Journal: Molecular Biotechnology

Article Title: Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase

doi: 10.1007/s12033-022-00642-6

Figure Lengend Snippet: Detection of SB100x coding sequences in genomic DNA. A Different amounts of plasmid CMV-SB100x were mixed with 60 ng genomic DNA of naïve HEK293 cells equivalent to 10,000 genomes to determine the sensitivity of detection. A PCR reaction mixture without plasmid DNA template served as a negative control (H 2 O). B Exemplarily, one representative data set of the detection of SB100x gene in genomic DNA of recombinant HEK293 cell pools performed in triplicate is shown. Genomic DNA was isolated from selected cell pools upon co-transfection of donor vector SB-VEGF-IpW and transposase expression construct CMV-SB100x at different days post transfection to investigate transposase gene integration. 0.001 pg of the transposase vector mixed with 60 ng genomic DNA of naïve HEK293 cells equivalent to 10,000 genomes served as a positive control (10 2 copies). Genomic DNA of naïve HEK293 cells and template-free samples (H 2 O) served as negative controls

Article Snippet: Adherent human embryonic kidney HEK293 cells (ATCC CRL-3216) were grown in high glucose (4.5 g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM l -Glutamine and 10% fetal bovine serum (FBS); Gibco, Dreieich, Germany).

Techniques: Plasmid Preparation, Negative Control, Recombinant, Isolation, Cotransfection, Expressing, Construct, Transfection, Positive Control

Journal: Molecular Biotechnology

Article Title: Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase

doi: 10.1007/s12033-022-00642-6

Figure Lengend Snippet: Detection of SB100x transcripts in HEK293 cell pools. RNA of selected cell pools was isolated 17 and 48 days post transfection (dpt) and served as templates for cDNA synthesis. Equivalent volumes of synthesized cDNA were used for the amplification of DNA fragments of the SB100x coding sequences. GADPH amplification served as an internal and positive control. RNA isolated from naïve HEK293 cells and template-free samples (H 2 O) served as negative controls. Data shown are representative results of experiments conducted in triplicate

Article Snippet: Adherent human embryonic kidney HEK293 cells (ATCC CRL-3216) were grown in high glucose (4.5 g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM l -Glutamine and 10% fetal bovine serum (FBS); Gibco, Dreieich, Germany).

Techniques: Isolation, Transfection, cDNA Synthesis, Synthesized, Amplification, Positive Control

Journal: Molecular Biotechnology

Article Title: Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase

doi: 10.1007/s12033-022-00642-6

Figure Lengend Snippet: Detection of the transposase coding region in genomic DNA and RNA preparations of HEK293 cells after co-transfection. A Different ratios of donor vector SB-EGFP-IpW to SB100x-mRNA were transfected into HEK293 cells in duplicate and genomic DNA was isolated 2 and 16 days post transfection (dpt) for subsequent sensitive PCR analysis. In addition, co-transfections of the donor vector and the transposase plasmid CMV-SB100x and transfection with the donor vector exclusively were conducted. Transposase plasmid mixed with 60 ng genomic DNA of naïve HEK293 cells equivalent to 10,000 genomes served as positive control (10 2 copies). Genomic DNA of naïve HEK293 cells and template-free samples (H 2 O) served as negative controls. B Detection of SB100x transcripts in HEK293 cells. RNA of cell pools was isolated 16 dpt. Equivalent volumes of synthesized cDNA were used for the amplification of DNA fragments of the SB100x coding sequence. Amplification of GAPDH served as an internal positive control. Naïve HEK293 cells and template-free samples (H 2 O) served as negative controls. Data shown are representative results of experiments conducted in duplicate

Article Snippet: Adherent human embryonic kidney HEK293 cells (ATCC CRL-3216) were grown in high glucose (4.5 g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM l -Glutamine and 10% fetal bovine serum (FBS); Gibco, Dreieich, Germany).

