Molecular genetic system for regenerative studies using newts

Abstract

Urodele newts have the remarkable capability of organ regeneration, and have been used as a unique experimental model for more than a century. However, the mechanisms underlying regulation of the regeneration are not well understood, and gene functions in particular remain largely unknown. To elucidate gene function in regeneration, molecular genetic analyses are very powerful. In particular, it is important to establish transgenic or knockout (mutant) lines, and systematically cross these lines to study the functions of the genes. In fact, such systems have been developed for other vertebrate models. However, there is currently no experimental model system using molecular genetics for newt regenerative research due to difficulties with respect to breeding newts in the laboratory. Here, we show that the Iberian ribbed newt (Pleurodeles waltl) has outstanding properties as a laboratory newt. We developed conditions under which we can obtain a sufficient number and quality of eggs throughout the year, and shortened the period required for sexual maturation from 18 months to 6 months. In addition, P. waltl newts are known for their ability, like other newts, to regenerate various tissues. We revealed that their ability to regenerate various organs is equivalent to that of Japanese common newts. We also developed a method for efficient transgenesis. These studies demonstrate that P. waltl newts are a suitable model animal for analysis of regeneration using molecular genetics. Establishment of this experimental model will enable us to perform comparable studies using these newts and other vertebrate models.

Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl

“…Sustained expression from genomic integration is extremely important for the study of salamander limb regeneration. Successful germline transgenesis has been reported for axolotl (Sobkow et al., 2006) and has been applied to the Japanese newt and Iberian ribbed newt (Casco-Robles et al., 2010; Hayashi et al., 2013). Moreover, germline EGFP and nuclear Cherry-expressing transgenic axolotls were used in combination with tissue grafting to provide important proof that the regenerating limb blastema is a mixed population of tissue-restricted progenitors (Kragl et al., 2009).…”

Application of local gene induction by infrared laser‐mediated microscope and temperature stimulator to amphibian regeneration study

“…The heat‐shock‐inducible GFP F0 newt was prepared as previously reported (Hayashi et al . ). We obtained heat‐shock‐inducible GFP F2 animals by crossing a sexually mature F1 male newt with WT female newts.…”

“…; Hayashi et al . ). Several approaches have been tried for reproducible analyses of gene function with transgenesis (e.g, Cre‐lox, FLP‐FRT, heat shock promoter, tet regulatable promoter and GAL4‐UAS; reviewed in Blitz et al .…”

“…Recently, an inducible system with a heat shock promoter has been shown to work in a urodele amphibian, the Iberian ribbed newt (Hayashi et al . ). The heat shock inducible system has also been widely used for teleost fishes (Halloran et al .…”

“…Development of a transgenic (Tg) system for Xenopus has enabled us to start manipulating regeneration in an amphibian (Kroll & Amaya 1996;reviewed in Ogino & Ochi 2009). A Tg system for urodele amphibians subsequently became available (Ueda et al 2005;Sobkow et al 2006;Casco-Robles et al 2011;Hayashi et al 2013). Several approaches have been tried for reproducible analyses of gene function with transgenesis (e.g, Cre-lox, FLP-FRT, heat shock promoter, tet regulatable promoter and GAL4-UAS; reviewed in Blitz et al 2006).…”