By David W. Murhammer
The 3rd version of this quantity expands upon the former versions with new and updated equipment and protocols. Chapters contain step by step tactics interested in quantifying phone development, baculovirus an infection and phone metabolism, tips on how to isolate new cellphone strains and advance your individual serum-free medium, and regimen upkeep and garage of insect cellphone traces and baculoviruses, small- and large-scale recombinant protein creation with the BEVS in either insect and mammalian phone tradition and in insect larvae, creation and characterization of baculoviruses, eco-friendly fluorescent protein, tubular reactors and RNAi, and baculovirus/insect mobilephone approach to check apoptosis and producing envelop-modified baculovirus for gene supply into mammalian cells. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and key tips about troubleshooting and fending off identified pitfalls.
Authoritative and useful, Baculovirus and bug mobile Expression Protocols, 3rd Edition goals not to merely reduction the consumer in effectively finishing the initiatives defined, but additionally stimulate the improvement of more suitable strategies and new purposes of baculoviruses and bug cellphone culture.
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Extra info for Baculovirus and Insect Cell Expression Protocols
Unspliced transcripts encode IE-1 itself, while spliced transcripts encode another immediate-early transregulator, IE-0, identical to IE-1 except for 54 additional amino acids at its N-terminus [71, 99]. While IE-0 is expressed only during the early phase of infection, IE-1 RNA is expressed in both the early and late phases . Transient expression assays have shown IE-1 to have a negative regulatory effect on ie-0 promoter expression, while IE-0 transactivates the ie-1 promoter . Deletion of ie1/ie-0 from the virus genome using an Escherichia coli-based system prevented virus replication in insect cells, although restoration of the mutant with either gene largely restored production of infectious virus progeny .
Overexpressed and purified VLF1 added to transcription assays containing baculovirus RNA polymerase stimulated transcription of the polyhedrin gene promoter, but not 39K . Baculovirus Molecular Biology 41 Serial passage of Nucleopolyhedroviruses through cultured cell lines results in the appearance of a spontaneous mutant termed the ‘few polyhedra’ (FP) mutant [171, 172]. With continued passage this FP phenotype becomes dominant . Fourteen passages of Trichoplusia ni (Tn)MNPV in T. ni cells was found to result in a purely FP mutant population .
However, there are differences in morphology, timing and cellular site of maturation, structural proteins, source of viral envelopes, antigenicity, and infectivity [9–13]. BV particles possess spike-like structures known as peplomers, composed of the glycoprotein GP64 for group I NPVs, at one end of the virion . The GP64 protein is incorporated throughout the virus envelope, albeit at lower concentrations than at the peplomers . During infection, GP64 localizes to discrete areas of the plasma membrane at which points budding of virions takes place .
Baculovirus and Insect Cell Expression Protocols by David W. Murhammer