PUBLICATION
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
- Authors
- Lin, C.Y., Lee, H.C., Wu, J.H., Tsai, H.J.
- ID
- ZDB-PUB-181012-2
- Date
- 2018
- Source
- Biochemical and Biophysical Research Communications 505(3): 850-857 (Journal)
- Registered Authors
- Tsai, Huai-Jen
- Keywords
- none
- MeSH Terms
-
- Genetic Vectors/genetics*
- Neovascularization, Pathologic/drug therapy
- Green Fluorescent Proteins
- Vascular Endothelial Growth Factor A/antagonists & inhibitors
- Vascular Endothelial Growth Factor A/genetics
- Zebrafish/embryology
- RNA, Small Interfering/biosynthesis*
- RNA, Small Interfering/physiology
- 3' Flanking Region/genetics*
- Cell Line, Tumor
- Gene Knockdown Techniques/methods*
- Humans
- Animals
- PubMed
- 30301529 Full text @ Biochem. Biophys. Res. Commun.
Citation
Lin, C.Y., Lee, H.C., Wu, J.H., Tsai, H.J. (2018) 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. Biochemical and Biophysical Research Communications. 505(3):850-857.
Abstract
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
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping