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https://www.readbyqxmd.com/read/29625294/overexpressing-human-gpr109a-leads-to-pronounced-reduction-in-plasma-triglyceride-levels-in-bac-transgenic-rats
#1
Yusuke Masuda, Nobuya Kurikawa, Tomohiro Nishizawa
BACKGROUND AND AIMS: Nicotinic acid administration causes plasma non-esterified fatty acid (NEFA) reduction and plasma lipid changes, including reduced triglyceride levels. GPR109A, which is expressed mainly in adipose tissue and has anti-lipolytic activity, was reported to be a molecular target for nicotinic acid. However, recent clinical reports have shown that most GPR109A agonists failed to induce clinically meaningful plasma lipid changes. In addition, a recent study has shown that the TG lowering effect of nicotinic acid was not diminished in Gpr109a deficient mice, which is different from the original finding...
March 24, 2018: Atherosclerosis
https://www.readbyqxmd.com/read/29565902/cost-effective-high-throughput-single-haplotype-iterative-mapping-and-sequencing-for-complex-genomic-structures
#2
Daniel W Bellott, Ting-Jan Cho, Jennifer F Hughes, Helen Skaletsky, David C Page
The reference sequences of structurally complex regions can be obtained only through highly accurate clone-based approaches. We and others have successfully used single-haplotype iterative mapping and sequencing (SHIMS) 1.0 to assemble structurally complex regions across the sex chromosomes of several vertebrate species and to allow for targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 is expensive and time consuming, requiring resources that only a genome center can provide...
April 2018: Nature Protocols
https://www.readbyqxmd.com/read/29565577/generation-of-a-synthetic-human-chromosome-with-two-centromeric-domains-for-advanced-epigenetic-engineering-studies
#3
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
https://www.readbyqxmd.com/read/29549001/expression-of-recombinant-human-lysozyme-in-bacterial-artificial-chromosome-transgenic-mice-promotes-the-growth-of-bifidobacterium-and-inhibits-the-growth-of-salmonella-in-the-intestine
#4
Lu Dan, Shen Liu, Shengzhe Shang, Huihua Zhang, Ran Zhang, Ning Li
Targeted gene modification is a novel intervention strategy to increase disease resistance more quickly than traditional animal breeding. Human lysozyme, a natural, non-specific immune factor, participates in innate immunity, exerts a wide range of antimicrobial activities against pathogens, and has immuneregulatory effects. Therefore, it is a candidate gene for improved disease resistance in animals. In this study, we successfully generated a transgenic mouse model by microinjecting a modified bacterial artificial chromosome containing a recombinant human lysozyme (rhLZ) gene into the pronuclei of fertilized mouse embryos...
March 13, 2018: Journal of Biotechnology
https://www.readbyqxmd.com/read/29505588/development-of-a-multiple-gene-loading-method-by-combining-multi-integration-system-equipped-mouse-artificial-chromosome-vector-and-crispr-cas9
#5
Kazuhisa Honma, Satoshi Abe, Takeshi Endo, Narumi Uno, Mitsuo Oshimura, Tetsuya Ohbayashi, Yasuhiro Kazuki
Mouse artificial chromosome (MAC) vectors have several advantages as gene delivery vectors, such as stable and independent maintenance in host cells without integration, transferability from donor cells to recipient cells via microcell-mediated chromosome transfer (MMCT), and the potential for loading a megabase-sized DNA fragment. Previously, a MAC containing a multi-integrase platform (MI-MAC) was developed to facilitate the transfer of multiple genes into desired cells. Although the MI system can theoretically hold five gene-loading vectors (GLVs), there are a limited number of drugs available for the selection of multiple-GLV integration...
2018: PloS One
https://www.readbyqxmd.com/read/29501423/3d-fish-to-analyse-gene-domain-specific-chromatin-re-modeling-in-human-cancer-cell-lines
#6
Silvia Kocanova, Isabelle Goiffon, Kerstin Bystricky
Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35kb to 300kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains...
February 28, 2018: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/29424036/bioluminescence-based-cytotoxicity-assay-for-simultaneous-evaluation-of-cell-viability-and-membrane-damage-in-human-hepatoma-hepg2-cells
#7
Katsuhiro Uno, Kazutoshi Murotomi, Yasuhiro Kazuki, Mitsuo Oshimura, Yoshihiro Nakajima
We have developed a bioluminescence-based non-destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)-targeted copepod luciferase (GLuc-KDEL), respectively, by using multi-integrase mouse artificial chromosome (MI-MAC) vector. We have demonstrated that the time-dependent concentration response curves of ELuc luminescence intensity and WST-1 assay, and GLuc-KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant-treated ELuc- and GLuc-KDEL-expressing HepG2 stable cell lines...
February 8, 2018: Luminescence: the Journal of Biological and Chemical Luminescence
https://www.readbyqxmd.com/read/29398301/human-and-mouse-artificial-chromosome-technologies-for-studies-of-pharmacokinetics-and-toxicokinetics
#8
REVIEW
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)...
