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Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins


Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3–4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.

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The data that support the findings of this study are available from the corresponding authors upon reasonable request.


  1. 1.

    Aricescu, A. R. & Owens, R. J. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology. Curr. Opin. Struct. Biol. 23, 345–356 (2013).

  2. 2.

    Chang, V. T. et al. Glycoprotein structural genomics: solving the glycosylation problem. Structure 15, 267–273 (2007).

  3. 3.

    Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D Biol. Crystallogr. 62, 1243–1250 (2006).

  4. 4.

    Chaudhary, S. et al. Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells. Nat. Protoc. 7, 453–466 (2012).

  5. 5.

    Naldini, L., Trono, D. & Verma, I. M. Lentiviral vectors, two decades later. Science 353, 1101–1102 (2016).

  6. 6.

    Benabdellah, K. et al. Development of an all-in-one lentiviral vector system based on the original TetR for the easy generation of Tet-ON cell lines. PLoS ONE 6, e23734 (2011).

  7. 7.

    De Groote, P. et al. Generation of a new Gateway-compatible inducible lentiviral vector platform allowing easy derivation of co-transduced cells. Biotechniques 60, 252–259 (2016).

  8. 8.

    Campeau, E. et al. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 4, e6529 (2009).

  9. 9.

    Bandaranayake, A. D. et al. Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Res. 39, e143 (2011).

  10. 10.

    Chang, V. T. et al. Initiation of T cell signaling by CD45 segregation at ‘close contacts’. Nat. Immunol. 17, 574–582 (2016).

  11. 11.

    Chang, V. T., Spooner, R. A., Crispin, M. & Davis, S. J. Glycan remodeling with processing inhibitors and lectin-resistant eukaryotic cells. Methods Mol. Biol. 1321, 307–322 (2015).

  12. 12.

    Cronin, J., Zhang, X. Y. & Reiser, J. Altering the tropism of lentiviral vectors through pseudotyping. Curr. Gene Ther. 5, 387–398 (2005).

  13. 13.

    Finkelshtein, D. et al. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proc. Natl. Acad. Sci. USA 110, 7306–7311 (2013).

  14. 14.

    Demaison, C. et al. High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum. Gene Ther. 13, 803–813 (2002).

  15. 15.

    Zufferey, R. et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15, 871–875 (1997).

  16. 16.

    Lin, Y. C. et al. Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat. Commun. 5, 4767 (2014).

  17. 17.

    Qin, J. Y. et al. Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS ONE 5, e10611 (2010).

  18. 18.

    Reeves, P. J., Kim, J. M. & Khorana, H. G. Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc. Natl. Acad. Sci. USA 99, 13413–13418 (2002).

  19. 19.

    Foecking, M. K. & Hofstetter, H. Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene 45, 101–105 (1986).

  20. 20.

    Gorman, C. M., Gies, D., McCray, G. & Huang, M. The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins. Virology 171, 377–385 (1989).

  21. 21.

    Donello, J. E., Loeb, J. E. & Hope, T. J. Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J. Virol. 72, 5085–5092 (1998).

  22. 22.

    Garcia-Nafria, J., Watson, J. F. & Greger, I. H. IVA cloning: a single-tube universal cloning system exploiting bacterial In Vivo Assembly. Sci. Rep. 6, 27459 (2016).

  23. 23.

    Schmidt, T. G. et al. Development of the Twin-Strep-tag and its application for purification of recombinant proteins from cell culture supernatants. Protein Expr. Purif. 92, 54–61 (2013).

  24. 24.

    Schmidt, T. G. & Skerra, A. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat. Protoc. 2, 1528–1535 (2007).

  25. 25.

    Kumar, M., Keller, B., Makalou, N. & Sutton, R. E. Systematic determination of the packaging limit of lentiviral vectors. Hum. Gene Ther. 12, 1893–1905 (2001).

  26. 26.

    Knox, R. et al. A streamlined implementation of the glutamine synthetase-based protein expression system. BMC Biotechnol. 13, 74 (2013).

  27. 27.

    Mancia, F. et al. Optimization of protein production in mammalian cells with a coexpressed fluorescent marker. Structure 12, 1355–1360 (2004).

  28. 28.

    Oberbek, A., Matasci, M., Hacker, D. L. & Wurm, F. M. Generation of stable, high-producing CHO cell lines by lentiviral vector-mediated gene transfer in serum-free suspension culture. Biotechnol. Bioeng. 108, 600–610 (2011).

  29. 29.

    Shaner, N. C., Patterson, G. H. & Davidson, M. W. Advances in fluorescent protein technology. J. Cell Sci. 120, 4247–4260 (2007).

  30. 30.

    Lam, A. J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).

  31. 31.

    Goedhart, J. et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat. Commun. 3, 751 (2012).

  32. 32.

    Elegheert, J. et al. Structural basis for integration of GluD receptors within synaptic organizer complexes. Science 353, 295–299 (2016).

  33. 33.

    Elegheert, J. et al. Structural mechanism for modulation of synaptic neuroligin-neurexin signaling by MDGA proteins. Neuron 95, 896–913.e10 (2017).

  34. 34.

    Reeves, P. J., Callewaert, N., Contreras, R. & Khorana, H. G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

  35. 35.

    Yao, F. et al. Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum. Gene Ther. 9, 1939–1950 (1998).

  36. 36.

    Brooks, A. R. et al. Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle. J. Gene Med. 6, 395–404 (2004).

  37. 37.

    Hsu, C. C. et al. Targeted methylation of CMV and E1A viral promoters. Biochem. Biophys. Res. Commun. 402, 228–234 (2010).

  38. 38.

    He, J., Yang, Q. & Chang, L. J. Dynamic DNA methylation and histone modifications contribute to lentiviral transgene silencing in murine embryonic carcinoma cells. J. Virol. 79, 13497–13508 (2005).

  39. 39.

    Kim, J. H. et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE 6, e18556 (2011).

  40. 40.

    Li, Z. et al. Simple piggyBac transposon-based mammalian cell expression system for inducible protein production. Proc. Natl. Acad. Sci. USA 110, 5004–5009 (2013).

  41. 41.

    Matasci, M., Baldi, L., Hacker, D. L. & Wurm, F. M. The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability. Biotechnol. Bioeng. 108, 2141–2150 (2011).

  42. 42.

    Boyce, F. M. & Bucher, N. L. Baculovirus-mediated gene transfer into mammalian cells. Proc. Natl. Acad. Sci. USA 93, 2348–2352 (1996).

  43. 43.

    Dukkipati, A. et al. BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies. Protein Expr. Purif. 62, 160–170 (2008).

  44. 44.

    Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).

  45. 45.

    Morales-Perez, C. L., Noviello, C. M. & Hibbs, R. E. Manipulation of subunit stoichiometry in heteromeric membrane proteins. Structure 24, 797–805 (2016).

  46. 46.

    Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).

  47. 47.

    Otto, E. et al. Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences. Hum. Gene Ther. 5, 567–575 (1994).

  48. 48.

    Miyoshi, H. et al. Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150–8157 (1998).

  49. 49.

    Zufferey, R. et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873–9880 (1998).

  50. 50.

    Schermelleh, L., Spada, F. & Leonhardt, H. Visualization and measurement of DNA methyltransferase activity in living cells. Curr. Protoc. Cell Biol. Chapter 22, Unit 22.12 (2008).

  51. 51.

    Hattori, M., Hibbs, R. E. & Gouaux, E. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20, 1293–1299 (2012).

  52. 52.

    Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

  53. 53.

    Davis, H. E., Rosinski, M., Morgan, J. R. & Yarmush, M. L. Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation. Biophys. J. 86, 1234–1242 (2004).

  54. 54.

    Backliwal, G. et al. Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. Biotechnol. Bioeng. 101, 182–189 (2008).

  55. 55.

    Bell, C. H. et al. Structure of the repulsive guidance molecule (RGM)-neogenin signaling hub. Science 341, 77–80 (2013).

  56. 56.

    Healey, E. G. et al. Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein. Nat. Struct. Mol. Biol. 22, 458–465 (2015).

  57. 57.

    Byrne, E. F. X. et al. Structural basis of Smoothened regulation by its extracellular domains. Nature 535, 517–522 (2016).

  58. 58.

    Miller, P. S. & Aricescu, A. R. Crystal structure of a human GABAA receptor. Nature 512, 270–275 (2014).

  59. 59.

    Rueden, C. T. et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529 (2017).

  60. 60.

    Meissner, P. et al. Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol. Bioeng. 75, 197–203 (2001).

  61. 61.

    Kutner, R. H., Zhang, X. Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 4, 495–505 (2009).

  62. 62.

    Telford, W. G. et al. Flow cytometry of fluorescent proteins. Methods 57, 318–330 (2012).

  63. 63.

    Robinson, J. P. & Roederer, M. History of science. Flow cytometry strikes gold. Science 350, 739–740 (2015).

  64. 64.

    Davies, D. Cell separations by flow cytometry. Methods Mol. Biol. 878, 185–199 (2012).

  65. 65.

    Andrell, J. et al. Generation of tetracycline-inducible mammalian cell lines by flow cytometry for improved overproduction of membrane proteins. Methods Mol. Biol. 1432, 63–78 (2016).

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We thank G. Davies and C. Green (University of Oxford) for assistance with flow cytometry, S. Padilla-Parra and L.A.J. Alvarez (University of Oxford) for assistance with microscopic imaging, D. Laverty (MRC-LMB) for the modified pEZT-BM vector, J. Watson (MRC-LMB) for advice on IVA cloning, and S. Ressl (Indiana University Bloomington) for comments on the manuscript. pMD2.G (Addgene plasmid no.12259) and psPAX2 (Addgene plasmid no. 12260) were a gift from D. Trono (École Polytechnique Fédérale de Lausanne (EPFL)). This work was supported by a Marie-Curie (FP7-328531) long-term postdoctoral fellowship (to J.E.); UK Medical Research Council grants MR/L009609/1 and MC_UP_1201/15 (to A.R.A.) and MR/L017776/1 (to C.S.); a UK Biotechnology and Biological Sciences Research Council grant (BB/M024709/1, to A.R.A.); Cancer Research UK grants C20724/A14414 (to C.S.) and C375/A10976 (to E.Y.J.); a European Research Council (ERC) grant (647278, to C.S.); and Wellcome Trust studentships (105247/Z/14/Z, to S.S., and 203726/Z/16/Z, to R.E.W.). The Wellcome Centre for Human Genetics is funded by Wellcome Trust Core Award 203852/Z/16/2.

Author information

J.E. and E.B. designed and constructed the pHR-CMV-TetO2 plasmid suite. J.E., E.B., B.B. and V.T.C. established and optimized the protocol. J.E., B.B. and E.B. performed the cloning, western blotting, flow cytometry and microscopic imaging. S.S. performed the GABAA-β3FL comparative expression tests. R.E.W. performed the SMOXTAL expression tests. S.C.G. performed the NET1/NEO1 large-scale expressions. E.F.X.B. performed the generation of the inducible HEK293lenti-TetR cell lines. D.I.S., E.Y.J., C.S. and A.R.A. provided funding and oversaw the research. J.E. and A.R.A. conceptualized the research. J.E. wrote the paper. All authors provided feedback on the final version of the manuscript.

Correspondence to Jonathan Elegheert or Christian Siebold or A. Radu Aricescu.

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Chang, V. T. et al. Nat. Immunol. 17, 574–582 (2016):

Integrated supplementary information

Supplementary Figure 1 The pHR-CMV-TetO2_3C-Avi-His6 transfer plasmid.

LTR: long-terminal repeat; ψ: psi packaging signal; RRE: Rev response element; cPPT: central polypurine tract; CMV-MIE: major immediate early cytomegalovirus enhancer/promoter; SS: signal sequence; MCS: multiple cloning site; 3C: Human Rhinovirus (HRV) 3C protease cleavage site; Avi: Avi-tag; His6: His6-tag; WPRE: Woodchuck Hepatitis Virus posttranscriptional regulatory element; ΔU3: U3 deletion in the 3ʹ LTR; AmpR: Ampicillin resistance gene; ori: origin of replication; SV40: Simian vacuolating virus 40; UTR: untranslated region. The genetic elements flanked by the 5ʹ and 3ʹ LTRs will be stably integrated into the host cell genome as proviral DNA, and are highlighted in pink. The gene encoding E. coli xanthine-guanine phosphoribosyltransferase (XGPRT, indicated with a star symbol) has been inactivated by deletion of a single base pair, likely to remove an internal KpnI site, and resulting introduction of a stop codon. This modification was already present in the pHR-SIN-CSGW lentiviral transfer plasmid on which the pHR-CMV-TetO2 plasmid suite is based. The dominant selectable marker XGPRT is normally used to permit isolation of stably transfected cells using mycophenolic acid, an immunosuppressant drug. We did not correct the open reading frame since our procedure does not require stable maintenance of the lentiviral transfer plasmid in HEK293T Lenti-X cells. The figure was prepared using the SnapGene software (from GSL Biotech; available at

Supplementary Figure 2 The pHR-CMV-TetO2 multiple cloning site.

