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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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.
The authors declare no competing financial interests.
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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
Nature Protocols (2011)