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human artificial chromosome HAC

Elisa Pesenti, Natalay Kouprina, Mikhail Liskovykh, Joan Aurich-Costa, Vladimir Larionov, Hiroshi Masumoto, William C Earnshaw, Oscar Molina
It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC...
March 22, 2018: ACS Synthetic Biology
Daisuke Satoh, Satoshi Abe, Kaoru Kobayashi, Yoshihiro Nakajima, Mitsuo Oshimura, Yasuhiro Kazuki
In the earliest stage of drug discovery/development, various cell-based models and animal models were used for the prediction of human pharmacokinetics and toxicokinetics. Unfortunately, drugs under development are often discontinued because their nonclinical results do not extrapolate to human clinical studies in relation to either safety or efficacy. Therefore, it is important to improve the time- and cost-effectiveness of drug development. This might be achieved by developing new technologies including pharmacokinetics and toxicokinetics models that use human and mouse artificial chromosome vectors (HACs/MACs)...
February 2018: Drug Metabolism and Pharmacokinetics
Sara Benedetti, Narumi Uno, Hidetoshi Hoshiya, Martina Ragazzi, Giulia Ferrari, Yasuhiro Kazuki, Louise Anne Moyle, Rossana Tonlorenzi, Angelo Lombardo, Soraya Chaouch, Vincent Mouly, Marc Moore, Linda Popplewell, Kanako Kazuki, Motonobu Katoh, Luigi Naldini, George Dickson, Graziella Messina, Mitsuo Oshimura, Giulio Cossu, Francesco Saverio Tedesco
Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC)...
February 2018: EMBO Molecular Medicine
Nicholas C O Lee, Jung-Hyun Kim, Nikolai S Petrov, Hee-Sheung Lee, Hiroshi Masumoto, William C Earnshaw, Vladimir Larionov, Natalay Kouprina
The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoidtetO -HAC)...
January 19, 2018: ACS Synthetic Biology
Oscar Molina, Natalay Kouprina, Hiroshi Masumoto, Vladimir Larionov, William C Earnshaw
Centromeres are the site of assembly of the kinetochore, which directs chromosome segregation during cell division. Active centromeres are characterized by the presence of nucleosomes containing CENP-A and a specific chromatin environment that resembles that of active genes. Recent work using human artificial chromosomes (HAC) sheds light on the fine balance of different histone post-translational modifications and transcription that exists at centromeres for kinetochore assembly and maintenance. Here, we review the use of HAC technology to understand centromere assembly and function...
October 2017: Chromosoma
Kaoru Kobayashi, Chihiro Abe, Mika Endo, Yasuhiro Kazuki, Mitsuo Oshimura, Kan Chiba
BACKGROUND: Cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme that is expressed in the liver and small intestine of humans. Various factors influence the expression of CYP3A4, but gender difference in CYP3A4 expression remains debatable. OBJECTIVE: To clarify gender difference of hepatic and intestinal CYP3A4 in CYP3A-humanized mice generated by a human artificial chromosome (HAC) vector system. The CYP3A-humanized (CYP3AHAC) mice have essential regulatory regions, including promoters and enhancers, and unknown elements affecting the expression of CYP3A4...
November 17, 2017: Drug Metabolism Letters
Harmen J G van de Werken, Josien C Haan, Yana Feodorova, Dominika Bijos, An Weuts, Koen Theunis, Sjoerd J B Holwerda, Wouter Meuleman, Ludo Pagie, Katharina Thanisch, Parveen Kumar, Heinrich Leonhardt, Peter Marynen, Bas van Steensel, Thierry Voet, Wouter de Laat, Irina Solovei, Boris Joffe
The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome...
June 2017: Genome Research
Toru Takenaka, Kanako Kazuki, Naomoto Harada, Jiro Kuze, Masato Chiba, Takahiro Iwao, Tamihide Matsunaga, Satoshi Abe, Mitsuo Oshimura, Yasuhiro Kazuki
The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers...
February 2017: Drug Metabolism and Pharmacokinetics
David M Brown, Yujia A Chan, Prashant J Desai, Peter Grzesik, Lauren M Oldfield, Sanjay Vashee, Jeffrey C Way, Pamela A Silver, John I Glass
The delivery of large DNA vectors (>100 000 bp) remains a limiting step in the engineering of mammalian cells and the development of human artificial chromosomes (HACs). Yeast is commonly used to assemble genetic constructs in the megabase size range, and has previously been used to transfer constructs directly into cultured cells. We improved this method to efficiently deliver large (1.1 Mb) synthetic yeast centromeric plasmids (YCps) to cultured cell lines at rates similar to that of 12 kb YCps. Synchronizing cells in mitosis improved the delivery efficiency by 10-fold and a statistical design of experiments approach was employed to boost the vector delivery rate by nearly 300-fold from 1/250 000 to 1/840 cells, and subsequently optimize the delivery process for multiple mammalian, avian, and insect cell lines...
April 20, 2017: Nucleic Acids Research
Ltk Do, M Wittayarat, T Terazono, Y Sato, M Taniguchi, F Tanihara, T Takemoto, Y Kazuki, K Kazuki, M Oshimura, T Otoi
The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs...
