Abstract
RNA interference (RNAi) is a specific and efficient method to silence gene expression in mammalian cells. However, genetic manipulation of primary cells including human hepatocytes by RNAi remained challenging. Therefore an efficient gene transfer protocol to modify gene expression in primary cells by using VSV-G-pseudotyped, EGFP-expressing lentiviral vectors was established. The protocol comprises the production of lentiviral vectors as well as the steps for efficient delivery of short-hairpin RNAs (shRNAs), microRNAs, or antagomiRs to human hepatocytes. With this method the amount of preparative work is reduced, by achieving high transduction efficiencies with low multiplicity of infection (MOI). Depending on the laboratory equipment, we provide two alternative workflows. The procedure of lentiviral vector production with subsequent titer determination takes approx. 6–10 working days.
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Acknowledgements
The EGFP-expressing lentiviral vector pLenti6-GFP was kindly provided by Dr. Martin Kriebel, NMI, Reutlingen. We also thank Igor Liebermann and Britta Klumpp, Stuttgart, for expert technical assistance. This study was supported by the German Federal Ministry of Education and Research (Virtual Liver Network Grant 0315755) and by the Robert Bosch Foundation, Stuttgart, Germany.
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Rieger, J.K., Thomas, M. (2016). Introduction of shRNAs, miRNAs, or AntagomiRs into Primary Human Liver Cells Through Lentiviral Vectors. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_6
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DOI: https://doi.org/10.1007/978-1-4939-3753-0_6
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3751-6
Online ISBN: 978-1-4939-3753-0
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