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https://www.readbyqxmd.com/read/28925083/selective-knockout-of-astrocytic-na-h-exchanger-isoform-1-reduces-astrogliosis-bbb-damage-infarction-and-improves-neurological-function-after-ischemic-stroke
#1
Gulnaz Begum, Shanshan Song, Shaoxia Wang, Hanshu Zhao, Mohammad Iqbal H Bhuiyan, Eric Li, Rachel Nepomuceno, Qing Ye, Ming Sun, Michael Joseph Calderon, Donna B Stolz, Claudette St Croix, Simon C Watkins, Yinhuai Chen, Pingnian He, Gary E Shull, Dandan Sun
Stimulation of Na(+) /H(+) exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1(flox/flox) (Nhe1(f/f) ) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1(f/f) mice with Gfap-Cre(ERT2) Cre-recombinase mice. Gfap-Cre(ERT2+/-) ;Nhe1(f/f) mice at postnatal day 60-90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post-injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke...
September 19, 2017: Glia
https://www.readbyqxmd.com/read/28912069/generation-of-a-novel-human-cytomegalovirus-bacterial-artificial-chromosome-tailored-for-transduction-of-exogenous-sequences
#2
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 11, 2017: Virus Research
https://www.readbyqxmd.com/read/28910443/spatiotemporally-controlled-ablation-of-klf5-results-in-dysregulated-epithelial-homeostasis-in-adult-mouse-corneas
#3
Chelsea L Loughner, Anil Tiwari, Doreswamy Kenchegowda, Sudha Swamynathan, Shivalingappa K Swamynathan
Purpose: Corneal epithelial (CE) homeostasis requires coordination between proliferation and differentiation. Here we examine the role of cell proliferation regulator Krüppel-like factor 5 (Klf5) in adult mouse CE homeostasis. Methods: Klf5 was ablated in a spatiotemporally restricted manner by inducing Cre expression in 8-week-old ternary transgenic Klf5LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre (Klf5Δ/ΔCE) mouse CE by administering doxycycline via chow. Normal chow-fed ternary transgenic siblings served as controls...
September 1, 2017: Investigative Ophthalmology & Visual Science
https://www.readbyqxmd.com/read/28894268/recycling-of-a-selectable-marker-with-a-self-excisable-plasmid-in-pichia-pastoris
#4
Cheng Li, Ying Lin, Xueyun Zheng, Qingyan Yuan, Nuo Pang, Xihao Liao, Yuanyuan Huang, Xinying Zhang, Shuli Liang
Pichia pastoris is a widely used heterologous protein production workhorse. However, with its multiple genetic modifications to solve bottlenecks for heterologous protein productivity, P. pastoris lacks selectable markers. Existing selectable marker recycling plasmids have drawbacks (e.g., slow growth and conditional lethality). Here, zeocin-resistance marker recycling vectors were constructed using the Cre/loxP recombination system. The vectors were used to (i) knock in heterologous phytase, xylanase and lipase expression cassettes, (ii) increase the phytase, xylanase and lipase gene copy number to 13, 5, and 5, respectively, with vector introduction and (iii) engineer the secretion pathway by co-overexpressing secretion helper factors (Sly1p and Sec1p) without introducing selectable markers, giving a phytase field of 0...
September 11, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28893911/molecular-and-functional-characterization-of-the-endothelial-atp-sensitive-potassium-channel
#5
Qadeer Aziz, Yiwen Li, Naomi Anderson, Leona Ojake, Elena Tsisanova, Andrew Tinker
ATP-sensitive potassium (KATP) channels are widely expressed in the cardiovascular system where they regulate a range of biological activities by linking cellular metabolism with membrane excitability. KATP channels in vascular smooth muscle have a well-defined role in regulating vascular tone. KATP channels are also thought to be expressed in vascular endothelial cells (ECs), but their presence and function in this context is less clear. As a result, we aimed to investigate the molecular composition and physiological role of endothelial KATP channels...
September 11, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28888474/recent-advances-in-the-genomic-and-molecular-biology-of-giardia
#6
M Guadalupe Ortega-Pierres, Aaron R Jex, Brendan R E Ansell, Staffan G Svärd
Giardia duodenalis is the most common gastrointestinal protozoan parasite of humans and a significant contributor to the global burden of both diarrheal disease and post-infectious chronic disorders. Robust tools for analyzing gene function in this parasite have been developed and a range of genetic tools are now available. These together with public databases have provided insights on the function of different genes in Giardia. In this review we provide a current perspective on different molecular aspects of Giardia related to genomics, regulation of encystation, trophozoite transcriptional responses to physiological and xenobiotic (drug-induced) stress, and mechanisms of drug resistance...
September 6, 2017: Acta Tropica
https://www.readbyqxmd.com/read/28884202/gcg-creert2-knockin-mice-as-a-tool-for-genetic-manipulation-in-pancreatic-alpha-cells
#7
Chiyo Shiota, Krishna Prasadan, Ping Guo, Joseph Fusco, Xiangwei Xiao, George K Gittes
AIMS/HYPOTHESIS: The Cre/loxP system, which enables tissue-specific manipulation of genes, is widely used in mice for diabetes research. Our aim was to develop a new Cre-driver mouse line for the specific and efficient manipulation of genes in pancreatic alpha cells. METHODS: A Gcg (CreERT2) knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new Gcg (CreERT2) mouse line was crossed to the Rosa26 (tdTomato) (R26 (tdTomato) ) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of Gcg (CreERT2) -mediated recombination...
September 7, 2017: Diabetologia
https://www.readbyqxmd.com/read/28883902/sirt1-activation-by-resveratrol-alleviates-cardiac-dysfunction-via-mitochondrial-regulation-in-diabetic-cardiomyopathy-mice
#8
Sai Ma, Jing Feng, Ran Zhang, Jiangwei Chen, Dong Han, Xiang Li, Bo Yang, Xiujuan Li, Miaomiao Fan, Congye Li, Zuhong Tian, Yabin Wang, Feng Cao
BACKGROUND: Diabetic cardiomyopathy (DCM) is a major threat for diabetic patients. Silent information regulator 1 (SIRT1) has a regulatory effect on mitochondrial dynamics, which is associated with DCM pathological changes. Our study aims to investigate whether resveratrol, a SRIT1 activator, could exert a protective effect against DCM. METHODS AND RESULTS: Cardiac-specific SIRT1 knockout (SIRT1(KO)) mice were generated using Cre-loxP system. SIRT1(KO) mice displayed symptoms of DCM, including cardiac hypertrophy and dysfunction, insulin resistance, and abnormal glucose metabolism...
2017: Oxidative Medicine and Cellular Longevity
https://www.readbyqxmd.com/read/28871269/a-chemical-induced-seed-soaking-activation-procedure-for-regulated-gene-expression-in-rice
#9
Zaijie Chen, Qianqian Cheng, Chanquan Hu, Xinrui Guo, Ziqiang Chen, Yan Lin, Taijiao Hu, Maria Bellizzi, Guodong Lu, Guo-Liang Wang, Zonghua Wang, Songbiao Chen, Feng Wang
Inducible gene expression has emerged as a powerful tool for plant functional genomics. The estrogen receptor-based, chemical-inducible system XVE has been used in many plant species, but the limited systemic movement of inducer β-estradiol in transgenic rice plants has prohibited a wide use of the XVE system in this important food crop. Here, we constructed an improved chemical-regulated, site-specific recombination system by employing the XVE transactivator in combination with a Cre/loxP-FRT system, and optimized a seed-soaking procedure for XVE induction in rice...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28854700/comparison-of-spinocerebellar-ataxia-type-3-mouse-models-identifies-early-gain-of-function-cell-autonomous-transcriptional-changes-in-oligodendrocytes
#10
Biswarathan Ramani, Bharat Panwar, Lauren R Moore, Bo Wang, Rogerio Huang, Yuanfang Guan, Henry L Paulson
Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine-encoding CAG repeat expansion in the ATXN3 gene. This expansion leads to misfolding and aggregation of mutant ataxin-3 (ATXN3) and degeneration of select brain regions. A key unanswered question in SCA3 and other polyglutamine diseases is the extent to which neurodegeneration is mediated through gain-of-function versus loss-of-function. To address this question in SCA3, we performed transcriptional profiling on the brainstem, a highly vulnerable brain region in SCA3, in a series of mouse models with varying degrees of ATXN3 expression and aggregation...
September 1, 2017: Human Molecular Genetics
https://www.readbyqxmd.com/read/28842873/revealing-sister-chromatid-interactions-with-the-loxp-cre-recombination-assay
#11
Elise Vickridge, Charlène Planchenault, Olivier Espéli
Sister chromatid cohesion is a transient state during replication in bacteria. It has been recently demonstrated that the extent of contact between cohesive sisters during the cell cycle is dependent on topoisomerase IV activity, suggesting that topological links hold sister chromatids together. In the present protocol, we describe a simple method to quantify the frequency of the contacts between two cohesive sister chromatids. This method relies on a site specific recombination assay between loxP sites upon Cre induction...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28840657/a-new-type-of-gene-disruption-cassette-with-a-rescue-gene-for-pichia-pastoris
#12
Tatsuro Shibui, Hiroyoshi Hara
Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene...
August 25, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28840320/cre-lox-based-multiple-markerless-gene-disruption-in-the-genome-of-the-extreme-thermophile-thermus-thermophilus
#13
Yoichiro Togawa, Tatsuo Nunoshiba, Keiichiro Hiratsu
Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP-htk-loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination...
