title: Use of the viral 2A peptide for bicistronic expression in transgenic mice.
abstract: BACKGROUND
  Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner.
  
  
  RESULTS
  To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line.
  
  
  CONCLUSIONS
  The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.
authors.affiliation: Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, UK. georgios.trichas@dpag.ox.ac.uk
authors.forename: Georgios
authors.initials: G
authors.lastname: Trichas
authors.forename: Jo
authors.initials: J
authors.lastname: Begbie
authors.forename: Shankar
authors.initials: S
authors.lastname: Srinivas
mesh_terms: D000818:Animals; D002642:Chick Embryo; D015870:Gene Expression; D005796:Genes / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014162:Transfection; D019076:Transgenes; D014780:Viruses / Q000737:chemistry*; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D010455:Peptides; D000097573:Red Fluorescent Protein
references.citation: Nat Biotechnol. 2004 May;22(5):589-94
references.pmid: 15064769
references.citation: BMC Biotechnol. 2006 Jan 12;6:4
references.pmid: 16409632
references.citation: Traffic. 2004 Aug;5(8):616-26
references.pmid: 15260831
references.citation: Gene Ther. 2001 Jun;8(11):864-73
references.pmid: 11423934
references.citation: J Gen Virol. 2001 May;82(Pt 5):1013-1025
references.pmid: 11297676
references.citation: Nat Biotechnol. 2004 Dec;22(12):1567-72
references.pmid: 15558047
references.citation: Nat Protoc. 2006;1(1):406-17
references.pmid: 17406263
references.citation: J Virol. 1985 Jun;54(3):651-60
references.pmid: 2987518
references.citation: J Gen Virol. 2001 May;82(Pt 5):1027-1041
references.pmid: 11297677
references.citation: Trends Biotechnol. 2006 Feb;24(2):68-75
references.pmid: 16380176
references.citation: J Virol. 1988 Aug;62(8):2636-43
references.pmid: 2839690
references.citation: EMBO J. 1994 Feb 15;13(4):928-33
references.pmid: 8112307
references.citation: FEBS Lett. 1997 May 5;407(3):313-9
references.pmid: 9175875
references.citation: Mol Cell Biol. 2008 Jul;28(13):4227-39
references.pmid: 18458056