Build A Better Mouse Model

Build a Better Mouse Model, with 2A Peptide Strategies

conditional knockout

Researchers often ask us about ways to track a knockout or knockin allele in a new mouse model, or how to express multiple proteins in a single mouse model. Utilizing the 2A peptide is often an ideal solution.

The 2A peptide system

Several 2A peptide sequences are commonly used, each consisting of about 20 residues, which can serve as a linker between two different proteins. During translation a break is created in the protein product resulting in two proteins being produced from a single bicistronic mRNA.

This co-expression paradigm enables creation of more versatile mouse models. With the 2A system Cre or CreERT2 can be co-expressed along with a targeted gene, rather than requiring a knockout of that allele. New options are also available for co-expressing a fluorescent reporter gene, for example when making a Rosa26-targeted knockin or a conditional knockout model.

Co-expression considerations

A key question regarding co-expression or reporter knockin relates to expression levels. Again, the 2A peptide represents an ideal solution, offering 1:1 stoichiometry for the co-expressed proteins, which are produced from continuous translation of a transcript.

Another method for achieving bicistronic expression involves using an IRES, or Internal Ribosome Entry Site, for secondary translation initiation within an mRNA. However, IRES elements are bulky, generally about 600 to 700 bp in size, and are known to produce lower expression levels of the downstream gene.