By L. A. King
The selection to jot down a booklet concerning the sensible elements of the baculovirus expression procedure stems from the varied cell demands support we have now had, and from the numerous viewers to our labora tories requiring guidance to discover the elusive polyhedrin-negative virus containing their favorite gene. now we have additionally geared up expression method workshops and from the manuals we wrote for those, it appeared a logical development to increase them into ebook shape. We savour that people who find themselves 'old-hands' on the baculovirus expression procedure could have differing perspectives on a few of our systems, however the equipment during this publication are provided within the mild of our personal reports within the laboratory and from our sensible workshops, and we are hoping that the booklet could be specially important to these new to the procedure. the 1st 3 chapters provide the history info to the baculovirus expression method, and comprises suggestion on the right way to pick out the proper move vector and discusses some of the equipment which are to be had to choose recombinant viruses. the sensible chapters be aware of these points that are novel to the baculovirus method (insect mobile tradition, virus amplification and titration, and so on. ) and, mostly, depart the traditional molecular organic suggestions to the opposite first-class laboratory manuals which are on hand. despite the fact that, for completeness sake and to prevent consistent connection with different manuals, we now have integrated short info of a few common ideas the place they're essential to the luck of the baculovirus protocols.
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Extra info for The Baculovirus Expression System: A laboratory guide
3 RECOMBINANT VIRUS SELECTION USING DOT-BLOT HYBRIDIZATION A useful alternative to the above methods is to identify recombinant viruses using dot-blot hybridization. This may done in two ways. In the first, putative recombinant plaques are identified with one of the above methods and used to establish an infection in cells growing in a 96-well microtitre plate (Summers and Smith, 1987). , 1988). The DNA is then extracted from cells in both methods and hybridized with a radiolabelled copy of the foreign gene coding sequences.
A. King, unpublished data). (c) pJVNheI and pAcDZI Another interesting and useful group of polyhedrin promoter-based transfer vectors are those which also have a copy of the E. coli IaeZ coding sequences, under the control of another promoter, inserted upstream of the polyhedrin gene. , 1990). In pJVNheI the IaeZ sequences are under the control of a copy of the pl0 promoter and in pAcDZl they are under the control of a Drosophila melanogaster heat-shock promoter, which is active in Sf cells. These vectors provide for the selection of recombinant viruses which have both the desired foreign gene and the IaeZ coding sequences inserted; the viruses may be identified by staining plaque-assays with Xgal which results in the formation of blue, polyhedrin-negative plaques.
This is particularly advantageous if modifications need to be made to the inserted foreign gene coding sequences after the initial construction has been made. Single-stranded DNA may be produced and used in sitedirected mutagenesis techniques with synthetic oligonucleotides. The pAcCL29 transfer vectors are also slightly smaller than pAcYM1 and should, therefore, be able to accept more foreign DNA before becoming unstable. The vectors pAcALl-2 were derived from pAcRP23 and are similar to the pAcCL29-series, in that they also have single-stranded capability (based on pT7T3), but these vectors are smaller and have been Baculovirus transfer vectors / 25 shown to be useful in accommodating large foreign gene codingsequences (A.