Techniques: Cotransfection, Plasmid Preparation, Transfection, Isolation, Positive Control, Synthesized, Amplification, Sequencing

Journal: Molecular Biotechnology

Article Title: Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase

doi: 10.1007/s12033-022-00642-6

Figure Lengend Snippet: Flow cytometric analysis of HEK293 cells and determination of vector copy number (VCN) per genome from different cell pools 16 days post transfection after constant selection performed in duplicate. A HEK293 cells were co-transfected with different ratios of donor vector SB-EGFP-IpW to SB100x-mRNA and were analyzed for mean fluorescence intensity of EGFP expression (EGFP-MFI) employing flow cytometry. In addition, cells transfected with the donor vector only and co-transfected with the vector CMV-SB100x were analyzed. Mean values are indicated and error bars represent standard deviations. (*) A P value ≤ 0.05 is indicated for the ratios Donor + mRNA 1:1 and Donor + mRNA 1:1/5. The difference of mean EGFP-MFI values of ratios Donor + mRNA 1:1 and Donor + mRNA 1:1/2 is not statistically significant (n.s.; using the one-way ANOVA analysis). B Determination of VCN 16 days post transfection. Genomic DNA was isolated of HEK293 cell pools transfected with different amounts of SB100x-mRNA or transposase expression plasmid CMV-SB100x and subjected to Taqman®-qPCR using oligonucleotides allowing for the amplification of DNA fragments of WPRE in the donor vector and ampicillin (Amp R ) present in the backbones of all constructs used. An additional primer/probe set amplifying the ptbp2 gene was used as an internal reference. Mean values are shown and error bars represent standard deviations resulting from two independent experiments

Article Snippet: Adherent human embryonic kidney HEK293 cells (ATCC CRL-3216) were grown in high glucose (4.5 g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM l -Glutamine and 10% fetal bovine serum (FBS); Gibco, Dreieich, Germany).

Techniques: Plasmid Preparation, Transfection, Selection, Fluorescence, Expressing, Flow Cytometry, Isolation, Amplification, Construct

Journal: bioRxiv

Article Title: Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

doi: 10.1101/2024.06.14.599118

Figure Lengend Snippet: ( A ) Live cell count, ( B ) cell viability and ( C ) AAV9 capsid titer was measured for 10 days after triple transfection in 293F and AC1P. n = 3 replicates per sample. Data are shown as mean ± SD. ( C ) Representative single-cell derived clonal colony in a 96-well plate. Dashed lines show colony boundary at 3 (green), 5 (purple), 7 (pink), and 10 (blue) days after single-cell seeding. Scale bar, 1000 µm. ( D ) Cell viability and ( F ) live cell count of top 24 clones was measured during the suspension adaptation phase. After 9 days of suspension adaptation, the top 12 clones (based on viability and cell count) were selected and progressed to 125mL shake flasks.

Article Snippet: Adherent polyclonal HEK293 cells (“AC1P” for short) (Cytion) were expanded in DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a 37°C, 5% CO 2 incubator.

Techniques: Cell Counting, Transfection, Derivative Assay, Clone Assay, Suspension

Journal: bioRxiv

Article Title: Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

doi: 10.1101/2024.06.14.599118

Figure Lengend Snippet: ( A ) Schematic of workflow for single-cell cloning and isolation of HEK293 clones with improved AAV production capacity. The Solentim VIPS™ single cell seeder was used to isolate clonal HEK293 cells using a two-step clonality verification process. ( B ) Subclone selection strategy for identification of top AAV9 producer clonal cells. ( C ) Approximately 300 single-cell derived HEK293 clones were identified based on growth, and top 24 performing colonies were selected based on transfection efficiency and AAV9 production. ( D ) After suspension adaptation, AAV productivity of the best suspension adapted clones was measured relative to AC1P (parental polyclonal cell line). n = 1–2 replicates per cell line. Data are shown as mean ± SD fold-change from AC1P (control).

Article Snippet: Adherent polyclonal HEK293 cells (“AC1P” for short) (Cytion) were expanded in DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a 37°C, 5% CO 2 incubator.

