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

The electroporation technique is widely used in developmental biology to deliver foreign DNA into cells and study gene function. The chick embryos exhibit a remarkable easy access to perform electroporation and follow in ovo development.

Electroporation of limb somites allows the misexpression of genes in limb somite derivatives, like myogenic and endothelial cells, while electroporation of the lateral plate targets other limb cells like cartilage, bone and tendons.

We previously performed limb somite electroporation using two plasmids, one containing the gene-of-interest and one containing the reporter gene. However, this approach does not allow analysis at the cellular level, which constitutes a limitation of the technique.

To overcome this limitation and perform analysis at the cellular level, we aimed to design bicistronic vectors to misexpress, in the same cell, the gene-of-interest and the reporter gene. To do this, we took advantage of the 2A peptide that allows expression of a bicistronic mRNA. In these vectors, a single peptide is produced by the bicistronic mRNA, and auto-cleavage of the 2A peptide subsequently produces equal amounts of the two proteins. To test our vectors, we used control plasmids expressing two fluorescent proteins, Tomato and GFP, separated by the 2A peptide. We further used the Tol2 transposon system to allow genomic integration of the construct and enable analysis at late developmental stages.

One focus of our research is skeletal muscle and tendon formation. Therefore, we designed a set of stable bicistronic vectors containing different promoters to target muscle cells at different stages of differentiation. After performing limb somite electroporation using these vectors, we observed a simultaneous cellular expression of Tomato (membrane) and GFP (nuclei) at the different stages of muscle differentiation. In electroporated limbs, the ubiquitously expressed CMV/βactin promoter targeted both muscle progenitors (Pax7+ cells) and differentiated cells (myosin+ cells). The p57MRE/βactin promoter, which drives expression in differentiated myoblasts, targeted mononucleated (myosin-) cells and muscle fibres (myosin+). Finally, the MLC (myosin light chain) promoter targeted differentiated cells (myosin+). Lateral plate electroporation with the vector containing the CMV/βactin promoter allowed Tomato and GFP co-expression in cartilage, tendons and connective tissue of the limbs, but never in myogenic (Pax7+ or myosin+) cells.

We believe that this set of tools can be used to efficiently misexpress genes at different time points of myogenic cell differentiation and analyse the consequences for muscle development. Moreover, because these vectors can be integrated into the genome, the analysis at late developmental time points can be performed. Finally, the combination of limb somite and lateral plate electroporation can provide us with a tool to study the molecular and cellular interactions between the different components of the musculoskeletal system.

Full article at : http://www.biomedcentral.com/1471-213X/15/39

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