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https://www.readbyqxmd.com/read/29424036/bioluminescence-based-cytotoxicity-assay-for-simultaneous-evaluation-of-cell-viability-and-membrane-damage-in-human-hepatoma-hepg2-cells
#1
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
#2
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
#3
Steve 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...
January 8, 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
#4
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
#5
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...
December 15, 2017: 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
#6
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)...
December 14, 2017: EMBO Molecular Medicine
https://www.readbyqxmd.com/read/29206867/gnb3-overexpression-causes-obesity-and-metabolic-syndrome
#7
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
#8
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
#9
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
#10
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
#11
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 (SCCmec)...
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
#12
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...
November 6, 2017: 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
#13
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
#14
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
https://www.readbyqxmd.com/read/29020088/expression-level-of-human-tlr4-rather-than-sequence-is-the-key-determinant-of-lps-responsiveness
#15
Adeline M Hajjar, Robert K Ernst, Jaehun Yi, Cathy S Yam, Samuel I Miller
To address the role of Toll-like receptor 4 (TLR4) single nucleotide polymorphisms (SNP) in lipopolysaccharide (LPS) recognition, we generated mice that differed only in the sequence of TLR4. We used a bacterial artificial chromosome (BAC) transgenic approach and TLR4/MD-2 knockout mice to specifically examine the role of human TLR4 variants in recognition of LPS. Using in vitro and in vivo assays we found that the expression level rather than the sequence of TLR4 played a larger role in recognition of LPS, especially hypoacylated LPS...
2017: PloS One
https://www.readbyqxmd.com/read/28989973/cloning-assembly-and-modification-of-the-primary-human-cytomegalovirus-isolate-toledo-by-yeast-based-transformation-associated-recombination
#16
Sanjay Vashee, Timothy B Stockwell, Nina Alperovich, Evgeniya A Denisova, Daniel G Gibson, Kyle C Cady, Kristofer Miller, Krishna Kannan, Daniel Malouli, Lindsey B Crawford, Alexander A Voorhies, Eric Bruening, Patrizia Caposio, Klaus Früh
Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes...
September 2017: MSphere
https://www.readbyqxmd.com/read/28989096/exploiting-2a-peptides-to-elicit-potent-neutralizing-antibodies-by-a-multi-subunit-herpesvirus-glycoprotein-complex
#17
Felix Wussow, Flavia Chiuppesi, Zhuo Meng, Joy Martinez, Jenny Nguyen, Peter A Barry, Don J Diamond
Neutralizing antibodies (NAb) interfering with glycoprotein complex-mediated virus entry into host cells are thought to contribute to the protection against herpesvirus infection. However, using herpesvirus glycoprotein complexes as vaccine antigens can be complicated by the necessity of expressing multiple subunits simultaneously to allow efficient complex assembly and formation of conformational NAb epitopes. By using a novel bacterial artificial chromosome (BAC) clone of the clinically deployable Modified Vaccinia Ankara (MVA) vector and exploiting ribosomal skipping mediated by 2A peptides, MVA vectors were generated that expressed self-processing subunits of the human cytomegalovirus (HCMV) pentamer complex (PC) composed of gH, gL, UL128, UL130, and UL131A...
January 2018: Journal of Virological Methods
https://www.readbyqxmd.com/read/28986875/gene-targeted-mice-with-conditional-knock-in-out-of-nmdar-mutations
#18
Rolf Sprengel, Ahmed Eltokhi, Frank N Single
For the genetic alterations of NMDA receptor (NMDAR) properties like Ca(2+)-permeability or voltage-dependent gating in mice and for the experimental analysis of nonsense or missense mutations that were identified in human patients, single nucleotide mutations have to be introduced into the germ line of mice (Burnashev and Szepetowski, Curr Opin Pharmacol 20:73-82, 2015; Endele et al., Nat Genet 42:1021-1026, 2010). This can be done with very high precision by the well-established method of gene replacement, which makes use of homologous recombination in pluripotent embryonic stem (ES) cells of mice...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28986519/crispr-cas9-induced-transgene-insertion-and-telomere-associated-truncation-of-a-single-human-chromosome-for-chromosome-engineering-in-cho-and-a9-cells
#19
Narumi Uno, Kei Hiramatsu, Katsuhiro Uno, Shinya Komoto, Yasuhiro Kazuki, Mitsuo Oshimura
Chromosome engineering techniques including gene insertion, telomere-associated truncation and microcell-mediated chromosome transfer (MMCT) are powerful tools for generation of humanised model animal, containing megabase-sized genomic fragments. However, these techniques require two cell lines: homologous recombination (HR)-proficient DT40 cells for chromosome modification, and CHO cells for transfer to recipient cells. Here we show an improved technique using a combination of CRISPR/Cas9-induced HR in CHO and mouse A9 cells without DT40 cells following MMCT to recipient cells...
October 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28965926/exposure-to-4100k-fluorescent-light-elicits-sex-specific-transcriptional-responses-in-xiphophorus-maculatus-skin
#20
William T Boswell, Mikki Boswell, Dylan J Walter, Kaela L Navarro, Jordan Chang, Yuan Lu, Markita G Savage, Jianjun Shen, Ronald B Walter
It has been reported that exposure to artificial light may affect oxygen intake, heart rate, absorption of vitamins and minerals, and behavioral responses in humans. We have reported specific gene expression responses in the skin of Xiphophorus fish after exposure to ultraviolet light (UV), as well as, both broad spectrum and narrow waveband visible light. In regard to fluorescent light (FL), we have shown that male X. maculatus exposed to 4100K FL (i.e. "cool white") rapidly suppress transcription of many genes involved with DNA replication and repair, chromosomal segregation, and cell cycle progression in skin...
September 29, 2017: Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP
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