Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system.
A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 microg/microl. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.
Wang
Na
N
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, 7# Donghu South Road, Wuhan, Hubei 430072, China.
Sun
Yong-Hua
YH
Liu
Jing
J
Wu
Gang
G
Su
Jian-Guo
JG
Wang
Ya-Ping
YP
Zhu
Zuo-Yan
ZY
D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005326:Fetal Proteins; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D022661:RNA, Untranslated / Q000235:genetics* / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000098908:Brachyury Protein
D005326:Fetal Proteins; D049452:Green Fluorescent Proteins; D022661:RNA, Untranslated; D020825:T-Box Domain Proteins; D029961:Zebrafish Proteins; D000098908:Brachyury Protein