Preservation of hepatic phenotype in lentiviral-transduced primary human hepatocytes.
Lentiviral vectors effectively transduce both dividing and non-dividing cells and stably integrate into the genome of the host cell. In this study, we evaluated the usefulness of a lentiviral system for genetic modulation of primary human hepatocyte cultures. Infection with GFP-expressing lentivectors shows that Huh7 and HepG2 cell lines, as well as primary cultures of human hepatocytes, are efficiently transduced by lentiviral vectors. Real-time RT-PCR analyses demonstrate that infection with lentivectors does not alter hepatic hallmarks such as the expression of the nuclear receptors CAR, PXR, RXR alpha, or HNF4 alpha, or expression of the secretory protein, albumin. Additionally, infected hepatocytes retain the capacity for CYP3A4 induction in response to treatment with phenobarbital, a uniquely sensitive indicator of hepatic differentiation status. Lentivectors may be used for both over-expression and knockdown analyses in primary hepatocytes, as demonstrated in this study by >200-fold CAR over-expression and knockdown of CAR to less than 40% of endogenous levels, with corresponding effects on CYP2B6 expression. In summary, lentiviral vectors provide a novel methodology by which primary human hepatocytes may be stably genetically manipulated, with minimal effects on the differentiated hepatic phenotype. These approaches offer considerable advantage over current methodologies, providing a valuable alternative for use in pharmacological and toxicological investigations involving primary human hepatocyte models and potentially for cell-based therapeutics to treat hepatic dysfunction in vivo.
Zamule
Stephanie M
SM
Center for Molecular Toxicology & Carcinogenesis and Department of Veterinary & Biomedical Sciences, Pennsylvania State University, 101 Life Sciences Building, University Park, PA 16802, USA.
Strom
Stephen C
SC
Omiecinski
Curtis J
CJ
D000328:Adult; D000368:Aged; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics; D022781:Hepatocytes / Q000502:physiology* / Q000821:virology; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics; D010641:Phenotype; D000077297:Pregnane X Receptor; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000096:biosynthesis; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics; D047490:Retinoid X Receptor alpha / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012709:Serum Albumin / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D014161:Transduction, Genetic
D000090702:Constitutive Androstane Receptor; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D047490:Retinoid X Receptor alpha; D012709:Serum Albumin; D014157:Transcription Factors; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating