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https://www.readbyqxmd.com/read/29043639/methods-to-study-repeat-fragility-and-instability-in-saccharomyces-cerevisiae
#1
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
#2
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
#3
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
#4
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...
October 6, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/28986875/gene-targeted-mice-with-conditional-knock-in-out-of-nmdar-mutations
#5
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
#6
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
#7
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 28, 2017: Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP
https://www.readbyqxmd.com/read/28934140/applications-of-pna-based-artificial-restriction-dna-cutters
#8
REVIEW
Narumi Shigi, Jun Sumaoka, Makoto Komiyama
More than ten years ago, artificial restriction DNA cutters were developed by combining two pseudo-complementary peptide nucleic acid (pcPNA) strands with either Ce(IV)/EDTA or S1 nuclease. They have remarkably high site-specificity and can cut only one predetermined site in the human genome. In this article, recent progress of these man-made tools have been reviewed. By cutting the human genome site-selectively, desired fragments can be clipped from either the termini of chromosomes (telomeres) or from the middle of genome...
September 21, 2017: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
https://www.readbyqxmd.com/read/28912069/generation-of-a-novel-human-cytomegalovirus-bacterial-artificial-chromosome-tailored-for-transduction-of-exogenous-sequences
#9
William L Close, Amit Bhandari, Marwa Hojeij, Philip E Pellett
The study of herpesviruses, including human cytomegalovirus (HCMV), is complicated by viral genome complexity and inefficient methods for genetic manipulation in tissue culture. Reverse genetics of herpesviruses has been facilitated by propagating their genomes in E. coli as bacterial artificial chromosomes (BACs), which enables complex and precise genetic manipulation using bacterial recombinational systems. Internal capsid volume imposes a strict limit on the length of genome that can be packaged efficiently...
September 12, 2017: Virus Research
https://www.readbyqxmd.com/read/28904200/epstein-barr-virus-bkrf4-gene-product-is-required-for-efficient-progeny-production
#10
H M Abdullah Al Masud, Takahiro Watanabe, Masahiro Yoshida, Yoshitaka Sato, Fumi Goshima, Hiroshi Kimura, Takayuki Murata
Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics...
September 13, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28899976/core-concept-human-artificial-chromosomes-offer-insights-therapeutic-possibilities-and-challenges
#11
Amber Dance
No abstract text is available yet for this article.
September 12, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28894962/ring-chromosomes-from-formation-to-clinical-potential
#12
REVIEW
Inna E Pristyazhnyuk, Aleksei G Menzorov
Ring chromosomes (RCs) are circular DNA molecules, which occur rarely in eukaryotic nuclear genomes. Lilian Vaughan Morgan first described them in the fruit fly. Human embryos very seldom have RCs, about 1:50,000. Carriers of RCs may have varying degrees of symptoms, from healthy phenotype to serious pathologies in physical and intellectual development. Many authors describe common symptoms of RC presence: short stature and some developmental delay that could be described as a "ring chromosome syndrome." As a rule, RCs arise de novo through the end-joining of two DNA double-strand breaks, telomere-subtelomere junction, or inv dup del rearrangement in both meiosis and mitosis...
September 12, 2017: Protoplasma
https://www.readbyqxmd.com/read/28878310/large-scale-production-of-recombinant-human-lactoferrin-from-high-expression-marker-free-transgenic-cloned-cows
#13
Ming Wang, Zhaolin Sun, Tian Yu, Fangrong Ding, Ling Li, Xi Wang, Mingbo Fu, Haiping Wang, Jinming Huang, Ning Li, Yunping Dai
Human lactoferrin (hLF) is a valuable protein for pharmaceutical products and functional foods, and worldwide demand for this protein has steadily increased. However, large-scale recombinant human lactoferrin (rhLF) production using current animal bioreactor techniques is limited by the low expression of foreign proteins, the use of antibiotic resistance genes and the down-regulation of endogenous milk proteins. Here, we generated a herd of marker-free, hLF bacterial artificial chromosome (BAC) transgenic cloned cows, as confirmed by Polymerase chain reaction, Southern blot and Western blot analyses...
September 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28809151/the-pentameric-complex-of-human-cytomegalovirus-cell-tropism-virus-dissemination-immune-response-and-vaccine-development
#14
REVIEW
Giuseppe Gerna, Maria Grazia Revello, Fausto Baldanti, Elena Percivalle, Daniele Lilleri
Between the 1980s and 1990s, three assays were developed for diagnosis of human cytomegalovirus (HCMV) infections: leuko (L)-antigenemia, l-viremia and l-DNAemia, detecting viral protein pp65, infectious virus and viral DNA, respectively, in circulating leukocytes Repeated initial attempts to reproduce the three assays in vitro using laboratory-adapted strains and infected cell cultures were consistently unsuccessful. Results were totally reversed when wild-type HCMV strains were used to infect either fibroblasts or endothelial cells...
