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https://www.readbyqxmd.com/read/27743290/the-combination-of-calcium-ionophore-a23187-and-gm-csf-can-safely-salvage-aged-human-unfertilized-oocytes-after-icsi
#1
Konstantinos A Economou, Dimitra Christopikou, Erika Tsorva, Stephen Davies, Minas Mastrominas, Haris Cazlaris, Michael Koutsilieris, Panagoula Angelogianni, Dimitris Loutradis
PURPOSE: Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol...
October 14, 2016: Journal of Assisted Reproduction and Genetics
https://www.readbyqxmd.com/read/27709566/mutagenesis-and-genome-engineering-of-epstein-barr-virus-in-cultured-human-cells-by-crispr-cas9
#2
Kit-San Yuen, Chi-Ping Chan, Kin-Hang Kok, Dong-Yan Jin
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27706134/de-novo-assembly-and-phasing-of-a-korean-human-genome
#3
Jeong-Sun Seo, Arang Rhie, Junsoo Kim, Sangjin Lee, Min-Hwan Sohn, Chang-Uk Kim, Alex Hastie, Han Cao, Ji-Young Yun, Jihye Kim, Junho Kuk, Gun Hwa Park, Juhyeok Kim, Hanna Ryu, Jongbum Kim, Mira Roh, Jeonghun Baek, Michael W Hunkapiller, Jonas Korlach, Jong-Yeon Shin, Changhoon Kim
Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17...
October 5, 2016: Nature
https://www.readbyqxmd.com/read/27639575/the-kaposi-s-sarcoma-associated-herpesvirus-orf35-gene-product-is-required-for-efficient-lytic-virus-reactivation
#4
Shir Bergson, Inbal Itzhak, Talya Wasserman, Anastasia Gelgor, Inna Kalt, Ronit Sarid
Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the etiology of several human malignancies. KSHV open reading frame (orf) 35 encodes a conserved gammaherpesvirus protein with an, as yet, unknown function. Employing the bacterial artificial chromosome (BAC) system, we generated a recombinant viral clone that fails to express ORF35 (BAC16-ORF35-stop) but preserves intact adjacent and overlapping reading frames. Using this construct, we studied the role of this previously uncharacterized gene product during lytic reactivation of KSHV...
September 15, 2016: Virology
https://www.readbyqxmd.com/read/27608724/transcription-of-telomeric-dna-leads-to-high-levels-of-homologous-recombination-and-t-loops
#5
Anirban Kar, Smaranda Willcox, Jack D Griffith
The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex...
November 2, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27602518/m-6-a-rna-methylation-promotes-xist-mediated-transcriptional-repression
#6
Deepak P Patil, Chun-Kan Chen, Brian F Pickering, Amy Chow, Constanza Jackson, Mitchell Guttman, Samie R Jaffrey
The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N(6)-methyladenosine (m(6)A) residues-a reversible base modification of unknown function in long non-coding RNAs. We show that m(6)A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m(6)A-methylation complex and recruit it to specific sites in RNA...
September 7, 2016: Nature
https://www.readbyqxmd.com/read/27591740/a-kidney-injury-molecule-1-kim-1-gene-reporter-in-a-mouse-artificial-chromosome-the-responsiveness-to-cisplatin-toxicity-in-immortalized-mouse-kidney-s3-cells
#7
Kenji Kokura, Yasushi Kuromi, Takeshi Endo, Naohiko Anzai, Yasuhiro Kazuki, Mitsuo Oshimura, Tetsuya Ohbayashi
BACKGROUND: Kidney injury molecule-1 (Kim-1) has been validated as a urinary biomarker for acute and chronic renal damage. The expression of Kim-1 mRNA is also activated by acute kidney injury induced by cisplatin in rodents and humans. To date, the measurement of Kim-1 expression has not fully allowed the detection of in vitro cisplatin nephrotoxicity in immortalized culture cells such as human kidney-2 cells and immortalized proximal tubular epithelial cells. METHODS: We measured the augmentation of Kim-1 mRNA expression after cisplatin addition using immortalized S3 cells established from the kidneys of transgenic mice harboring temperature-sensitive large T antigen from Simian virus 40...
