Hammerhead ribozyme-mediated knockdown of mRNA for fibrotic growth factors: transforming growth factor-beta 1 and connective tissue growth factor

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 820; p. 117
• Main Authors: Robinson, Paulette M, Blalock, Timothy D, Yuan, Rong, Lewin, Alfred S, Schultz, Gregory S
• Format: Journal Article
• Language: English
• Published: United States 01.01.2012
• Subjects: Cell Differentiation; Cells, Cultured; Connective Tissue Growth Factor - metabolism; Extracellular Matrix - metabolism; Fibroblasts - cytology; Fibroblasts - metabolism; Fibrosis - pathology; Gene Knockdown Techniques - methods; Humans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Transforming Growth Factor beta1 - metabolism
• ISSN: 1940-6029
• DOI: 10.1007/978-1-61779-439-1_8

Excessive scarring (fibrosis) is a major cause of pathologies in multiple tissues, including lung, liver, kidney, heart, cornea, and skin. The transforming growth factor-β (TGF-β) system has been shown to play a key role in regulating the formation of scar tissue throughout the body. Furthermore, connective tissue growth factor (CTGF) has been shown to mediate most of the fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. Currently, no approved drugs selectively and specifically regulate scar formation. Thus, there is a need for a drug that selectively targets the TGF-β cascade at the molecular level and has minimal off-target side effects. This chapter focuses on the design of hammerhead ribozymes, measurement of kinetic activity, and assessment of knockdown mRNAs of TGF-β and CTGF in cell cultures.