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Controlling gene loss of function in newts with emphasis on lens regeneration


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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Figure 1: Pax-6 morpholinos decrease expression of pax-6 in cultured PECs and during in vivo lens regeneration in the newt N. viridescens.
Figure 2: Effects of B4 knockdown.


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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.

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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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Correspondence to Panagiotis A Tsonis.

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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).

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