January 11, 2018: Drug Metabolism and Pharmacokinetics
https://www.readbyqxmd.com/read/29311146/impact-of-broad-regulatory-regions-on-gdf5-expression-and-function-in-knee-development-and-susceptibility-to-osteoarthritis
#9
Steven K Pregizer, Ata M Kiapour, Mariel Young, Hao Chen, Michael Schoor, Zun Liu, Jiaxue Cao, Vicki Rosen, Terence D Capellini
OBJECTIVES: Given the role of growth and differentiation factor 5 ( GDF5 ) in knee development and osteoarthritis risk, we sought to characterise knee defects resulting from Gdf5 loss of function and how its regulatory regions control knee formation and morphology. METHODS: The brachypodism ( bp ) mouse line, which harbours an inactivating mutation in Gdf5 , was used to survey how Gdf5 loss of function impacts knee morphology, while two transgenic Gdf5 reporter bacterial artificial chromosome mouse lines were used to assess the spatiotemporal activity and function of Gdf5 regulatory sequences in the context of clinically relevant knee anatomical features...
March 2018: Annals of the Rheumatic Diseases
https://www.readbyqxmd.com/read/29301233/analyses-of-tissue-culture-adaptation-of-human-herpesvirus-6a-by-whole-genome-deep-sequencing-redefines-the-reference-sequence-and-identifies-virus-entry-complex-changes
#10
Joshua G Tweedy, Eric Escriva, Maya Topf, Ursula A Gompels
Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for related betaherpesviruses, Roseolovirus, human herpesvirus 6A (HHV-6A) strain U1102. This virus is also naturally "cloned" by germline subtelomeric chromosomal-integration in approximately 1% of human populations, and accurate references are key to understanding pathological relationships between exogenous and endogenous virus...
December 31, 2017: Viruses
https://www.readbyqxmd.com/read/29244209/generation-of-a-kshv-k13-deletion-mutant-for-vflip-function-study
#11
Fei Wang, Yuanyuan Guo, Wan Li, Chun Lu, Qin Yan
Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral Fas-associated death domain-like IL-1-converting enzyme inhibitory protein (vFLIP) is one of the latently expressed genes and plays a key role in cell survival and maintenance of latent infection by activating the NF-κB pathway. To obtain a genetic system for studying KSHV vFLIP mutation in the context of the viral genome, we generated recombinant viruses lacking the coding sequence (CDS) of vFLIP gene (K13/ORF71) by bacterial artificial chromosome (BAC) technology and the Escherichia coli Red recombination system...
April 2018: Journal of Medical Virology
https://www.readbyqxmd.com/read/29242210/reversible-immortalisation-enables-genetic-correction-of-human-muscle-progenitors-and-engineering-of-next-generation-human-artificial-chromosomes-for-duchenne-muscular-dystrophy
#12
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
https://www.readbyqxmd.com/read/29206867/gnb3-overexpression-causes-obesity-and-metabolic-syndrome
#13
Alev Cagla Ozdemir, Grace M Wynn, Aimee Vester, M Neale Weitzmann, Gretchen N Neigh, Shanthi Srinivasan, M Katharine Rudd
The G-protein beta subunit 3 (GNB3) gene has been implicated in obesity risk; however, the molecular mechanism of GNB3-related disease is unknown. GNB3 duplication is responsible for a syndromic form of childhood obesity, and an activating DNA sequence variant (C825T) in GNB3 is also associated with obesity. To test the hypothesis that GNB3 overexpression causes obesity, we created bacterial artificial chromosome (BAC) transgenic mice that carry an extra copy of the human GNB3 risk allele. Here we show that GNB3-T/+ mice have increased adiposity, but not greater food intake or a defect in satiety...
2017: PloS One
https://www.readbyqxmd.com/read/29185920/a-tb40-e-derived-human-cytomegalovirus-genome-with-an-intact-us-gene-region-and-a-self-excisable-bac-cassette-for-immunological-research
#14
Kerstin Laib Sampaio, Anja Weyell, Narmadha Subramanian, Zeguang Wu, Christian Sinzger
For immunological research on the human cytomegalovirus (HCMV), a virus that combines the broad cell tropism of clinical isolates, efficient replication in cell culture, the complete set of MHC-I modulator genes, and suitability for genetic engineering is desired. Here, we aimed to generate a genetically complete derivative of HCMV strain TB40/E as a bacterial artificial chromosome (BAC) with a self-excisable BAC cassette. The BAC cassette was inserted into the US2-US6 gene region (yielding TB40-BACKL7), relocated into the UL73/UL74 region with modifications that favor excision of the BAC cassette during replication in fibroblasts, and finally the US2-US6 region was restored, resulting in BAC clone TB40-BACKL7-SE When this BAC clone was transfected into fibroblasts at efficiencies >0...
