Generation of 2A-linked multicistronic cassettes by recombinant PCR

Cold Spring Harb Protoc. 2012 Feb 1;2012(2):251-4. doi: 10.1101/pdb.prot067884.


The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.

MeSH terms

  • Gene Expression
  • Genes
  • Genetic Engineering / methods*
  • Genetic Vectors*
  • Open Reading Frames
  • Peptides / genetics*
  • Peptides / metabolism*
  • Polymerase Chain Reaction / methods*
  • Protein Biosynthesis
  • Protein Processing, Post-Translational
  • Proteolysis
  • Ribosomes / metabolism


  • Peptides