MSALMINEN@finnphi.bitnet (MIKA SALMINEN) (06/01/90)
I have the following type of problem i hope somebody on the board can help me
to solve. We are currently involved in a project concerning expression of
a mammalian protein in various eucryotic vectors (virus-based). One of these is
called pM22 and is a BPV-based expression vector containing a MMTV
glucocorticoid regulatable promoter. The bacterial ORI is from pBR 322
containing to my understanding almost the whole pBR 322 plasmid (bases
378-4363).
This vector is derived from the plasmid pM 22 which was constructed by
Michael C. Ostrowski et al. (Mol. Cell. Biol. 1983, 3:2045-2057) and i recieved
it as a kind gift throgh a third person.
Though the vector is an elegant construct with many appealing features (that's
why we are using it!) there is one problem. As most of you surely know, pBR 322
contains in it's sequence so called "poison" sequences. The name comes entirely
from these sequences property to inhibit transformation and replication of the
episome in a mammalian cell. This is circumvented in this construct by exising
almost the entire pBR with Sal I (the Eco RI site in pBR 322 has been converted
to a Sal I site) and religating the episomal fragment before transfection.
Even though i could do this (the cDNA lacks Sal I sites), it still seems like a
less elegant system to me than transfecting with a supercoiled plasmid
directly. If the plasmid is supercoiled, its much smaller and presumably enters
cells more easily than the relaxed circular forms produced by religation, and
this means better transfection-efficiency and a smaller risc of rearrangements.
So here i come to the point (or as we say in Finland, to the core of the
poodle): does anyone know of a way of exicing these "poison" sequences from pBR
322 by direct or partial digestion, in such a way that the AMPr phenotype and
replicational ability of the plasmid remain unaltered ?
Another question, has anyone by any chance the whole sequence of pM 22 in
computer-readable form ? I would also like to get the sequence of plasmid
pAcYM1 (Y. Matsuura et al. J. Gen. Virol. 1987, 68:1233-1250) if someone has
it (its a Baculovirus vector).
I would be extremely grateful for any help in these matters.
Mika Salminen
National Public Health Institute
HIV-lab.
Mannerheimintie 166
00300 Helsinki
FINLAND
e-mail: MSALMINEN@FINNPHI.BITNET
FAX: +358-0-4744408
PHONE: +358-0-4744454