human embryonic kidney cell line hek293t  (ATCC)

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: RNF149 expression is upregulated by EMCV. (A–D) A549 and HEK293T cells were infected with EMCV at 0.1 MOI were harvested post infection 24 hours, RT-qPCR was performed to detect the mRNA levels of RNF6, RNF19B, RNF24, RNF149, RNF157, RNF182, RNF186, RNF123, RNF14, RNF43, RNF152, RNF207, RNF208 , and RNF224 mRNA. (E-F) A549 and HEK293T cells were infected with EMCV at 0.1 MOI were harvested post infection 0 h, 6 h, 9 h, 12 h, and 24 h, protein level measured using anti-RNF149 and anti-EMCV-VP1 antibodies. β-actin was used as a loading control. The results are presented as means ± sd. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Expressing, Infection, Quantitative RT-PCR, Control

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: RNF149 promotes EMCV replication. (A) HEK293T cells were transfected with negative control siRNA (siRNA-ctrl) or siRNA-1#, −2#, −3#, and −4# against RNF149. Western blotting was used to analysis the protein expression level with anti-RNF149. β-actin was used as a loading control. (B) A549 cells were transfected with negative control siRNA-ctrl, siRNA-4# for 48 h and then infected with EMCV at 0.1 MOI were harvested at 24 hpi, RT-qPCR detect EMCV mRNA. Virus titers were analyzed by TCID 50 assay. (C) HEK293T cells were transfected with negative control siRNA-ctrl, siRNA-4# for 48 h and then infected with EMCV at 0.1 MOI were harvested at 24 hpi, RT-qPCR detect EMCV mRNA. Virus titers were analyzed by TCID 50 assay. (D) HEK293T cells were transfected with negative control siRNA-ctrl, siRNA-4# for 48 h and then the cells were infected with VSV-GFP at 0.01 MOI for 18 h, and the cells were observed with fluorescence microscopy. Scale bars: 300 µm. (E) VSV mRNA level was detected by RT-qPCR. (F) A549 cells were transfected with pcDNA3.1-Flag-RNF149 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg) or empty vector (0.5 or 1 μg) for 24 h and then infected with EMCV at 0.1 MOI, the cells were harvested at 24 hpi, RT-qPCR detect EMCV mRNA, protein level measure using anti-RNF149 antibodies. (G) virus titers were analyzed by a TCID 50 assay. (H) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg) or empty vector (0.5 or 1 μg) for 24 h and then infected with EMCV at 0.1 MOI, the cells were harvested at 24 hpi, RT-qPCR detect EMCV mRNA, protein level measure using anti-RNF149 antibodies. (I) virus titers were analyzed by TCID 50 assay. The results are presented as means ± sd. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Transfection, Negative Control, Western Blot, Expressing, Control, Infection, Quantitative RT-PCR, Virus, Fluorescence, Microscopy, Plasmid Preparation

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: RNF149 inhibits type I IFN-mediated signaling. (A–B) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg), pISRE-luc (200 ng), pSTAT1-luc (200 ng), and pRL-TK (10 ng). After 24 h, the cells were treated with IFN-α or IFN-β for 12 h. Then, luminescence was detected using dual-luciferase report. (C-F) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg), and empty vector. After 24 h, the cells were treated with IFN-β for 12 h. Then, MX1, ISG15, OASL and ISG56 mRNA levels were detected using RT-qPCR. (G) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (1 μg) or empty vector. Then, the cells were infected with VSV-GFP at 0.01 MOI for 12 h followed by treatment with IFN-α or IFN-β for 12 h. The cells were observed microscopically. Scale bars: 300 µm. The results are presented as means ± sd. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Transfection, Luciferase, Plasmid Preparation, Quantitative RT-PCR, Infection, Control

