Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes.
OBJECTIVE
Lentiviral vectors were designed to obtain efficient transduction of primary quiescent hepatocytes.


METHODS
A hepatitis B virus (HBV) fragment containing enhancers and posttranscriptional regulatory element was used to increase expression levels. The human immunodeficiency virus (HIV) central polypurine tract (PPT) was used to increase transduction of quiescent cells. HBV elements were incorporated downstream and the HIV PPT was incorporated upstream of green fluorescent protein expression cassettes in third generation self inactivating lentiviral vectors.


RESULTS
The HBV fragment increased mean fluorescence of transduced HepG2 hepatoma cells 4.3+/-1.7-fold and 2.3-6.0-fold in various other cell types. A role of HBV x protein in the function of the HBV element was excluded. The HBV element increased the number of transducing units per pg of HIV p24 twofold. The unmodified lentiviral vector transduced 5+/-1% of cultured quiescent primary rat hepatocytes, HBV elements increased transduction to 54+/-13% and increased fluorescence 2.8+/-0.6-fold. The PPT increased transduction to 47+/-11% and increased fluorescence 2.3+/-0.4-fold. The vector with PPT and HBV elements transduced 68+/-10% of hepatocytes and increased fluorescence synergistically, 17+/-6 fold.


CONCLUSIONS
This study shows that HBV elements or HIV PPT are required for efficient transduction of primary hepatocytes.
Seppen
Jurgen
J
Department of Experimental Hepatology, F0-116, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. j.seppen@amc.uva.nl
Rijnberg
Martijn
M
Cooreman
Michel P
MP
Oude Elferink
Ronald P J
RP
D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics; D022781:Hepatocytes / Q000201:enzymology / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008856:Microscopy, Fluorescence; D011685:Purine Nucleotides / Q000235:genetics; D012313:RNA / Q000378:metabolism; D051381:Rats; D015534:Trans-Activators / Q000502:physiology; D054334:Viral Regulatory and Accessory Proteins; D014771:Virion / Q000235:genetics
D011685:Purine Nucleotides; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D012313:RNA; D002222:Carbamoyl-Phosphate Synthase (Ammonia)