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. 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