Abstract
Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.
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Acknowledgments
The authors thank past and present members of the laboratory whose work has contributed to studies that are described in this chapter. This research was supported by the Deutsche Forschungsgemeinschaft (STA357/7-1), the Wilhelm Sander-Stiftung (2016.087.1), the Kompetenznetzwerk Zytomegalie Baden-Württemberg (KSKV002), and the International Graduate School Ulm (IGradU). Anne-Charlotte Stilp and Patrick König contributed equally to this work.
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Stilp, AC., König, P., Scherer, M., Stamminger, T. (2021). Stable and Inducible Gene Knockdown in Primary Human Fibroblasts: A Versatile Tool to Study the Role of Human Cytomegalovirus Host Cell Factors. In: Yurochko, A.D. (eds) Human Cytomegaloviruses. Methods in Molecular Biology, vol 2244. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1111-1_7
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DOI: https://doi.org/10.1007/978-1-0716-1111-1_7
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