hek 293t human embryonic kidney cells  (ATCC)

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: 3P SARS2 VLPs are superior to 4P SARS2 VLPs. ( A ) 4P and 3P SARS2 Luc-PS9 VLPs were produced by co-transfection of either four or three plasmids into 293T producer cells. These plasmids encode for the viral structural proteins, along with a luciferase reporter coupled to PS9 packaging signal (Luc-PS9). ( B ) Luciferase assay showed ∼7-fold higher luminescence intensity in 293T-hACE2 cells upon using 3P versus 4P VLPs. ( C ) VLP concentrate (10 μl for M and E, 2 μl for S2 and N) was loaded in each lane. Western blot analysis suggests incorporation of all SARS2 structural components in VLPs, with 3P SARS2 Luc-PS9 VLPs displaying more intense protein bands compared with 4P VLPs. ( D ) Higher Luc-PS9 transcript levels were observed using RT-PCR in the case of 3P SARS2 Luc-PS9 VLPs. ( E ) Cryo-TEM images of 3P SARS2 Luc-PS9 VLPs showed spherical ∼100-nm-sized VLPs with double-layered membrane structures. ( F ) 3P SARS2 EGFP-PS9 VLPs produced with EGFP reporter efficiently infected 293T-hACE2 and A549-hACE2-TMPRSS2 cells. Fluorescence images were acquired 24 h post-infection. ( G ) Flow cytometry VLP entry time-course showed peak fluorescence for 3P SARS2 EGFP-PS9 VLPs at 24 h followed by decrease at larger times. This was observed both for 293T-hACE2 and A549-hACE2-TMPRSS2 cells. Abbreviations: aa, amino acid; nt, nucleotide. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: Human embryonic kidney 293T Lenti-X cells (“293T”) (Catalog #: 632180) were purchased from Clontech/Takara Bio (Mountain View, CA).

Techniques: Produced, Cotransfection, Luciferase, Western Blot, Reverse Transcription Polymerase Chain Reaction, Membrane, Infection, Fluorescence, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Tuning VLP viral tropism by altering viral glycoprotein. ( A ) Different types of VLPs were produced using the 3P system by varying the viral glycoprotein (VSV-G, SARS2 spike, SARS spike, or MERS spike) and reporter genes (luciferase or EGFP). ( B ) One microgram of the viral glycoprotein was used to create various 3P Luc-PS9 VLPs and these were used to infect five cell types: WT 293T (293T), 293T-hACE2, A549-hACE2-TMPRSS2, Calu-3, and 293T-DPP4. SARS2 and SARS spike VLPs displayed similar tropism and entered only hACE2-expressing cells (293T-hACE2, A549-hACE2-TMPRSS2, and Calu-3), with SARS exhibiting higher luminescence intensity compared with SARS2. MERS VLPs infected Calu-3 cell at low level and efficiently entered 293T-DPP4 cells. VSV-G VLPs entered all cell types. ( C ) 3P EGFP-PS9 VLPs were produced with 4 μg VSV-G, 1 μg SARS2 spike, or 1 μg MERS spike plasmid. VSV-G VLPs entered all cell types. SARS2 VLPs only entered ACE2 cells. MERS VLPs only entered DPP4 cells. Methods provide detailed steps for VLP production. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: Human embryonic kidney 293T Lenti-X cells (“293T”) (Catalog #: 632180) were purchased from Clontech/Takara Bio (Mountain View, CA).