The pHR-CMV-TetO2 multiple cloning site (MCS) is fully compatible with subcloning of inserts from pHLsec; XbaI/EcoRI – cRPTPσ signal sequence – AgeI – insert – KpnI – tag + stop codons – XhoI, as well as with in vivo assembly (IVA) cloning. (A) The pHR-CMV-TetO2_3C-Avi-His6(_IRES-FP) plasmids contain the 3C-Avi-His6 tag inserted between KpnI and XhoI. (B) The pHR-CMV-TetO2_3C-Twin-Strep(_IRES-FP) plasmids contain the 3C-Twin-Strep tag inserted between KpnI and XhoI. The figure was prepared using the SnapGene software.

Supplementary Figure 3 Proviral DNA elements of the pHR-CMV-TetO2 plasmids.

Schematic representation of the empty pHR-CMV-TetO2 (4.2 kb proviral DNA), pHR-CMV-TetO2_IRES-FP (5.5 kb proviral DNA), pHR-CMV-TetO2_3C-mVenus-Twin-Strep (5.0 kb proviral DNA), pHR-CMV-TetO2_3C-Avi-His6_HA-BirA-ER (5.9 kb proviral DNA), pHR-SFFV (4.0 kb proviral DNA) and pHR-CAG (5.2 kb proviral DNA) transfer plasmid variants. ψ: psi packaging signal; RRE: Rev response element; PPT: polypurine tract; CMV-MIE: major immediate early cytomegalovirus enhancer/promoter; SFFV: Spleen Focus Forming Virus; CAG: CMV enhancer, chicken β-actin promoter, rabbit β-globin splice acceptor; TRE: tetracycline response element; MCS: multiple cloning site; IRES: internal ribosomal entry site; FP: fluorescent protein; WPRE: Woodchuck Hepatitis Virus posttranscriptional regulatory element; LTR: long-terminal repeat; 3C: Human Rhinovirus (HRV) 3C protease site; mV: mVenus; TS: Twin-Strep. The figure was prepared using the SnapGene software.

Supplementary Figure 4 Bicistronic expression using IRES-EmGFP.

(A) Schematic representation of the mRuby2-IRES-EmGFP expression construct (2.1 kb insert, 6.0 kb proviral DNA). (B) Non-concentrated lentiviral particles encoding mRuby2-IRES-EmGFP were used to infect HEK293T cells. IRES-EmGFP mean fluorescence intensity is ~30-fold lower at identical 488 nm laser power as compared to free EmGFP expression. Scale bar: 100 μm. (C) EmGFP and mRuby2 co-localize and their fluorescence intensities show a Pearson correlation coefficient (Pearson’s r) of 0.89 between pixel values, implying that IRES-EmGFP is predictive of the presence and expression level of the preceding transgene. Free and bicistronic mRuby2 mean fluorescence intensities are comparable, indicating that IRES-EmGFP does not negatively affect expression of the preceding transgene. Fluorescence images were taken on a Leica SP8 SMD X confocal microscope as sequential scans to avoid spectral crosstalk of EmGFP and mRuby fluorescence into the red and green detection channels, respectively. Fluorescence emission was detected using hybrid detectors operated in photon-counting mode, meaning that every incident photon gave rise to one gray value. Scale bar: 100 μm. (D) Non-concentrated lentiviral particles encoding mRuby2-IRES-EmGFP were used to infect HEK293S GnTI TetR cells. Expression was induced using 1 μg/mL Dox for 12 h. Using flow cytometry, we measured a Pearson’s r of 0.85 between EmGFP and mRuby2 compensated fluorescence intensities, based on a total of 35,546 single, gated cells.

Supplementary Figure 5 Endpoint dilution assay to determine functional lentiviral titer.

