Mulcos: a vector for amplification and simultaneous expression of two foreign genes in mammalian cells

Gene
H IkedaI Saito

Abstract

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 is...Continue Reading

References

Dec 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·D J Charron, H O McDevitt
Nov 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·I Saito, G R Stark
Apr 1, 1973·Virology·F L Graham, A J van der Eb
Sep 19, 1980·Science·R C Mulligan, P Berg
May 1, 1981·Gene·J L Hartley, T J Gregori
Dec 15, 1962·Proceedings of the National Academy of Sciences of the United States of America·E H SZYBALSKA, W SZYBALSKI

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Citations

Jan 1, 1990·Immunologic Research·D M AltmannJ Trowsdale
Dec 1, 1996·Genetic Analysis : Biomolecular Engineering·J H LeeS C Kim
Aug 1, 1992·Molecular and Cellular Biology·M MatsudaM Shibuya

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