Abstract
Here we describe a protocol for gene loss of function during regeneration in newts, specifically applied to lens regeneration. Knockdown with the use of morpholinos can be achieved both in vitro and in vivo, depending on the experimental design. These methods achieve desirable levels of gene knockdown, and thus can be compared with methods developed for use in other animals, such as zebrafish. The technology has been applied to study molecular mechanisms during the process of lens regeneration by knocking down genes at specific stages and examining their effects on other genes and lens differentiation. The protocol can take a few days or up to 20 d to complete, depending on the duration of the experiment.
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References
Sánchez Alvarado, A. & Tsonis, P.A. Bridging the regeneration gap: genetic insights from diverse animal models. Nat. Rev. Genet. 7, 873–884 (2006).
Tsonis, P.A. Regeneration in vertebrates. Dev. Biol. 221, 273–284 (2000).
Kragl, M. et al. Cells keep a memory of their tissue origin during axolotl limb regeneration. Nature 460, 60–65 (2009).
Del Rio-Tsonis, K. & Tsonis, P.A. Eye regeneration at the molecular age. Dev. Dyn. 226, 211–224 (2003).
Tsonis, P.A. & Del Rio-Tsonis, K. Lens and retina regeneration: transdifferentiation, stem cells and clinical applications. Exp. Eye Res. 78, 161–172 (2004).
Maki, N. et al. Expression of stem cell pluripotency factors during regeneration in newts. Dev. Dyn. 238, 1613–1616 (2009).
Casco-Robles, M.M. et al. Expressing exogenous genes in newts by transgenesis. Nat. Protoc. 6, 600–608 (2011).
Madhavan, M. et al. The role of Pax-6 in lens regeneration. Proc. Natl Acad. Sci. USA 103, 14848–14853 (2006).
Maki, N. et al. Oocyte-type linker histone B4 is required for transdifferentiation of somatic cells in vivo. FASEB J. 24, 3462–3467 (2010).
Spector, D.L. & Goldman, R.D. Basic Methods in Microscopy (Cold Spring Harbor Laboratory Press, 2006).
Grogg, M.W. et al. BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration. Nature 438, 858–862 (2005).
Tsonis, P.A. Limb Regeneration (Cambridge University Press, 1996).
Acknowledgements
This work was supported by a Grant-in-Aid for Challenging Exploratory Research (20650060) and a Grant-in-Aid for Scientific Research (B) (21300150) from the Japan Society for the Promotion of Science (JSPS) to C.C. and by NIH grant EY10540 to P.A.T.
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P.A.T. and K.D.R.-T. designed, directed and analyzed data pertaining to loss of function experiments. T.H. and N.M. performed experiments and wrote part of the protocols. M.M.C.-R., S.Y., T.M. and K.N. wrote part of the protocols. P.A.T. and C.C. co-wrote the final version of the paper.
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Tsonis, P., Haynes, T., Maki, N. et al. Controlling gene loss of function in newts with emphasis on lens regeneration. Nat Protoc 6, 593–599 (2011). https://doi.org/10.1038/nprot.2011.341
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DOI: https://doi.org/10.1038/nprot.2011.341
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