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

Centlivre, Mireille; Zhou, Xue; Pouw, Stephan M.; Weijer, Kees; Kleibeuker, Wendy; Das, Atze T.; Blom, Bianca; Seppen, Jurgen; Berkhout, Ben; Legrand, Nicolas

doi:10.1038/gt.2009.109

Cited by 28 publications

(26 citation statements)

References 44 publications

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“…To make the pCHMWS series lentivector constructs, wtCD38-EGFP, EGFP-sCD38, and EGFP-sCD38(DM) were subcloned from the corresponding pEGFP vector into pCHMWS-EGFP, which was a gift from the Pasteur Research Centre of The University of Hong Kong. For the tetracycline-inducible expression lentivector, EGFP-sCD38 and EGFP-sCD38(DM) were amplified by PCR and ligated with AgeI/PstI into TREAuto-V14, which was a gift from N. Legrand (University of Amsterdam) (34). pDsRed was purchased from Clontech.…”

Section: Methodsmentioning

confidence: 99%

“…This tetracycline-inducible vector is an improvement over the widely used commercial Tet-On system. It contains an optimized reverse tetracycline-responsive transactivator and combines all the elements required for Tet-On regulation into a single cassette with an autoregulatory loop for the expression of reverse tetracycline-responsive transactivator (34,47). The autoregulatory expression of the reverse tetracycline-responsive transactivator was shown to be superior to the commercial Tet-On system, resulting in higher viral titers, low background, improved induction kinetics, and increased induction levels (47).…”

Section: Lane)mentioning

confidence: 99%

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Cytosolic CD38 Protein Forms Intact Disulfides and Is Active in Elevating Intracellular Cyclic ADP-ribose

Zhao

Zhang

Lam

et al. 2011

Journal of Biological Chemistry

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CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca 2؉ messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca 2؉ signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254 -Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism. Mobilization of Ca2ϩ from intracellular stores is of fundamental importance in virtually all aspects of cellular activity. The major intracellular Ca 2ϩ stores are in the endoplasmic reticulum (ER) 2 and are mobilized by specific messenger molecules, inositol trisphosphate and cyclic ADP-ribose (cADPR). The latter is a novel cyclic nucleotide derived from NAD. It was first described in sea urchin eggs (1, 2) but has since been established as a second messenger molecule responsible for regulating a wide range of physiological functions as diverse as abscisic acid signaling in plants (3) and sponges (4) and social behavior in mice (Ref. 5; reviewed in Refs. 6 and 7). It targets the ryanodine receptor of the endoplasmic Ca 2ϩ stores. The synthesis and hydrolysis of cADPR in mammalian cells are catalyzed by CD38 (8), a transmembrane protein ubiquitously expressed in virtually all tissues (reviewed in Ref. 9). Gene knock-out studies have established that CD38 plays a critical role in a wide range of physiological functions, including insulin secretion (10), susceptibility to bacterial infection (11), and social behavior of mice through modulating neuronal oxytocin secretion (5).CD38 is a membrane protein with a short N-terminal tail, a single transmembrane segment, and a large C-terminal domain containing all the enzymatic activities (8, 12, 13). The crystal structure of the catalytic domain of CD38 has been solved, and the mechanism of how it catalyzes the multiple reactions responsible for metabolizing cADPR has also been elucidated to atomic resolution by x-ray crystallography (14 -16). It is established that the s...

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Section: Methodsmentioning

confidence: 99%

Section: Lane)mentioning

confidence: 99%

Cytosolic CD38 Protein Forms Intact Disulfides and Is Active in Elevating Intracellular Cyclic ADP-ribose

Zhao

Zhang

Lam

et al. 2011

Journal of Biological Chemistry

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“…The major subsets of the human immune system in the blood are also analyzed for their frequency and absolute number in the human GFP+ and GFP-population. Adapted from [24,70] . a P < 0.05.…”

Section: Cytotoxicity In Vivomentioning

confidence: 99%

Selection of RNAi-based inhibitors for anti-HIV gene therapy

Knoepfel

Centlivre²,

Liu³

et al. 2012

WJV

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In the last decade, RNA interference (RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAibased inhibitors against the chronic infection with human immunodeficiency virus type 1 (HIV-1). A lentiviral gene therapy is proposed for HIV-infected patients that will protect and reconstitute the vital immune cell pool. The RNAi-based inhibitors that have been developed are short hairpin RNA molecules (shRNAs), of which multiple are needed to prevent viral escape. In ten distinct steps, we describe the selection process that started with 135 shRNA candidates, from the initial design criteria, via testing of the in vitro and in vivo antiviral activity and cytotoxicity to the final design of a combinatorial therapy with three shRNAs. These shRNAs satisfied all 10 selection criteria such as targeting conserved regions of the HIV-1 RNA genome, exhibiting robust inhibition of HIV-1 replication and having no impact on cell physiology. This combinatorial shRNA vector will soon move forward to the first clinical studies.

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“…For H2B-GFP pulse-chase experiments, doxycycline was incorporated in food (2 g/kg; Harlan Laboratories) or in drinking water (2 mg/ml doxycycline supplemented with 5% sucrose) (27,28). All procedures were in accordance with the Canadian Council on Animal Care guidelines and approved by the Comité de Déontologie et Expérimentation Animale de l'Université de Montréal.…”

Section: Micementioning

confidence: 99%

Adult Thymic Epithelium Contains Nonsenescent Label-Retaining Cells

Dumont-Lagacé

Brochu

St-Pierre

et al. 2014

The Journal of Immunology

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Progress in our understanding of thymic epithelial cell (TEC) renewal and homeostasis is hindered by the lack of markers for TEC progenitors. Stem and progenitor cell populations display remarkable diversity in their proliferative behavior. In some but not all tissues, stemness is associated with quiescence. The primary goal of our study was to discover whether quiescent cells were present in neonatal and adult TECs. To this end, we used a transgenic label-retaining cell (LRC) assay in which a histone H2B-GFP fusion protein is expressed under the control of the reverse tetracycline-controlled transactivator and the tetracycline operator minimal promoter. In adult mice, we found that both cortical and medullary TECs (cTECs and mTECs) proliferated more actively in females than males. Moreover, we observed three main differences between neonatal and adult TECs: 1) neonatal TECs proliferated more actively than adult TECs; 2) whereas cTECs and mTECs had similar turnover rates in young mice, the turnover of mTECs was more rapid than that of cTECs in adults; and 3) although no LRCs could be detected in young mice, LRCs were detectable after a 16-wk chase in adults. In female mice, LRCs were found almost exclusively among cTECs and expressed relatively low levels of p16INK4a, p19ARF, and Serpine1, and high levels of Bmi1, Foxn1, Trp63, and Wnt4. We conclude that LRCs in adult TECs are not senescent postmitotic cells and may represent the elusive progenitors responsible for TEC maintenance in the adult thymus.

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

Cited by 28 publications

References 44 publications

Cytosolic CD38 Protein Forms Intact Disulfides and Is Active in Elevating Intracellular Cyclic ADP-ribose

Cytosolic CD38 Protein Forms Intact Disulfides and Is Active in Elevating Intracellular Cyclic ADP-ribose

Selection of RNAi-based inhibitors for anti-HIV gene therapy

Adult Thymic Epithelium Contains Nonsenescent Label-Retaining Cells

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