Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes

Seppen, Jurgen; Rijnberg, Martijn; Cooreman, Michael P.; Elferink, Ronald P.J. Oude

doi:10.1016/s0168-8278(01)00308-7

Cited by 114 publications

(107 citation statements)

References 34 publications

Supporting

Mentioning

104

Contrasting

Order By: Relevance

“…11 These two constructs belong to the third generation of lentiviral selfinactivating vectors, 21,22 with the central polypurine tract for enhanced transduction efficiency and the hepatitis B virus post-transcriptional regulatory element for enhanced gene expression. 23 In these two vectors, the rtTA2 S -S2 (TRECMVR2) and rtTA3 (TREAutoR3) were replaced by the improved rtTA-V14 variant that shows increased dox sensitivity and contributes to better viral replication in the HIV-rtTA backbone, as compared with rtTA2 S -S2. 10 However, the two elements of the Tet-On system (TRE and rtTA) are combined in a different manner in these vectors.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

et al. 2009

View full text Add to dashboard Cite

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated 'human immune system' (HIS) mice by transplanting newborn BALB/c Rag2 À/À IL-2Rg c À/À immunodeficient mice with human hematopoietic stem cells transduced with a doxycyclineinducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

show abstract

Section: Resultsmentioning

confidence: 99%

“…23,24 The virus stocks were concentrated using Amicon Ultra concentrator, MWCO 100 000 (Millipore Corporation, Bedford, MA, USA), and aliquots of 0.4 mL were stored at À80 1C. Virus production was determined by CA-p24 enzyme-linked immunosorbent assay.…”

Section: Lentiviral Vector Preparation and Virus Titer Determinationmentioning

confidence: 99%

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

et al. 2009

View full text Add to dashboard Cite

show abstract

“…Plasmids encoding for short hairpin RNA (shRNA) against human SLC4A2 (AE2) or nontargeting shRNA, and containing a puromycin resistance gene, were obtained from the Sigma Mission shRNA library (Sigma-Aldrich, St. Louis, MO), and recombinant lentivirus was produced as previously described. 36 For lentiviral transduction, H69 cells were grown to 30%-40% confluence and incubated with virus-containing supernatants/DMEM (1:1) supplemented with 10 lg/mL of diethylaminoethyl-dextran for 4 hours. Subsequently, medium was refreshed, cells were cultured for 48 hours, and then selected for cells that integrated the viral DNA by adding 10 ug/mL of puromycin.…”

Section: Methodsmentioning

confidence: 99%

A biliary HCO₃⁻umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes

et al. 2011

View full text Add to dashboard Cite

Human cholangiocytes are continuously exposed to millimolar levels of hydrophobic bile salt monomers. We recently hypothesized that an apical biliary HCO À 3 umbrella might prevent the protonation of biliary glycine-conjugated bile salts and uncontrolled cell entry of the corresponding bile acids, and that defects in this biliary HCO À 3 umbrella might predispose to chronic cholangiopathies. Here, we tested in vitro whether human cholangiocyte integrity in the presence of millimolar bile salt monomers is dependent on (1) pH, (2) adequate expression of the key HCO À 3 exporter, anion exchanger 2 (AE2), and (3) an intact cholangiocyte glycocalyx. To address these questions, human immortalized cholangiocytes and cholangiocarcinoma cells were exposed to chenodeoxycholate and its glycine/taurine conjugates at different pH levels. Bile acid uptake was determined radiochemically. Cell viability and apoptosis were measured enzymatically. AE2 was knocked down by lentiviral short hairpin RNA. A cholangiocyte glycocalyx was identified by electron microscopy, was enzymatically desialylated, and sialylation was quantified by flow cytometry. We found that bile acid uptake and toxicity in human immortalized cholangiocytes and cholangiocarcinoma cell lines in vitro were pH and AE2 dependent, with the highest rates at low pH and when AE2 expression was defective. An apical glycocalyx was identified on cholangiocytes in vitro by electron microscopic techniques. Desialylation of this protective layer increased cholangiocellular vulnerability in a pH-dependent manner. Conclusion: A biliary HCO À 3 umbrella protects human cholangiocytes against damage by bile acid monomers. An intact glycocalyx and adequate AE2 expression are crucial in this process. Defects of the biliary HCO À 3 umbrella may lead to the development of chronic cholangiopathies.

show abstract

“…cDNAs were cloned into a phosphoglycerate kinase promoter-containing lentiviral transfer vector, and a recombinant lentivirus was produced as described. 25 Lentiviral Transduction of Cells and Preparation of Cell Lysates. UPS-1 or WIF-B9 cells, grown to 50%-60% confluence, were incubated with virus-containing supernatants/DMEM (1:1) supplemented with 10 g/mL diethylaminoethyl-dextran for 4 hours.…”

Section: Methodsmentioning

confidence: 99%

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

et al. 2007

View full text Add to dashboard Cite

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1. Previously, we have shown in mice that Atp8b1 deficiency leads to enhanced biliary excretion of phosphatidylserine, and we hypothesized that ATP8B1 is a flippase for phosphatidylserine. However, direct evidence for this function is still lacking. In Saccharomyces cerevisiae, members of the Cdc50p/Lem3p family are essential for proper function of the ATP8B1 homologs. We have studied the role of two human members of this family, CDC50A and CDC50B, in the routing and activity of ATP8B1. When only ATP8B1 was expressed in Chinese hamster ovary cells, the protein localized to the endoplasmic reticulum. Coexpression with CDC50 proteins resulted in relocalization of ATP8B1 from the endoplasmic reticulum to the plasma membrane. Only when ATP8B1 was coexpressed with CDC50 proteins was a 250%-500% increase in the translocation of fluorescently labeled phosphatidylserine observed. Importantly, natural phosphatidylserine exposure in the outer leaflet of the plasma membrane was reduced by 17%-25% in cells coexpressing ATP8B1 and CDC50 proteins in comparison with cells expressing ATP8B1 alone. The coexpression of ATP8B1 and CDC50A in WIF-B9 cells resulted in colocalization of both proteins in the canalicular membrane. Conclusion: Our data indicate that CDC50 proteins are pivotal factors in the trafficking of ATP8B1 to the plasma membrane and thus may be essential determinants of ATP8B1-related disease. In the plasma membrane, ATP8B1 functions as a flippase for phosphatidylserine. Finally, CDC50A may be the potential ␤-subunit or chaperone for ATP8B1 in hepatocytes. (HEPATOLOGY 2008;47: 268-278.)

show abstract

Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes

Cited by 114 publications

References 34 publications

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

A biliary HCO₃⁻umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

Contact Info

Product

Resources

About

Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes

Cited by 114 publications

References 34 publications

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

A biliary HCO3−umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

Contact Info

Product

Resources

About

A biliary HCO₃⁻umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes