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High efficiency transduction and stable gene expression of primary human hepatocytes with lentiviral vectors
Author(s) -
Zamule Stephanie M.,
Strom Stephen C.,
Omiecinski Curtis J.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a442
Subject(s) - transduction (biophysics) , biology , viral vector , green fluorescent protein , hepatocyte , microbiology and biotechnology , cell culture , gene expression , gene , rna interference , transgene , virology , genetics , rna , in vitro , biochemistry , recombinant dna
Lentiviral vectors are retroviral‐derived constructs that effectively transduce mammalian cells and integrate stably into the host's genome. We hypothesized that methodologies could be developed enabling the use of lentivectors for the genetic manipulation of primary human hepatocytes in non‐dividing cultures. Infection of hepatocytes with green fluorescence protein (GFP)‐expressing lentivectors (MOI=5) resulted in >80% GFP positive cells, with GFP expression retained for >2 weeks in primary culture. Real‐time RT‐PCR analyses demonstrated that lentivectors engineered to express siRNAs successfully knocked down specific nuclear receptor targets. Further, lentiviral infection did not result in detectable alteration of assessed hepatocyte expression markers, including the phenobarbital (PB) induction response – a hallmark feature of the differentiated hepatocyte. Real‐time RT‐PCR indicated that 500 uM PB treatment produced ~20‐fold induction of both CYP2B6 and CYP3A4 mRNA transcripts, independent of viral infection status. In summary, we demonstrate that lentiviral methodology enables highly efficient transduction and stable gene expression in primary human hepatocytes. These approaches establish a powerful platform for conducting toxicological and pharmacological research in a culture model, and offer the potential for clinical cell‐based therapeutics to treat hepatic disease.