2A used in the Production of Induced Pluripotent Stem Cells (iPS cells or iPSCs)

iPSCs are typically derived from differentiated adult somatic cells (non-pluripotent cell) by co-expression of certain genes. IPSCs were first produced in 2006 from mouse cells [1] and in 2007 from human cells [2,3]. Such technology enables iPS cells to be produced from individual patients and has great significance in the fields of regenerative medicine, gene therapy and drug discovery/testing.

In the mouse study, four genes (Oct3/4 - or Pou5f1-, Sox2, c-Myc, and Klf4) needed to be co-expressed to bring about this transformation. In the case of human cells, multiple genes were also co-expressed: in one study Oct4, Sox2, NANOG, and Lin28 were used [2], whilst in the other report genes encoding Oct3/4, Sox2, Klf4, and c-Myc were used [3].

One way of introducing the four different genes into a single cell is by the use of multiple, different, viruses. A single cell may be co-infected with four viruses each encoding a single transgene. This approach has been adopted using two different types of virus gene-delivery system.

In one series of studies, multiple different retroviral vectors were constructed encoding genes such as Oct4, Sox2, Klf4 or c-Myc [4-7]. Cells were transfected with multiple retroviruses and puripotent stem cells were produced highly efficiently. In another study, cells were transformed by infection with four different (non-integrating) adenoviruses encoding either Oct4, Sox2, Klf4, or c-Myc [8]. Again, this approach requires a cell to be infected by each of the four different (adeno-)viruses, but in this case does not lead to potential genetic damage by the integration of the vector sequences into the host cell chromosome - each gene is only transiently expressed.

Using 2A to Improve the Efficiency of Co-expression of Oct4, Sox2, Klf4, and c-Myc.

Initial studies were concerned with identification of the 'repertoire' of genes required for the establishment of the puripotent state. In the studies outlined above, each of these genes was expressed individually - a single cell needed to be transformed with each of four genes - and each cell required the co-expression of each of these (trans)genes at suitable levels.

2A has been used in a very wide range of biotechnological and biomedical applications for the efficient co-expression of multiple genes. In the case of iPSCs, four different genes need to be co-expressed within a single cell to induce the puripotent state. Two recent reports have utilised the 2A system for the co-expression of these genes.

Using a plasmid DNA (non-viral) system, the Yamanaka group were able to produce iPS cells by transfection with two plasmids. The first encoded c-Myc. In the second plasmid, sequences encoding Oct3/4, Sox2 and Klf4 were assembled (in all permutations) into a single open reading frame (ORF) using 2A linkers [9]. Transformation into the puripotent state could be achieved without integration of the plasmids into the host-cell chromosome - and potentially harmful effects of'collateral' genetic damage which could be caused by integration at random sites.

In another study a single lentivirus was constructed encoding two ORFs, separated by an internal ribosome entry sequences (IRES) [10]. The first ORF encoded Oct4 and Klf4 linked via a 2A sequence from foot-and-mouth disease virus (F2A). The second ORF, downstream of the IRES, encoded Sox2 and c-Myc linked via a 2A sequence from equine rhinitis A virus (E2A). Notably, all four genes are translated from a single transcript mRNA. This technology provides a very efficient method of generating iPS cells: the transgenes are delivered highly efficiently using a lentivirus particle and co-expression of all four genes is achieved by a single virus construct with a single integration event.

Groups at the Whitehead Institute for Biomedical Research and the Department of Biology, M.I.T. constructed a single lentivirus encoding Oct4, Sox2, c-Myc, and Klf4 - this time all linked into a single ORF using a range of 2A-like sequences (F2A, T2A, E2A, or P2A). Post-natal human keratinocytes plus embryonic and adult murine fibroblasts were induced to form pluripotent iPS cells [11]. A similar approach was used - along with co-expression of a GFP marker - by the groups in Maine and Chicago [12]. This work suggests that with sufficient expression of these four factors - from a single proviral copy of a 2A-based polycistronic vector, it may be possible to reprogram cells using a stably expressing single transgene.

Recent developments include the use of piggyBac (PB) transposition system. PB is host-factor independent and was used to demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition [13,14]. Stable iPS cells thus generated express characteristic pluripotency markersand take advantage of the natural propensity of thePBsystem for seamless excision such that the individual PB insertions can be removed from established iPS cell lines.


[1] Takahashi, K. & Yamanaka, S. (2006). .Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.

[2] Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I. & Thomson, J.A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920.

[3] Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872.

[4] Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E. & Jaenisch, R. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-24.

[5] Aasen, T., Raya, A., Barrero, M.J., Garreta, E., Consiglio, A., Gonzalez, F., Vassena, R., Bilic, J., Pekarik, V., Tiscornia, G., Edel, M., Boué, S. & Belmonte J.C. (2008). Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nature Biotech. 11, 1276-1284.

[6] Brambrink, T., Foreman, R., Welstead, G.G., Lengner, C.J., Wernig, M., Suh, H. & Jaenisch, R. (2008). Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151-159.

[7] Hanna, J., Markoulaki, S., Schorderet, P., Carey, B.W., Beard, C., Wernig, M., Creyghton, M.P., Steine, E.J., Cassady, J.P., Foreman, R., Lengner, C.J., Dausman, J.A. & Jaenisch, R. (2008). Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell. 133, 250-64.

[8] Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G. & Hochedlinger K. (2008). Induced pluripotent stem cells generated without viral integration. Science 322, 945-949.

References citing the use of 2A;

[9] Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T. & Yamanaka, S. (2008). Generation of mouse induced puripotent stem cells without viral vectors. Science 322, 949-953.

[10] Sommer, C.A., Stadtfeld, M., Murphy, G.J., Hochedlinger, K. Kotton, D.N. & Mostoslavsky, G. (2008). iPS cell generation using a single lentiviral stem cell cassette. Stem Cells e-pub doi:10.1634/stemcells.2008-1075.

[11] Carey, B.W., Markoulaki, S., Hanna, J., Saha, K., Gao, Q., Mitalipova, M. & Jaenisch, R. (2009). Reprogramming of murine and human somatic cells using a single polycistronic vector. PNAS 106, 157-162.

[12] Shao, L., Feng, W., Sun, Y., Bai, H., Liu, J., Currie, C., Kim, J., Gama, R., Wang, Z., Qian, Z., Liaw, L. & Wu, W-S. (2009). Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame. Cell Research 19, 296-306.

[13] Woltjen, K., Michael1, I.P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., Cowling, R., Wang, W., Liu, P., Gertsenstein, M., Kaji, K., Sung,H-K. & Nagy, A. (2009). piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature doi:10.1038/nature07863

[14] Kaji, K., Norrby, K., Paca, A., Mileikovsky, M., Mohseni, P. & Woltjen. K. (2009). Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature doi:10.1038/nature07864

Further References citing the use of 2A;

Chang, C.W., Lai, Y.S., Pawlik, K.M., Liu, K., Sun, C.W., Li, C., Schoeb, T.R. & Townes, T.M. (2009). Polycistronic lentiviral vector for "hit and run'' reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells 27, 1042-1049

Gonzalez, F., Monasterio, M.B., Tiscornia, G., Pulido, N.M., Vassena, R., Morera, L.B., Piza,I.R. & Belmonte, J.C.I. (2009). Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector. PNAS 106, 8918-8922.

Papapetrou, E.P., Tomishima, M.J., Chambers, S.M., Mica, Y., Reed, E., Menon, J., Tabar, V., Mo, Q., Studer, L. & Sadelain, M. (2009). Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation. PNAS 106, 12759-12764.


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