( Site-specific recombinase-based v...)Mammalian Expression Vectors with Interchangeable Selectable Markers

Site-specific recombinase-based vector system

Lisa Marsh Carsten Carstens
Stratagene

Stratagenes Exchanger system of eukaryotic expression vectors offers unparalleled convenience. The drug-resistance genes of these vectors can be exchanged without subcloning. Through site-specific recombination, hygromycin, puromycin, and neomycin functional cassettes are quickly and efficiently inserted into the pExchange core vectors. Insertion is carried out in vitro by a Cre-mediated recombination of LoxP sites, and recombination products are selected via drug resistance. The components of the Exchanger system include Cre recombinase; one of the six different pExchanger core vectors; linearized insertion modules, which are flanked by LoxP sites and provide hygromycin, puromycin, or neomycin resistance for selection in mammalian cells; the corresponding luciferase core-vector control; and supercompetent Epicurian Coli XL1-Blue cells. The entire site-specific recombination procedure consists of a 30-minute recombination reaction, a 15-minute heat inactivation, and transformation of suitable hosts with the recombination reaction. Desired reaction products are obtained with greater than 90% efficiency.

Stratagenes pCMV-Script and pCMV-Tag mammalian expression vectors^1,2,### offer easy cloning and high-level expression; accordingly, they are used in a variety of standard molecular biology applications. These vectors, in addition to containing an expression cassette for inserting the gene of interest into a multiple cloning site, usually include a cassette that contains a selectable marker. However, when it is necessary for the expression vector to carry a different selectable marker, the most common method is to subclone the gene of interest into a different vector, which is very inconvenient. Therefore,
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