A Targeted Neuroglial Reporter Line Generated by Homologous Recombination in Human Embryonic Stem Cells

Han, Xue; Wu, Sen; Papadeas, Sophia T.; Spusta, Steve; Swistowska, Anna Maria; MacArthur, Chad C.; Mattson, Mark P.; Maragakis, Nicholas J.; Capecchi, Mario R.; Rao, Mahendra S.; Zeng, Xianmin; Liu, Ying

doi:10.1002/stem.129

Cited by 68 publications

(59 citation statements)

References 57 publications

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“…WA09 (WiCell Research Institute) and the platform line derived from WA09 [7] (46, XX) were maintained as described [7,17]. Briefly, cells were cultured on a layer of mitomycin C (Sigma)-inactivated mouse embryonic fibroblast cells (MitC-MEFs) in hESC medium containing DMEM-F12 with glutamax, 20% knockout serum replacement, 1% nonessential amino acid, and 55 mM 2-mercaptoethanol, supplemented with 4 ng/mL basic fibroblast growth factor (bFGF).…”

Section: Cell Culturementioning

confidence: 99%

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

MacArthur

Han

Hoof

et al. 2012

Stem Cells and Development

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Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1a) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5¢ and 3¢ of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1a or CMV early enhancer/chicken b actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1a-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain-and loss-of-function experiments, and human disease modeling using hESCs.

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Section: Cell Culturementioning

confidence: 99%

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

MacArthur

Han

Hoof

et al. 2012

Stem Cells and Development

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“…It is not unreasonable to assume that such a repair should be feasible in humans as well. For more details see Xue et al [18]. As with other gene manipulation approaches, several improvements have been reported with homologous recombination, and strategies to perform repairs have evolved.…”

Section: Issues Related To Cells As Delivery Vehiclesmentioning

confidence: 99%

Cell-Based Therapy for Neural Disorders — Anticipating Challenges

Chiu

Rao

2011

Neurotherapeutics

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Neurological syndromes, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, and lysosomal storage disorders, such as Battens disease, are devastating because they result in increasing loss of cognitive and physical function. Sadly, no drugs are currently available to halt their progression. The relative paucity of curative approaches for these and other conditions of the nervous system have led to a widespread evaluation of alternative treatment modalities including cell-based interventions.Several cell types have been tested successfully in animal models where safety and efficacy have been demonstrated. Early clinical trials have also been initiated in humans, and some have shown a degree of success albeit on a more limited scale than in animal experiments. Recent demonstrations that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, can differentiate into a variety of specific neural phenotypes has stimulated worldwide enthusiasm for developing cell-based intervention of neurological disease. Indeed, several groups are preparing investigational new drug applications to treat disorders as diverse as macular degeneration, lysosomal storage diseases, and Parkinson's disease. It is noteworthy that cell replacement therapies for neurological conditions face key challenges, some of which are unique, because of the development and organization of the nervous system, its metabolism, and connectivity. Choice of the cell (or cells), the process of manufacturing them, defining the delivery pathway, developing and testing in an appropriate preclinical model, selecting a patient population, and visualizing and following or monitoring patients all pose specific issues as related to the central and peripheral nervous systems. In this review, we address a myriad of challenges that are solvable, but require careful planning and attention to the special demands of the human nervous system.

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“…The difficulties in targeting hESCs by HR is illustrated by the observation that, so far, <20 genes have been successfully targeted in hESCs (Thomson et al 1998;Zwaka and Thomson 2003;Urbach et al 2004;Costa et al 2007;Irion et al 2007;Davis et al 2008a,b;Suzuki et al 2008;Ruby and Zheng 2009;Xue et al 2009;Song et al 2010;see below). This is in contrast to the thousands of examples of successful gene targeting in mESCs.…”

Section: Gene Targeting In Mouse Embryonic Stem Cellsmentioning

confidence: 99%

“…Conventional gene targeting approaches have also been used to generate cell lines that report in real time the specific cell-fate decisions of hESCs during differentiation (Davis et al 2008b;Ruby and Zheng 2009;Xue et al 2009). Targeting of the Mixl1 (Davis et al 2008b) and Olig2 (Xue et al 2009) loci exemplify this experimental approach.…”

Section: Conventional Gene Targeting In Human Pluripotent Cellsmentioning

confidence: 99%

Gene Targeting in Human Pluripotent Cells

Hockemeyer

Jaenisch²

2010

Cold Spring Harbor Symposia on Quantitative Biology

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Mouse embryonic stem cells (mESCs) have the ability to differentiate into any cell type and can generate chimeric mice when transplanted into a host blastocyst. This remarkable potential, together with the development of robust gene targeting strategies in mESCs, were essential for establishing the mouse as the most widely used model organism in biomedical research. Recent advances have allowed the isolation of human embryonic stem cells and the derivation of induced pluripotent stem cells. Genetic tools similar to those proven routine in the mouse system are needed to realize the full potential of human pluripotent cells as disease models and putative therapeutics. Gene targeting in human cells, however, has proven to be more difficult, more time-consuming, and less robust than in mESCs. In this chapter, we discuss the strategies that have been used to allow specific genetic modifications in human pluripotent cells. We focus on the novel application of custom-engineered zinc-finger nucleases for gene targeting, which has promise to become a robust tool for efficient genetic manipulation of human pluripotent cells.

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A Targeted Neuroglial Reporter Line Generated by Homologous Recombination in Human Embryonic Stem Cells

Cited by 68 publications

References 57 publications

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

Cell-Based Therapy for Neural Disorders — Anticipating Challenges

Gene Targeting in Human Pluripotent Cells

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