and displayed higher and levels of expression of and respectively. the cells by with the displayed the highest and levels of transgene expression Similar results were observed these cell lines were by or by the of which an approximately 10-fold in the expression of in the BleoR cell line to the or cell lines, with levels of expression in the and cell data for cell lines in a of HEK293 cells with HEK293 cells with the in were selected for in blasticidin, hygromycin, or of these cell lines were then on with show and the of the These experiments were in of HEK293 cells HEK293 cells with the in were selected for in blasticidin, hygromycin, or of cell was using for the is to the of and markers and show and the of signals of the and were using and The data used in these is also is the W. of on the of and on by human Biol. Full Text Full Text PDF PubMed Scopus Google and has as a for exosome into is by level of expression in cells, we cells of HEK293 with the PuroR- and expression vectors and A we selected for antibiotic-resistant which were as of and These cell lines were into in and for The cells were then the and were by a combination of low and The exosome were then by using a D. M. A. F. M. E. with an and between and 2019; 8: PubMed Scopus Google to the and of in each of the of which the of of and that we and the of The cell which levels of a of in which displayed levels of In contrast, the cell which higher levels of a of in which levels of a than in exosome which is consistent with the stochastic of exosome biogenesis 2019; PubMed Scopus (583) Google and data as by cell of in a These a hypothesis in which selectable markers a threshold of transgene expression below which no cell can this threshold was by the of transgene we a of DNA vectors that express the recombinant protein different transcriptional control M. Weber F. Jahn G. Dorsch-Hasler K. Fleckenstein B. Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus.Cell. 1985; 41: 521-530Abstract Full Text PDF PubMed Scopus (836) Google or long terminal A. Yamamoto Y. Bernstein A. Retrovirus long terminal repeats activate expression of coding sequences for the herpes simplex virus thymidine kinase gene.Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1573-1577Crossref PubMed Scopus (14) Google by vector and and of a and in human cells.Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, E. B. A. A. K. J. E. D. of a gene in Genet. 41: PubMed Scopus Google Scholar), or B. of virus in mammalian 1985; PubMed Scopus Google Scholar), or or A and HEK293 cells were with the DNA vectors and with the by selection of clones to cell lines. These were by that cell lines displayed data for HEK293 cells or with different vector in a results observed a expression in the of puromycin we HEK293 cells with the DNA vectors and expression at As these cell many cells, a level of and higher cell-to-cell in expression. these results that expression was higher than expression These results that the in in expression in the cell lines was to the of puromycin also data on the of expression, which that transgene expression in the of antibiotic selection the gene that we used in this data for HEK293 cell in a choice of selectable marker has a on transgene expression in mammalian cell lines, we the simian virus African green monkey kidney cell line with the to express linked by a to the NeoR (3Southern P.J. Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google BsdR M. T. of the gene and use as a selectable marker for and Genet. PubMed Scopus Google Scholar), of a bacterial resistance gene by transcriptional and and by DNA 1983; PubMed Scopus Google Scholar), J. F. A. and expression of a puromycin gene in and Escherichia 1985; 33: PubMed Scopus Google Scholar), and BleoR P. A. M. G. resistance as a dominant selectable marker in Cell Mol. Genet. PubMed Scopus Google markers cell lines were generated each of cells by in antibiotic for clones each and then under selection for These cell lines were then and by which the of linked highest in the cell lines selected in lowest in cell lines selected with or and in cell lines selected with or puromycin The creation of transgenic mammalian cells is a in many However, is no and rapid for transgenic cell lines that express and levels of linked recombinant we the impact that the choice of selectable marker has on the levels of a linked recombinant protein and that can have to a 10-fold on expression Moreover, the choice of selectable marker also has a on the cell-to-cell in transgene expression, with highest with the lowest expression and the lowest cell-to-cell observed in the cell lines. The of these is that each selectable establishes a threshold of transgene expression below which no cell can that these thresholds are at in by each marker of and the that these are to for is that each a threshold of transgene expression. This that and selectable marker antibiotic at very low levels of expression. As a result, use of markers results in the of cells that express level of the linked recombinant protein, which in cell lines that a low level of transgene expression and a of cell-to-cell in transgene expression These well to those we observed for cell lines generated using the NeoR or BsdR markers, that the NeoR and BsdR selectable marker or As for the higher levels of transgene expression in or cell lines, we that the and or the that the BleoR marker yielded cell lines with the highest and levels of transgene expression that has the lowest of selectable marker This is consistent with of zeocin as the BleoR protein rather by in that have a of to protein A. H. G. resistance by a PubMed Scopus Google Scholar). 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R. M. and in of 2019; PubMed Scopus Google Scholar), and DNA can in cell lines M. M. F. to the antibiotic by expression of the gene DNA in human PubMed Scopus Google Scholar). However, also for antibiotics used to transgenic mammalian cell lines. are also to the of that express the very highest levels of linked recombinant Specifically, use of the BleoR marker is to an the to those that express the very highest levels of the the of that to in the for a cell line that levels of the protein of As for the markers, use is with the of that express levels of the the screening of This is for the NeoR which was the widely used dominant selectable marker gene and is still in than selectable to time, effort, and resources have been in mammalian to use of this selectable we can by vectors that the of this this we have a of and vectors that are on the we have of the experiments in this used as the protein of as one of the of this was to the that affect exosome experiments that the of a is to the levels of protein expression. This is consistent with the that exosome biogenesis is a stochastic process in which the of exosome is by the levels of exosome in the of the exosome budding 2019; PubMed Scopus (583) Google Scholar). Moreover, results that the choice of selectable marker is an in the of exosome with for the of and were in cells, in and bacterial using and DNA data were and using The and coding regions were for expression in human cells, in into mammalian cell expression and to The and of the vectors of a and in human cells.Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, E. B. A. A. K. J. E. D. of a gene in Genet. 41: PubMed Scopus Google and the cis-acting of a and M. Y. of vectors for transducing cells the Google were also in into bacterial and to The and sequences B. of virus in mammalian 1985; PubMed Scopus Google were by a combination of in gene and into a with of of and were into transgene vectors using recombinant DNA cloning of vectors used in this or in this are of used in this in a for transgene in a cells were with a of the a expression a expression and a expression vector M. Y. of vectors for transducing cells the Google Scholar). 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