2A used in the Creation of Transgenic Plants, Microbes & Animals.
The first types of genetically modified organisms created using 2A to co-express multiple proteins were plants. Initially as a research tool, but also to improve the hardiness (drought-resistance; Kwon et al., 2004) and nutritional qualities (Ralley et al., 2004).
Latterly, transgenic mice have been created - notably the coexpression of the multiple (6 different) chains required to create a functional T-cell receptor complex (Szymczak et al., 2004). Transgenic mice have been created expressing fluorescent proteins to study the transplantation of bone marrow cells (Cao et al., 2005) using whole-body imaging techniques.
Transgenic mice co-expressing different fluorescent proteins targeted to different sub-cellular sites have produced superb images of these proteins in a wide range of tissues (Trichas et al., 2008).
This technology has also been used to great effect in zebra fish (Provost et al., 2007) and will provide a very useful tool to study, for example, developmental processes in this species.
References citing the use of 2A;
Halpin, C., Cooke, S.E., Barakate, A., El Amrani, A. & Ryan, M.D. (1999). Self-processing polyproteins - a system for co-ordinate expression of multiple proteins in transgenic plants. Plant J. 17, 453-459.Halpin, C., Barakate, A., Askari, B., Abbott, J. & Ryan, M.D. (2001). Enabling technologies for manipulating multiple genes on complex pathways. Plant Mol. Biol. 47, 295-310.
François, I.E., De Bolle, M.F., Dwyer, G., Goderis, I.J., Woutors, P.F., Verhaert, P.D., Proost, P., Schaaper, W.M., Cammue, B.P. & Broekaert, W.F. (2002). Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins. Plant Physiol. 128, 1346-1358.
Ma, C. & Mitra, A. (2002) Expressing multiple genes in a single open reading frame with the 2A region of foot-and-mouth disease virus as a linker. Molecular Breeding 9, 191-199.
Ma, C & Mitra, A. (2002) Instrinsic direct repeats generate consistent post-transcriptional gene silencing in tobacco. The Plant Journal 31, 37-49.
El Amrani, A., Barakate, A., Askari, B.M., Li, X., Roberts, A.G., Ryan, M.D. & Halpin, C. (2004). Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene. Plant Physiol. 135, 16-24.
Francois, I.E.J.A., Van Hemelrijck, W., Aerts, A.M., Wouters, P.F.J., Proost, .P, Broekaert, W.F. & Cammue, B.P.A. (2004). Processing in Arabidopsis thaliana of a heterologous polyprotein resulting in differential targeting of the individual plant defensins. Plant Sci. 166, 113-121.
Kwon, S.J., Hwang, E.W. & Kwon, H.B. (2004). Genetic engineering of drought resistant potato plants by co-introduction of genes encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Zygosaccharomyces rouxii. Korean J. Genet. 26, 199-206.
Ralley, L., Enfissi, E.M.A., Misawa, N., Schuch, W., Bramley, P.M. & Fraser, P.D. (2004). Metabolic engineering of ketocarotenoid formation in higher plants. Plant J. 39 477-486.
Szymczak, A.L., Workman, C.J., Wang, Y., Vignali, K.M., Dilioglou, S., Vanin, E.F. & Vignali, D.A. (2004). Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nature Biotech. 22, 589-94.
Cao, Y.A., Bachmann, M.H., Beilhack, A., Yang, Y., Tanaka, M., Swijnenburg, R.J., Reeves, R., Taylor-Edwards, C., Schulz, S., Doyle, T.C., Fathman, C.G., Robbins, R.C., Herzenberg, L.A., Negrin, R.S. & Contag, C.H. (2005). Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation. Transplantation 80, 134-139.
Szymczak, A.L. & Vignali, D.A.A. (2005). Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opinion 5, 627-638.
Yasuda, H., Tada, Y., Hayashi, Y., Jomori, T. & Takaiwa, F. (2005). Expression of the small peptide GLP-1 in transgenic plants. Transgenic Res. 14, 677-684.
Holst, J., Szymczak-Workman, A.L., , K.M., Burton, A.R., Workman, C.J. & Vignali, D.A.A. (2006). Generation of T-cell receptor retrogenic mice. Nature Protocols 1, 406-417.
Samalova, M., Fricker, M. & Moore, I. (2006). Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins. Traffic 7, 1701-1723.
Provost, E., Rhee, J. & Leach, S.D. (2007). Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos. Genesis 45, 625-629.
Teh, O-K & Moore, I. (2007). An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells. Nature 448, 493-497.
Wang, S., Yao, Q., Tao, J., Qiao, Y. & Zhang, Z. (2007). Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris. App. Micro. & Biotech. 77, 891-899.
Park, M., Kang, K., Park, S., Kim, Y.S., Ha, S.H., Lee, S.W., Ahn, M.J., Bae, J.M. & Back, K. (2008). Expression of serotonin derivative synthetic genes on a single self-processing polypeptide and the production of serotonin derivatives in microbes. App. Microbiol. Biotech. 81, 43-49.
Samalova, M., Fricker, M. & Moore, I. (2008). Quantitative and qualitative analysis of plant membrane traffic using fluorescent proteins. Meth. Cell Biol. 85, 353-380.
Trichas, G., Begbie, J. & Srinivas, S. (2008). Use of the viral 2A peptide for bicistronic expression in transgenic mice. BMC Biology 6, Article #40.