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Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Publication Date: October 11, 2010
Journal: PLOS ONE
Authors: Florian Krammer, Jens Pontiller, Christopher Tauer, Dieter Palmberger, et al
Volume: 5
Issue: 10
Pages: e13265
DOI: https://dx.plos.org/10.1371/journal.pone.0013265
Publisher URL: http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0013265
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/20949004
PubMed Central: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952591
Europe PMC: http://europepmc.org/abstract/MED/20949004
Web of Science: 000282748100014
Scopus: 78149441000
Mendeley: http://www.mendeley.com/research/evaluation-influenza-replicon-transient-expression-recombinant-proteins-mammalian-cells-2

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{"title"=>"Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells", "type"=>"journal", "authors"=>[{"first_name"=>"Florian", "last_name"=>"Krammer", "scopus_author_id"=>"35362059600"}, {"first_name"=>"Jens", "last_name"=>"Pontiller", "scopus_author_id"=>"23095582000"}, {"first_name"=>"Christopher", "last_name"=>"Tauer", "scopus_author_id"=>"35747171700"}, {"first_name"=>"Dieter", "last_name"=>"Palmberger", "scopus_author_id"=>"35362633200"}, {"first_name"=>"Andreas", "last_name"=>"Maccani", "scopus_author_id"=>"35772776000"}, {"first_name"=>"Martina", "last_name"=>"Baumann", "scopus_author_id"=>"7202494627"}, {"first_name"=>"Reingard", "last_name"=>"Grabherr", "scopus_author_id"=>"6701521847"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"20949004", "sgr"=>"78149441000", "doi"=>"10.1371/journal.pone.0013265", "scopus"=>"2-s2.0-78149441000", "pui"=>"359930903", "isbn"=>"1932-6203", "issn"=>"19326203"}, "id"=>"8ce0e0ff-f985-34f1-812a-07823442bb8d", "abstract"=>"Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.", "link"=>"http://www.mendeley.com/research/evaluation-influenza-replicon-transient-expression-recombinant-proteins-mammalian-cells-2", "reader_count"=>13, "reader_count_by_academic_status"=>{"Student > Doctoral Student"=>2, "Researcher"=>5, "Student > Ph. D. Student"=>5, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Student > Doctoral Student"=>2, "Researcher"=>5, "Student > Ph. D. Student"=>5, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>11, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>11}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}}, "group_count"=>0}

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Figshare 13804 May 08:48 UTC

