title: Targeting nuclear receptors with lentivirus-delivered small RNAs in primary human hepatocytes.
abstract: BACKGROUND
  RNA interference (RNAi) has tremendous potential for investigating gene function and for developing new therapies. Primary human hepatocytes (PHH) are the "gold standard" for studying the regulation of hepatic metabolism in vitro. However, application of RNAi in PHH has some technical hurdles. The objective of this study was to develop effective and robust protocol for transduction of PHH with lentiviral vectors.
  
  
  METHODS
  We used lentiviral vectors to transduce PHH for introduction of short hairpin RNAs (shRNAs) targeting constitutive androstane receptor (CAR), peroxisome proliferator activated receptor alpha (PPARα), and microRNA, miR-143. Infection efficiency was quantitatively analyzed by flow cytometry and microscopy. Target gene expression was assessed using quantitative real-time (qRT-PCR) method.
  
  
  RESULTS
  Lentiviral vector transduction resulted in ≥95% of infected cells at low multiplicity of infection (MOI) of 3, which did not impair cellular viability. We demonstrated the feasibility of this technique in studies on targeting nuclear receptors, PPARα and CAR, with shRNAs as well as in lentivirus-mediated overexpression and knock-down of miRNA-143 experiments.
  
  
  CONCLUSIONS
  We developed an efficient and robust protocol with standardized procedures for virus production, method of titer determination, and infection procedure for RNAi in primary human hepatocytes based on delivery of shRNAs, microRNAs or anti-microRNAs in different laboratory settings. This approach should be useful to study not only the regulation via nuclear receptors but also other biological, pharmacological, and toxicological aspects of drug metabolism.
authors.forename: Maria
authors.initials: M
authors.lastname: Thomas
authors.forename: Jessica K
authors.initials: JK
authors.lastname: Rieger
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Kandel
authors.forename: Kathrin
authors.initials: K
authors.lastname: Klein
authors.forename: Ulrich M
authors.initials: UM
authors.lastname: Zanger
mesh_terms: D045744:Cell Line, Tumor; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008856:Microscopy, Fluorescence; D047493:PPAR alpha / Q000235:genetics; D061251:Primary Cell Culture; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic
chemical_list: D000090702:Constitutive Androstane Receptor; D047493:PPAR alpha; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins