Purchase this article with an account.
P. M. Kuznia, L. Glickman, R. Yuan, T. D. Blalock, W. W. Hauswirth, A. S. Lewin, G. S. Schultz; Hammerhead Ribozyme Mediated Knockdown of TGF-β1 in Cell Culture Using an scAAV Vector. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3385. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Corneal trauma, surgery or infection initiate a complex cascade of growth factors, cytokines, chemokines and proteases that may result in stromal scarring that impairs vision. Transforming growth factor- beta 1 (TGF- β1), a secreted growth factor, has been implicated in multiple roles in the corneal scaring pathway. Currently, there is no clinically available agent to reduce the activity of TGF- β1. We investigated whether a hammerhead ribozyme that targets TGF-β1 mRNA.can reduce the amount of TGF- β1 protein synthesized in tissue culture cells.
A ribozyme cleavage site in human TGF- β1 mRNA was identified, and the coding sequence for a 33 nucleotide hammerhead ribozyme was chemically constructed. The ribozyme gene was cloned into a self complementary Adeno associated virus (scAAV) expression plasmid and transfected into HEK293 cells. An inactive hammerhead ribozyme cloned into the same scAAV plasmid was used as a negative control. The effects of the ribozyme on expression of TGF-β1 protein levels in media were measured using an ELISA specific for TGF-β1.
Stable transfection of the TGF- β1 ribozyme into HEK293 cells showed a statistically significant (p<0.05) reduction of 77% of TGF- β1 protein in the media as compared to control cell media. The TGF- β1 ribozyme knocked down 46% more protein in the media than the inactive ribozyme. The difference between TGF-β1 levels in media from untransfected cells and cells transfected with inactive ribozyme plasmid was not statistically significant.
This PDF is available to Subscribers Only