December 2016: Reproduction in Domestic Animals, Zuchthygiene
Mikhail Liskovykh, Nicholas Co Lee, Vladimir Larionov, Natalay Kouprina
Microcell-mediated chromosome transfer (MMCT) technology enables individual mammalian chromosomes, megabase-sized chromosome fragments, or mammalian artificial chromosomes that include human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) to be transferred from donor to recipient cells. In the past few decades, MMCT has been applied to various studies, including mapping the genes, analysis of chromosome status such as aneuploidy and epigenetics. Recently, MMCT was applied to transfer MACs/HACs carrying entire chromosomal copies of genes for genes function studies and has potential for regenerative medicine...
2016: Molecular Therapy. Methods & Clinical Development
Teruhiko Suzuki, Yasuhiro Kazuki, Mitsuo Oshimura, Takahiko Hara
Microcell-mediated chromosome transfer (MMCT) is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs) as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses...
2016: PloS One
Sarine Markossian, Alexei Arnaoutov, Nakhle S Saba, Vladimir Larionov, Mary Dasso
Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure...
July 2, 2016: Cell Cycle
Jung-Hyun Kim, Hee-Sheung Lee, Nicholas C O Lee, Nikolay V Goncharov, Vadim Kumeiko, Hiroshi Masumoto, William C Earnshaw, Natalay Kouprina, Vladimir Larionov
Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene ("loss of signal" assay)...
March 22, 2016: Oncotarget
Hee-Sheung Lee, Nicholas C O Lee, Natalay Kouprina, Jung-Hyun Kim, Alex Kagansky, Susan Bates, Jane B Trepel, Yves Pommier, Dan Sackett, Vladimir Larionov
Whole chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a distinguishing feature of most cancer types. CIN is generally considered to drive tumorigenesis, but a threshold level exists whereby further increases in CIN frequency in fact hinder tumor growth. While this attribute is appealing for therapeutic exploitation, drugs that increase CIN beyond this therapeutic threshold are currently limited. In our previous work, we developed a quantitative assay for measuring CIN based on the use of a nonessential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene...
February 15, 2016: Cancer Research
Nuno M C Martins, Jan H Bergmann, Nobuaki Shono, Hiroshi Kimura, Vladimir Larionov, Hiroshi Masumoto, William C Earnshaw
Centromeres are characterized by the centromere-specific H3 variant CENP-A, which is embedded in chromatin with a pattern characteristic of active transcription that is required for centromere identity. It is unclear how centromeres remain transcriptionally active despite being flanked by repressive pericentric heterochromatin. To further understand centrochromatin's response to repressive signals, we nucleated a Polycomb-like chromatin state within the centromere of a human artificial chromosome (HAC) by tethering the methyltransferase EZH2...
January 1, 2016: Molecular Biology of the Cell
Nobuaki Shono, Jun-ichirou Ohzeki, Koichiro Otake, Nuno M C Martins, Takahiro Nagase, Hiroshi Kimura, Vladimir Larionov, William C Earnshaw, Hiroshi Masumoto
Although it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator (alphoid(tetO)). We also obtained cell lines bearing the alphoid(tetO) array at ectopic integration sites on chromosomal arms...
December 15, 2015: Journal of Cell Science
Yasuhiro Watanabe, Yasuhiro Kazuki, Kanako Kazuki, Mitsutaka Ebiki, Mami Nakanishi, Kazuomi Nakamura, Miho Yoshida Yamakawa, Hiroyuki Hosokawa, Tetsuya Ohbayashi, Mitsuo Oshimura, Kenji Nakashima
A human artificial chromosome (HAC) is maintained as an episome within a cell and avoids random integration into the host genome. It can transfer multiple and/or large transgenes along with their regulatory elements thereby resembling native chromosomes. Using this HAC system, we established mesenchymal stem cells (MSCs) that simultaneously expressed hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1, termed HAC-MSCs. This cell line provides an opportunity for stable transplantation and thorough analyses...
2015: Molecular Therapy. Nucleic Acids
Hiroaki Matsushita, Akiko Sano, Hua Wu, Zhongde Wang, Jin-An Jiao, Poothappillai Kasinathan, Eddie J Sullivan, Yoshimi Kuroiwa
Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle...
2015: PloS One
Masaharu Hiratsuka, Kana Ueda, Narumi Uno, Katsuhiro Uno, Sayaka Fukuhara, Hajime Kurosaki, Shoko Takehara, Mitsuhiko Osaki, Yasuhiro Kazuki, Yoshikazu Kurosawa, Takafumi Nakamura, Motonobu Katoh, Mitsuo Oshimura
BACKGROUND: Human artificial chromosome (HAC) vectors have some unique characteristics as compared with conventional vectors, carrying large transgenes without size limitation, showing persistent expression of transgenes, and existing independently from host genome in cells. With these features, HACs are expected to be promising vectors for modifications of a variety of cell types. However, the method of introduction of HACs into target cells is confined to microcell-mediated chromosome transfer (MMCT), which is less efficient than other methods of vector introduction...
2015: BMC Biotechnology
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