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: RNF149 deficiency promotes JAK-STAT signaling pathway activation. (A) RNF149 knockout HEK293T cells (RNF149 KO) were constructed using CRISPR-Cas9 technology. The RNF149 protein expression was detected by Western blotting. β-actin was used as a loading control. (B–C) RNF149-KO or RNF149-WT cells infected with EMCV at 0.1 MOI were harvested at 24 hpi, RT-qPCR detect EMCV mRNA. Virus titers were analyzed by TCID 50 assay. (D–E) RNF149-KO and RNF149-WT cells were transfected with pISRE-luc (200 ng), pSTAT1-luc (200 ng), or pR-TK (10 ng). After 24 h, the cells were treated with IFN-α or IFN-β for 12 h. Then, luminescence was detected using dual-luciferase report. (F) the heat map of the DEGs. (G) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis the DGEs of upregulated in RNA-seq. The results are presented as means ± sd. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Activation Assay, Knock-Out, Construct, CRISPR, Expressing, Western Blot, Control, Infection, Quantitative RT-PCR, Virus, Transfection, Luciferase, RNA Sequencing

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: RNF149 inhibits the JAK-STAT signaling pathway through JAK1. (A–E) HEK293T cells were co-transfected with Myc-JAK1 (0.5 μg), Myc-TYK2 (0.5 μg), Myc-STAT1 (0.5 μg), Myc-STAT2 (0.5 μg), Myc-IRF9 (0.5 μg), pcDNA3.1-Flag-RNF149 (1 μg), pISRE-luc (200 ng), and pRL-TK (10 ng). After 24 h, luminescence was detected using dual-luciferase report. (f) HEK293T cells were co-transfected with pcDNA3.1-Flag-RNF149 (1 μg) and Myc-JAK1 (1 μg), or empty vector. After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-Myc antibodies. (G) HEK293T cells were co-transfected with EGFP-RNF149 and mcherry-JAK1. At 24 h post transfected, co-localization was performed using confocal fluorescence microscopy. The nuclei were stained by DAPI. The fluorescence intensity profile of DAPI (blue), RNF149 (green) and JAK1 (red) was measured along the line drawn by image J. Scale bars, 10 μm. The results are presented as means ± sd. ns, not significant; **, p < 0.01 versus control.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Transfection, Luciferase, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Fluorescence, Microscopy, Staining, Control

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: RNF149 regulates viral replication through degrading JAK1. (A) HEK293T cells were co-transfected with pcDNA3.1-Flag-RNF149 (1 μg) or empty vector. After 24 h, the cells were treated with IFN-β for 12 h. Then, JAK1, TYK2, STAT1, STAT2 , and IRF9 mRNA levels were detected using RT-qPCR. (B–F) HEK293T cells were co-transfected with Myc-JAK1 (1 μg), Myc-TYK2 (1 μg), Myc-STAT1 (1 μg), Myc-STAT2 (1 μg), Myc-IRF9 (1 μg), and pcDNA3.1-Flag-RNF149 (1 μg). After 24 h, the cell lysate was harvested. The proteins level was analyzed by Western blotting. β-actin was used as a loading control. (G) HEK293T cells were co-transfected with Myc-JAK1 (1 μg), pcDNA3.1-Flag-RNF149 (1 μg) or pcDNA3.1-Flag-RNF149 (1.5 μg). After 24 h, the cell lysate was harvested. The proteins level was analyzed by Western blotting. β-actin was used as a loading control. (H) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (1 μg) or pcDNA3.1-Flag-RNF149 (1.5 μg). After 24 h, the cell lysate was harvested. The proteins level was analyzed by Western blotting using anti-JAK1 antibodies. β-actin was used as a loading control. (i) A549 cells were transfected with negative control siRNA-ctrl, siRNA-4#. After 48 h, the cells were treated with IFN-β for 12 h. Then, the cell lysate was harvested. The proteins level was analyzed by Western blotting using anti-JAK1 and anti-RNF149 antibodies. β-actin was used as a loading control. (J) RNF149 KO cells and RNF149 wt cells were treated with IFN-β for 12 h, following the lysate was harvested. The proteins level was analyzed by Western blotting using anti-JAK1 and anti-RNF149 antibodies. β-actin was used as a loading control. (K) HEK293T cells were infected with EMCV at 0.1 MOI were harvested post infection 0 h, 6 h, 9 h, 12 h, and 24 h, protein level was measured using anti-JAK1 antibodies. β-actin was used as a loading control. (L) RNF149-KO and RNF149-WT cells were infected with EMCV at 0.1 MOI were harvested post infection 12 h, protein level was measured using anti-JAK1 and anti-RNF149 antibodies. β-actin was used as a loading control. The results are presented as means ± sd. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control, Negative Control, Infection

