By William S. M. Wold, Ann E. Tollefson
Adenovirus equipment and Protocols, moment variation, now in volumes, is a necessary source for adenovirus (Ad) researchers starting within the box, and an inspirational start line for researchers trying to department into new parts of advert learn. as well as updating and increasing the 1st version, the authors have additional new chapters that handle leading edge components of emphasis in advert study, together with advert vector development and use, real-time PCR, use of latest animal versions, and strategies for quantification of advert virus or virus expression/interactions. all of the protocols provided in those volumes is written via trendsetting researchers.
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Additional info for Adenovirus Methods and Protocols: Adenoviruses, Ad Vectors, Quantitation, and Animal Models
DNA–protein complex is prepared from the virus (26) and then digested with EcoRI. To increase the probability of obtaining recombinant viruses relative to wt viruses, we added a restriction digest/Klenow fillin step to the preparation of DNA–protein complex prior to transfection (1,4). We also describe the preparation of MAV-1 viral DNA (see Note 6). In the second section we describe the transfection protocol. We have successfully used mouse 3T6 cells to obtain mutants. In some cases we have also used 3T6 cell derivatives, which inducibly express the gene region to be mutated (3,4).
27), but other methods may be used. Any of a variety of methods can be used to obtain the mutation in a plasmid containing the gene of interest, including oligonucleotide-directed mutagenesis, polymerase chain reaction (PCR) mutagenesis, and restriction fragment replacement cloning. In the third section we describe the identification and plaque purification of mutants. We have used preparation of viral DNA directly from plaques, from cell culture fluid, and by the Hirt method (28) for use in PCR.
47. Mohammadi, E. , Ketner, E. , Johns, D. , and Ketner, G. (2004) Expression of the adenovirus E4 34k oncoprotein inhibits repair of double strand breaks in the cellular genome of a 293-based inducible cell line. Nucleic Acids Res. 32, 2652–2659. 48. , and Pettersson, U. (1991) Adenovirus E4-dependent activation of the early E2 promoter is insufficient to promote the early-to-late-phase transition. J. Virol. 65, 1440–1449. Defective Adenovirus Deletion Mutants 19 2 Isolation, Growth, and Purification of Defective Adenovirus Deletion Mutants Gary Ketner and Julie Boyer Summary Defective adenovirus deletion mutants can be grown by complementation in the presence of helper viruses that supply essential functions missing in the deletion mutant.