By N.J. Clifton
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Extra info for Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications (Methods in Molecular Biology Vol 219)
3. E . coli Pulser (Bio-Rad, cat. no. 1652102). 4. 1-cm gap (Bio-Rad, cat. no. 165-2089). 5. 14-mL polypropylene round-bottomed tubes, 17 × 100 mm (Becton Dickinson Labware, Franklin Lakes, NJ, Falcon 4059). 6. C. medium (Gibco-BRL, #15544-034). Store at room temperature. 34 Duan, Yue, and Engelhardt 7. Amp selection LB agar plates (100 µg/mL ampicillin). Store at 4°C. 8. Standard materials for large-scale plasmid preparation. To achieve the best transfection efficiency for AAV production, we recommend preparing the cis proviral plasmids and helper plasmids by CsCl/ethidium bromide equilibrium centrifugation.
And Chamberlain, J. S. (2002) Packaging cell lines for gutted adenoviral vector growth using E1, E2b, and E3-deleted helper viruses. In press. Graham, F. , Russell, W. , and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59–72. , Begy, C. , and Chamberlain, J. S. (1996) Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors. Proc. Natl. Acad. Sci. USA 93, 3352–3356.
2. Generating Overlapping Vectors The approach for generating overlapping AAV vectors is much more simple and straightforward than that for trans-splicing AAV vectors (see Note 1). In this section, we will use the LacZ gene to illustrate procedures for constructing Upstream and Downstream AAV overlapping vectors (Fig. 4A). An alternative generic approach is also presented (Fig. 4B). Dual Vector Expansion of AAV 41 Fig. 4. Schematic outline of rAAV overlapping vector cloning strategies. (A) Construction of AAV LacZ overlapping vectors.
Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications (Methods in Molecular Biology Vol 219) by N.J. Clifton