Abstract
RNA interference has emerged as a powerful technique to down-regulate gene expression. The lentiviral vector-mediated expression of small hairpin RNAs (shRNAs) from polymerase III promoters allows permanent down-regulation of a specific gene in a wide range of cell types both in vitro and in vivo. In this chapter, we describe a method permitting the expression of shRNA from lentiviral vectors in primary murine myogenic cells. We designed shRNAs targeted to the muscular glycogen synthase isoform (shGYS1), a highly regulated enzyme responsible for glycogen synthesis, in order to modulate the muscle glycogen biosynthetic pathway and to improve the phenotype in primary myogenic cells from a murine model of glycogen storage disease type II (GSDII). This method based on shRNA-mediated down-regulation could be applied to other muscular disorders to evaluate new therapeutic options.
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References
Matzke, M.A., Birchler, J.A. (2005) RNAi-mediated pathways in the nucleus. Nat Rev Genet 6, 24–35.
Agrawal, N., Dasaradhi, P.V., Mohmmed, A., Malhotra, P, Bhatnagar, R.K., Mukherjee, S.K. (2003) RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 67, 657–685.
Naldini, L., Blömer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F.H., Verma, I.M., Trono, D. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267.
Zufferey, R., Dull, T., Mandel, R.J., Bukovsky, A., Quiroz, D., Naldini, L., Trono, D. (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72, 9873–9880.
Li, S., Kimura, E., Fall, B.M., Reyes, M., Angello, J.C., Welikson, R., Hauschka, S.D., Chamberlain, J.S. (2005) Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin. Gene Ther 12, 1099–1108.
Ikemoto, M., Fukada, S., Uezumi, A., Masuda, S., Miyoshi, H., Yamamoto, H., Wada, M.R., Masubuchi, N., Miyagoe-Suzuki, Y., Takeda, S. (2007) Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice. Mol Ther 15, 2178–2185.
Richard, E., Douillard-Guilloux, G., Batista, L, Caillaud, C. (2008) Correction of glycogenosis type 2 by muscle-specific lentiviral vector. In Vitro Cell Dev Biol Anim 44, 397–406.
Rubinson, D.A., Dillon, C.P., Kwiatkowski, A.V., Sievers C., Yang, L., Kopinja, J., Rooney, D.L., Zhang, M., Ihrig, M.M., McManus, M.T., Gertler, F.B., Scott, M.L., Van Parijs, L. (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33, 401–406. Erratum in: (2003) Nat Genet 34, 231.
Tiscornia, G., Singer, O., Ikawa, M., Verma, I.M. (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 100, 1844–1848.
Hirschhorn, R., Reuser, A. (2001) Glycogen storage disease type II: acid alpha-glucosidase (acid maltase) deficiency. In The Metabolic and Molecular Bases of Inherited Disease, McGraw Hill, New York, pp 3389–3420.
Kishnani, P.S., Steiner, R.D., Bali, D., Berger, K., Byrne, B.J., Case, L.E., Crowley, J.F., Downs, S., Howell, R.R., Kravitz, R.M., Mackey, J., Marsden, D., Martins, A.M., Millington, D.S., Nicolino, M., O’Grady, G., Patterson, M.C., Rapoport, D.M., Slonim, A., Spencer, C.T., Tifft, C.J., Watson, M.S. (2006) Pompe disease diagnosis and management guideline. Genet Med 8, 267–288.
Raben, N., Nagaraju, K., Lee, E., Kessler, P., Byrne, B., Lee, L., LaMarca, M., King, C., Ward, J., Sauer, B, Plotz P. (1998) Targeted disruption of the acid alpha-glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II. J Biol Chem 7, 53–62.
Bijvoet, AG., van de Kamp, E.H., Kroos, M.A., Ding, J.H., Yang, B.Z., Visser, P., Bakker, C.E., Verbeet, M.P., Oostra, B.A., Reuser, A.J., van der Ploeg, A.T. (1998) Generalized glycogen storage and cardiomegaly in a knockout mouse model of Pompe disease. Hum Mol Genet 7, 53–62.
Bruni, S., Loschi, L., Incerti, C., Gabrielli, O., Coppa, G.V. (2007) Update on treatment of lysosomal storage diseases. Acta Myol 26, 87–92.
Douillard-Guilloux, G., Raben, N., Takikita, S., Batista, L., Caillaud, C., Richard, E. (2008) Modulation of glycogen synthesis by RNA interference: towards a new therapeutic approach for glycogenosis type II. Hum Mol Genet 17, 3876–3886.
Mäkinen, P.I., Koponen, J.K., Kärkkäinen, A.M., Malm, T.M., Pulkkinen, K.H., Koistinaho, J., Turunen, M.P., Ylä-Herttuala, S. (2006) Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain. J Gene Med 8, 433–441.
Naito, Y., Yamada, T., Ui-Tei, K., Morishita, S., Saigo, K. (2004) siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res 32, W124–W129.
Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki H., Juni, A., Ueda, R., Saigo, K. (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32, 936–948.
Ohanna, M., Sobering, A.K., Lapointe, T., Lorenzo, L., Praud, C., Petroulakis, E., Sonenberg, N., Kelly, P.A., Sotiropoulos, A., Pende, M. (2005) Atrophy of S6K1(−/−) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control. Nat Cell Biol 7, 286–294.
Acknowledgments
This work was supported by INSERM and the Association Vaincre les Maladies Lysosomales (VML). ER was supported by postdoctoral fellowships from VML and the Association Française contre les Myopathies (AFM). GD was supported by doctoral fellowship from Genzyme (France) and AFM.
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Richard, E., Douillard-Guilloux, G., Caillaud, C. (2011). Lentiviral Vector Delivery of shRNA into Cultured Primary Myogenic Cells: A Tool for Therapeutic Target Validation. In: Duan, D. (eds) Muscle Gene Therapy. Methods in Molecular Biology, vol 709. Humana Press. https://doi.org/10.1007/978-1-61737-982-6_14
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DOI: https://doi.org/10.1007/978-1-61737-982-6_14
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