Sample results with the Tol2kit

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Here are some examples of results obtained with the Tol2kit. They are included in a methods paper that has recently (September 2007) been accepted at Developmental Dynamics.

[edit] Validation of IRES clones

Below are confocal images of 24 hpf embryos, injected at the one-cell stage with expression constructs made with the pISce-Dest destination vector. This gives mosaic expression in the skin and muscle cells of the trunk. All cells show both mCherry and EGFP fluorescence, demonstrating that the plasmids generate functional bicistronic messages.

Image:IRES_results.jpg

[edit] Other IRES clones

We have also generated a set of equivalent IRES constructs driving mCherry, but have not been able to visualize the mCherry protein, likely for three reasons: the relative dimness of mCherry compared to EGFP, the low expression level driven by the IRES, and suboptimal imaging using 543 nm excitation, far from the peak of mCherry excitation at 587 nm (Shaner et al., 2004).

If you are interested in testing these constructs, please contact Chi-Bin Chien. We also encourage others to generate other IRES constructs, for instance using CFP or red fluorescent proteins such as DsRedExpress or tdTomato.

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