Short fish-origin DNA elements served as flanking sequences in a knockdown cloning vector enabling the generation of a functional siRNA molecule in mammalian cells and fish embryos

Lin, C.Y., Lee, H.C., Wu, J.H., Tsai, H.J.
Biochemical and Biophysical Research Communications   505(3): 850-857 (Journal)
Registered Authors
Tsai, Huai-Jen
MeSH Terms
  • 3' Flanking Region/genetics*
  • Animals
  • Cell Line, Tumor
  • Gene Knockdown Techniques/methods*
  • Genetic Vectors/genetics*
  • Green Fluorescent Proteins
  • Humans
  • Neovascularization, Pathologic/drug therapy
  • RNA, Small Interfering/biosynthesis*
  • RNA, Small Interfering/physiology
  • Vascular Endothelial Growth Factor A/antagonists & inhibitors
  • Vascular Endothelial Growth Factor A/genetics
  • Zebrafish/embryology
30301529 Full text @ Biochem. Biophys. Res. Commun.
Improving the quality of a siRNA-knockdown cloning vector requires simpler, shorter, and more effective flanking sequences. In this study, we designed such flanking sequences based on those found in zebrafish pre-miR3906, namely, internal element (IE) 1 and IE2. We engineered a vegf-shRNA fragment flanked by an 80-bp IE1/IE2 and then inserted into the 3' UTR of GFP reporter cDNA driven by a cytomegalovirus promoter to obtain a plasmid containing gfp-IE-vegf-shRNA-polA. Upon microinjection of this plasmid into zebrafish embryos, we found that IE flanking sequences could effectively induce the production of vegf-shRNA fragment, which was then processed into a functional siRNA to silence the target vegf121 gene. Northern blot showed that the vegf-shRNA fragment was cleaved from gfp-IE-vegf-shRNA-polA, resulting in the loss of polyA tails, subsequently degrading the remaining RNA-containing GFP. Moreover, Western blot revealed that addition of IE-based vegf-shRNA fragment could markedly decrease the expression of VEGF. Finally, to facilitate a more versatile application of the IE-based knockdown vector, we generated an inducible expression vector in which IE-vegf-shRNA was constructed downstream in a Tet-on system to generate a Tet-on-IE-vegf-shRNA construct. After doxycycline induction, the protein level of VEGF in SW620 cells harboring the Tet-on-IE-vegf-shRNA construct was decreased 77%. Interestingly, when SW620 cells harboring Tet-on-IE-vegf-shRNA cells were induced and transplanted into zebrafish embryos, we found that abnormal branch of the sub-intestinal vessels was reduced in the recipient embryos, suggesting that vegf-shRNA cleaved from Tet-on-IE-vegf-shRNA-polA was processed into a functional vegf-siRNA in embryos suppressing endogenous VEGF and reducing tumor angiogenesis. Therefore, we conclude that fish-origin IEs are flanking sequences with short, simple, and effective DNA elements. This IE-based knockdown cloning vector provides a new alternative material to facilitate the generation of functional siRNA with which to perform loss-of-function experiments, both in vitro (mammalian cells) and in vivo (zebrafish embryos).
Genes / Markers
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes