OBJECTIVE OF THE EXPERIMENT The mechanisms that silence unwanted gene expression are critical for normal cellular function. Double-stranded RNA (dsRNA) induces gene silencing by the degradation of single-stranded (ssRNA) targets complementary to the dsRNA trigger. RNA interference (RNAi) effects activated by dsRNA have been demonstrated in a number of organisms including plants, protozoa, hydra, planarians, nemertines, annelids, nematodes, insects and mammals. In this experiment, we aim to introduce a methodology to produce and visualize the loss of function phenotype of dsRNA-injected planarians. In this experiment, two genes will be silenced by post-transcriptional gene silencing: Gtsix-1, a gene involved in the determination of the eye field and eye formation, and Tcen49, an invertebrate trophic factor involved in regional cell survival.

To achieve Six/tcen49 loss-of-function, several procedures will be used:

• plasmid linearization

• ribosynthesis

• dsRNA production

• dsRNA delivery by microinjection

DEGREE OF DIFFICULTY This experiment is moderately difficult. It requires some skills in ribosynthesis and microinjection, and the use of appropriate materials.

INTRODUCTION

Platyhelminthes are acoelomate members of the Bilateria, which share with other phyla like Gastrotricha and Acanthocephala a basal position in the Lophotrochozoa clade (Aguinaldo et al., 1997). Planarians (order Tricladida) are free-living platyhelminthes, well known for their ability for regeneration (Baguñà, 1998; Saló and Baguñà, 2002), which can be easily triggered by amputation of body fragments. In addition to the regenerative capacity of the planarian body, its morphological simplicity, which nevertheless includes all the basic characteristics of a bilaterian representative – cephalization, dorso–ventral and antero–posterior polarity – makes it a useful model for investigating the evolution of molecular developmental processes.