By L. A. King
The selection to jot down a e-book concerning the useful facets of the baculovirus expression method stems from the varied cellphone demands aid we have now had, and from the numerous viewers to our labora tories requiring information to discover the elusive polyhedrin-negative virus containing their favorite gene. now we have additionally prepared expression method workshops and from the manuals we wrote for those, it appeared a logical development to increase them into publication shape. We take pleasure in that those people who are 'old-hands' on the baculovirus expression process could have differing perspectives on a few of our strategies, however the tools during this ebook are offered within the gentle of our personal stories within the laboratory and from our functional workshops, and we are hoping that the publication can be specifically beneficial to these new to the process. the 1st 3 chapters provide the historical past info to the baculovirus expression process, and comprises suggestion on the best way to pick out the suitable move vector and discusses many of the tools which are on hand to pick recombinant viruses. the sensible chapters pay attention to these points that are novel to the baculovirus process (insect mobilephone tradition, virus amplification and titration, and so on. ) and, ordinarily, go away the normal molecular organic recommendations to the opposite very good laboratory manuals which are on hand. in spite of the fact that, for completeness sake and to prevent consistent connection with different manuals, we now have incorporated short info of a few typical innovations the place they're vital to the luck of the baculovirus protocols.
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Extra resources for The Baculovirus Expression System: A laboratory guide
A second method which uses an element of positive-selection was described by Weyer et al. (1990). A transfer vector was constructed with a copy of the pl0 promoter and a RE cloning site inserted upstream of the intact polyhedrin gene. When used in co-transfections with DNA from a polyhedrin-negative mutant virus, the resulting polyhedrin-positive recombinants were relatively easy to identify. The disadvantage of this method, as with the use of ~-galactosidase, is that polyhedrin protein will be a contaminant of cell extracts; although if the recombinant protein is located in the cytoplasm the nuclei containing the polyhedra may be easily removed using centrifugation, after lysing the cells with non-ionic detergents.
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In those viruses where the polyhedrin coding sequences have been replaced via homologous recombination, plaques lacking polyhedra are produced. With practice these virus mutants may be readily identified using two alternative methods. In the first, virus plaques without polyhedra are located by inspection using a good quality microscope. In the second method, the plaques are observed with the naked eye against a black background and illuminated with a bright light source (held at an acute angle).