Inhibition of no tail (ntl) gene expression in zebrafish by external guide sequence (EGS) technique.
External guide sequence (EGS) technique, a branch of ribozyme strategy, can be enticed to cleave the target mRNA by forming a tRNA-like structure. In the present study, no tail gene (ntl), a key gene participating in the formation of normal tail, was used as a target for ribonuclease (RNase) P-mediated gene disruption in zebrafish in vivo. Transient expression of pH1-m3/4 ntl-EGS or pH1-3/4 ntl-EGS produced the full no tail phenotype at long-pec stage in proportion as 24 or 35%, respectively. As is expected that the full-length ntl mRNA of embryos at 50% epiboly stage decreased relative to control when injected the embryos with 3/4 EGS or m3/4 EGS RNA. Interestingly, ntl RNA transcripts, including the cleaved by EGS and the untouched, increased. Taken together, these results indicate that EGS strategy can work in zebrafish in vivo and becomes a potential tool for degradation of targeted mRNAs.
Pei
De-Sheng
DS
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.
Sun
Yong-Hua
YH
Long
Yong
Y
Zhu
Zuo-Yan
ZY
D000818:Animals; D001483:Base Sequence; D005326:Fetal Proteins; D005786:Gene Expression Regulation; D020868:Gene Silencing; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D043262:Ribonuclease P / Q000378:metabolism; D020825:T-Box Domain Proteins / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism; D058727:RNA, Small Untranslated; D000098908:Brachyury Protein
D005326:Fetal Proteins; D012333:RNA, Messenger; D043262:Ribonuclease P; D020825:T-Box Domain Proteins; D029961:Zebrafish Proteins; D058727:RNA, Small Untranslated; D000098908:Brachyury Protein