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1.
Methods Mol Biol ; 434: 291-309, 2008.
Article in English | MEDLINE | ID: mdl-18470652

ABSTRACT

Drug-inducible systems allowing the control of transgene expression and knockdown in mammalian cells are invaluable tools for genetic research, and could also play important roles in translational research or gene therapy. We and others have developed a lentivector-based, conditional gene expression system for drug-controllable expression of transgenes and small hairpin RNAs (shRNAs). This system is highly robust and versatile, governing tightly controlled expression of transgenes and endogenous cellular genes (through shRNAs) in various primary and established cell lines in vitro, as well as in vivo in the central nervous system or in human cancer cells xenotransplanted into nude mice. The goal of this article is to provide a concise methodology for construction and manipulation of this conditional lentiviral-based system, and quantitative analyses of drug-inducible transgene expression and gene knockdown both in vitro and in vivo.


Subject(s)
Breast Neoplasms/therapy , Gene Silencing , Gene Targeting/methods , Genetic Vectors/genetics , Lentivirus/genetics , RNA, Small Interfering/genetics , Animals , Breast Neoplasms/genetics , Doxycycline/pharmacology , Female , Gene Expression Regulation, Neoplastic , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice , Mice, Nude , Transfection , Transgenes/physiology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays
2.
J Neurochem ; 104(4): 1101-15, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17986227

ABSTRACT

Delta-like 1 (Dlk1), a member of the Delta/Notch protein family, is expressed in the mouse ventral midbrain (VM) as early as embryonic day 11.5 (E11.5) followed by exclusive expression in tyrosine 3-monooxygenase (TH) positive neurons from E12.5 onwards. To further elucidate the yet unknown function of Dlk1 in VM neuron development, we investigated the effect of soluble Dlk1 protein as well as the intrinsic Dlk1 function in the course of VM progenitor expansion and dopaminergic (DA) neuron differentiation in vitro. Dlk1 treatment during expansion increased DA progenitor proliferation and the proportion of NR4A2+ neurons expressing TH after differentiation, whereas Dlk1 treatment during the course of DA precursor differentiation did not alter TH+ neuron counts. In contrast, silencing of endogenously expressed Dlk1 prior to DA precursor differentiation partially prevented the expression of DA neuron markers, which was not accompanied with alteration of overall or local proliferation. Due to the latter finding in combination with the absence of Dlk1 negative DA neurons in differentiated cultures, we suggest that Dlk1 expression might have a permissive effect on DA neuron differentiation in vitro. The study presented here is the first publication identifying Dlk1 effects on ventral midbrain-derived DA precursor differentiation.


Subject(s)
Cell Differentiation/physiology , Dopamine/physiology , Intracellular Signaling Peptides and Proteins/physiology , Membrane Proteins/physiology , Mesencephalon/metabolism , Neurons/metabolism , Stem Cells/metabolism , Animals , Cell Proliferation , Cells, Cultured , Dopamine/genetics , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Mesencephalon/cytology , Mice , NIH 3T3 Cells , Neurons/cytology , Pregnancy , Stem Cells/cytology
3.
J Biol Chem ; 282(47): 34535-41, 2007 Nov 23.
Article in English | MEDLINE | ID: mdl-17893143

ABSTRACT

The Krüppel-associated box (KRAB) domain is a transcriptional repression module responsible for the DNA binding-dependent gene silencing activity of hundreds of vertebrate zinc finger proteins. We previously exploited KRAB-mediated repression within the context of a tet repressor-KRAB fusion protein and of lentiviral vectors to create a method of external gene control. We demonstrated that with this system transcriptional silencing was fully reversible in cell culture as well as in vivo. Here we reveal that, in sharp contrast, KRAB-mediated repression results in irreversible gene silencing through promoter DNA methylation if it acts during the first few days of mouse development.


Subject(s)
Carrier Proteins/metabolism , DNA Methylation , Embryonic Development/physiology , Nuclear Proteins/metabolism , Promoter Regions, Genetic/physiology , Repressor Proteins/metabolism , Transcription, Genetic/physiology , Animals , Carrier Proteins/genetics , Gene Silencing/physiology , Lentivirus , Mice , Nuclear Proteins/genetics , Protein Structure, Tertiary/genetics , Repressor Proteins/genetics
4.
J Neurosci Methods ; 163(2): 338-49, 2007 Jul 30.
Article in English | MEDLINE | ID: mdl-17397931

ABSTRACT

Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.


Subject(s)
Gene Transfer Techniques/standards , Genetic Therapy/methods , Genetic Vectors/genetics , Lentivirus/genetics , Stem Cells/metabolism , Animals , Astrocytes/metabolism , Astrocytes/virology , Biomarkers/metabolism , Brain Tissue Transplantation/methods , Cell Differentiation/genetics , Cell Proliferation , Cells, Cultured , Fetus , Glial Cell Line-Derived Neurotrophic Factor/biosynthesis , Glial Cell Line-Derived Neurotrophic Factor/genetics , Humans , Neurons/metabolism , Neurons/virology , Parkinson Disease/genetics , Parkinson Disease/therapy , Promoter Regions, Genetic/genetics , Rats , Rats, Inbred Lew , Spheroids, Cellular/cytology , Spheroids, Cellular/physiology , Spheroids, Cellular/virology , Stem Cells/virology , Transgenes
5.
Nat Methods ; 3(9): 682-8, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16929312

ABSTRACT

RNA interference (RNAi) has emerged as a powerful tool to downregulate the expression of specific genes. Drug-inducible systems allowing for conditional RNAi that offer the unique potential to modulate expression of virtually any endogenous gene in the cell have been recently developed. Their applications are very broad, ranging from basic studies of gene function to translational research including modeling of human diseases, analysis of potential side effects of candidate drugs, testing of gene-based therapies and loss-of-function screens. Here we summarize the state of the art of systems allowing for drug-controllable knockdown, and provide a description of their current and future applications.


Subject(s)
RNA Interference , RNA, Small Interfering/genetics , Genetic Vectors , Humans , Lentivirus/genetics , RNA Polymerase III/metabolism
6.
Nat Methods ; 3(2): 109-16, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16432520

ABSTRACT

Drug-inducible systems allowing the control of gene expression in mammalian cells are invaluable tools for genetic research, and could also fulfill essential roles in gene- and cell-based therapy. Currently available systems, however, often have limited in vivo functionality because of leakiness, insufficient levels of induction, lack of tissue specificity or prohibitively complicated designs. Here we describe a lentiviral vector-based, conditional gene expression system for drug-controllable expression of polymerase (Pol) II promoter-driven transgenes or Pol III promoter-controlled sequences encoding small inhibitory hairpin RNAs (shRNAs). This system has great robustness and versatility, governing tightly controlled gene expression in cell lines, in embryonic or hematopoietic stem cells, in human tumors xenotransplanted into nude mice, in the brain of rats injected intraparenchymally with the vector, and in transgenic mice generated by infection of fertilized oocytes. These results open up promising perspectives for basic or translational research and for the development of gene-based therapeutics.


Subject(s)
Gene Expression Regulation/genetics , Gene Targeting/methods , RNA Interference , Animals , Bacterial Proteins/genetics , Brain/metabolism , Carrier Proteins/genetics , Cell Differentiation/genetics , Cell Line , Cell Line, Tumor , DNA Polymerase II/genetics , DNA Polymerase III/genetics , Doxycycline/pharmacology , Female , GATA1 Transcription Factor/genetics , Gene Expression Regulation/drug effects , Gene Silencing , Genetic Engineering/methods , Genetic Vectors/genetics , Glial Cell Line-Derived Neurotrophic Factor/genetics , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Lentivirus/genetics , Male , Mice , Mice, Inbred Strains , Mice, Nude , Mice, Transgenic , Promoter Regions, Genetic/genetics , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Rats, Wistar , Repressor Proteins/genetics , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/metabolism , Transfection , Transgenes/genetics , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
7.
Nat Med ; 11(4): 423-8, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15768028

ABSTRACT

Mutations in Cu/Zn superoxide dismutase (encoded by SOD1), one of the causes of familial amyotrophic lateral sclerosis (ALS), lead to progressive death of motoneurons through a gain-of-function mechanism. RNA interference (RNAi) mediated by viral vectors allows for long-term reduction in gene expression and represents an attractive therapeutic approach for genetic diseases characterized by acquired toxic properties. We report that in SOD1(G93A) transgenic mice, a model for familial ALS, intraspinal injection of a lentiviral vector that produces RNAi-mediated silencing of SOD1 substantially retards both the onset and the progression rate of the disease.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , RNA Interference , Superoxide Dismutase/genetics , Animals , Disease Models, Animal , Disease Progression , Genetic Vectors , Humans , Lentivirus , Mice , Mice, Transgenic , Molecular Sequence Data , Mutation , RNA, Small Interfering
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