We produced VSVg pseudotyped lentiviral vectors expressing the murine Fah complementary DNA (cDNA)

from an internal SFFV promoter. The enhanced green fluorescence protein (eGFP) was coexpressed either by an internal ribosomal entry site (IRES) or by a 2A proteolytic cleveage site (RRL.PPT.SFFV.eGFP.pre*, RRL.PPT.SFFV.Fah.ires.eGFP.pre*, RRL.PPT.SFFV.Fah.2a.eGFP.pre*).29 The vector supernatants were produced by 293T cells, concentrated with Centricon Plus-70 filter units http://www.selleckchem.com/products/Adriamycin.html (Millipore, Schwalbach, Germany) and titrated on HepA1.6 cells.30 Viral titers were in the range of 107 to 108 IU/mL. Hepatocytes were isolated from 3 to 6-month-old mice as described.6 For further enrichment of hepatocytes we used discontinuous Percoll (GE Healthcare) gradients. A 25% phase prevented dead cells and debris from entering the gradient. A lower 50% phase facilitated the enrichment of small, highly viable, and robust hepatocytes. For depletion of nonparenchymal liver cells, we used PE (Phycoerythrin)-labeled anti-CD45 and anti-CD31 antibodies (BD Pharmingen), anti-PE MicroBeads on an automated MACS Separator (Miltenyi Biotech, Bergisch Gladbach). Analysis of eGFP expression was performed on day 5 after in vitro transduction of primary murine hepatocytes

or directly after hepatocyte isolation during serial transplantations by FACS. For in vitro experiments, hepatocytes were cultured on Primaria dishes (1.1 × 106 cells/35 cm2) in HCM medium (Lonza). Fah(-/-) hepatocytes isolated from C57Bl/6-Fahtm1Mgo were cultured in the presence of NTBC (9.6 ng/mL) BTK inhibitors high throughput screening (Swedish Orphan). Transduction (multiplicity of infection [MOI] 10) was performed as previously described by our group.6 RNA from hepatocytes was hybridized on the Affymetrix Mouse 430 2.0 GeneChip.

Data can be found at Geo link http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE34731. LM-PCR was performed and analyzed as described.31, 32 (See also Supporting Material and Methods.) C57Bl/6-Fahtm1Mgo (Fah(-/-)) mice are defective for the Fah gene, which encodes the enzyme fumaryacetoacetate hydrolase (Fah).25 Mice were maintained by supplementation of drinking water with NTBC (4 mg mL-1, Swedish Orphan International).26, selleckchem 27 To allow engraftment of hepatocytes the medication was discontinued For in vivo gene transfer, lentiviral particles were injected in 250 μL phosphate-buffered saline (PBS) intrasplenically (∼1 infectious particle / parenchymal liver cell). For ex vivo gene therapy 1 × 106 cultured Fah(-/-) hepatocytes were transduced overnight (MOI 10). For serial hepatocyte transplantation the livers from gene-corrected mice were perfused with collagenase. Aliquots of 0.5 × 106 liver cells were injected in 250 μL PBS into the next generation of Fah(-/-) mice.