Mulcos: a vector for amplification and simultaneous expression of two foreign genes in mammalian cells.
A method was developed for amplification and expression of foreign genes in mammalian cells. This procedure exploits the fact that an SfiI cleavage site, GGCCGCCT/CGGCC (the recognition sequences are underlined), is present at the SV40 replication origin and the cleaved ends, CCT-3' and AGG-3', are not rotationally equivalent. Thus DNA fragments flanked by the SfiI sites can be ligated in head-to-tail tandem arrays and cloned in cosmids; the resulting construct is called a mulcos. The cosmid vector we have used, pCHD2L, contains the single SfiI site as well as HmBR and dhfr genes, selectable markers in mammalian cells. Cassette plasmid pmoRH contains two expression units, each of which consists of SV40 early promoter, EcoRI or HindIII cloning site, small T splicing region, and poly(A) signal, and the two units as a whole are flanked by the SfiI sites. A set of alpha- and beta-chain cDNAs of a human major histocompatibility class-II antigen were inserted into the EcoRI and HindIII sites, respectively. The purified SfiI fragment, containing both expression units, was then ligated with SfiI-linearized cosmid vector pCHD2L at a molar ratio of 20:1. A mulcos containing eight pairs of the alpha- and beta-chain expression units was isolated by in vitro packaging in phage lambda heads and subsequent transfection into Escherichia coli. Drug-selected cells transfected with the mulcos contained significantly higher copy numbers of the expression units and higher expression levels than those obtained using conventional plasmids. More than 85% of these cells expressed class-II antigen on their cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)
Ikeda
H
H
Human Immunogenetics Laboratory, Imperial Cancer Research Fund, London, U.K.
Trowsdale
J
J
Saito
I
I
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003360:Cosmids; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006681:HLA-D Antigens / Q000235:genetics; D006801:Humans; D014162:Transfection
D006681:HLA-D Antigens; D004247:DNA