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.
Li
Ming
M
Department of Neurosciences, Division of Research, Medical University of South Carolina, 167 Ashley Ave, SEI 511, Charleston, 29425, USA.
Rohrer
Baerbel
B
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