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Production of Cloned Calves Using Roscovitine-Treated Adult Somatic Cells as Donors

The stage of the donor cell cycle is a major factor in the success of cloning. Quiescent cells arrested in the G0/G1 phases of the cell cycle by either serum starvation or growth arrest when cultured cells reach confluence have been used as donors to produce cloned animals. Recently, we have developed a nov ...

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Optimization of Procedures for Cloning by Somatic Cell Nuclear Transfer in Mice

Cloning by somatic cell nuclear transfer is a complex procedure that is dependent on correct interactions between oocyte and donor cell genome. These interactions require minimal insult to either the oocyte or the transplanted nucleus. Available data also indicate that reprogrammi ...

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A Double Nuclear Transfer Technique for Cloning Pigs

The first round of double nuclear transfer (NT) procedure includes the following steps: transfer of somatic cell nuclei into enucleated recipient oocytes, fusion, activation, and culture of reconstructed oocytes. The next day, a second round of NT is performed by removing karyoplasts fr ...

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Nuclear Reprogramming: An Overview

Nuclear reprogramming is an intriguing phenomenon, in which specialized somatic cells reacquire pluripotency through the global resetting of epigenetic modifications without changes occurring to their nuclear DNA information. The nuclear reprogramming activity reta ...

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Nuclear Remodeling Assay in Xenopus Egg Extract

Xenopus egg extract is an ideal material to identify nuclear remodeling activities important for nuclear cloning. Since the protocol for egg extract preparation was established more than 20 yr ago, egg extract has been widely used as a source for the purification of factors associated with a nu ...

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In Vitro Reprogramming of Nuclei and Cells

Directly turning a somatic cell type into another would be beneficial for producing replacement cells for therapeutic purposes. To this end, novel cell reprogramming strategies are being developed. We describe here methods for functionally reprogramming a somatic cell using an ext ...

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Techniques for Nuclear Transfer to Mouse Embryonic Stem Cells

In this chapter, methods are described that permit the enucleation of mouse embryonic stem (ES) cells and the transfer of donor nuclei to these cells before or after enucleation has taken place. The small size and high nucleus-to-cytoplasm volume ratio of ES cells poses a challenge to their enucle ...

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Gene Expression and Methylation Patterns in Cloned Embryos

A considerable proportion of the offspring, in particular in ruminants and mouse, born from nuclear transfer (NT)-derived and in vitro-produced (IVP) embryos are affected by multiple abnormalities, of which a high birthweight and an extended gestation length are the predominant feat ...

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Are There Any Normal Clones

An exhaustive study of the fidelity of a clone to its parent is prohibitive because of cost and the necessary scope of experimental design. Therefore, these data must be gathered from existing observational evidence. This in itself cannot provide a definitive accounting of the abnormaliti ...

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The US and EU Regulatory Perspectives on the Clinical Use of Hematopoietic Stem/Progenitor Cells Genetically Modified Ex Vivo by Retroviral Vectors

A primary safety issue presented by human hematopoietic stem cells/progenitor cells (HS/PC) genetically modified by gammaretroviral or lentiviral vectors is the risk of oncogenesis. This risk is a potential consequence of either of the following events: (a) the possible unintended g ...

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Database Setup for Preclinical Studies of Gene-Modified Hematopoiesis

Murine safety studies are routinely used for gathering preclinical safety and efficacy data and, for Phase I studies, Good Laboratory Practice (GLP) compliance is not mandated. However, extensive amounts of data must be gathered and analyzed. An inter-relational database is an effecti ...

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Murine Hematopoietic Stem Cell Transduction Using Retroviral Vectors

Hematopoietic stem cells (HSCs) represent an important target cell population in bone marrow transplantation and gene therapy applications. Their progeny cells carry the genetic information of the HSCs and replenish the blood and immune system. Therefore, in the setting of inherited ...

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Genetic Modification of Human Hematopoietic Cells: Preclinical Optimization of Oncoretroviral-mediated Gene Transfer for Clinical Trials

This chapter provides information about the oncoretroviral transduction of human hematopoietic stem/ progenitor cells under clinically applicable conditions. We describe in detail a short −60 h transduction protocol which consistently yields transduction efficienci ...

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Short-Term Culture of Human CD34+ Cells for Lentiviral Gene Transfer

Haematopoietic Stem Cells (HSCs) are attractive targets for the gene therapy. Upon ex vivo gene transfer and transplant, they may generate a progeny of gene-corrected cells potentially for a lifespan. The viral vectors most often used for HSC gene transfer are gamma-retroviral vectors (RV ...

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T Cell Culture for Gammaretroviral Transfer

Gene transfer into mature T cells with gammaretroviral vectors requires prestimulation, as only mitotic cells are susceptible to integration of the gammaretroviral proviral genome. Costimulation via the CD3/ TCR complex and a second costimulatory molecule, such as CD28 was found to b ...

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Retroviral Transduction of Murine Primary T Lymphocytes

In comparison to human T cells, efficient retroviral gene transfer and subsequent expansion of murine primary T cells is more difficult to achieve. Herein, we describe an optimized gene transfer protocol utilizing an ecotropic viral vector to transduce primary murine T cells activated w ...

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Lentiviral Vector Gene Transfer into Human T Cells

Efficient gene transfer into T lymphocytes may allow the treatment of several genetic dysfunctions of the hematopoietic system, such as severe combined immunodeficiency, and the development of novel therapeutic strategies for diseases such as cancers and acquired diseases such as ...

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DNA Transposons for Modification of Human Primary T Lymphocytes

Genetic modification of peripheral blood T lymphocytes (PBL) or hematopoietic stem cells (HSC) has been shown to be promising in the treatment of cancer (Nat Rev Cancer 3:35–45, 2003), transplant complications (Curr Opin Hematol 5:478–482, 1998), viral infections (Science 285:546–551, 1 ...

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Capillary Electrophoresis for Pharmaceutical Analysis

This chapter describes the application of capillary electrophoresis (CE) to pharmaceutical analysis. The areas of pharmaceutical analysis covered are enantiomer separation, analysis of small molecules such as amino acids or drug counter-ions, pharmaceutical assay, related ...

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Capillary Electrophoresis of Neutral Carbohydrates: Mono-, Oligosaccharides, Glycosides

This chapter reports an overview of the recent advances in the analysis of neutral sugars by capillary electrophoresis (CE); furthermore, some relevant reviews and research articles in the field are tabulated. Comparison of CE with chromatography is also presented, with special atten ...

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