Nucleofection of Human Embryonic Stem Cells
Department of Biological Chemistry, Sue and Bill Gross Stem Cell Research Center, School of Medicine, University of California, Irvine, Irvine, CA, USA. Methods in molecular biology (Clifton, N.J.)
(Impact Factor: 1.29).
01/2011; 767:333-41. DOI: 10.1007/978-1-61779-201-4_24
The ability to realize the full potential of human pluripotent stem cells (hPSCs) as tools for -understanding human development and advancing the field of regenerative medicine is dependent on efficient methods to genetically manipulate these cells. There are several methods for introducing foreign DNA into cells such as electroporation, lipid-based transfection technology, and viral transduction. We describe here a method to transfect human embryonic stem cells (hESCs) using nucleofection technology. This unique method uses the Nucleofector II Device that combines the use of a cell type-specific Nucleofector Solution and preprogrammed electrical parameters to efficiently deliver DNA into the cell nucleus. The use of this technology allows high-efficiency transfer of nucleic acids into hESCs enabling both transient and stable manipulation of gene expression in these cells.
Available from: Karen Ring
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ABSTRACT: Tauopathies represent a group of neurodegenerative disorders characterized by the accumulation of pathological TAU protein in brains. We report a human neuronal model of tauopathy derived from induced pluripotent stem cells (iPSCs) carrying a TAU-A152T mutation. Using zinc-finger nuclease-mediated gene editing, we generated two isogenic iPSC lines: one with the mutation corrected, and another with the homozygous mutation engineered. The A152T mutation increased TAU fragmentation and phosphorylation, leading to neurodegeneration and especially axonal degeneration. These cellular phenotypes were consistent with those observed in a patient with TAU-A152T. Upon mutation correction, normal neuronal and axonal morphologies were restored, accompanied by decreases in TAU fragmentation and phosphorylation, whereas the severity of tauopathy was intensified in neurons with the homozygous mutation. These isogenic TAU-iPSC lines represent a critical advancement toward the accurate modeling and mechanistic study of tauopathies with human neurons and will be invaluable for drug-screening efforts and future cell-based therapies.
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ABSTRACT: Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals.
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