Figshare

{"files"=>["https://ndownloader.figshare.com/files/825664"], "description"=>"HEK-293 cells were either tranfected with pcDNA-luc (CMV), pGL3-control (SV40) or with the influenza replicon and pMono-lucNS (Flu Replicon) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. Four hours post transfection cells were transferred to 27°C or 40°C or remained at 37°C. Cells were harvested 48 hours post transfection and were subjected to luciferase assay. Firefly values have been normalized to Renilla values. Data represents arithmetic mean values and standard deviation.", "links"=>[], "tags"=>["dependence", "influenza"], "article_id"=>496032, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g009", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Temperature_dependence_of_the_influenza_replicon_/496032", "title"=>"Temperature dependence of the influenza replicon.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:00:03"}
{"files"=>["https://ndownloader.figshare.com/files/825324"], "description"=>"Cells transfected with the replicon system show two very distinct populations in the forward light scatter (EGFP), one completely negative, the other highly positive. pEGFP-N1 driven EGFP expressions shows a broad distribution spanning from completely negative to moderate and high expressing cells.", "links"=>[], "tags"=>["cells", "transfected", "pegfp-n1", "influenza", "replicon", "pmono-egfp", "77"], "article_id"=>495682, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g005", "stats"=>{"downloads"=>1, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_FACS_analysis_of_cells_transfected_with_either_pEGFP_N1_CMV_or_the_influenza_replicon_system_and_pMono_EGFP_Flu_Replicon_77_hours_post_transfection_/495682", "title"=>"FACS analysis of cells transfected with either pEGFP-N1 (CMV) or the influenza replicon system and pMono-EGFP (Flu Replicon) 77 hours post transfection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 22:58:12"}
{"files"=>["https://ndownloader.figshare.com/files/825799"], "description"=>"HEK-293 cells were either tranfected with pRC-LC and pRC-HC (CMV HC∶LC = 1∶1, CMV HC∶LC = 10∶1) or with the influenza replicon and pMono-LC and pMono-HC (Flu Replicon HC∶LC = 1∶1, Flu Replicon HC∶LC = 10∶1). Heavy chain to light chain ratios of 1∶1 and 10∶1 were tested. Antibody concentration in the culture supernatant was monitored for 101 hours by ELISA. Data represents arithmetic mean values and standard deviation.", "links"=>[], "tags"=>["monoclonal", "anti-gp41", "antibody"], "article_id"=>496163, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g011", "stats"=>{"downloads"=>1, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_a_the_monoclonal_anti_gp41_antibody_3D6_/496163", "title"=>"Expression of a the monoclonal anti-gp41 antibody 3D6.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:00:45"}
{"files"=>["https://ndownloader.figshare.com/files/825162"], "description"=>"HEK-293 cells were either tranfected with pEGFP-N1 (CMV) or with the influenza replicon and pMono-EGFP (Flu Replicon). Pictures were taken 56 and 98 hours post transfection. Non-transfected cells were used as negative control (Neg. Control).", "links"=>[], "tags"=>["biotechnology", "biotechnology/applied microbiology", "virology/viral replication and gene regulation"], "article_id"=>495525, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g004", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_EGFP_expression_/495525", "title"=>"EGFP expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 22:57:19"}
{"files"=>["https://ndownloader.figshare.com/files/825868"], "description"=>"HEK-293 cells were either tranfected with pRC-LC (CMV) or with the influenza replicon and pMono-LC (Flu Replicon). Light chain concentration in the culture supernatant was monitored for 101 hours by ELISA. Data represents arithmetic mean values and standard deviation.", "links"=>[], "tags"=>["biotechnology", "biotechnology/applied microbiology", "virology/viral replication and gene regulation"], "article_id"=>496232, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g012", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Light_chain_expression_/496232", "title"=>"Light chain expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:01:08"}
{"files"=>["https://ndownloader.figshare.com/files/825424"], "description"=>"HEK-293 cells either tranfected with pcDNA-luc (CMV) or with the influenza replicon and pMono-lucNS (Flu Replicon) were monitored over a period of 101 hours by luciferase assay. Data represents arithmetic mean values and standard deviation.", "links"=>[], "tags"=>["firefly", "luciferase"], "article_id"=>495785, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g006", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Time_course_of_firefly_luciferase_expression_/495785", "title"=>"Time course of firefly luciferase expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 22:58:47"}
{"files"=>["https://ndownloader.figshare.com/files/824941"], "description"=>"Bidirectional cassettes (A) were used for generation of the influenza replicon. Monodirectional cassettes (B) were used to generate vRNA of reporters or genes of interest.", "links"=>[], "tags"=>["bidirectional", "monodirectional", "transcription"], "article_id"=>495306, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g002", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schemata_of_bidirectional_and_monodirectional_transcription_cassettes_/495306", "title"=>"Schemata of bidirectional and monodirectional transcription cassettes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 22:56:10"}