November 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/29180668/regulation-of-human-and-mouse-telomerase-genes-by-genomic-contexts-and-transcription-factors-during-embryonic-stem-cell-differentiation
#15
De Cheng, Shuwen Wang, Wenwen Jia, Yuanjun Zhao, Fan Zhang, Jiuhong Kang, Jiyue Zhu
Differential regulation of telomerase reverse transcriptase (TERT) genes contribute to distinct aging and tumorigenic processes in humans and mice. To study TERT regulation, we generated mouse embryonic stem cell (ESC) lines containing single-copy bacterial artificial chromosome (BAC) reporters, covering hTERT and mTERT genes and their neighboring loci, via recombinase-mediated BAC targeting. ESC lines with chimeric BACs, in which two TERT promoters were swapped, were also generated. Using these chromatinized BACs, we showed that hTERT silencing during differentiation to embryoid bodies (EBs) and to fibroblast-like cells was driven by the human-specific genomic context and accompanied by increases of repressive epigenetic marks, H3K9me3 and H3K27me3, near its promoter...
November 27, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29162642/-abca4-midigenes-reveal-the-full-splice-spectrum-of-all-reported-noncanonical-splice-site-variants-in-stargardt-disease
#16
Riccardo Sangermano, Mubeen Khan, Stéphanie S Cornelis, Valerie Richelle, Silvia Albert, Alejandro Garanto, Duaa Elmelik, Raheel Qamar, Dorien Lugtenberg, L Ingeborgh van den Born, Rob W J Collin, Frans P M Cremers
Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells...
January 2018: Genome Research
https://www.readbyqxmd.com/read/29152580/chromosomal-targeting-by-the-type-iii-a-crispr-cas-system-can-reshape-genomes-in-staphylococcus-aureus
#17
Jing Guan, Wanying Wang, Baolin Sun
CRISPR-Cas (clustered regularly interspaced short palindromic repeat [CRISPR]-CRISPR-associated protein [Cas]) systems can provide protection against invading genetic elements by using CRISPR RNAs (crRNAs) as a guide to locate and degrade the target DNA. CRISPR-Cas systems have been classified into two classes and five types according to the content of cas genes. Previous studies have indicated that CRISPR-Cas systems can avoid viral infection and block plasmid transfer. Here we show that chromosomal targeting by the Staphylococcus aureus type III-A CRISPR-Cas system can drive large-scale genome deletion and alteration within integrated staphylococcal cassette chromosome mec (SCC mec )...
November 2017: MSphere
https://www.readbyqxmd.com/read/29115621/effects-of-shield1-on-the-viral-replication-of-varicella%C3%A2-zoster-virus-containing-fkbp%C3%A2-tagged-orf4-and-48
#18
Shuying Li, Zhanjun Liu, Ji Li, Aihua Liu, Lihua Zhu, Kui Yu, Ke Zhang
The present study aimed to explore the effects of a stabilizing ligand, Shield‑1, on the replication of recombinant varicella‑zoster virus (VZV) containing FK506 binding protein (FKPB) tags in essential open reading frames (ORF) 4 and 48. A specific galactokinase (galK) selection method was conducted, following the addition of galK labels to VZV ORF4 and 48, using a SW102 VZV bacterial artificial chromosome (BAC) system. Subsequently, recombinant VZV containing FKPB tags in ORF4 and 48 was constructed by counterselection and homologous recombination...
January 2018: Molecular Medicine Reports
https://www.readbyqxmd.com/read/29097363/interrogation-of-the-atherosclerosis-associated-sort1-sortilin-1-locus-with-primary-human-hepatocytes-induced-pluripotent-stem-cell-hepatocytes-and-locus-humanized-mice
#19
Xiao Wang, Avanthi Raghavan, Derek T Peters, Evanthia E Pashos, Daniel J Rader, Kiran Musunuru
OBJECTIVE: The noncoding single-nucleotide polymorphism rs12740374 has been hypothesized to be the causal variant responsible for liver-specific modulation of SORT1(sortilin 1) expression (ie, expression quantitative trait locus) and, by extension, the association of the SORT1 locus on human chromosome 1p13 with low-density lipoprotein cholesterol levels and coronary heart disease. The goals of this study were to compare 3 different hepatocyte models in demonstrating that the rs12740374 minor allele sequence is responsible for transcriptional activation of SORT1 expression...
November 2, 2017: Arteriosclerosis, Thrombosis, and Vascular Biology
https://www.readbyqxmd.com/read/29043639/methods-to-study-repeat-fragility-and-instability-in-saccharomyces-cerevisiae
#20
Erica J Polleys, Catherine H Freudenreich
Trinucleotide repeats are common in the human genome and can undergo changes in repeat length. Expanded CAG repeats have been linked to over 14 human diseases and are considered hotspots for breakage and genomic rearrangement. Here, we describe two Saccharomyces cerevisiae based assays that evaluate the rate of chromosome breakage that occurs within a repeat tract (fragility), and a PCR-based assay to evaluate tract length changes (instability). The first fragility assay utilizes end-loss and subsequent telomere addition as the main mode of repair of a yeast artificial chromosome (YAC)...
2018: Methods in Molecular Biology
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