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Additional resources for Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications (Methods in Molecular Biology Vol 219)

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71, 3293–3298. , Hartigan-O’Connor, D. , Scott, J. , 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.

3). An SV40 polyA sequence is included in this plasmid to facilitate the expression of the trans-spliced gene product. The KpnI/SnaBI sites in pDD295 are designed for introducing the splicing acceptor signal. 4. PCR is used to introduce the splicing donor sequence into the pre-Donor vector. Donor vector is composed of a 20–25-mer oligonucleotide located upstream of a unique restriction site in the cDNA used for cloning the final product. The design of this primer should follow the general PCR primer designing principles.

3. Helper plasmid for type 1 and type 5 AAV: p5E18(2/1) for rAAV-1 packaging (33). pAV2-Rep and pAV5-Trans for rAAV-5 packaging (34). These plasmids provide viral replication and structural proteins for pseudo-packaging a type 2 rAAV genome into type 1 and type 5 capsids (see Note 7). 4. 5 M CaCl2. Sterilize by filtration and store at –20°C. 5. 05. Sterilize by filtration and store at –20°C. 05 in order to achieve high transfection efficiency. 6. DNAse I (Sigma D4513, 11 mg protein/vial, total 33 K [kuniz] units) (see Note 8).

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