Techniques: Produced, Luciferase, Expressing, Infection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Streamlining VLP technology using a 2P system. ( A ) Four constructs were developed with two independent promoters driving expression of reporter gene and SARS2 structural proteins. The promoters were separated by insulator and terminator sequences to minimize promoter interference: synthetic polyA (spa); a G-rich sequence from β-actin (Tactb); chicken hypersensitive site 4 (cHS4); and a synthetic MAR sequence 8 (sMAR8) at the end of the E protein. (B, C) Each of these constructs was transfected into 293T cells along with spike plasmid to produce four different 2P SARS2 Luc-PS9 VLPs. 2P.2 VLPs displayed highest luminescence intensity ( B ). Its signal was comparable to 4P VLP but lower than 3P VLP ( C ). ( D ) Western blots of SARS2 structural proteins showed different patterns of protein expression for different 2P plasmids. 2P.2 SARS2 Luc-PS9 VLPs displayed more intense protein bands compared with 2P.3 and 2P.4, but this was lower than 2P.1. ( E ) 2P.2.EGFP VLPs were created by replacing the luciferase reporter with EGFP. VLP entry of 2P.2.EGFP SARS2 VLPs into A549-hACE2-TMPRSS2 and 293T-hACE2 cells was measured using flow cytometry. Data are mean ± STD. *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: Human embryonic kidney 293T Lenti-X cells (“293T”) (Catalog #: 632180) were purchased from Clontech/Takara Bio (Mountain View, CA).

Techniques: Construct, Expressing, Sequencing, Transfection, Plasmid Preparation, Western Blot, Luciferase, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: VLPs deliver functional Cas9 mRNA into target cells to achieve gene editing. ( A ) 3P Cas9-P2A-dTo-T20 VLPs (dTo: dTomato) were used for gene editing studies, with surface glycoprotein encoding for either VSV-G or SARS2 spike. ( B ) General workflow of gene editing study performed in panels (C)–(E). sgRNAs were transfected into cells on day −1 using plasmids carrying BFP reporter. VLP-carrying spCas9 mRNA was introduced into cells on day 0. Tropism of the VLP depends on surface glycoprotein. Gene editing efficiency was quantified on day 6. Editing efficiency quantified percentage of BFP(+) population that either turned EGFP(−) (panels C and E) or lost ACE2 expression based on anti-hACE2 binding (panel D). ( C ) sgRNAs targeting EGFP were introduced into 293T-hACE2-EGFP and spCas9 mRNA was delivered using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. ( D ) sgRNAs against hACE2 were introduced to knockout the receptor in 293T-hACE2 cells using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. ( E ) sgRNAs targeting EGFP were introduced in 293T-EGFP cells, with genome editing being performed using 3P Cas9-P2A-dTo-T20 VLPs bearing either VSV-G or SARS2 spike. In all panels, the target gene ( EGFP or hACE2 ) was knocked out in 20%–35% of cells expressing sgRNA. Higher VLP amount resulted in greater editing. ( F ) 293T-hACE2 stably expressed sgRNAs against SLC35A1 were infected with 3P SARS2 Cas9-P2A-dTo-T20 VLPs (1.885 μg/μl N protein equivalent) or without SARS2 spike (1.385 μg/μl N protein equivalent). Gene editing efficiency was evaluated based on increase in fluorescent peanut agglutinin lectin (PNA) binding to cells. More than 70% gene editing was observed upon using 3P VLPs to edit endogenous genes. Volume of VLP used in each assay is specified in individual panels. Data are mean ± STD. *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: Human embryonic kidney 293T Lenti-X cells (“293T”) (Catalog #: 632180) were purchased from Clontech/Takara Bio (Mountain View, CA).

Techniques: Functional Assay, Transfection, Expressing, Binding Assay, Knock-Out, Stable Transfection, Infection