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: RNF149 RING-finger domain antagonizes IFN signaling. (A) schematic representation of the mutants of RNF149. (B) HEK293T cells were co-transfected with Flag-tag empty vector, Flag-RNF149 FL (1 μg), Flag-RNF149 1–221 (1 μg), Flag-RNF149 67–310 (1 μg), Flag-RNF149 176–400 (1 μg) and Myc-JAK1 (1 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-Myc antibodies. (C–D) HEK293T cells were co-transfected with Flag-RNF149 FL (1 μg), Flag-RNF149 1–221 (1 μg), Flag-RNF149 67–310 (1 μg), Flag-RNF149 176–400 (1 μg), pISRE-luc (200 ng), pSTAT1-luc (200 ng), and pRL-TK (10 ng). After 24 h, the cells were treated with IFN-α or IFN-β for 12 h. Then, luminescence was detected using dual-luciferase report. (e) HEK293T cells were transfected with Flag-tag empty vector, Flag-RNF149 wt (1 μg), Flag-RNF149-C269A/C272A (1 μg) or Flag-RNF149-H289A (1 μg), the proteins were analyzed by Western blotting. β-actin was used as a loading control. (f) HEK293T cells were co-transfected with Myc-JAK1 (1 μg), Flag-tag empty vector, Flag-RNF149 wt (1 μg), Flag-RNF149-C269A/C272A (1 μg) or Flag-RNF149-H289A (1 μg), the proteins were analyzed by Western blotting. β-actin was used as a loading control. (G-H) HEK293T cells were co-transfected with Flag-RNF149 wt (0.5 μg), Flag-RNF149-C269A/C272A (0.5 μg), Flag-RNF149-H289A (0.5 μg), pISRE-luc (200 ng), pSTAT1-luc (200 ng), and pRL-TK (10 ng). After 24 h, the cells were treated with IFN-α or IFN-β for 12 h. Then, luminescence was detected using dual-luciferase report. The results are presented as means ± sd. ns, not significant; **, p < 0.01; ***, p < 0.001 versus control.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Transfection, FLAG-tag, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Luciferase, Control

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: RNF149 promotes the K27-linked and K33-linked ubiquitination of JAK1. (A) HEK293T cells were co-transfected with pcDNA3.1-Flag-RNF149 (1 µg) and Myc-JAK1 (1 µg). After 24 h, treated with dimethyl sulfoxide (DMSO), 3-MA (10 mM), MG-132 (10 μM), baf A1 (100 nM), and Z-VAD (20 μM) for 12 h. Protein expression was measured using Western blotting. β-actin was used as a loading control. (B) HEK293T cells were transfected with pcDNA3.1-Flag-RNF149 (1 µg) or empty vector for 24 h, then cells were treated with DMSO or MG-132 for 12 h. Protein expression was measured using Western blotting. β-actin was used as a loading control. (C) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1 (1 μg) and His-Ub (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-His antibodies. (D–E) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1 (1 μg) and His-Ub or the indicated Ub mutant plasmids (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-His antibodies.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Western Blot, Control, Plasmid Preparation, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: The 1–559 aa region of JAK1 is critical for efficient RNF149-mediated degradation. (A) schematic representation of the mutants of JAK1. (B) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1 FL (1 μg), Myc-JAK1 1–559 (1 μg), Myc-JAK1 560–1154 (1 μg) and Myc-JAK1 851–1154 (1 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-Myc antibodies. (C) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1 FL (1 μg), Myc-JAK1 1–559 (1 μg), Myc-JAK1 560–1154 (1 μg) and Myc-JAK1 851–1154 (1 μg). After 24 h, protein expression was measured using Western blotting. β-actin was used as a loading control. (d) HEK293T cells were co-transfected with Flag-RNF149 (1 μg), Myc-JAK1 1–559 (1 μg) and the indicated Ub mutant plasmids (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-His antibodies.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Control, Mutagenesis

Journal: Virulence

Article Title: Encephalomyocarditis virus evades IFN-mediated antiviral response by RNF149 targeting JAK1 for ubiquitination and degradation

doi: 10.1080/21505594.2026.2679319

Figure Lengend Snippet: A working model of the role of RNF149 protein in the regulation of IFNs-mediated JAK-STAT signaling pathway to promote EMCV replication.

