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Introduction of shRNAs, miRNAs or anti-microRNAs into primary human hepatocytes with lentivirus

Created: 4th Apr 2011 at 17:06
Last updated: 21st Jan 2013 at 12:16
Last used: 24th Aug 2019 at 02:51

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Here we used VSV-G-pseudotyped, EGFP-expressing lentiviral vectors to develop an efficient gene transfer protocol to modify gene expression in primary human hepatocytes (by RNAi). The protocol comprises the production of recombinant viruses as well as the steps for efficient delivery of short-hairpin RNA (shRNAs), microRNAs or anti-microRNAs to human hepatocytes. On average infection efficiencies of over 95% are achieved at relatively low multiplicity of infection (MOI), which effectively reduces the amount of preparative work required per experiment. Depending on the laboratory equipment available, we provide here two alternative workflows, which can be easily adapted in the lab. The procedure of virus production with subsequent titer determination takes approx. 6 to 10 working days. The procedure of viral infection of hepatocytes until effects can be measured takes approx. 3 to 5 days. This protocol should be helpful to study many aspects of functional genomics in primary human hepatocytes.

Filename: Lentiviral production and infection_SOP_04042011.pdf

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