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: In vivo pulmonary gene delivery using VLPs. ( A ) VLPs bearing either VSV-G or maSARS2 were instilled into mice at time = 0. Twenty-four hours post-instillation, luciferase activity was measured in tissue extracts from left lung, right lung, or trachea. Protein concentration in lysate was used to normalize luminescence signal, with untreated/mock values being set to 1.0 for all in vivo studies. ( B ) VSV-G VLPs were instilled via either o.p.a. (100 μl) or i.n. (50 μl) routes. o.p.a. resulted in VLP administration to mouse lung. ( C ) Q493K and N501Y mutations were introduced into SARS2 spike to generate maSARS2 spike. 3P VLPs with VSV-G, SARS2, maSARS2, and no spike were produced with N protein equivalents of 1.859, 1.149, 1.334, and 2.024 μg/μl, respectively. A total of 50 μl of VLP was used to infect three target cell types: 293T, 293T-hACE2, and 293T-mACE2. VSV-G VLPs infected all three cell types, SARS2 spike only infected 293T-hACE2 (hACE2), whereas maSARS2 spike was permissive to both 293T-hACE2 and 293T-mACE2. VSV-G luminescence was set to 1.0 in this panel. ( D ) Hundred microliters of VSV-G VLPs or maSARS2 VLPs, both 0.820 μg/μl N protein equivalent, were instilled via o.p.a. into mice. Whole lung tissue was harvested. Mice without VLPs served as negative control. Both VSV-G and maSARS2 VLPs enabled luciferase signal in mouse lung with VSV-G being more efficient. Data are mean ± STD. N = 5–6 for each mouse treatment group. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: Human embryonic kidney 293T Lenti-X cells (“293T”) (Catalog #: 632180) were purchased from Clontech/Takara Bio (Mountain View, CA).

Techniques: In Vivo, Luciferase, Activity Assay, Protein Concentration, Produced, Infection, Negative Control

Journal: Cell Chemical Biology

Article Title: Ligand-induced assembly of antibody variable fragments for the chemical regulation of biological processes

doi: 10.1016/j.chembiol.2025.01.007

Figure Lengend Snippet: Application of Fv-based CIDs for inducible regulation of gene expression (A) Scheme of an inducible CRISPRa-based system for regulation of gene expression. The transcriptional activator domain VPR and the catalytically inactive Cas9 protein (dCas9) are fused Fv variable domains (V L or V H ). Upon ligand addition, the VPR and dCas9 domains are brought together and activate the transcription of the firefly luciferase gene (fLuc) encoded on a reporter plasmid with 10 binding sites for gRNA upstream of the minimal promoter (pMIN). (B) Inducible activation of luciferase reporter gene using FvNIC (left) and FvEST (right) systems in HEK293T cells stimulated with 1 mM nicotine and 10 μM estradiol, respectively. (C and D) Construction and validation of OR gate (C) and AND gate (D) logic circuits using FvNIC and FvEST systems in combination with dCas9 DNA-binding domain. Cells were stimulated with 10 μM estradiol and/or 1 mM nicotine as indicated. Results in (B–D) are shown as fold activation in reporter gene expression compared to vehicle control mock cells transfected with reporter plasmid only. Plots in (B–D) show the means ± SD of n = 8 biological replicates combined from two independent experiments. (E) Activation of endogenous genes by FvNIC (cyan) and FvEST (orange) systems. Cells were transfected with individual gRNAs targeting TTN, ASCL1, and NEUROD1 genes and stimulated with indicated ligands. Cells transfected only with constitutively expressed dCas9:VPR fusion and indicated gRNAs served as a positive control to determine the maximum expected level of gene activation (dark gray bars). Results are shown as fold activation in reporter gene expression compared to vehicle control mock cells transfected with an empty plasmid (pcDNA3.1). Plots in (E) show the means ± SD of n = 6 biological replicates combined from two independent experiments. Conditions were compared using a two-sided unpaired t test with Welch correction (∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns > 0.05).

Article Snippet: The cell lines Human embryonic kidney (HEK) 293T cells (ATCC, HEK293T) and Lenti-X™293T cells (Takara-Bio, LentiX293) were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 10 % v/v FBS (Thermo Fisher Scientific).

Techniques: Gene Expression, Luciferase, Plasmid Preparation, Binding Assay, Activation Assay, Control, Transfection, Positive Control

Journal: Cell Chemical Biology

Article Title: Ligand-induced assembly of antibody variable fragments for the chemical regulation of biological processes

doi: 10.1016/j.chembiol.2025.01.007

Figure Lengend Snippet:

Article Snippet: The cell lines Human embryonic kidney (HEK) 293T cells (ATCC, HEK293T) and Lenti-X™293T cells (Takara-Bio, LentiX293) were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 10 % v/v FBS (Thermo Fisher Scientific).

