drg at prophet.pharm.pitt.edu
Mon Nov 8 14:08:23 EST 1993
In article <2blpmh$k5t at mserv1.dl.ac.uk>, phillipsa at afrc.ac.uk (Andy
Phillips, Long Ashton, Bristol, UK) wrote:
> Any baculovirus experts out there?
> I am initiating a project to express plant cytochrome P450
> enzymes in insect cells using baculovirus vectors. My problem
> is that the new UK Genetic Manipulation Regulations require
> catagorization of the genetically modified organism as either
> Type I (non harmfull to man, animals, plants and environment)
> or Type II (potentially harmfull to above). A categorization of Type
> II will result in a 60-day delay of the work (and much more paperwork).
> The baculovirus kits sold by both Baculovirus Systems and Clontech have
> a deletion which removes the polyhedrin protein. The polyhedrin
> is required for protection of the virus in the environment, so
> polyhedrin-deficient virus should not be viable outside the
> tissue culture flask....true or false?
> If any UK researchers have already submitted baculovirus
> proposals to the HSE, I'd be grateful for advice on categorization.
> Andy Phillips (PHILLIPSA at AFRC.AC.UK)
Most likely, your recombinant baculovirus would be catagorized as a Type I
organism (but, this being a purely governmental designation, all bets are
off). In a review of baculovirus biology by Lois K. Miller (Ann. Rev.
Microbiol. 1988, vol 42, p. 177-199), she writes (p. 191 and I quote):
"Using a dual marker gene recombinant of AcMNPV, it was possible to
demonstrate that AcMNPV enters dipteran cells (e.g. Drosophila and Aedes)
and efficiently expresses the CAT gene under the control of the RSV-LTR,
which behaves at least in part as an early promoter in permissive S.
frugiperda cells (6). Late genes, however, are not expressed in these
recombinant viruzs-infected dipteran cells, and the infection is not
productive (6,60). Expression of the RSV-LTR CAT gene is not ovserved in
recombinant AcMNPV-infected mammalian cells, which indicated that there is
a block in the ability of AcMNPV to deliver its DNA genome to the mammalian
nucleus (7). Virus entry into the cytoplasm of mammalian cells has been
confirmed by recombinant virus studies (7), but the efficiency of entrance
appears to be low, and the progress of the virus is apparently blocked,
perhaps in the lysosomes, prior to nuclear entry or uncoating. These
observations support the view that baculovirus vector systems are
relatively safe with respect to mammals."
Sounds like a Type I to me. I don't know of any references that describe
the inactivation pathways of unprotected AcMNPV on a laboratory benchtop.
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