Introduction of shRNAs, miRNAs, or AntagomiRs into Primary Human Liver Cells Through Lentiviral Vectors.
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.
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376, Stuttgart, Germany.|University of Tuebingen, Tuebingen, Germany.
Jessica K
JK
Rieger
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376, Stuttgart, Germany. Maria.Thomas@ikp-stuttgart.de.|University of Tuebingen, Tuebingen, Germany. Maria.Thomas@ikp-stuttgart.de.
Maria
M
Thomas
D000818:Animals; D000070416:Antagomirs / Q000235:genetics; D005822:Genetic Vectors; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*
D000070416:Antagomirs; D035683:MicroRNAs; D034741:RNA, Small Interfering
AntagomiRs; Lentiviral vector transduction; Primary human hepatocytes; RNAi; miRNAs