The assay was performed as described in Box 2 of the main Protocol. Functional lentiviral titer (and thus MOI) decreases with increasing insert size. Images were taken on a Nikon wide-field TE2000U Microscope. Exposure time was 500 ms and every image was individually enhanced using ImageJ to reveal all cells with fluorescence. Scale bar: 200 μm.

Supplementary Figure 6 Determination of transduction efficiency using flow cytometry.

Non-concentrated lentiviral particles encoding transgene-IRES-EmGFP were used to infect HEK293T cells. The plots show the forward and side scatter area (A), height (H) and width (W), and fluorescence gates used to analyse cells expressing EmGFP. Functional lentiviral titer (and thus MOI) decreases with increasing insert size, leading to decreasing transduction efficiency and also a lower number of integrated gene copies per cell.

Supplementary Figure 7 GABAAR-β3FL: comparison of BacMam and lentiviral transduction.

(A) The mVenus-GABAAR-β3FL-1D4 construct was cloned into the pHR-CMV-TetO2 transfer plasmid. The FSEC analysis shows lentivirus-mediated protein expression in function of expression time (24, 48 h or 72 h), expression temperature (30°C or 37°C), concentration of Dox (0, 5 or 10 μg/mL), and concentration of VPA or sodium butyrate (0 or 5 mM). (B) The mVenus-GABAAR-β3FL-1D4 construct was cloned into a modified pEZT-BM plasmid. The FSEC analysis shows BacMam-mediated protein expression in function of expression time (24, 48 h or 72 h), expression temperature (30°C or 37°C), concentration of VPA or sodium butyrate (0 or 5 mM), and multiplicity of infection (MOI; 0, 1, 4 or 10). Note the difference in scale for the fluorescence emission between panels A and B.

Supplementary Figure 8 Multi-color FACS to enrich co-transduced cells.

(A) Non-concentrated lentiviral particles encoding either mTurquoise2 (mT2; 0.7 kb insert, 4.7 kb proviral DNA), mVenus (mV; 0.7 kb insert, 4.7 kb proviral DNA) or mRuby2 (mR2; 0.7 kb insert, 4.7 kb proviral DNA) were used to co-infect HEK293S GnTI TetR cells. Fluorescence microscopic imaging of separately infected or co-infected cells, before and after induction with 10 μg/mL Dox, and after 24 h of expression. DIC; differential interference contrast. Fluorescence images were taken on a Leica SP8 SMD X confocal microscope as sequential scans to avoid spectral crosstalk of fluorophores into the separate detection channels. (B) The plots show the forward and side scatter area (A), height (H) and width (W), and fluorescence gates used to isolate cells expressing mTurquoise2, mVenus and mRuby2. Scale bar: 200 μm.

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Fig. 1: Large-scale expression of soluble or membrane proteins using lentiviral transduction.
Fig. 2: The pHR-CMV-TetO2 transfer plasmid for stable lentiviral expression in HEK293 cells.
Fig. 3: The pHR-CMV-TetO2_IRES-EmGFP transfer plasmid for determination of transduction efficiency and enrichment of transduced cells using FACS.
Fig. 4: Lentiviral transduction, inducible expression and FSEC screening of the GPCR Smoothened in HEK293S GnTI TetR cells.
Fig. 5: Lentiviral transduction and inducible expression of the GABAA receptor β3 homopentamer (GABAAR-β3FL) in HEK293S GnTI TetR cells, and comparison with BacMam.
Fig. 6: Tools for multi-color FACS, in vivo biotinylation and the generation of inducible cell lines.
Supplementary Figure 1: The pHR-CMV-TetO2_3C-Avi-His6 transfer plasmid.
Supplementary Figure 2: The pHR-CMV-TetO2 multiple cloning site.
Supplementary Figure 3: Proviral DNA elements of the pHR-CMV-TetO2 plasmids.
Supplementary Figure 4: Bicistronic expression using IRES-EmGFP.
Supplementary Figure 5: Endpoint dilution assay to determine functional lentiviral titer.
Supplementary Figure 6: Determination of transduction efficiency using flow cytometry.
Supplementary Figure 7: GABAAR-β3FL: comparison of BacMam and lentiviral transduction.
Supplementary Figure 8: Multi-color FACS to enrich co-transduced cells.


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