August 24, 2017: Molecular Genetics and Genomics: MGG
https://www.readbyqxmd.com/read/28839100/platelet-clec-2-protects-against-lung-injury-via-effects-of-its-ligand-podoplanin-on-inflammatory-alveolar-macrophages-in-the-mouse
#14
Sian Lax, Julie Rayes, Surasak Wichaiyo, Elizabeth J Haining, Kate Lowe, Beata Grygielska, Ryan Laloo, Per Flodby, Zea Borok, Edward D Crandall, David Thickett, Steve P Watson
There is no therapeutic intervention proven to prevent the acute respiratory syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes which occur during ARDS, however the mechanisms remain elusive. The platelet receptor C-type lectin-like 2 (CLEC-2) has been shown to regulate vascular integrity at sites of acute inflammation. Therefore, the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS...
August 24, 2017: American Journal of Physiology. Lung Cellular and Molecular Physiology
https://www.readbyqxmd.com/read/28833628/neural-androgen-receptor-overexpression-affects-cell-number-in-the-spinal-nucleus-of-the-bulbocavernosus
#15
Lindsay A Coome, Ashlyn Swift-Gallant, Firyal Ramzan, Lindsay Melhuish Beaupre, Tanya Brkic, D Ashley Monks
The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic neuromuscular system in which masculinization of cell number is thought to depend on perinatal androgen action in non-neural targets, but masculinization of cell size is thought to depend primarily on adult androgen action on SNB cells themselves. In order to test these hypotheses, we characterized the SNB of Cre/loxP transgenic mice that overexpress androgen receptor (AR) throughout the body (CMV-AR) or in neural tissue only (Nestin-AR)...
August 20, 2017: Journal of Neuroendocrinology
https://www.readbyqxmd.com/read/28829806/genome-engineering-in-bacillus-anthracis-using-tyrosine-site-specific-recombinases
#16
Andrei P Pomerantsev, Rita M McCall, Margaret Chahoud, Nathan K Hepler, Rasem Fattah, Stephen H Leppla
Tyrosine site-specific recombinases (T-SSR) are polynucleotidyltransferases that catalyze cutting and joining reactions between short specific DNA sequences. We developed three systems for performing genetic modifications in Bacillus anthracis that use T-SSR and their cognate target sequences, namely Escherichia coli bacteriophage P1 Cre-loxP, Saccharomyces cerevisiae Flp-FRT, and a newly discovered IntXO-PSL system from B. anthracis plasmid pXO1. All three tyrosine recombinase systems were used for creation of a B...
2017: PloS One
https://www.readbyqxmd.com/read/28819420/loss-of-complement-factor-h-in-plasma-increases-endothelial-cell-migration
#17
Ju Liu, Josephine Hoh
Tumor growth depends on angiogenesis, the growth of new blood vessels. Complement factor H (CFH) is a plasma glycoprotein that functions as a regulator of the complement system. The aim of this study is to delineate the role of CFH in angiogenesis. A conditional null allele of the Cfh gene was generated in C57BL/6J mice by flanking the exon 3 with loxP sites. The Cfh(flox/flox) mice were crossed with Rosa26-Cre mice to obtain the mice homozygotes of Cfh deletion (Cfh(-/-)). The Cfh(-/-) mice were examined by in vivo angiogenesis assays...
2017: Journal of Cancer
https://www.readbyqxmd.com/read/28815506/preparing-fosmid-mate-paired-libraries-using-cre-loxp-recombination
#18
Ze Peng, Jeff L Froula, Jan-Fang Cheng
Fosmid end sequencing has been widely utilized in genome sequence assemblies and genome structural variation studies. We have developed a new approach to construct fosmid paired-end libraries that is suitable for Illumina sequencing platform. This approach employs a newly modified fosmid vector (pFosClip) which contains two loxP sites with identical orientation and two inverse Illumina adaptor priming sites flanking the cloning site. DNA prepared from the fosmid library constructed with pFosClip can be treated with the Cre recombinase to remove most of the vector DNA, leaving only 107 bp of the vector sequence with insert DNA...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815505/preparing-mate-paired-illumina-libraries-using-cre-recombinase
#19
Ze Peng, Nandita Nath, Zhiying Zhao, Jeff L Froula, Jan-Fang Cheng, Feng Chen
Large insert mate pair reads have been used in de novo assembly and discovery of structural variants. We developed a new approach, Cre-LoxP inverse PCR paired end (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends. We have successfully created CLIP-PE libraries of up to 22 kb jumping pairs and demonstrated their ability to improve genome assemblies...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815500/nanoparticle-mediated-recombinase-delivery-into-maize
#20
Susana Martin-Ortigosa, Brian G Trewyn, Kan Wang
We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a reporter gene. The release of the Cre recombinase protein inside the plant cell leads to recombination at the loxP sites, eliminating both genes. Visual screening is used to identify recombination events, which can be regenerated to mature and fertile plants...
2017: Methods in Molecular Biology
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