Article Snippet: RNF149 knockout HEK293T cell lines were produced by Ubigene Biosciences.

Techniques:

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: Identification of the ubiquitination site on RIG-I targeted by FGF8. (a) diagram illustrating the truncated constructs of RIG-I. (b) HEK-293T cells were co-transfected with specified plasmids and exposed to MG132 for 6 hours. Western blot analysis was conducted to assess the ubiquitination of various RIG-I truncation constructs. (C) Western blot analysis identified the ubiquitination site on RIG-I targeted by FGF8, and band intensities were quantified by densitometry to assess the degradation of each mutant. (d) a dual-luciferase assay was conducted in HEK293T cells co-transfected with specified RIG-I mutants and FGF8 to evaluate the impact of FGF8 on IFN-β promoter activity. Error bars indicate the mean ± SEM from three independent experiments. Two-tailed unpaired Student’s t-tests were used. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0016), human embryonic kidney cell line HEK293T (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0005), and Madin-Darby canine kidney cell line MDCK (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0154) were used for virus infection experiments, protein interaction validation experiments, and virus titration assays, respectively.

Techniques: Ubiquitin Proteomics, Construct, Transfection, Western Blot, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Engineering high-titer lentiviral vectors for robust expression of RNA-based gene circuits

doi: 10.64898/2026.05.12.724401

Figure Lengend Snippet: A. Viral vectors with low titer or poor expression require larger doses to elicit the desired behavior in target cells. Optimized vectors can improve production scale-up. B. Lentiviral vectors are packaged in producer cell lines transfected with a transfer plasmid to express the viral genome. The viral genome is a single-stranded RNA species spanning from the 5’ long terminal repeat sequence (LTR) to the 3’ LTR that contains the transgenic cargo along with other viral sequences that facilitate packaging. This transcript is exported from the nucleus and packaged with viral proteins into particles that bud from the membrane of producer cells. The viral particles are collected and then applied to target cells. During transduction, the envelope protein mediates fusion of the particle with the target cell membrane, releasing the particle contents into the cytoplasm. The viral genome is reverse transcribed into proviral double-stranded DNA (dsDNA), which is randomly integrated into the genome of the target cell. The target cell then expresses the transgenic cargo. C. Workflow to measure functional titer and expression for various vector designs. Single-gene vectors containing a fluorescent protein expressed by either the human elongation factor 1 alpha (EF1α) or EF1α short (EFS) promoter were transduced at varying amounts into HEK293T cells. Left: Expression of the single-gene vectors measured via fluorescence in arbitrary units (AU, log scale) from a flow cytometer. Light gray vertical line depicts the expression gate, used to calculate the fraction of expressing cells in each condition. Distributions show one representative batch of virus (one biological replicate). Middle: For the same vectors, fraction expressing as a function of virus volume was fit to a Poisson distribution to obtain titer in transducing units (TU) per µL virus. Points represent technical replicates for a representative batch of virus, and lines depict the Poisson curve fits. The computed titer is annotated on the plot, and virus volume is plotted on a log scale. Right: To quantify cargo expression, HEK293T cells were transduced at a low multiplicity of infection (MOI). Expression is the geometric mean of expressing cells (AU, log scale). Solid light gray line depicts the expression gate. Points represent means ±standard error for n = 3 biological replicates. Statistic is a two-sided Student’s t-test, **** p < 0.0001. Annotation shows fold change between indicated points.

Article Snippet: HEK293T cells (ATCC, CRL-3216), Lenti-X HEK293T cells (Takara Bio, 632180), and Plat-E retroviral packaging HEK293T cells (Cell Biolabs, RV-101) were cultured using DMEM (Genesee Scientific, 25-500) plus 10% FBS (Genesee Scientific, 25-514H) and incubated at 37°C with 5% CO 2 .