Techniques: Clone Assay, Isolation, Selection, Derivative Assay, Transfection, Suspension, Control

Journal: bioRxiv

Article Title: Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

doi: 10.1101/2024.06.14.599118

Figure Lengend Snippet: ( A ) Workflow and experimental design for RNA-sequencing analysis during AAV production. 293F, AC1P (polyclonal cell line) and 3 clonally derived cell lines from AC1P were used for the analysis. Samples were collected before transfection (0 hr) and at 24 and 72 hr after triple-plasmid transfection. 3 small-scale shake flasks were used per condition. ( B ) AAV9 vector genome titer was measured at 72 hr from the 5 cell lines. 293F and AC1P cells were designated as “base producers” with an average titer of ∼2.4E9 vg/mL. AC112 and AC230 were designated as high producers with an average titer of ∼1.1E10 vg/mL. n = 3 replicates per cell line. Data are shown as mean ± SD. ( C ) Uniform manifold approximation and projection (UMAP) and ( D ) gene correlation matrix of the 5 HEK293 cell lines show high reproducibility among replicates. RNA expression differences between cell lines are greater drivers of variability than differences between timepoints after transfection. ( E ) Heatmap of differentially enriched genes and functional categories in higher AAV producer cells (AC230 and AC122) compared to baseline producer cells (AC1P and 293F). Differentially enriched transcripts were manually categorized into cell cycle modulators, cell signaling, Extracellular matrix / adhesion, Ion transport, mitochondrial, heat shock protein, and cell fate and differentiation.

Article Snippet: Adherent polyclonal HEK293 cells (“AC1P” for short) (Cytion) were expanded in DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a 37°C, 5% CO 2 incubator.

Techniques: RNA Sequencing Assay, Derivative Assay, Transfection, Plasmid Preparation, RNA Expression, Functional Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of cell-based assay for detecting replication-competent adeno-associated virus by qPCR

doi: 10.1016/j.omtm.2025.101529

Figure Lengend Snippet: Schematic describing the finalized rcAAV assay workflow For the first round of transduction (T1), HEK293 cells are transduced with AAV in the presence of Ad5 at MOI 3 with viral combinations listed in . R2C8 is replication competent and contains rep2-cap8 genes. After 72 ± 2 h, supernatant is harvested from T1. For the second round of transduction (T2), fresh HEK293 cells are seeded and transduced in the presence of fresh Ad5 at MOI 3 with T1 supernatant. After 72 ± 2 h, supernatant is harvested from T2. Similarly, third round of transduction (T3) is set up. After each round, DNA is extracted from the cell pellet, normalized to 5 ng/μL, and then qPCR is set up to measure rep2 . In parallel, the “no transduction control” (T0) and other positive, negative, and spike controls are set up.

Article Snippet: Adherent HEK293 cells (ATCC, P/N: CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, P/N: 10564-011) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, P/N: 10100147), penicillin, and streptomycin (Life Technologies, P/N 15140-122) at 37°C in a 5% CO 2 atmosphere.

Techniques: Transduction, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Development of cell-based assay for detecting replication-competent adeno-associated virus by qPCR

doi: 10.1016/j.omtm.2025.101529

Figure Lengend Snippet: Schematic of the workflow for defining transduction conditions for AAV HEK293 cells were transduced with R2C8 in the presence of Ad5 at MOI 2 for T1. After 48 and 72 ± 2 h, supernatant and lysate were harvested from T1. A small aliquot of supernatant at each time point was set aside for measuring via qPCR. For the second round of transduction (T2), fresh HEK293 cells were seeded and transduced in the presence of fresh Ad5 at MOI 2 either with T1 supernatant or T1 lysate, from both 48 and 72 h time points. Supernatants were harvested after 48 ± 2 h from T2 for both sources of transduction (48 h supernatant T1 and 48 h lysate T1). Similarly, supernatants were harvested after 72 ± 2 h from T2 for both sources of transduction (72 h supernatant T1 and 72 h lysate T1).

