1. ¹Departments of Neurosciences, Division of Research, Medical University of South Carolina, 167 Ashley Ave, SEI 511, Charleston, SC 29425, USA
2. ²Departments of Ophthalmology, Medical University of South Carolina, 167 Ashley Ave, SEI 511, Charleston, SC 29425, USA

Correspondence: Baerbel Rohrer, Tel: +843-792-5086; Fax: +843-792-1723; E-mail: rohrer@musc.edu

Received 26 September 2005; Revised 27 October 2005; Accepted 29 October 2005.

Abstract

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase III. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by 60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for 'repression of gene function' studies in vertebrate model systems.