Journal: bioRxiv

Article Title: An improved workflow for rapid, large-scale protein production in HEK293 cells via antibiotic enrichment after lentiviral transduction

doi: 10.64898/2026.03.07.710266

Figure Lengend Snippet: a , Vector architecture of pHR-AB-CMV-TetO 2 (Puro R variant). The CMV-MIE promoter drives GOI expression, whereas a compact EF-1 α promoter independently drives expression of the antibiotic-resistance cassette to enable stringent enrichment of transduced cells before induction. All genetic elements are flanked by unique restriction sites. b , Antibiotic variants of the pHR-AB-CMV-TetO 2 system, available with puromycin, blasticidin, hygromycin or zeocin cassettes, permitting single-vector selection or orthogonal co-selection when co-infecting with multiple constructs. c , Inducible expression mechanism of the pHR-AB-CMV-TetO 2 vectors. In TetR-expressing cell lines, TetR binds the two TetO sites and represses the CMV-MIE promoter; addition of doxycycline (Dox) releases TetR and activates GOI transcription. In cell lines lacking TetR, CMV-MIE functions as a strong constitutive promoter. d , Vector architecture of pHR-AIO-AB (Puro R variant). A compact EF-1 α promoter constitutively drives expression of the bicistronic open reading frame encoding rtTA-V16 and the antibiotic-resistance marker linked by a T2A peptide, providing both the transactivator and selection marker from a single transcript. The GOI is placed under the TRE3GS promoter for Dox-inducible expression. All genetic elements are flanked by unique restriction sites. e , Antibiotic variants of the pHR-AIO-AB system, available with puromycin, blasticidin, hygromycin or zeocin cassettes, permitting single-vector selection or orthogonal co-selection when co-infecting with multiple constructs. f , Inducible expression mechanism of the pHR-AIO-AB vectors. In the presence of Dox, rtTA-V16 binds TRE3GS to activate GOI transcription; in the absence of Dox, TRE3GS exhibits very low basal activity, enabling tight control of induction. g , Schematic overview of inducible expression after proviral integration (Puro R variant). Left: The pHR-AB-CMV-TetO 2 proviral DNA is integrated in the genome of HEK293S GnTI − TetR LENTI or HEK293T TetR LENTI cell lines constitutively expressing TetR (from a previously integrated proviral TetR–Blast R cassette). TetR represses the CMV-MIE promoter until Dox is added, which releases repression and triggers GOI transcription. Constitutive expression of the EF-1 α cassette enables antibiotic selection. Right: In regular HEK293S GnTI − or HEK293T cells transduced with pHR-AIO-AB vectors, the EF-1 α cassette constitutively produces rtTA-V16 and the antibiotic-resistance marker, enabling enrichment of transduced cells and subsequent Dox-dependent induction of the GOI. Abbreviations: TKpA, thymidine kinase polyadenylation signal; CTE, constitutive transport element; Puro R , puromycin resistance; EF-1 α , Human elongation factor-1 alpha promoter; cHS4, chicken hypersensitive site 4 insulator; CMV-MIE, human cytomegalovirus major immediate-early promoter; TetO 2 , two tandem Tet (tetracycline) operator sites; GOI, gene of interest; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; Zeo R , zeocin resistance; Hygro R , hygromycin resistance; Blast R , blasticidin resistance; TetR, Tet Repressor protein; Dox, doxycycline; T2A, Thosea asigna virus 2A peptide; c-myc NLS, nuclear localisation signal derived from the c-Myc protein; rtTA V16, reverse tetracycline transactivator V16; TRE3GS, minimal CMV promoter with seven TetO operator sequences; HEK293, human embryonic kidney 293; GnTI − , N-acetylglucosaminyltransferase I-deficient; dsDNA, double-stranded DNA.

Article Snippet: HEK293T cells (G418 R ; ATCC, cat. no. CRL-3216) HEK293S GnTI − cells (ATCC, cat. no. CRL-3022) HEK293T TetR LENTI cells (G418 R , Blast R ; obtainable by request to J.E.)

Techniques: Plasmid Preparation, Variant Assay, Expressing, Selection, Construct, Marker, Activity Assay, Control, Transduction, Virus, Derivative Assay

Journal: bioRxiv

Article Title: An improved workflow for rapid, large-scale protein production in HEK293 cells via antibiotic enrichment after lentiviral transduction

doi: 10.64898/2026.03.07.710266

Figure Lengend Snippet: a , HEK293T benchmark. Cells were infected at low MOI to yield a ∼5–15% mNG–positive cell pool before selection. Flow cytometry histograms show enrichment of the sparsely infected populations to >90% mNG-positive with a tight, unimodal distribution. Because HEK293T cells lack TetR, CMV-MIE-TetO 2 drives constitutive expression. The magenta line marks the positivity threshold used for gating. b , HEK293S GnTI⁻ TetR benchmark. Low-MOI infection produced a ∼5 % mNG-positive cell pool that increased to >90% after two selection cycles. In the TetR background, basal expression in the enriched population was low-leak (∼1–5% positive without Dox); addition of Dox (1 µg/mL) produced a uniform and robust right-shift, indicating tight repression and synchronous induction. Gating as in (a). Abbreviations: AB, antibiotic; CMV, human cytomegalovirus; TetO₂, tandem Tet (tetracycline) operator sites; HEK293, human embryonic kidney 293; mNG, mNeonGreen; GnTI − , N-acetylglucosaminyltransferase I-deficient; TetR, Tet (tetracycline) Repressor protein; BlastR, blasticidin resistance; MOI, multiplicity of infection; Dox, doxycycline.

Article Snippet: HEK293T cells (G418 R ; ATCC, cat. no. CRL-3216) HEK293S GnTI − cells (ATCC, cat. no. CRL-3022) HEK293T TetR LENTI cells (G418 R , Blast R ; obtainable by request to J.E.)

Techniques: Infection, Selection, Flow Cytometry, Expressing, Produced