Abstract
The use of RNase P ribozyme (M1GS catalytic RNA) for inhibition of murine cytomegalovirus (MCMV) propagation in mice is described in this chapter. General information about RNase P based technology is included and followed by detailed protocols focused on (1) construction and in vitro cleavage assay of the customized M1GS ribozyme, (2) stable expression of the M1GS RNA and evaluation of its activity in inhibition of viral gene expression and growth in cultured cells, and (3) investigation of M1GS-mediated inhibition of viral infection and pathogenesis in animals. Using these methods, we have successfully constructed catalytic M1-1 RNA against the MCMV assembly protein (mAP) and M80 mRNA. Our recent study has demonstrated that an 80% reduction in the expression of mAP and M80 and a 2,000-fold reduction in viral growth were observed in cells expressing the ribozyme. Furthermore, after the functional ribozyme-expressing constructs were delivered into MCMV-infected SCID mice, a significant reduction of viral gene expression and infection was detected, and the survival of the infected animals was significantly improved. Collectively, our data demonstrate the feasibility of the use of RNase P ribozyme for inhibition of viral gene expression in animals and support the utility of RNase P ribozyme for gene-targeting applications in vivo.
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Acknowledgments
We are grateful to Dr. Phong Trang for helpful discussion. Yong Bai was partially supported by a predoctoral Block grant from University of California at Berkeley. This research has been supported by NIH (AI041927 and DE014842).
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Bai, Y., Rider, P.J.F., Liu, F. (2010). Catalytic M1GS RNA as an Antiviral Agent in Animals. In: Sioud, M. (eds) RNA Therapeutics. Methods in Molecular Biology, vol 629. Humana Press. https://doi.org/10.1007/978-1-60761-657-3_22
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DOI: https://doi.org/10.1007/978-1-60761-657-3_22
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