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https://www.readbyqxmd.com/read/28819420/loss-of-complement-factor-h-in-plasma-increases-endothelial-cell-migration
#1
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
#2
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
#3
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
#4
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
https://www.readbyqxmd.com/read/28815499/marker-removal-in-transgenic-plants-using-cre-recombinase-delivered-with-potato-virus-x
#5
Lilya Kopertekh, Joachim Schiemann
In this chapter we present an alternative method to develop marker-free transgenic plants. It makes use of the Cre/loxP recombination system from bacteriophage P1 and consists of two essential components. The first component is the transgenic plant containing a loxP-flanked marker gene. The second component is a cre transient expression vector based on potato virus X. The great benefit of this transient delivery method consists in the avoidance of stable integration of the cre recombinase gene into the plant genome...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815496/injection-based-delivery-of-cell-permeable-peptide-tagged-cre
#6
Wei-Ming Chien, Yonggang Liu, Ana A Dinca, Michael T Chin
The technique of Cre-mediated DNA recombination at loxP sites has been used widely in manipulation of the genome in cultured cells and in living organisms. Local delivery of Cre recombinase protein tagged with a cell-penetrating (or permeable) peptide (Cre-CPP) has the advantage of additional spatial and temporal control when compared to genetic delivery methods. In this chapter, we describe protocols for injection-based intramuscular delivery of Cre-CPP dissolved in hydrogel to skeletal muscle and by ultrasound-guided injection to cardiac muscle in mice...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815492/building-cre-knockin-rat-lines-using-crispr-cas9
#7
Yuanwu Ma, Lianfeng Zhang, Xingxu Huang
Conditional gene inactivation strategy helps researchers to study the gene functions that are critical in embryogenesis or in defined tissues of adulthood. The Cre/loxP system is widely used for conditional gene inactivation/activation in cells or organisms. Cre knockin animal lines are essential for gene expression or inactivation in a spatially and temporally restricted manner. However, to generate a Cre knockin line by traditional approach is laborious. Recently, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) has been proven as a simple and efficient genome-editing tool...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28815491/direct-generation-of-conditional-alleles-using-crispr-cas9-in-mouse-zygotes
#8
Colin E J Pritchard, Lona J Kroese, Ivo J Huijbers
Conditional alleles in genetically modified mice allow for the deletion of a gene of interest in a target tissue when combined with a tissue-specific Cre recombinase. A conditional allele is achieved by introducing LoxP sites around a critical exon, a gene, or a cluster of genes. Previously, conditional alleles were introduced in the mouse germline by classic gene targeting in embryonic stem cells, a challenging and time-consuming procedure. Now, conditional alleles can be generated directly in fertilized mouse eggs (zygotes) using the CRISPR/Cas9 technology...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28813413/polylox-barcoding-reveals-haematopoietic-stem-cell-fates-realized-in-vivo
#9
Weike Pei, Thorsten B Feyerabend, Jens Rössler, Xi Wang, Daniel Postrach, Katrin Busch, Immanuel Rode, Kay Klapproth, Nikolaus Dietlein, Claudia Quedenau, Wei Chen, Sascha Sauer, Stephan Wolf, Thomas Höfer, Hans-Reimer Rodewald
Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system...
August 16, 2017: Nature
https://www.readbyqxmd.com/read/28811218/wnt-signaling-positively-regulates-endothelial-cell-fate-specification-in-the-fli1a-positive-progenitor-population-via-lef1
#10
Kathleen Hübner, Kathrin S Grassme, Jyoti Rao, Nina K Wenke, Cordula L Zimmer, Laura Korte, Katja Mu Ller, Saulius Sumanas, Boris Greber, Wiebke Herzog
During vertebrate embryogenesis, vascular endothelial cells (ECs) and primitive erythrocytes become specified within close proximity in the posterior lateral plate mesoderm (LPM) from a common progenitor. However, the signaling cascades regulating the specification into either lineage remain largely elusive. Here, we analyze the contribution of β-catenin dependent Wnt signaling to EC and erythrocyte specification during zebrafish embryogenesis. We generated novel β-catenin dependent Wnt signaling reporters which, by using destabilized fluorophores (Venus-Pest, dGFP), specifically allow us to detect Wnt signaling responses in narrow time windows as well as in spatially restricted domains, defined by Cre recombinase expression (Tg(axin2BAC:Venus-Pest)(mu288); Tg(14TCF:loxP-STOP-loxP-dGFP)(mu202))...
