A method allowing for efficient and quantitative coexpression of multiple heterologous proteins in neurons in vivo would be highly valuable for many applications in neuroscience. To date, different approaches, such as internal ribosomal entry site (IRES) elements (Douin et al., 2004), bidirectional or double promoters (Baron et al., 1995), or coinfection with multiple viral vectors (Mastakov et al., 2002), are commonly used. These strategies, however, mostly suffer from the fundamental problem that coexpression of the heterologous proteins is unreliable and far from quantitative. In the case of IRES-mediated coexpression, for example, the upstream elements in these bicistronic mRNA open reading frames (ORFs) are more strongly transcribed than the IRES-controlled downstream protein (Hennecke et al., 2001).

An alternative and more promising approach involves the use of self-processing viral peptide bridges. So-called 2A or 2A-like peptide sequences separate different protein coding sequences in a single ORF transcription unit of Picornaviridae (Ryan et al., 1991). The 2A peptide sequences from different members of the picornavirus family share a highly conserved motif of only 18 aa, mediating the cleavage between the C-terminal glycine and the N-terminal proline of the downstream 2B sequence (Ryan et al., 1991). Initially, the 2A peptide cleavage was thought to be mediated by an autoproteolytic event (Ryan et al., 1991), and 2A peptides were called “self-cleaving peptides.” Ultimately, a ribosomal-skip mechanism was proposed, and 2A and 2A-like sequences are now referred to as CHYSELs (cis-acting hydrolase elements) rather than selfcleaving peptides (Donnelly et al., 2001). Linking proteins with 2A or 2A-like peptide sequences results in cellular expression of multiple, discrete proteins (in essentially equimolar quantities) derived from a single ORF (de Felipe et al., 2006). The 2A peptide-mediated cleavage has previously been shown to result in coexpression of functional heterologous pro-