Techniques: Virus, Recombinant, Expressing, Lysis, Membrane, Affinity Chromatography, Luciferase, Cell Isolation, Enzyme-linked Immunosorbent Assay, Isolation, SYBR Green Assay, Software

Journal: bioRxiv

Article Title: A rapid and efficient red-light-activated Cre recombinase system for genome engineering in mammalian cells and transgenic mice

doi: 10.1101/2025.03.16.643490

Figure Lengend Snippet: a , Schematic for the photoactivatable Cre system (REDMAP Cre ). Cre recombinase was split into two fragments: CreN59 (residues 1-59) fused to FHY1 and CreC60 (residues 60-343) fused to ΔPhyA, with the expression of each driven by a constitutive promoter. In the presence of the photosensitive pigment PCB, ΔPhyA and FHY1 dimerize upon 660 nm light illumination, resulting in the restoration of Cre recombinase catalytic activity, enabling excision of DNA sequences (STOP) flanked by loxP sites. b , Screening the indicated combinations of ΔPhyA/FHY1 domains and split Cre (59/60) fragments. HEK-293T cells (6×10 4 ) co-transfected with the various fusion protein expression vectors and the Cre-dependent SEAP reporter (pGY125, P hCMV - loxP -STOP- loxP -SEAP-pA), were supplied with PCB (5 μM) and then illuminated with red light (660 nm, 1 mW cm - ) for 48 hours. SEAP production in the culture supernatant was quantified after illumination. c , Optimization for recombination efficiency using the different linkers (L1 to L5) between the FHY1 and CreN domains. HEK-293T cells (6×10 4 ) were co-transfected with ΔPhyA-L5-CreC expression vector (pYZ208), pGY125, and FHY1-CreN fusion protein expression vector with different linkers (L1 to L5), with treatment as described in b . SEAP production was quantified 48 hours after illumination. d , REDMAP Cre -catalyzed DNA recombination is dependent on red light and on PCB. HEK-293T cells (6×10 4 ) co-transfected with ΔPhyA-L5-CreC (pYZ208), FHY1-L3-CreN (pYZ247), and pGY125, were supplied with or without PCB, followed with or without red light illumination (660 nm, 1 mW cm - ). SEAP production was profiled at 48 hours after illumination. Black block, with treatment; white block, without treatment. e , REDMAP Cre -catalyzed DNA recombination for fluorescent (EGFP) reporter expression. 6×10 4 HEK-293T cells were co-transfected with pYZ208, pYZ247, and the Cre-dependent EGFP reporter (pDL78, P hCMV - loxP -STOP- loxP -EGFP-pA). Then, the transfected cells were supplied with PCB, followed by red light illumination (660 nm, 1 mW cm - ) for 48 hours. EGFP levels were profiled by fluorescence microscopy after illumination (using Image J for quantification). Representative images from 5 biological replicates; scale bar, 200 μm. Data in b , c , and e are expressed as means ± SD. Student’s t- tests were used for comparison. n = 3 or 5 independent experiments. Data in d is expressed as the mean ± SD. One-way ANOVA was used for multiple comparisons. n = 3 independent experiments. NS, not significant.

Article Snippet: Human embryonic kidney 293T cells (HEK-293T, CRL-11268, ATCC), human cervical adenocarcinoma cells (HeLa, CCL-2, ATCC), HEK-293-derived Hana3A cells engineered for constitutive expression of RTP1, RTP2, REEP1, and Gαoλϕ, Baby hamster kidney cell line (BHK-21, CCL-10, ATCC), mouse Neuro-2a cells (N2A, CCL-131, ATCC) and telomerase-immortalized human mesenchymal stem cells (hMSC-TERT) were cultured in Dulbecco′s Modified Eagle Medium (DMEM; C11995500BT, Gibco) containing 10% (v/v) fetal bovine serum (FBS; FBSSA500-S, AusGeneX) and 1% (v/v) penicillin/streptomycin solution (ST488, Beyotime).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Blocking Assay, Fluorescence, Microscopy, Comparison

