Journal: Methods in Molecular Biology

Article Title: Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools

doi: 10.1007/978-1-4939-3753-0

Figure Lengend Snippet: Fig. 3 PTEN shRNA triage, Gateway cloning, and in vitro validation . ( a ) Luciferase assay triage of PTEN specifi c shRNAs. ( b ) A Multisite Gateway LR recombination reaction to create a lentiviral vector expressing the dsRed fl uorophore, a puromycin resistance gene, and a PTEN targeting shRNA (pLEG-dsRed-iPuro-shPTEN2). ( c ) dsRed expression in WM1617 melanoma cells infected with pLEG-dsRed-iPuro-shRNA lentivirus (post- puromycin selection). ( d ) Western blot analysis of PTEN specifi c knockdown in WM1617 pLEG-dsRed-iPuro- shRNA cells

Article Snippet: Second-generation lentiviral packaging system plasmids: pGLTR shRNA expression vector plasmid, pSPAX2 ( http:// www.addgene.org/12260 ), or any other second-generation packaging plasmid, pMD.G ( http://www.addgene. org/12259/ ), or any other envelope plasmid expressing VSV-G envelope protein for pseudotyping.

Techniques: shRNA, Cloning, In Vitro, Biomarker Discovery, Luciferase, Plasmid Preparation, Expressing, Infection, Selection, Western Blot, Knockdown

Journal: Methods in Molecular Biology

Article Title: Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools

doi: 10.1007/978-1-4939-3753-0

Figure Lengend Snippet: Fig. 3 Transduction of CD30+ lymphoma cells with chemical adjuvants, spinoculation, and scFv-αCD30-added LV. Quantifi cation of transduction of CD30+ lymphoma cells KARPAS-299 ( a ), SUP-M2 ( b ), and SUDHL-1 ( c ) with MOI 10 and 1 of GFP-coding lentiviral particles (100 % wt-VSV-G and 33 % scFv-αCD30-VSV-G) with or without spinoculation and a mix of polybrene and P338 (three different experiments, mean ± SD, * p < 0.05, ** p < 0.01 by t -test; adapted from [ 18 ])

Article Snippet: Second-generation lentiviral packaging system plasmids: pGLTR shRNA expression vector plasmid, pSPAX2 ( http:// www.addgene.org/12260 ), or any other second-generation packaging plasmid, pMD.G ( http://www.addgene. org/12259/ ), or any other envelope plasmid expressing VSV-G envelope protein for pseudotyping.

Techniques: Transduction

Journal: Methods in Molecular Biology

Article Title: Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools

doi: 10.1007/978-1-4939-3753-0

Figure Lengend Snippet: Fig. 2 Estimation of gene transfer/expression levels and background activity of the lentiviral T11/EGFP vector in transduced (Lin − ) Rosa26rtTA cells. ( a , b ) T11 background activity (at 0 µg/mL of DOX) and transduction rate (percentage of EGFP + cells; MFI of EGFP), when different concentrations of DOX (0.01, 0.1, 1.0 µg/mL) were applied for 24 h to induce EGFP expression in (Lin − ) Rosa26rtTA cells transduced with the T11/EGFP vector using MOI = 0.1 ( a ) and MOI = 1.0 ( b ). Selected contour plots from three biological replicates are presented. MFI mean fl uorescence intensity of EGFP, DOX doxycycline, MOI multiplicity of infection

Article Snippet: Second-generation lentiviral packaging system plasmids: pGLTR shRNA expression vector plasmid, pSPAX2 ( http:// www.addgene.org/12260 ), or any other second-generation packaging plasmid, pMD.G ( http://www.addgene. org/12259/ ), or any other envelope plasmid expressing VSV-G envelope protein for pseudotyping.

Techniques: Expressing, Activity Assay, Plasmid Preparation, Transduction, Infection

Journal: Methods in Molecular Biology

Article Title: Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools

doi: 10.1007/978-1-4939-3753-0

Figure Lengend Snippet: Fig. 3 T11 promoter inducibility in (Lin − ) Rosa26rtTA cells transduced with lentiviral T11/EGFP vector. Gene transfer (percentage of EGFP + cells) ( a ) and gene expression (MFI of EGFP) ( b ) levels, when different concen- trations of DOX (0, 0.01, 0.1, 1.0 µg/mL) were applied for 24 h to induce EGFP expression in (Lin − ) Rosa26rtTA cells transduced with the T11/EGFP vector using MOI = 0.1 and MOI = 1.0. Data are summarized from experi- ments performed in biological replicates and presented as mean ± SD, n = 3. MFI mean fl uorescence intensity of EGFP, DOX doxycycline, MOI multiplicity of infection

Article Snippet: Second-generation lentiviral packaging system plasmids: pGLTR shRNA expression vector plasmid, pSPAX2 ( http:// www.addgene.org/12260 ), or any other second-generation packaging plasmid, pMD.G ( http://www.addgene. org/12259/ ), or any other envelope plasmid expressing VSV-G envelope protein for pseudotyping.

