RNA Interference on Regenerating Holothurian Gut Tissues

Abstract

Functional studies on echinoderms have been reduced to the use of pharmacological treatments. The ability to modulate the genetic expression of regenerating tissues can elucidate potential effectors during this process. Here we describe an effective transfection protocol that allows the introduction of Dicer-substrate interference RNAs (DsiRNAs) for the modulation of gene expression and its characterization during regeneration.

Keywords: DsiRNA; Echinoderm; Electroporation; Gut rudiment; RNAi; Regeneration; Sea Cucumber.

Figure 1.

Electroporation of tetramethylrhodamine-conjugated anionic dextran in explants of the sea cucumber H. glaberrima. Nuclei are stained with DAPI (green) and tetramethylrhodamine-conjugated anionic dextran stained the cytosol (red). (A) Electroporation of the explant with 0.1 M PBS. (B) Tetramethylrhodamine-conjugated anionic dextran alone (without electroporation). (C) Electroporation of the explant with tetramethylrhodamine-conjugated anionic dextran. (D) Higher magnification of (C).

Figure 2

RNA interference promotes β-catenin downregulation. To evaluate the efficiency of the interference of β-catenin transcript, qRT-PCR of intestinal explants was performed after 2 days of DsiRNA electroporation. Expression values were plotted as fold change relative to DsiRNA-targeting GFP (negative control) treatment in log₂ scale. N = 4.