Techniques: Expressing, Transfection, Plasmid Preparation, Sequencing, Transgenic Assay, Membrane, Transduction, Reverse Transcription, Functional Assay, Fluorescence, Flow Cytometry, Virus, Infection

Journal: bioRxiv

Article Title: Engineering high-titer lentiviral vectors for robust expression of RNA-based gene circuits

doi: 10.64898/2026.05.12.724401

Figure Lengend Snippet: A. Schematic of the RNA species transcribed in producer cells for vectors with cargo on the sense or antisense strand relative to the viral genome. The transfer plasmid expresses the viral genome, as well as cargo transcripts and spliced or truncated forms of the viral transcript. The viral genome can be packaged into productive particles, while the other species are unproductive, meaning they do not form viral particles that integrate the intended sequence into target cells. Additionally, antisense cargo transcripts can form double-stranded RNA (dsRNA) by binding to the viral genome, potentially inhibiting proper packaging. B. Expression in target cells is influenced by biophysical coupling defined by the syntax of the encoded genes, resulting in attenuated or amplified expression. C. Schematic of two-gene vectors with tandem or divergent syntax that express a fluorescent protein from the strong, synthetic CAG promoter or from the relatively weak human phosphoglycerate kinase (hPGK) promoter, and a second fluorescent protein from a downstream EFS promoter. These vectors are compared to a single-gene vector containing only the downstream gene from Fig. 1 . The upstream gene includes the bovine growth hormone (bGH) polyadenylation signal (PAS), and the downstream gene includes the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) in the 3’ UTR. Viral titer is in transducing units (TU) per µL (log scale). D. Expression of the upstream and downstream genes for each vector in HEK293T cells. Expression is the geometric mean fluorescence in arbitrary units (AU, log scale). Solid light gray lines represent titers calculated for untransduced cells or expression gates. Dashed dark gray lines depict values for the single-gene vector for reference. Points represent means ±standard error for n ≥3 biological replicates. Statistics are two-sided Student’s t-tests, n.s. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Annotations show the fold change between indicated points.

Article Snippet: HEK293T cells (ATCC, CRL-3216), Lenti-X HEK293T cells (Takara Bio, 632180), and Plat-E retroviral packaging HEK293T cells (Cell Biolabs, RV-101) were cultured using DMEM (Genesee Scientific, 25-500) plus 10% FBS (Genesee Scientific, 25-514H) and incubated at 37°C with 5% CO 2 .

Techniques: Plasmid Preparation, Sequencing, Binding Assay, Expressing, Amplification, Virus, Fluorescence

Journal: bioRxiv

Article Title: Engineering high-titer lentiviral vectors for robust expression of RNA-based gene circuits

doi: 10.64898/2026.05.12.724401

Figure Lengend Snippet: A. Schematic of transcripts produced from a divergent vector with the upstream, antisense gene driven by a Tet-ON inducible promoter (TRE3G). In producer cells cultured with-out inducer, the viral genome is transcribed from the transfer plasmid, and some unproductive downstream transcripts may also be produced. After integration in target cells, addition of the inducer doxycycline (dox) and the reverse tetracycline transactivator (rtTA) protein leads to strong expression of the inducible gene, along with strong expression of the downstream gene due to biophysical coupling. B. Two-gene vectors with an inducible upstream gene and tan-dem (T) or divergent (D) syntax were delivered to HEK293T cells containing a separately integrated rtTA cassette. Titer is in transducing units (TU) per µL (log scale), and expression is the geometric mean fluorescence in arbitrary units (AU, log scale). Cells were treated with 1 µg/mL dox (filled points) or untreated (open points). Solid light gray lines represent titers calculated for untransduced cells or expression gates. Dashed lines depict values for several vectors from Fig. 2 for reference. Annotations show the fold change between indicated points. C. Performance of vectors in B is compared to vectors lacking WPRE in the 3’ UTR of the downstream gene. Titer and expression of the downstream gene for the vectors with WPRE are normalized to values for vectors without WPRE, plotted on a log scale. Solid gray line indicates a fold change of 1, and gray shading spans a two-fold change in either direction (0.5 to 2) for reference. Annotations show the values of the points, and statistics compare unnormalized values. Points represent means ±standard error for n ≥ 3 biological replicates. Statistics are two-sided Student’s t-tests, n.s. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: HEK293T cells (ATCC, CRL-3216), Lenti-X HEK293T cells (Takara Bio, 632180), and Plat-E retroviral packaging HEK293T cells (Cell Biolabs, RV-101) were cultured using DMEM (Genesee Scientific, 25-500) plus 10% FBS (Genesee Scientific, 25-514H) and incubated at 37°C with 5% CO 2 .

