Viral Gene Techniques by Kenneth W. Adolph

By Kenneth W. Adolph

Viral Gene suggestions is a pragmatic laboratory consultant to present concepts of molecular biology and genetics. the amount is worried with tools for the research of viral genes and chromosomes: DNA viruses and RNA viruses together with HIV are mentioned. * tools provided for ease of use and reasdy edition to new structures* distinct experimental protocols incorporated for:* Viral vectors - building and use of DNA virus vectors (adenovirus, adeno-associated virus, vaccinia virus, Epstein - Barr virus)* DNA viruses - virus/host interactions, viral chromosomes , transcription law (viruses mentioned contain herpes simplex, hepatitis B, SV40, JC, Epstein-Barr, adenovirus)* Human Immunodeficiency Virus / retroviruses -quantitation of HIV-1 virus inventory and RNA, retrovirus opposite transcription / integration, retrovirus-mediated mobile fusion, use as cellphone lineage markers* RNA viruses - RNA virus meeting, research of RNA genomes, assays for RNA-binding proteins (viruses mentioned comprise poliovirus, influenza virus, hepatitis delta virus)

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Pick two or more colonies off the plate, resuspend each in 5 ml of LB plus antibiotics, and shake at 37 ~C for several hours. 3. Add each 5-ml culture to 500 ml of LB plus antibiotics and continue shaking overnight. 4. Purify the plasmid DNA by alkaline lysis of the bacteria and CsC1 banding as described in standard cloning manuals (14). Plasmid DNA that has not undergone any detectable rearrangement (as determined by restriction and agarose gel electrophoresis) is suitable for use in cotransfections.

67, 5911 (1993). 8. R. Spessot, K. Inchley, T. M. Hupel, and S. Bacchetti, Virology 168, 378 (1989). 9. F. L. Graham and J. Lu, unpublished observations (1992). 10. C. L. Addison, A. J. Bett, and E L. Graham, unpublished observations (1992). 11. M. Boshart, E Weber, G. Jahn, K. Dorsch-Hasler, B. Fleckenstein, and W. ) 41, 521 (1985). 12. P. Gunning, J. Leavitt, G. -Y. Ng, and L. Kedes, Proc. Natl. Acad. Sci. A. 83, 4831 (1987). 13. D. Davidson and J. A. Hassell, J. Virol. 61, 1226 (1987). 14. J.

S. 3 kbp in length. In general, these systems rely on in vivo recombination between shuttle plasmids containing the gene of interest flanked by Ad E1 or E3 sequences and a second plasmid containing essentially the entire Ad genome in a circular form (Fig. 2). A large number of shuttle plasmids have been generated that differ in the size and location of the Ad deletion, the regulatory sequences present, and the unique restriction sites available for insertion of the foreign gene (Table I). The Ad genome plasmids (Table II) have all been designed to give low or no background of nonrecombinant infectious progeny upon cotransfection: pJM17 (3) is based on Ad5 dl309 (4) and has a deletion/substitution in E3 and an insertion of pBR322 DNA in El, which exceeds the packaging constraints of Ad; the pBHG plasmids (5) are missing the Ad sequences from nucleotides (nt) 1881339, which includes the essential viral packaging signal; and pFG173 (6) carries a lethal deletion in E3 (from about nt 27,320 to 30,560).

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