The simultaneous expression of multiple proteins in mammalian cells is a key technology in modern biology. We developed MultiLabel, a modular plasmid-based mammalian expression system that allows the expression of several proteins. Independent expression vectors are assembled by a Cre/LoxP reaction into a single plasmid with multiple expression cassettes. Up to five proteins can be expressed simultaneously in a transient transfection and stable cell lines can be generated in a single step.

Many applications in cell biology need the modification of mammalian cells with multiple genes. Examples include the reprogramming of somatic cells into stem cells by the expression of four transcription factors (Oct3/4, Sox2, c-Myc, and Klf4) [1], the expression of protein complexes for structural studies, or the use of fluorescently tagged sensor proteins that allow the simultaneous monitoring of multiple parameters in living cells [2]. Numerous technologies have been developed in the last years to express multiple genes in mammalian cells. The easiest way is the cotransfection of several plasmids each containing an independent expression cassette. This leads to a cell population with heterogeneous expression levels, which is sometimes a disadvantage, especially for biochemical studies. Coinfection with multiple viruses is an alternative to cotransfection, but it harbors the disadvantage that special safety requirements have to be fulfilled [3]. Multiple proteins can also be produced as large precursor proteins that are then divided into individual proteins by (self-) cleavage [4]. This approach often leads to proteins with modified N- and C-termini, which might interfere with their cellular function.

The assembly of expression vectors by Cre/LoxP recombination is an elegant way to express several proteins from a single plasmid. This technology was initially developed to express multiprotein complexes in insect cells and in E.coli [5,6]. We have now adapted this technology for the transient and stable expression of multiple proteins in mammalian cells [7].

Experimental Design
We designed a set of acceptor and donor vectors based on commonly used regulatory elements.

The overall structure of these vectors is standardized to allow for rapid exchange of the genes of interest between acceptor and donor vectors (fig. 1A). Acceptor vectors contain a ColE1-derived origin of replication that allows propagation of the plasmid in all E.coli strains, and a kanamycin or ampicillin resistance gene. In addition, they contain eukaryotic selection markers for the generation of stable cell lines. Donor vectors contain the R6Kγ origin of replication and an ampicillin, spectinomycin, chloramphenicol, or gentamycin resistance gene. These plasmids need pir+ E.coli strains for replication. Acceptor and donor vectors are fused by in vitro Cre/LoxP recombination and the reaction mixture is then transformed into an E.coli strain like DH10β or TopTen (fig. 1B). Donor vectors alone can not propagate due to the lack of the pir gene in these cells. Therefore, selection with appropriate antibiotics leads to the isolation of the fused plasmid. These vectors are then used to transiently or stably modify mammalian cells.

Results and Discussion
Cotransfection of multiple plasmids into mammalian cells often leads to a cell population with heterogeneous expression levels, including cells that do not express all desired genes. This becomes especially prominent if the cell line is difficult to transfect or if primary cells are used. We could show that cells which were transiently transfected with a MultiLabel plasmid encoding five expression cassettes for fluorescent proteins always express all five proteins (fig. 2). The use of MultiLabel is therefore a good strategy to simultaneously express several proteins in individual mammalian cells. Sometimes it is necessary to stably modify the genome of a mammalian cell. Our acceptor vectors therefore contain eukaryotic selection markers and a unique homing endonuclease site for linearization of the plasmids. These two features allow the generation of stable cell lines. Linearization is crucial since we never obtained a cell line expressing four or five transgenes when using circular plasmids. An example of a cell line expressing five fluorescently tagged subcellular markers is shown in figure 3.

Drug development and screening very often relies on tailor-made cell lines. These cell lines are either used directly for drug screening or enable the visualization of disease processes. The generation of stable cell lines with multiple modifications was so far a sequential, time consuming process. MultiLabel will simplify this process and thereby contribute to drug development program.