Techniques: Produced, Plasmid Preparation, Cell Culture, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Engineering high-titer lentiviral vectors for robust expression of RNA-based gene circuits

doi: 10.64898/2026.05.12.724401

Figure Lengend Snippet: An “all-in-one” inducible vector contains both an inducible gene and a constitutive gene that expresses the required activator, which can be arranged with tandem (T) or divergent (D) syntax. Here, the dox-inducible TRE3G promoter (Tet-ON system) controls expression of a fluorescent protein in the upstream gene, and in the downstream position EFS drives expression of rtTA and a second fluorescent protein, separated by a “self-cleaving” 2A peptide. The upstream gene includes the bGH PAS, and the downstream gene includes WPRE. Left: Viral titer is shown normalized to the tandem vector for each batch of virus. Points represent means ±standard error for n ≥3 biological replicates. Statistic is a two-sided Student’s t-test, ** 0.05 ≤ p < 0.01. Right: 2D density distributions depict upstream and downstream expression via fluorescence in arbitrary units (AU, log scale) for a representative biological replicate of HEK293T cells transduced with each vector and treated with 1 µg/mL dox. Populations are gated on cells expressing both genes. Solid gray lines show expression gates.

Article Snippet: HEK293T cells (ATCC, CRL-3216), Lenti-X HEK293T cells (Takara Bio, 632180), and Plat-E retroviral packaging HEK293T cells (Cell Biolabs, RV-101) were cultured using DMEM (Genesee Scientific, 25-500) plus 10% FBS (Genesee Scientific, 25-514H) and incubated at 37°C with 5% CO 2 .

Techniques: Plasmid Preparation, Expressing, Virus, Fluorescence, Transduction

Journal: bioRxiv

Article Title: Engineering high-titer lentiviral vectors for robust expression of RNA-based gene circuits

doi: 10.64898/2026.05.12.724401

Figure Lengend Snippet: A. ComMAND is a compact, post-transcriptional circuit that reduces dosage-associated variability in expression. ComMAND uses a synthetic microRNA within an intron of the output coding sequence to target a matched microRNA target site in the 3’ UTR of the same transcript, resulting in an incoherent feedforward loop. Because ComMAND operates at the RNA level, circuit activity can interfere with virus production when encoded on the sense strand of the viral genome. “All-in-one” inducible vectors as in Fig. 4 were constructed with ComMAND (light purple) or the output gene alone (base gene, dark blue) as the upstream cargo. Left: Titer is normalized to the base gene vector with tandem syntax for each batch of virus (log scale). Right: Standard deviation of circuit (upstream gene) expression measured via fluorescence in arbitrary units (AU, log scale). B. A ribozyme switch, consisting of a ligand-responsive RNA aptamer fused with a self-cleaving ribozyme, controls expression of an output gene when placed in the 3’ UTR. In the absence of ligand, the ribozyme switch cleaves, leading to degradation of the mRNA and a reduction in protein levels (OFF). Binding of the ligand changes the folding of the switch to minimize ribozyme cleavage and restore protein expression (ON). When encoded on the sense strand of the viral genome, ribozymes can interfere with virus production by cleaving and truncating transcripts. “All-in-one” inducible vectors as in Fig. 4 were constructed with a guanine (gua)- responsive ribozyme switch or a constitutively active ribozyme added to the upstream gene. Left: Titer is normalized to a vector lacking a ribozyme (not shown) for each batch of virus (log scale). Right: Upstream expression is the geometric mean fluorescence (AU, log scale) of cells transduced with vectors containing the ribozyme switch and treated without (light purple) or with (pink) 100 µM guanine. Solid light gray line shows the expression gate. Dashed teal lines depict values for a divergent vector lacking a ribozyme, and dashed dark blue line represents the value for the divergent vector with the constitutively active ribozyme. To compare optimal designs for each syntax, tandem vectors lack a PAS on the upstream gene, while divergent vectors include a bGH PAS. In all plots, HEK293T cells were transduced and treated with 1 µg/mL dox. Points represent means ±standard error for n ≥3 biological replicates. Statistics are two-sided Student’s t-tests, n.s. p ≥0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Annotations show the fold change between indicated points.

