Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis.
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 approximately 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.
Department of Neurosciences, Division of Research, Medical University of South Carolina, 167 Ashley Ave, SEI 511, Charleston, 29425, USA.
Ming
M
Li
Baerbel
B
Rohrer
D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000235:genetics; D002460:Cell Line; D004247:DNA; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D012160:Retina / Q000378:metabolism; D014162:Transfection; D014982:Xenopus laevis / Q000235:genetics*
D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D004247:DNA