Techniques: Transduction, Plasmid Preparation, Gene Expression, Expressing, Infection

Journal: Methods in Molecular Biology

Article Title: Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools

doi: 10.1007/978-1-4939-3753-0

Figure Lengend Snippet: Fig. 1 Schematic workfl ow for the generation of antibody-secreting cell lines using lentiviral vectors. A selected cell line is transduced by LV encoding the antibody LC and HC. Individual clones with high secretion rate are isolated. The production of the recombinant antibody by the implantable cell line is assessed both quantitatively and qualitatively

Article Snippet: Second-generation lentiviral packaging system plasmids: pGLTR shRNA expression vector plasmid, pSPAX2 ( http:// www.addgene.org/12260 ), or any other second-generation packaging plasmid, pMD.G ( http://www.addgene. org/12259/ ), or any other envelope plasmid expressing VSV-G envelope protein for pseudotyping.

Techniques: Clone Assay, Isolation, Recombinant

Journal: Methods in Molecular Biology

Article Title: Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools

doi: 10.1007/978-1-4939-3753-0

Figure Lengend Snippet: Fig. 2 Lentiviral vector constructs used for antibody production. The DNA constructs encoding the antibody LC and HC are derived from the pRRLSIN.cPPT.PGK-GFP.WPRE shuttle plasmid. The LV genome produced from this con- struct contains two self-inactivating long terminal repeats. Abbreviations: RSV Rous Sarcoma Virus promoter; LTR long terminal repeats; SIN self-inactivating; cPPT central polypurine tract; huPGK human phosphoglycerate kinase 1 promoter; Kz optimized Kozak sequence; WPRE woodchuck hepatitis posttranscriptional regulatory element

Article Snippet: Second-generation lentiviral packaging system plasmids: pGLTR shRNA expression vector plasmid, pSPAX2 ( http:// www.addgene.org/12260 ), or any other second-generation packaging plasmid, pMD.G ( http://www.addgene. org/12259/ ), or any other envelope plasmid expressing VSV-G envelope protein for pseudotyping.

Techniques: Plasmid Preparation, Construct, Derivative Assay, Produced, Virus, Sequencing

Journal: Molecular medicine reports

Article Title: Propranolol suppresses the proliferation and induces the apoptosis of liver cancer cells.

doi: 10.3892/mmr.2018.8476

Figure Lengend Snippet: Figure 1. Propranolol hydrochloride treatment inhibits the proliferation of HepG2 and HepG2.2.15 cells. (A) Following 48 h of incubation with 80 µmol/l propranolol hydrochloride, the confluence of HepG2 cells was decreased, based on the analysis performed using the IncuCyte ZOOM live‑cell imaging and analysis system. Scale bar, 200 µm. (B) In HepG2 and HepG2.2.15 cells, the cell proliferation ratio was decreased at 48 h of incubation with 80 µmol/l propranolol. However, propranolol exhibited no effect on the proliferation ratio of HL‑7702 cells. (C) Propranolol hydrochloride suppressed the proliferation of HepG2 and HepG2.2.15 cells in a time‑ and concentration‑dependent manner, while the proliferation of HL‑7702 cells remained unaltered. *P<0.05 vs. 0 µmol/l.

Article Snippet: The IncuCyte ZOOM second-generation live-cell imaging and analysis system was purchased from Essen Bioscience (Ann Arbor, MI, USA).

Techniques: Incubation, Live Cell Imaging

Journal: Cells

Article Title: Modification of Hinge/Transmembrane and Signal Transduction Domains Improves the Expression and Signaling Threshold of GXMR-CAR Specific to Cryptococcus spp.

doi: 10.3390/cells11213386

Figure Lengend Snippet: GXMR-CAR expression in Jurkat cells is enhanced by CD8α molecule as hinge/transmembrane domain. Schematic protocol for production of lentiviral particles carrying the GXMR-CAR sequence, the steps in the transfection of HEK 293-FT cells for GXMR-CAR lentiviral packaging, and the procedure to calculate the titer of lentiviral particles harvested and concentrated ( a ). DNA sequence for each GXMR-CAR construct indicating the recognition portion of GXM (scFv obtained from anti-GXM antibody, clone 18B7) that is similar for all constructs, and hinge/transmembrane and intracellular domains as specified in the scheme ( b ). Lentiviral particle titer of distinct GXMR-CAR constructs representative of five independent batches ( c ). Jurkat cells were transduced with GXMR-CAR at an MOI of 1 and 3, and after 3 days, the cells were analyzed using flow cytometry to measure the percentage of GXMR-CAR-positive cells ( d ) and the expression of GXMR-CAR ( e , f ). Data are expressed as mean ± SD. Each experiment was performed in 3 to 5 replicates and repeated at least three times. ** p < 0.01, *** p < 0.001, according to one-way ANOVA. ns, not significant.

Article Snippet: CAR-encoding sequences were subcloned into a second-generation lentiviral plasmid backbone (pLentiCas9-EGFP, GenScript, NJ, USA) containing the GFP sequence as a reporter, and the plasmid backbone containing the GFP sequence alone was considered as a control lentiviral (Lenti-mock) vector.

Techniques: Expressing, Sequencing, Transfection, Construct, Transduction, Flow Cytometry