Cell Research (2006)16:99-105© 2006 IBCB, SIBS, CAS All rights reserved 1001-0602/06 $ 30.00
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Gene silencing in Xenopuslaevis by DNA vector-based RNA interference and transgenesis

Ming Li¹, Baerbel Rohrer^{1, 2}

Departments of ¹Neurosciences and ²Ophthalmology, Division of Research, 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

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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 con-struct-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.

Cell Research (2006) 16:99-105. doi:10.1038/sj.cr.7310013; published online 16 January 2006

Keywords: RNAi, Xenopus U6 promoter, transgenic, Xenopus laevis, GFP