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Fig. 1 | BMC Developmental Biology

Fig. 1

From: Stable and bicistronic expression of two genes in somite- and lateral plate-derived tissues to study chick limb development

Fig. 1

Schematic representation of the 2A peptide system in Tol2-based vectors. Schematic representation of a vector containing the transposase under the control of a ubiquitous CMV/βactin promoter and a vector containing a cassette with two reporter fluorescent genes TdTomato and EGFP separated by the T2A peptide under the control of a CMV/βactin promoter, between the minimal Tol2 transposons. The transfection into chick cells of both vectors allows the stable transposition of the transgene (CMV/βactin promoter-TdTomato-T2A-EGFP cassette) into the chick genome. The TdTomato-T2A-EGFP cassette is transcribed under the control of the CMV/βactin promoter and then translated. At the level of the translation process, the T2A peptide will be self-cleaved between the two amino acids, Gly and Pro (double arrow), following a consensus sequence (boxed). The 19 first amino acids of the cleaved T2A peptide remains fused to the C-terminus of the TdTomato, while the Pro amino acid is added to the N-terminus of the GFP. With the 2A peptide system, one single mRNA is transcribed that produces two proteins in stoichiometric proportions. TdTomato is targeted to the membrane due to a myristoylation signal and EGFP is targeted to the nucleus due to a H2B sequence. This leads to expression of both proteins in two different subcellular compartments

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