Article Snippet: Adherent HEK293 cells (ATCC, P/N: CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, P/N: 10564-011) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, P/N: 10100147), penicillin, and streptomycin (Life Technologies, P/N 15140-122) at 37°C in a 5% CO 2 atmosphere.

Techniques: Transduction

Journal: eLife

Article Title: Structure of mouse protocadherin 15 of the stereocilia tip link in complex with LHFPL5

doi: 10.7554/eLife.38770

Figure Lengend Snippet:

Article Snippet: Adherent HEK293 tsa201 cells (ATCC CRL- 11268) were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum at 37°C.

Techniques: Recombinant, Software

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: TEM8 co-localizes with endosome markers and associates to transferrin receptor-containing protein complexes. A Immunofluorescence staining for TEM8 (HA) demonstrating co-localization with Alexa 488 labeled human transferrin (TF), with TFR- and Rab11a- but not Rab4-positive intracellular compartments. To label intracellular compartments with transferrin, serum deprived Hek298 cells were incubated with 50 μg/ml Alexa 488-labeled human transferrin for 1h prior to fixation. Size bar is 10μm. B Quantification of co-localization of the endosomal markers TF, TFR, Rab11 and Rab4 with TEM8. The number of analyzed cells is indicated at the bottom of the bar. P value was determined with a t-test C Detection of TEM8-associated to multiprotein complexes containing transferrin receptor after chemical cross-linking using DSP in TEM8 expressing Hek293 cells. Cells were treated in the absence (odd lanes) or presence of DSP (even lanes). Detergent extracts were immunoprecipitated with magnetic beads alone (lanes 1-2) or beads decorated with TFR antibodies (lanes 3-4). Immune complexes were analyzed by immunoblot to detect TEM8, TFR and actin. Inputs represent 5%. 1 of 8 independent experiments is shown. D Co-immunoprecipitation of TEM8 with transferrin receptor after chemical cross-linking in Hek293 cells treated with vehicle or with 2μM monensin for the indicated time. The experimental design is similar to C, except that cells were treated in the absence or presence of monensin (2μM) at 37°C before incubation with DMSO vehicle control (DSP- lanes) or DSP (DSP + lanes). 1 of 3 experiments is shown.

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Immunofluorescence, Staining, Labeling, Incubation, Expressing, Immunoprecipitation, Magnetic Beads, Western Blot

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: PA addition does not affect TEM8 recycling and induces receptor degradation. A TEM8-expressing Hek293 cells were surface biotinylated at 4°C with 0.5 mM EZ-Link Sulfo-NHS-Biotin. Unbound biotin was washed and cells were shifted for the indicated times to 37°C in the absence (PA-) or presence (PA+) of 1μg/ml PA. Cells were incubated with GSH to remove remaining surface biotin, washed and lysed at 4°C. Biotinylated proteins were isolated with streptavidin conjugated agarose beads and bound complexes resolved by SDS-PAGE and analyzed by Western Blot with antibodies against TEM8 (1 of 2 experiments). B Graphic representation of the quantification of the experiment shown in A. C TEM8 expressing Hek293 cells were treated or not with 1μg/ml PA for 30min. Cell were washed and treated at 4°C with DMSO vehicle control (DSP- lanes) or with DSP (DSP+ lanes). Detergent extracts were immunoprecipitated with magnetic beads decorated with LAMP1 antibodies or beads decorated with TFR antibodies. Immune complexes were analyzed by immunoblot to detect TEM8, TFR and LAMP1. Inputs represent 5%. 1 of 3 independent experiments is shown. D TEM8 expressing Hek293 cells were labeled overnight with 100μCi/ml [S35]-methionine and chased in media containing cold methionine either in the absence or presence of 1μg/ml PA for the indicated times. Chase was ended by cell lysis and TEM8 and TFR were immunoprecipitated. Radiolabeled immunocomplexes were resolved by SDS-PAGE and radioactive bands detected by fluorography.1 of 3 independent experiments is shown.

