Journal:

Article Title: A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide

doi: 10.1002/jgm.1345

Figure Lengend Snippet: (a) Schematic representation of chimeric Sindbis virus envelope proteins. 2.2 1L1L was derived from the chimeric 2.2 envelope protein [18]. 2.2 1L1L has two flexible linkers (Gly-Gly-Gly-Gly-Ser) and AVR II-Bst E II cloning sites at amino acid 71 of the E2 protein. BAP Sindbis contains a biotin acceptor peptide (BAP) derived from Escherichia coli biotin holoenzyme synthetase between the two flexible linkers. BBAPH contains BAP at the first amino acid of the E2 protein, and BAP II SINDBIS contains two BAP at both positions of the E2 protein. (b) The schematic strategy to conjugate virus with anti-human or rat transferrin receptor 1 and transferrin. BAP II SINDBIS envelope proteins were covalently conjugated with biotin. Anti-human or rat transferrin receptor antibodies were fused with avidin, designated anti-huTfR IgG-Av or anti-ratTfR IgG3-Av, respectively. Anti-huTfR IgG-Av or anti-ratTfR IgG3-Av can be conjugated with the BAP II SINDBIS envelope protein through the interaction of avidin and the biotin of the envelope protein. Neutravidin has four biotin binding sites. Thus, one neutravidin can bind both the biotinylated BAP II SINDBIS envelope protein and biotinlynated transferrin, which results in bridging the pseudotyped virus with transferrin

Article Snippet: Jurkat and Y3 cells (5 × 10 5 ) were incubated with 100 µl (5 µg/ml) of either mouse anti-rat transferrin receptor 1 (BD Biosciences, San Diego, CA, USA) or mouse antihuman transferrin receptor 1 (BD Biosciences) for 1 h at 4 °C, followed by incubation with 100 µl (200-fold dilution in PBS) of rabbit anti-mouse IgG conjugated with Alexa 488 (Invitrogen) for 1 h at 4 °C.

Techniques: Derivative Assay, Clone Assay, Avidin-Biotin Assay, Binding Assay

Journal:

Article Title: A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide

doi: 10.1002/jgm.1345

Figure Lengend Snippet: Gene transduction mediated by fusion proteins between avidin and anti-human or transferrin receptor antibody fusion proteins. (a) Expression of human and rat transferrin receptor 1 on Jurkat (human T-cell line) and Y3 (rat myeloma cell line) cells. Each cell type was stained either with isotype control control antibody (black line), mouse anti-human (red line), or rat (blue line) transferrin receptor 1 antibody, followed by staining with Alexa 488-conjugated secondary antibody. (b) Jurkat, was infected with the BAP II SINDBIS pseudotype in the presence or absence of fusion protein between avidin and either anti-human (anti-huTfR IgG-Av) or anti-rat (anti-ratTfR IgG3-Av) transferrin receptor 1 antibody. EGFP expression was analysed by flow cytometry 3 and 10 days post-transduction. Representive flow cutometric profiles are shown. Percent EGFP-positive is shown as the mean ± SD of triplicate experiments. (c) Y3-Ag 1.2.3, was infected with the BAP II pseudotype in the presence or absence of anti-huTfR IgG-Av or anti-ratTfR IgG3-Av. EGFP expression was analysed by flow cytometry 3 and 10 days post-transduction. Representive flow cutometric profiles are shown. Percent EGFP-positive is shown as the mean ± SD of triplicate experiments

Article Snippet: Jurkat and Y3 cells (5 × 10 5 ) were incubated with 100 µl (5 µg/ml) of either mouse anti-rat transferrin receptor 1 (BD Biosciences, San Diego, CA, USA) or mouse antihuman transferrin receptor 1 (BD Biosciences) for 1 h at 4 °C, followed by incubation with 100 µl (200-fold dilution in PBS) of rabbit anti-mouse IgG conjugated with Alexa 488 (Invitrogen) for 1 h at 4 °C.

Techniques: Transduction, Avidin-Biotin Assay, Expressing, Staining, Infection, Flow Cytometry