Journal: bioRxiv

Article Title: A rapid and efficient red-light-activated Cre recombinase system for genome engineering in mammalian cells and transgenic mice

doi: 10.1101/2025.03.16.643490

Figure Lengend Snippet: a , REDMAP Cre -mediated SEAP expression in the indicated mammalian cell lines. Five mammalian cell lines (6×10 4 ) co-transfected with pYZ208, pYZ247, and pGY125 were supplied with PCB and then illuminated with red light (660 nm, 1 mW cm - ) for 48 hours. SEAP production was quantified after illumination. b , Assessment of illumination-intensity-dependent REDMAP Cre activity. HEK-293T cells (6×10 4 ) transfected as described in a were supplied with PCB and then illuminated with red light (660 nm) at the indicated light intensities (0 to 2 mW cm - ) for one second. SEAP production was quantified 48 hours after illumination. c , Exposure-time-dependent REDMAP Cre activity. HEK-293T cells (6×10 4 ) transfected as described in a were supplied with PCB and then illuminated with red light (660 nm, 0.2 mW cm - ) for the indicated time (0 to 60 seconds). SEAP production was quantified 48 hours after illumination. d , Evaluation of the spatial control of REDMAP Cre -mediated recombination activity. HEK-293T cells (3×10 6 ) were seeded into a 10-cm tissue culture dish and transfected with pYZ208, pYZ247, and pDL78. The dish was placed onto a smartphone screen projecting a pattern and illuminated with red light for 5 minutes (display brightness set to 100%, 10 μW cm - ) (schematic, left and middle). Fluorescence micrographs assessing EGFP production were taken 48 hours after illumination using fluorescence microscopy (bottom). Scale bar, 2 cm. e , Schematic representation of the rapamycin inducible Dre system (RAPA Dre ). In the absence of rapamycin, Dre recombinase is divided into two inactive parts. In the presence of rapamycin, split Dre (1-246 aa/247-343 aa) reassociates to form a complete Dre based on rapamycin-dependent dimerization of FKBP and FRB, enabling excision of DNA sequences (STOP) flanked by rox sites. f , REDMAP Cre and RAPA Dre mediated SEAP expression (AND gate). The reporter was constructed with a loxP flanked STOP sequence followed by a rox flanked STOP sequence positioned upstream of SEAP , and SEAP should only be expressed when both STOP cassettes were successfully excised. HEK-293T cells (6×10 4 ) transfected with plasmids encoding REDMAP Cre and RAPA Dre were induced by PCB plus red light or rapamycin as indicated. SEAP production was quantified 48 hours after induction. g , REDMAP Cre and RAPA Dre mediated SEAP expression (OR gate). The reporter was constructed with a STOP sequence flanked by loxP and rox sequences upstream of SEAP , and SEAP should be expressed when STOP cassettes were successfully excised. HEK-293T cells (6×10 4 ) transfected with plasmids encoding REDMAP Cre and RAPA Dre system were induced by PCB plus red light or rapamycin as indicated. SEAP production was quantified 48 hours after induction. Data in a , f , and g are expressed as means ± SD. Student’s t -tests were used for comparison. n = 3 independent experiments. Data in b, c is expressed as the mean ± SD. One-way ANOVA was used for multiple comparisons. n = 3 independent experiments.

Article Snippet: Human embryonic kidney 293T cells (HEK-293T, CRL-11268, ATCC), human cervical adenocarcinoma cells (HeLa, CCL-2, ATCC), HEK-293-derived Hana3A cells engineered for constitutive expression of RTP1, RTP2, REEP1, and Gαoλϕ, Baby hamster kidney cell line (BHK-21, CCL-10, ATCC), mouse Neuro-2a cells (N2A, CCL-131, ATCC) and telomerase-immortalized human mesenchymal stem cells (hMSC-TERT) were cultured in Dulbecco′s Modified Eagle Medium (DMEM; C11995500BT, Gibco) containing 10% (v/v) fetal bovine serum (FBS; FBSSA500-S, AusGeneX) and 1% (v/v) penicillin/streptomycin solution (ST488, Beyotime).

Techniques: Expressing, Transfection, Activity Assay, Control, Fluorescence, Microscopy, Construct, Sequencing, Comparison