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Expressing, Incubation, Isolation, SDS Page, Western Blot, Immunoprecipitation, Magnetic Beads, Labeling, Lysis

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: Recycling of cell surface labeled TEM8. Cell surface proteins in TEM8 expressing Hek293 cells were surface labeled by biotinylation at 4°C with 0.5 mM EZ-Link Sulfo-NHS-Biotin. Protein trafficking was allowed by incubating the cells at 16°C or 37°C for the indicated times. Cells were returned to 4°C to remove cell surface biotin by two GSH washes (GSH+ lanes) or were maintained in buffer in the absence of GSH (GSH- lanes). Proteins remaining biotinylated were recovered by streaptavidin-agarose beads precipitation from cell detergent lysates. Proteins associated to streptavidin beads were analyzed by western blot and probed for HA and epidermal growth factor receptor (EGFR).

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Labeling, Expressing, Western Blot

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: TEM8 and transferrin receptor distribution is altered by agents that perturb recycling endosomes. TEM8 expressing Hek293 cells were transfected with wild type EGFP-Rab11a, dominant negative EGFP-Rab11S25N, or a dominant negative EGFP-Myosin Vb tail construct. Fixed cells were double stained with antibodies against GFP and either HA to detect TEM8 or transferrin receptor. Size bar is 10μm.

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Expressing, Transfection, Dominant Negative Mutation, Construct, Staining

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: Receptor recycling leads to increased PA accumulation, but not to ligand targeting to recycling endosomes. A Specific binding of FITC labeled PA63 to non-transfected or TEM8 expressing Hek293 cells at 4°C or 22°C for 60 min. B FITC-PA63 binding to TEM8 expressing Hek293 cells untreated or pre-incubated for 15 min with 2μM monensin or for 60 min with 10μg/ml with cycloheximide respectively (1 of 4 experiments). C Immunoflurescence staining with anti FITC and Alexa 555 secondary antibody in TEM8-Hek293 cells transfected with the indicated EGFP constructs. Cell were fixed after a 1h incubation with 1μg/ml FITC-PA63 at 37°C. One of two experiments is shown. Size bar is 10μm.

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Binding Assay, Labeling, Transfection, Expressing, Incubation, Staining, Construct

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: Myosin Vb tail does not affect lethal toxin delivery to the cytosol, but interferes with TEM8-mediated cell spreading on PA coated surfaces. A Control or TEM8 expressing Hek293 either transfected with GFP or EGFP-Myosin Vb tail were incubated in the absence or presence of Lethal Toxin (1ug/ml PA, 0.2ug/ml Lethal Factor) for the indicated time. Cell intoxication was assessed by Western Blot analysis for N-terminus MEK-1 cleavage. B EGFP-Myosin Vb tail transfected TEM8-Hek293 cells were plated on PA or Matrigel (MA) coated glass coverslips and stained with Alexa labeled phalloidin to score for spreading area and GFP expression in the same cell. Size bar is 10μm. C Box plot graph shows the spreading area distribution of 30 cells from each indicated condition. The middle line in each condition represents the mean, the boxes represent the distribution of 50% of the cells, the lines represent the maximum and minimum values and the white dots represent outliers as determined by the graphing software. p value was determined using a t-test. D TEM8 expressing Hek293 cells either transfected with GFP or EGFP-Myosin Vb tail were plated on 0.1%BSA or 10μg/ml PA coated dishes in media containing 0.5 mM EZ-Link Sulfo-NHS-Biotin for 60 minutes at 37°C. Cells were washed and detergent soluble extracts were incubated with streptavidin conjugated agarose beads and bound complexes resolved by SDS-PAGE. Biotinylated proteins were analyzed by Western Blot using antibodies against TEM8 and EGF receptor (EGFR).

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Expressing, Transfection, Incubation, Western Blot, Staining, Labeling, Software, SDS Page