{"files"=>["https://ndownloader.figshare.com/files/824857"], "description"=>"pTripolis and pBi-NP generate the influenza replicon (mRNA/protein and vRNA) whereas pMono plasmids with various genes of interest or reporter genes drive transcription of vRNA.", "links"=>[], "tags"=>["drawings", "plasmids", "influenza", "replicon"], "article_id"=>495217, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g001", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_drawings_of_plasmids_used_to_generate_the_influenza_replicon_based_expression_system_/495217", "title"=>"Schematic drawings of plasmids used to generate the influenza replicon based expression system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 22:55:42"}
{"files"=>["https://ndownloader.figshare.com/files/825046"], "description"=>"HEK-293 cells either tranfected with pEGFP-N1 (CMV) or with the influenza replicon and pMono-EGFP (Flu Replicon) were monitored over a period of 101 hours by FACS analysis. Data represents arithmetic mean values and standard deviation.", "links"=>[], "tags"=>["biotechnology", "biotechnology/applied microbiology", "virology/viral replication and gene regulation"], "article_id"=>495410, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g003", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transfection_efficiency_time_course_/495410", "title"=>"Transfection efficiency time course.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 22:56:43"}
{"files"=>["https://ndownloader.figshare.com/files/825591"], "description"=>"HEK -293, MDCK or CHO-K1 cells were either tranfected with pcDNA-luc (CMV) or with the influenza replicon and pMono-lucNS (Influenza Replicon). The HEK-293 negative control has been transfected with pMono-lucNS and pBi-NP only. Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. In case of MDCK and CHO-K1, untransfected cells have been used as negative controls and can therefore not be normalized, absolute values can be found in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013265#s2\" target=\"_blank\">Results</a> section. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Data represents arithmetic mean values and standard deviation.", "links"=>[], "tags"=>["influenza", "replicon", "canine", "rodent"], "article_id"=>495956, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g008", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Activity_of_the_influenza_replicon_in_a_human_a_canine_and_a_rodent_cell_line_/495956", "title"=>"Activity of the influenza replicon in a human, a canine and a rodent cell line.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 22:59:38"}
{"files"=>["https://ndownloader.figshare.com/files/825506"], "description"=>"HEK-293 cells were either tranfected with pcDNA-luc (CMV), pGL3-control (SV40) or with the influenza replicon and pMono-lucNS (Influenza Replicon) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Firefly values have been normalized to Renilla values. Data represents arithmetic mean values and standard deviation.", "links"=>[], "tags"=>["firefly", "luciferase", "driven", "sv40", "influenza"], "article_id"=>495862, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g007", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_firefly_luciferase_expression_driven_by_CMV_SV40_or_by_the_influenza_replicon_/495862", "title"=>"Comparison of firefly luciferase expression driven by CMV, SV40 or by the influenza replicon.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 22:59:08"}
{"files"=>["https://ndownloader.figshare.com/files/825726"], "description"=>". HEK-293 cells were tranfected with the influenza replicon and either pMono-lucNS (NCR NS) or pMono-lucM (NCR M) or pMono-lucPB1 (NCR PB1) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for Renilla luciferase, was cotransfected in all cases. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Firefly values have been normalized to Renilla values. Data represents arithmetic mean values and standard deviation.", "links"=>[], "tags"=>["ncrs"], "article_id"=>496088, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.g010", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Influence_of_different_NCRs_on_the_expression_level_/496088", "title"=>"Influence of different NCRs on the expression level", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 23:00:22"}
{"files"=>["https://ndownloader.figshare.com/files/825980"], "description"=>"pEGFP-N1, pcDNA luc, pRC-LC and pRC-HC carry the CMV immediate-early promoter, pGL3-control and pRL carry SV40 promoter elements, pTripolis and pBi-NP drive bidirectional transcription (mRNA and vRNA) of the influenza polymerases and the NP protein. pMono-EGFP, pMono-lucNS, pMono-LC, pMono-HC, pMono-lucM and pMono-lucPB1 drive monodirectional (vRNA) transcription of the gene of interest. AB stands for antibody, LC stands for light chain experiments.", "links"=>[], "tags"=>["plasmid", "amounts"], "article_id"=>496345, "categories"=>["Biotechnology", "Infectious Diseases"], "users"=>["Florian Krammer", "Jens Pontiller", "Christopher Tauer", "Dieter Palmberger", "Andreas Maccani", "Martina Baumann", "Reingard Grabherr"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013265.t001", "stats"=>{"downloads"=>3, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Plasmids_and_plasmid_amounts_used_for_various_experiments_/496345", "title"=>"Plasmids and plasmid amounts used for various experiments.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 23:01:41"}

PMC Usage Stats 116417 Sep 14:33 UTC

PMC Usage Stats | Further Information

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Relative Metric 45648810 May 16:22 UTC

Relative Metric

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F1000Prime23 Sep 11:59 UTC

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