September 3, 2016: Journal of Gene Medicine
https://www.readbyqxmd.com/read/27568550/effects-of-duration-of-electric-pulse-on-in-vitro-development-of-cloned-cat-embryos-with-human-artificial-chromosome-vector
#8
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
https://www.readbyqxmd.com/read/27503586/brain-enhancer-activities-at-the-gene-poor-5p14-1-autism-associated-locus
#9
Yukiko U Inoue, Takayoshi Inoue
Due to the vast clinical and genetic heterogeneity, identification of causal genetic determinants for autism spectrum disorder (ASD) has proven to be complex. Whereas several dozen 'rare' genetic variants for ASD susceptibility have been identified, studies are still underpowered to analyse 'common' variants for their subtle effects. A recent application of genome-wide association studies (GWAS) to ASD indicated significant associations with the single nucleotide polymorphisms (SNPs) on chromosome 5p14.1, located in a non-coding region between cadherin10 (CDH10) and cadherin9 (CDH9)...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27502218/spliced-synthetic-genes-as-internal-controls-in-rna-sequencing-experiments
#10
Simon A Hardwick, Wendy Y Chen, Ted Wong, Ira W Deveson, James Blackburn, Stacey B Andersen, Lars K Nielsen, John S Mattick, Tim R Mercer
RNA sequencing (RNA-seq) can be used to assemble spliced isoforms, quantify expressed genes and provide a global profile of the transcriptome. However, the size and diversity of the transcriptome, the wide dynamic range in gene expression and inherent technical biases confound RNA-seq analysis. We have developed a set of spike-in RNA standards, termed 'sequins' (sequencing spike-ins), that represent full-length spliced mRNA isoforms. Sequins have an entirely artificial sequence with no homology to natural reference genomes, but they align to gene loci encoded on an artificial in silico chromosome...
September 2016: Nature Methods
https://www.readbyqxmd.com/read/27502217/representing-genetic-variation-with-synthetic-dna-standards
#11
Ira W Deveson, Wendy Y Chen, Ted Wong, Simon A Hardwick, Stacey B Andersen, Lars K Nielsen, John S Mattick, Tim R Mercer
The identification of genetic variation with next-generation sequencing is confounded by the complexity of the human genome sequence and by biases that arise during library preparation, sequencing and analysis. We have developed a set of synthetic DNA standards, termed 'sequins', that emulate human genetic features and constitute qualitative and quantitative spike-in controls for genome sequencing. Sequencing reads derived from sequins align exclusively to an artificial in silico reference chromosome, rather than the human reference genome, which allows them them to be partitioned for parallel analysis...
September 2016: Nature Methods
https://www.readbyqxmd.com/read/27464947/assessment-of-protein-binding-of-5-hydroxythalidomide-bioactivated-in-humanized-mice-with-human-p450-3a-chromosome-or-hepatocytes-by-two-dimensional-electrophoresis-accelerator-mass-spectrometry
#12
Hiroshi Yamazaki, Hiroshi Suemizu, Yasuhiro Kazuki, Ken Oofusa, Shunji Kuribayashi, Makiko Shimizu, Shinichi Ninomiya, Toru Horie, Norio Shibata, F Peter Guengerich
Bioactivation of 5-hydroxy-[carbonyl-(14)C]thalidomide, a known metabolite of thalidomide, by human artificial or native cytochrome P450 3A enzymes, and nonspecific binding in livers of mice was assessed using two-dimensional electrophoresis combined with accelerator mass spectrometry. The apparent major target proteins were liver microsomal cytochrome c oxidase subunit 6B1 and ATP synthase subunit α in mice containing humanized P450 3A genes or transplanted humanized liver. Liver cytosolic retinal dehydrogenase 1 and glutathione transferase A1 were targets in humanized mice with P450 3A and hepatocytes, respectively...
August 15, 2016: Chemical Research in Toxicology
https://www.readbyqxmd.com/read/27432859/vascular-smooth-muscle-sirtuin-1-protects-against-diet-induced-aortic-stiffness
#13
Jessica L Fry, Leona Al Sayah, Robert M Weisbrod, Isabelle Van Roy, Xiang Weng, Richard A Cohen, Markus M Bachschmid, Francesca Seta
Arterial stiffness, a major cardiovascular risk factor, develops within 2 months in mice fed a high-fat, high-sucrose (HFHS) diet, serving as a model of human metabolic syndrome, and it is associated with activation of proinflammatory and oxidant pathways in vascular smooth muscle (VSM) cells. Sirtuin-1 (SirT1) is an NAD(+)-dependent deacetylase regulated by the cellular metabolic status. Our goal was to study the effects of VSM SirT1 on arterial stiffness in the context of diet-induced metabolic syndrome. Overnight fasting acutely decreased arterial stiffness, measured in vivo by pulse wave velocity, in mice fed HFHS for 2 or 8 months, but not in mice lacking SirT1 in VSM (SMKO)...
