2A used in Bioimaging.
The discovery and development of fluorescent proteins has lead to a revolution in bioimaging. Many of these proteins tolerate N- or C-terminal extensions - still maintaining their fluorescent properties. Fusions can be created with proteins of interest to study expression, localisation etc.
Using 2A, the gene(s) of interest can be linked to fluorescent proteins such that the fluorescent protein will 'report' on the level of expression in each individual cell expressing the protein(s).
Co-expression of multiple, different, fluorescent proteins, in combination with different signal (leader) sequences (co- and post-translational) may be used to target different fluorecent proteins to different sub-cellular sites. In this manner, organelles or other cellular structures can be monitored in a real-time, non-invasive manner, to study normal cellular processes, or, the effects of infection, drugs, stress, etc.
2A has been been used to co-express different fluorecent proteins targeted to different sub-cellular sites. In the example shown on the right, a cDNA construct was assembled to encode a single, long, open reading frame - an artificial polyprotein - comprising; enhanced yellow fluoresent protein (EYFP), linked via 2A to enhanced cyan fluoresent protein (ECFP) targeted to mitochondria (MT-ECFP], in turn linked by another 2A to PAC (puromycin resistance). In this manner, one can establish fluoresent cell-lines (co-expressing both EYFP and ECFP) quickly and very easily using puromycin selection.
de Felipe, P. & Ryan, M.D. (2004) Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences. Traffic 5, 616-626.
EYFP2A - distributed evenly throughout the cytoplasm and nucleus MT-ECFP2A - localises to mitochondria Merge of the two images
(see also http://www.st-andrews.ac.uk/ryanlab/Translocon/Index.htm)
2A can also be used to monitor the expression of transgenes: a single ORF is created to encode the gene of interest, linked via 2A to a fluorescent protein. This enables one to visualise, in a non-invasive manner, both the individual cells which are expressing the transgene(s), and the tisues within the body. This technique has been used to great effect to show, for example, the reconstitution of the hematopoietic system following the transfer of transgenic bone marrow cells (expressing luciferase and GFP, linked via 2A) from the transgenic mouse donor animal into lethally irradiated syngeneic recipient animals.
The expression of a therapeutic transgene can be monitored by linking the gene, via 2A, to a marker protein, or, a selectable marker.
Transgenic animals and plants have be created with different fluoresent proteins targeted to different sub-cellular sites.
References citing the use of 2A
Furler, S., Paterna, J.C., Weibel, M., & Bueler H. (2001). Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons. Gene Therapy 8, 864-873.
Blankenberg, F.G., Mandl, S., Cao, Y.A., O'Connell-Rodwell, C., Contag, C., Mari, C., Gaynutdinov, T.I., Vanderheyden, J.L., Backer, M.V. & Backer, J.M. (2004). Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor. J. Nucl. Med. 45, 1373-80.
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.
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.
Osborn, M.J., Panoskaltsis-Mortari, A., McElmurry, R.T., Bell, S.K., Vignali, D.A.A., Ryan, M.D., Wilber, A., McIvor, R.S., Tolar, J. & Blazar, B.R. (2005). A picornaviral ‘2A-like’ sequence based tricistronic vector allowing for high level therapeutic gene expression coupled to a dual reporter system. Mol. Ther. 12, 569-574.
de Felipe, P., Luke, G.A., Hughes, L.E., Gani, D., Halpin, C. & Ryan, M.D. (2006). E unum pluribus: multiple proteins from a self-processing polyprotein. Trends in Biotech. 24, 68-75.
Samalova, M., Fricker, M. & Moore, I. (2006). Ratiometric fluorescence-imaging assays of plant membrane traffic using polyproteins. Traffic 7, 1701-1723.
Contag, C.H. (2007). In vivo pathology: Seeing with molecular specificity and cellular resolution in the living body Ann. Rev. Pathology-Mechansims of Dis. 2, 277-305.
Miloud T, Henrich C, Hämmerling GJ. (2007). Quantitative comparison of click beetle and firefly luciferases for in vivo bioluminescence imaging. J. Biomed. Opt. 12, 054018.
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.
Stephan, M.T., Ponomarev, V., Brentjens, R.J., Chang, A.H., Dobrenkov, K.V., Heller, G. & Sadelain, M. (2007). T cell-encoded CD80 and 4-1BBL induce auto- and transcostimulation, resulting in potent tumor rejection. Nature Med. 13, 14401449.
Wang, Q., Ilves, H., Chu, P., Contag, C.H., Leake, D., Johnston, B.H. & Kaspar, R.L. (2007). Delivery and inhibition of reporter genes by small interfering RNAs in a mouse skin model. J. Invest. Dermatology 127, 2577-2584.
Hickerson, R.P., Smith, F.J.D., Reeves, R.E., Contag, C.H., Leake, D., Leachman, S.A., Milstone, L.M., Leonard, M., McLean, W.H.I. & Kaspar, R.L. (2008). Single-nucleotide-specific siRNA targeting in a dominant-negative skin model. J. Invest. Dermatol. 128, 594-605.
Lee, Z.H., Dennis, J.E. & Gerson, S.L. (2008). Imaging stem cell implant for cellular-based therapies. Exp. Biol. Med. 233, 930-940.
O'Connell-Rodwell, C.E., Mackanos, M.A., Simanovskii, D., Cao, Y.A., Bachmann, M.H., Schwettman, H.A. & Contag, C.H. (2008). In vivo analysis of heat-shock-protein-70 induction following pulsed laser irradiation in a transgenic reporter mouse. J. Biomed. Optics 13, Article # 030501.
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.
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