Journal: Nature protocols

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

doi: 10.1038/s41596-018-0075-9

Figure Lengend Snippet: List of transfer plasmids.

Article Snippet: Mouse anti-His6 primary antibody (Takara/Clontech, cat. no. 631212).

Techniques: Plasmid Preparation, In Vivo, Expressing, Transduction

Journal: Nature protocols

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

doi: 10.1038/s41596-018-0075-9

Figure Lengend Snippet: (A) Genetic elements between the 5’ and 3’ long terminal repeats (LTRs) are packaged in lentiviral particles and integrated in the expression cell genome as proviral DNA. Total proviral DNA size in the empty pHR-CMV-TetO2 plasmid is 4.2 kb. ψ: psi packaging signal; RRE: Rev response element; PPT: polypurine tract; CMV-MIE: major immediate early cytomegalovirus enhancer and promoter; TRE: tetracycline response element; MCS: multiple cloning site; WPRE: Woodchuck Hepatitis Virus posttranscriptional regulatory element. (B) The pHR-CMV-TetO2 multiple cloning site (MCS). (C) Non-concentrated lentiviral particles were used to infect HEK293T cells and protein was expressed for 72 h. His6-tagged proteins were detected using Western blotting (mouse anti-His6 primary antibody: 1:5,000 dilution, HRP-conjugated goat anti-mouse secondary antibody: 1:5,000 dilution, rabbit anti-Cyclophilin A (CypA) primary antibody: 0.5 μg/mL, HRP-conjugated goat anti-rabbit secondary antibody: 1:5,000 dilution). Proviral DNA sizes are noted in brackets. (D) Comparative size-exclusion chromatograms of secreted NET1ΔNTR and NEO1FN456, showing improved secreted protein yields from lentiviral transduction (blue curves) as compared to transient transfection (red curves) of adherent HEK293T cells.

Article Snippet: Mouse anti-His6 primary antibody (Takara/Clontech, cat. no. 631212).

Techniques: Expressing, Plasmid Preparation, Clone Assay, Western Blot, Transduction, Transfection

Journal: Nature protocols

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

doi: 10.1038/s41596-018-0075-9

Figure Lengend Snippet: (A) Schematic representation of the pHR-CMV-TetO2_IRES-EmGFP proviral DNA (5.5 kb) for bicistronic expression of the transgene of interest and free cytosolic EmGFP. (B) Schematic representation of the β-NRX1LNS6 and α-NRX1ECTO-FL constructs. (C) Endpoint dilution assay to estimate functional titer of non-concentrated lentiviral particles encoding β-NRX1LNS6_IRES-EmGFP and α-NRX1ECTO-FL_IRES-EmGFP. Every image was individually enhanced using ImageJ 59 to reveal all cells with fluorescence. Scale bar: 100 μm. (D) Non-concentrated lentiviral particles were used to infect HEK293T cells and protein was expressed for 72 h. Comparison of transduction efficiency of β-NRX1LNS6_IRES-EmGFP and α-NRX1ECTO-FL_IRES-EmGFP. (E) Western blot detection of His6-tagged proteins produced using the pHR-CMV-TetO2_3C-Avi-His6 or pHR-CMV-TetO2_3C-Avi-His6_IRES-EmGFP transfer plasmids (mouse anti-His6 primary antibody: 1:5,000 dilution, HRP-conjugated goat anti-mouse secondary antibody: 1:5,000 dilution, rabbit anti-Cyclophilin A (CypA) primary antibody: 0.5 μg/mL, HRP-conjugated goat anti-rabbit secondary antibody: 1:5,000 dilution). Proviral DNA sizes are noted in brackets. (F) MDGA1ECTO-FL_IRES-EmGFP: forward and side scatter area (A), height (H) and width (W), and fluorescence gates. (G) Comparative size-exclusion chromatograms show secreted MDGA1ECTO-FL yields from 1.0 L (4 roller bottles) of adherent HEK293T cell culture, either transiently transfected (red curve; 6.7 mg total protein) or stably transduced using lentivirus (blue curve; 46.3 mg total protein). Transduced cells were enriched using FACS by applying sorting gates A, B and C from panel F.

Article Snippet: Mouse anti-His6 primary antibody (Takara/Clontech, cat. no. 631212).

Techniques: Expressing, Construct, Endpoint Dilution Assay, Functional Assay, Fluorescence, Transduction, Western Blot, Produced, Cell Culture, Transfection, Stable Transfection

Journal: Nature protocols

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

doi: 10.1038/s41596-018-0075-9

Figure Lengend Snippet: (A-D) Multi-color FACS. (A) Non-concentrated lentiviral particles encoding β-NRX1LNS6_IRES-EmGFP (6.1 kb proviral DNA) and Cbln1FL_IRES-mRuby2 (6.0 kb proviral DNA) were used to co-infect HEK293T cells. (B) Fluorescent imaging indicated a mixture of non-, single- and double-transduced cells. The image was taken on a Leica SP8 SMD X confocal microscope. Scale bar: 50 μm. (C) Two-dimensional compensated fluorescence plot. (D) Western blot detection of His6-tagged β-NRX1LNS6(+SS4) and Cbln1FL secreted from co-infected cells after cell sorting (mouse anti-His6 primary antibody: 1:5,000 dilution, HRP-conjugated goat anti-mouse secondary antibody: 1:5,000 dilution). (E-F) In vivo biotinylation. (E) Non-concentrated lentiviral particles encoding either HA-BirA-ER (5.1 kb proviral DNA) or 3C-Avi-His6-tagged NL1ECTO (5.9 kb proviral DNA) were used to co-infect HEK293T cells. (F) Non-concentrated lentiviral particles encoding 3C-Avi-His6-tagged NL1ECTO and IRES_HA-BirA-ER (7.6 kb proviral DNA) were used to infect HEK293T cells. A D-biotin concentration of 100 μM was maintained in the DMEM/F-12/2%FBS medium. Anti-D-biotin western blot detection of in vivo biotinylated NL1ECTO (high sensitivity streptavidin-HRP conjugate; 1:4000 dilution). Anti-HA western blot detection of HA-tagged TetR (mouse anti-HA primary antibody: 1:5,000 dilution, HRP-conjugated goat anti-mouse secondary antibody: 1:5,000 dilution). (G-H) Generation of inducible cell lines. (G) Non-concentrated lentiviral particles encoding TetR-HA-NLS-P2A-BSD-Myc (5.1 kb proviral DNA) and mVenus (4.7 kb proviral DNA) were used to infect HEK293T and HEK293S GnTI– cells. (H) Flow cytometry analysis of the resulting HEK293Tlenti-TetR and HEK293S GnTI–lenti-TetR inducible cell lines, and comparison with the HEK293S GnTI– TetR cell line 34. MFI: mean fluorescence intensity.

Article Snippet: Mouse anti-His6 primary antibody (Takara/Clontech, cat. no. 631212).

Techniques: Imaging, Microscopy, Fluorescence, Western Blot, Infection, FACS, In Vivo, Concentration Assay, Flow Cytometry