Subterranean Fishes of the World

3217

2013

AIM 2013

Transgenesis techniques in Astyanax mexicanus have been poorly used in the past years although this fish has become an increasingly popular microevolution model (cf Yamamoto et al. Dev Biol. 2009). It is however a fruitful way to analyze gene functions or to visualize expression patterns in live animals. Here, we compared the two main methods of transgenesis usually used in zebrafish. We used two vectors provided by the AMAGEN platform (aquatic animals transgenesis platform). One is a βcrystallin promoter driving CFP, the other is a cardiac actin promoter driving RFP. First we used Involin, an I-SceI meganuclease-mediated method. Meganucleases are restriction enzymes which cut 18bp recognition sites, typically absent of most genomes. The vector carrying ISceI site was co-injected together with the meganuclase protein in one cell stage surface fish and cavefish embryos. Second we used the Tol2 system. The Tol2 transposable element was originally found in the genome of the Japanese medaka fish, and contains a gene encoding an active transposase that catalyzes DNA transposition in vertebrate cells. Vectors carrying the transgene sided by the transposon LTR (Long Terrminal Repeat) were co-injected together with Tol2 transposase RNA in one cell stage embryos. Forty-one batches of surface and cave fish embryos (total 3095) were successfully injected with these two methods. Between 8.70 to 57.70 % of the surviving injected larvae expressed the reporter. A good overall survival rate after injection was achieved showing no statistical difference between injected eggs and controls. As expected, various types of mosaic expression were observed in F0 animals, from monolateral to bilateral and from strong to low expression. We established the first Astyanax surface fish trangenic lines expressing β-crystallin in the lens with the Involin method. Two FO (founders) males transmitted transgene to 14.60% and 23.60% of their progeny. Some transgene extinction was observed in a family but not in the other, for which F3 individuals still express strongly CFP in the lens. A new method started to be used for fish transgenesis, named TALEN (Transcription Activator- Like Effector Nucleases). TALEN-mediated gene knock-out and knock-in should be feasible. We will start investigating this new technique in Astyanax. All observations made so far let us think that surface and cave Astyanax will be very amenable to transgenesis. Work supported by ANR [ASTYCO] and DECA group-AMAGEN collaborative FRC grant.

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