September 2017: Journal of General Virology
https://www.readbyqxmd.com/read/28799737/method-to-assemble-genomic-dna-fragments-or-genes-on-human-artificial-chromosome-with-regulated-kinetochore-using-a-multi-integrase-system
#15
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 (alphoid(tetO)-HAC)...
August 24, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28768473/a-de-novo-missense-mutation-of-fgfr2-causes-facial-dysplasia-syndrome-in-holstein-cattle
#16
Jørgen S Agerholm, Fintan J McEvoy, Steffen Heegaard, Carole Charlier, Vidhya Jagannathan, Cord Drögemüller
BACKGROUND: Surveillance for bovine genetic diseases in Denmark identified a hitherto unreported congenital syndrome occurring among progeny of a Holstein sire used for artificial breeding. A genetic aetiology due to a dominant inheritance with incomplete penetrance or a mosaic germline mutation was suspected as all recorded cases were progeny of the same sire. Detailed investigations were performed to characterize the syndrome and to reveal its cause. RESULTS: Seven malformed calves were submitted examination...
August 2, 2017: BMC Genetics
https://www.readbyqxmd.com/read/28763890/-oncolytic-property-of-hsv-1-recombinant-viruses-carrying-the-human-il-12
#17
X J Liu, X Y Wang, J X Guo, H J Zhu, C R Zhang, Z H Ma
Objective: Constructed the recombinant HSV-1 deleted ICP47 and inserted human IL-12, and investigate the virus' replication ability and oncolytic property in vitro and vivo. Methods: The recombinant HSV-1 deleting ICP47 (MH1005) and then inserting human IL-12 (MH1006) were obtained with bacterial artificial chromosome technology.The replication ability and the efficiency of inhibiting tumor were detected in several nerve tumor cell lines infected with HSV-wt, MH1005 and MH1006 respectively.The murine tumor model was established by subcutaneous inoculation Neuro-2a cells on both sides of mice back respectively...
July 18, 2017: Zhonghua Yi Xue za Zhi [Chinese medical journal]
https://www.readbyqxmd.com/read/28754135/retrofitting-the-bac-cloning-vector-pbelobac11-by-the-insertion-of-a-mutant-loxp-site
#18
Jonathon S Coren
OBJECTIVE: Human genomic libraries constructed in bacterial artificial chromosome vectors were utilized to make physical maps of all 23-chromosome pairs and as the templates for DNA sequencing to aid in the completion of the Human Genome Project. The goal of this study was to modify the BAC vector pBeloBAC11 so that genomic inserts contained in this vector could be subjected to bidirectional transposon-mediated nested deletions from the wild-type and mutant loxP sites present. RESULTS: An oligonucleotide containing a mutant loxP 2272 site and a XhoI restriction enzyme sequence was designed and inserted at the SfiI restriction site located approximately 200 basepairs upstream of the lacZ gene in pBeloBAC11...
July 28, 2017: BMC Research Notes
https://www.readbyqxmd.com/read/28751472/parkinson-disease-associated-lrrk2-g2019s-transgene-disrupts-marrow-myelopoiesis-and-peripheral-th17-response
#19
Jeongho Park, Jang-Won Lee, Scott C Cooper, Hal E Broxmeyer, Jason R Cannon, Chang H Kim
Parkinson's disease (PD) is a neurodegenerative disease, whereas Crohn's disease is an inflammatory bowel disease. Interestingly, polymorphisms in the LRRK2 gene have been identified as risk factors for both diseases. LRRK2 G2019S is the most prevalent mutation found in PD. To gain insights into the role of the LRRK2 G2019S gene on the development and activation of the immune system in the brain-gut axis, we investigated the effect of LRRK2 G2019S on bone marrow myeloid progenitors and myeloid cell function in the periphery...
October 2017: Journal of Leukocyte Biology
https://www.readbyqxmd.com/read/28717161/development-and-characterization-of-a-novel-prox1-egfp-lymphatic-and-schlemm-s-canal-reporter-rat
#20
Eunson Jung, Daniel Gardner, Dongwon Choi, Eunkyung Park, Young Jin Seong, Sara Yang, Jorge Castorena-Gonzalez, Antoine Louveau, Zhao Zhou, Gene K Lee, David P Perrault, Sunju Lee, Maxwell Johnson, George Daghlian, Maria Lee, Yeo Jin Hong, Yukinari Kato, Jonathan Kipnis, Michael J Davis, Alex K Wong, Young-Kwon Hong
The lymphatic system plays a key role in tissue fluid homeostasis, immune cell trafficking, and fat absorption. We previously reported a bacterial artificial chromosome (BAC)-based lymphatic reporter mouse, where EGFP is expressed under the regulation of the Prox1 promoter. This reporter line has been widely used to conveniently visualize lymphatic vessels and other Prox1-expressing tissues such as Schlemm's canal. However, mice have a number of experimental limitations due to small body size. By comparison, laboratory rats are larger in size and more closely model the metabolic, physiological, and surgical aspects of humans...
July 17, 2017: Scientific Reports
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