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The modification of mammalian cells by the expression of multiple genes is a crucial technology in modern biological research. MultiLabel allows the modular assembly of independent expression units in a single plasmid which can be used for transient and stable modification of cells. In contrast to other methods, the assembly of the expression cassettes does not require restriction enzymes since it is recombination based. Here we describe strategies how genes of interest are efficiently cloned into MultiLabel vectors and how these expression vectors can be assembled by a Cre/LoxP reaction to a multigene plasmid. In addition, we describe how such plasmids are used to generate stable cell lines. Cloning and assembling of up to three plasmids is possible within two weeks, the insertion of a fourth and fifth expression cassette within another two weeks, and the generation of a stable cell line takes one to two months.

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