Adenovirus Methods and Protocols , 1st Edition by William S. M. Wold

By William S. M. Wold

A state-of-the-art selection of without difficulty reproducible tools for accomplishing learn with adenoviruses, the most appropriate and most generally used version in telephone and molecular biology. The equipment variety from how one can develop and titer adenoviruses and the way to build particular changes within the adenovirus genome, to tips on how to degree apoptosis caused through cells of the immune approach, cytokines, and intrinsic apoptosis effectors. moreover, there are ways to check transcription and splicing with in vitro platforms and for the adenovirus-mediated transformation of cells to a malignant country. each one strategy is written by way of a well known investigator well-versed within the approach and incorporates a short heritage dialogue and attempted, in addition to real step by step directions.

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64,2702-27 10 28. Berkner, K. L. and Sharp, P. A. (1983) Generation of adenovuus by transfectlon of plasmids. Nucleic Acids Res. 11,6003-6020. 29. Challberg, S. S. and Ketner, G (1981) Deletion mutants of adenovuus 2. lsolatlon and initial characterization of virus carrying mutattons near the right end of the viral genome Vzrology 114, 196-209. 30 Robmson, A J. and Bellett, A. D. J (1974) A circular DNA-protein complex from adenovnuses and its possible role m DNA replication. Cold Spring Harbor Symp Quant Blol 39,523-53 1.

Cytoplasmic, polyadenylated RNA is isolated from approx 5 x 10’ mfected 293 or HeLa cells at early (6 h) and late (18 or 24 h) times after viral infection. RNA isolation and Northern blots are performed as described in ref. 14). 3. Viral DNA Accumulation and Replication Rates The adenovirus-packaging elements lie in close proximity to the inverted terminal repeat (Fig. 1) that contains sequences required for viral DNA replication. It 1s important, therefore, to verify that mutations m the packagmg domain do not reduce viral DNA replication.

Digest 15 c(g of pBN27 with BumHI and EcoRI and the mutant E4 plasmld with EcoRI. Precipitate the digested DNAs as described above and redissolve m small volumes of TE 8. Add the digested plasmld DNAs to the dissolved digested DNAPC (see Note 1) Add concentrated hgase buffer to 1X final concentration, and 5-7 U of T4 DNA hgase. Total volume should be 70 $ 9 Incubate overnight at 4°C 10 Transfect the ligated DNA mto W 162 or other complementmg cells or, if the mutant 1slikely to be viable, mto 293 cells.

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