Application of RNAi technology to the inhibition of zebrafish GtHalpha, FSHbeta, and LHbeta expression and to functional analyses.
Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHalpha, common alpha), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHalpha RNAi expression compared with the other two subunits. A GtHalpha morpholino analysis showed that the GtHalpha morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHalpha -shRNA. Taken together, these results show that GtHalpha-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.
Huang
Wei-Tung
WT
Institute of Molecular Biotechnology, Da-Yeh University, Changhua, Taiwan.
Hsieh
Jung-Chen
JC
Chiou
Ming-Jyun
MJ
Chen
Jyh-Yih
JY
Wu
Jen-Leih
JL
Kuo
Ching-Ming
CM
D000818:Animals; D015139:Blotting, Southern; D017931:DNA Primers / Q000235:genetics; D047108:Embryonic Development / Q000187:drug effects* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006065:Gonadotropins, Pituitary / Q000378:metabolism* / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008845:Microinjections; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015027:Zebrafish / Q000196:embryology* / Q000378:metabolism*
D017931:DNA Primers; D006065:Gonadotropins, Pituitary; D049452:Green Fluorescent Proteins; D012313:RNA