Stable maintenance of <i>de novo</i> assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice

Liskovykh, Mikhail; Ponomartsev, Sergey V.; Popova, Elena; Bäder, Michael; Kouprina, Natalay; Larionov, Vladimir; Alenina, Natalia; Tomilin, Alexey

doi:10.1080/15384101.2015.1014151

Cited by 22 publications

(41 citation statements)

References 27 publications

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“…The HAC loss rate is roughly 10-fold higher when compared with the native chromosomes (Nakano et al 2008), making the assay very sensitized and allowing a statistically significant number of events in a realistic sample size when studying the CIN phenotype in human cells. Previously, the HAC was used for low-throughput identification of drugs that elevate chromosome instability (CIN) in cancer cells (Lee et al 2013aKim et al 2016), as a gene delivery vector for the efficient and regulated expression of exogenous full-length genes in mammalian cells (Iida et al 2010;Kim et al 2011;Kouprina et al 2012Kouprina et al , 2013Kouprina et al , 2014Kononenko et al 2014;Liskovykh et al 2015;Lee et al 2018), and for studies of the epigenetic regulation of human kinetochores (Bergmann et al 2012;Ohzeki et al 2015;Molina et al 2017).…”

Section: Experimental System To Identify Novel Human Genes Controllinmentioning

confidence: 99%

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission

et al. 2019

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One of the hallmarks of cancer is chromosome instability (CIN), which leads to aneuploidy, translocations, and other chromosome aberrations. However, in the vast majority of human tumors the molecular basis of CIN remains unknown, partly because not all genes controlling chromosome transmission have yet been identified. To address this question, we developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses a human artificial chromosome (HAC) expressing the GFP transgene. When this assay was applied to screen an siRNA library of protein kinases, we identified PINK1, TRIO, IRAK1, PNCK, and TAOK1 as potential novel genes whose knockdown induces various mitotic abnormalities and results in chromosome loss. The HAC-based assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, and transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.

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Section: Experimental System To Identify Novel Human Genes Controllinmentioning

confidence: 99%

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission

et al. 2019

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“…The metaphase spread was prepared as previously described [20,21,25]. Exponentially growing HAC-carrying CHO or iPSCs were treated for 4 or 12 h at 37 • C with 0.1 µg/mL Colcemid (Sigma-Aldrich) in a 5% CO 2 incubator.…”

Section: Preparation Of Metaphase Spreadsmentioning

confidence: 99%

“…The HAC was also shown to be unstable in some human cell lines and induced pluripotent stem cells (iPSCs) [20,24]. Apart from this, the alphoid tetO -HAC has become an attractive gene delivery vector that can be stably maintained in murine embryonic stem cells (ESCs) and their derivatives throughout mouse ontogeny [25] and can also be maintained as a 47th chromosome in human iPSCs [20]. The HAC-based gene therapy approach consists of an ex vivo genetic modification step of the autologous patient-derived stem cells, and consequent infusion of the modified cells into a patient [26,27].…”

Section: Introductionmentioning

confidence: 99%

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

Ponomartsev

Sinenko

Skvortsova

et al. 2020

Cells

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Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/− mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/– iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector—the alphoidtetO-HAC.

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“…A gene of interest may be inserted into one acceptor site, while the enhancer element is inserted into another (Suzuki et al 2014). Several studies have demonstrated the efficacy of de novo assembled HACs for delivery and expression of full-size genes, including those isolated by TAR cloning, in human or embryonic cells with their further differentiated progeny in mice (Kim et al 2011; Kononenko et al 2014; Liskovykh et al 2015; Oshimura et al 2015). …”

Section: Construction Of Human Artificial Chromosomes (Hacs) For Genementioning

confidence: 99%

Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

2016

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Transformation-associated recombination (TAR) cloning represents a unique tool for isolation and manipulation of large DNA molecules. The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. So far, TAR cloning is the only method available to selectively recover chromosomal segments up to 300 kb in length from complex and simple genomes. In addition, TAR cloning allows the assembly and cloning of entire microbe genomes up to several Mb as well as engineering of large metabolic pathways. In this review, we summarize applications of TAR cloning for functional/structural genomics and synthetic biology.

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Stable maintenance of de novo assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice

Cited by 22 publications

References 27 publications

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

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