Article Snippet: HEK293T cells (ATCC, CRL-3216), Lenti-X HEK293T cells (Takara Bio, 632180), and Plat-E retroviral packaging HEK293T cells (Cell Biolabs, RV-101) were cultured using DMEM (Genesee Scientific, 25-500) plus 10% FBS (Genesee Scientific, 25-514H) and incubated at 37°C with 5% CO 2 .

Techniques: Expressing, Sequencing, Activity Assay, Virus, Construct, Plasmid Preparation, Standard Deviation, Gene Expression, Fluorescence, Binding Assay, Transduction

Journal: bioRxiv

Article Title: Engineering high-titer lentiviral vectors for robust expression of RNA-based gene circuits

doi: 10.64898/2026.05.12.724401

Figure Lengend Snippet: A. A guanine-responsive splicing switch consists of two introns and an alternative exon (alt. exon) containing a stop codon. Addition of the splicing switch within the coding sequence of a gene of interest enables tunable control of protein expression. In the absence of guanine (gua), the main spliced isoform includes the alternative exon, leading to truncated protein product. In the presence of guanine, the RNA aptamer changes structure and alters the splicing pattern to exclude the alternative exon and enable production of full-length protein. Switches are encoded within the upstream gene of the best “all-in-one” inducible vectors identified previously (Fig. 4 ). The tandem vector (T) does not include a PAS on the upstream gene while the divergent vector (D) includes the bGH PAS. Left: Titer is measured in transducing units (TU) per µL (log scale). Right: Upstream expression is geometric mean fluorescence in arbitrary units (AU, log scale) for HEK293T cells treated with 1 µg/mL dox and without (teal) or with (pink) 100 µM guanine. Solid light gray line shows the expression gate. B. Gel depicts PCR products amplified from genomic DNA (gDNA) of cells transduced with vectors containing the splicing switch. Producer cells were cultured with or without 100 µM guanine during virus production. The amplicon spans the site where the splicing switch was added. Lengths of the ladder bands are labeled in base pairs (bp), and the most prominent bands in the conditions are annotated with their predicted splice products. The uncropped gel is displayed in Fig. S6E . C. Schematic of direct conversion of primary mouse embryonic fibroblasts to Hb9 ::GFP+ induced motor neurons (iMNs) via viral delivery of conversion factors. Hb9::GFP is a transgenic reporter of iMN fate used to identify converted cells. The splicing switch regulates expression of the oncogenic HRAS mutant HRAS G12V , which is fused to the fluorescent protein mCherry and delivered via the the best-performing divergent “all-in-one” inducible vector. Vectors contain the splicing switch within the mCherry coding sequence (circles) or lack the switch (diamonds) and were transduced into mouse embryonic fibroblasts alongside the remaining conversion factors. Cells were treated with 1 µg/mL dox (filled points) or without (open points), and additionally treated with 0–100 µM guanine for the duration of the conversion process. At 14 days post-transduction, iMN yield was quantified as the number of cells expressing Hb9::GFP per cell seeded, normalized to the same count for the untreated condition (neither dox nor guanine added) for each vector for each biological replicate. Cargo expression is the geometric mean of mCherry fluorescence for the iMN population (AU, log scale). D. Images show fluorescence of Hb9 ::GFP (top) and mCherry-HRAS G12V (bottom) at 14 days post-transduction for cells transduced with the vector containing the splicing switch, treated with dox, and treated with varying concentrations of guanine. Scale bar represents 100 µm. Points represent means ±standard error for n ≥ 3 biological replicates. Statistics are two-sided Student’s t-tests, n.s. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Annotations show the fold change between indicated points.

Article Snippet: HEK293T cells (ATCC, CRL-3216), Lenti-X HEK293T cells (Takara Bio, 632180), and Plat-E retroviral packaging HEK293T cells (Cell Biolabs, RV-101) were cultured using DMEM (Genesee Scientific, 25-500) plus 10% FBS (Genesee Scientific, 25-514H) and incubated at 37°C with 5% CO 2 .

Techniques: Sequencing, Control, Expressing, Plasmid Preparation, Fluorescence, Amplification, Transduction, Cell Culture, Virus, Labeling, Transgenic Assay, Mutagenesis