September 2016: Hypertension
https://www.readbyqxmd.com/read/27382603/moving-toward-a-higher-efficiency-of-microcell-mediated-chromosome-transfer
#14
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
https://www.readbyqxmd.com/read/27378146/a-chromosome-16p13-11-microduplication-causes-hyperactivity-through-dysregulation-of-mir-484-protocadherin-19-signaling
#15
M Fujitani, S Zhang, R Fujiki, Y Fujihara, T Yamashita
Chromosome 16p13.11 microduplication is a risk factor associated with various neurodevelopmental disorders such as attention-deficit/hyperactivity disorder, intellectual disabilities, developmental delay and autistic spectrum disorder. The underlying molecular mechanism of this genetic variation remained unknown, but its core genetic locus-conserved across mice and humans-contains seven genes. Here, we generated bacterial artificial chromosome-transgenic mice carrying a human 16p13.11 locus, and these mice showed the behavioral hyperactivity phenotype...
July 5, 2016: Molecular Psychiatry
https://www.readbyqxmd.com/read/27343755/induction-of-genomic-instability-and-activation-of-autophagy-in-artificial-human-aneuploid-cells
#16
Kentaro Ariyoshi, Tomisato Miura, Kosuke Kasai, Yohei Fujishima, Mitsuo Oshimura, Mitsuaki A Yoshida
Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability...
August 2016: Mutation Research
https://www.readbyqxmd.com/read/27310263/generation-of-meiomaps-of-genome-wide-recombination-and-chromosome-segregation-in-human-oocytes
#17
Christian S Ottolini, Antonio Capalbo, Louise Newnham, Danilo Cimadomo, Senthilkumar A Natesan, Eva R Hoffmann, Filippo M Ubaldi, Laura Rienzi, Alan H Handyside
We have developed a protocol for the generation of genome-wide maps (meiomaps) of recombination and chromosome segregation for the three products of human female meiosis: the first and second polar bodies (PB1 and PB2) and the corresponding oocyte. PB1 is biopsied and the oocyte is artificially activated by exposure to calcium ionophore, after which PB2 is biopsied and collected with the corresponding oocyte. The whole genomes of the polar bodies and oocytes are amplified by multiple displacement amplification and, together with maternal genomic DNA, genotyped for ∼300,000 single-nucleotide polymorphisms (SNPs) genome-wide by microarray...
July 2016: Nature Protocols
https://www.readbyqxmd.com/read/27271046/highly-efficient-transfer-of-chromosomes-to-a-broad-range-of-target-cells-using-chinese-hamster-ovary-cells-expressing-murine-leukemia-virus-derived-envelope-proteins
#18
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
https://www.readbyqxmd.com/read/27207956/large-deletions-at-the-shox-locus-in-the-pseudoautosomal-region-are-associated-with-skeletal-atavism-in-shetland-ponies
#19
Nima Rafati, Lisa S Andersson, Sofia Mikko, Chungang Feng, Terje Raudsepp, Jessica Pettersson, Jan Janecka, Ove Wattle, Adam Ameur, Gunilla Thyreen, John Eberth, John Huddleston, Maika Malig, Ernest Bailey, Evan E Eichler, Göran Dalin, Bhanu Chowdary, Leif Andersson, Gabriella Lindgren, Carl-Johan Rubin
Skeletal atavism in Shetland ponies is a heritable disorder characterized by abnormal growth of the ulna and fibula that extend the carpal and tarsal joints, respectively. This causes abnormal skeletal structure and impaired movements, and affected foals are usually killed. In order to identify the causal mutation we subjected six confirmed Swedish cases and a DNA pool consisting of 21 control individuals to whole genome resequencing. We screened for polymorphisms where the cases and the control pool were fixed for opposite alleles and observed this signature for only 25 SNPs, most of which were scattered on genome assembly unassigned scaffolds...
2016: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/27206972/host-induced-aneuploidy-and-phenotypic-diversification-in-the-sudden-oak-death-pathogen-phytophthora-ramorum
#20
Takao Kasuga, Mai Bui, Elizabeth Bernhardt, Tedmund Swiecki, Kamyar Aram, Liliana M Cano, Joan Webber, Clive Brasier, Caroline Press, Niklaus J Grünwald, David M Rizzo, Matteo Garbelotto
BACKGROUND: Aneuploidy can result in significant phenotypic changes, which can sometimes be selectively advantageous. For example, aneuploidy confers resistance to antifungal drugs in human pathogenic fungi. Aneuploidy has also been observed in invasive fungal and oomycete plant pathogens in the field. Environments conducive to the generation of aneuploids, the underlying genetic mechanisms, and the contribution of aneuploidy to invasiveness are underexplored. We studied phenotypic diversification and associated genome changes in Phytophthora ramorum, a highly destructive oomycete pathogen with a wide host-range that causes Sudden Oak Death in western North America and Sudden Larch Death in the UK...
2016: BMC Genomics
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