August 12, 2017: Developmental Biology
https://www.readbyqxmd.com/read/28801604/recombineering-using-recet-in-corynebacterium-glutamicum-atcc14067-via-a-self-excisable-cassette
#11
Yuanyuan Huang, Lu Li, Shan Xie, Nannan Zhao, Shuangyan Han, Ying Lin, Suiping Zheng
Gene manipulation is essential for metabolic engineering and synthetic biology, but the current general gene manipulation methods are not applicable to the non-model strain Corynebacterium glutamicum (C. glutamicum) ATCC14067, which is used for amino acid production. Here, we report an effective and sequential deletion method for C. glutamicum ATCC14067 using the exonuclease-recombinase pair RecE + RecT (RecET) for recombineering via a designed self-excisable linear double-strand DNA (dsDNA) cassette, which contains the Cre/loxP system, to accomplish markerless deletion...
August 11, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28767511/protein-kinase-a-regulates-inflammatory-pain-sensitization-by-modulating-hcn2-channel-activity-in-nociceptive-sensory-neurons
#12
Stefan Herrmann, Hamsa Rajab, Irina Christ, Christoph Schirdewahn, Daniel Höfler, Michael Jm Fischer, Ariane Bruno, Stefanie Fenske, Christian Gruner, Felix Kramer, Tassilo Wachsmann, Christian Wahl-Schott, Juliane Stieber, Martin Biel, Andreas Ludwig
Several studies implicated cAMP as an important second messenger for regulating nociceptor sensitization, but downstream targets of this signaling pathway which contribute to neuronal plasticity are not well understood. We used a Cre/loxP based strategy to disable the function of either HCN2 or PKA selectively in a subset of peripheral nociceptive neurons and analyzed the nociceptive responses in both transgenic lines. A near complete lack of sensitization was observed in both mutant strains when peripheral inflammation was induced by intradermal injection of 8br-cAMP...
July 31, 2017: Pain
https://www.readbyqxmd.com/read/28763160/crispr-cas9-mediated-targeting-of-the-rosa26-locus-produces-cre-reporter-rat-strains-for-monitoring-cre-loxp-mediated-lineage-tracing
#13
Yuanwu Ma, Lei Yu, Shuo Pan, Shan Gao, Wei Chen, Xu Zhang, Wei Dong, Jing Li, Rui Zhou, Lan Huang, Yunlin Han, Lin Bai, Li Zhang, Lianfeng Zhang
The rat is an important laboratory animal for physiological, toxicological and pharmacological studies. CRISPR/Cas9 is a simple and efficient tool to generate precise genetic modifications in rats, which will promote the accumulation of rat genetic resources and enable more precise studies of gene function. To monitor Cre/loxP-mediated excision in vivo, we generated a Cre reporter rat strain (Rosa26-imCherry) by knockin of a Cre reporter cassette at Rosa26 locus using CRISPR/Cas9. Rosa26-imCherry rats exhibited inducible expression of the mCherry cassette (imCherry) using the Cre/loxP system, whereas normal rats exhibited ubiquitous expression of eGFP but not mCherry in the whole body...
August 1, 2017: FEBS Journal
https://www.readbyqxmd.com/read/28759006/re-expression-of-sall1-in-podocytes-protects-against-adriamycin-induced-nephrosis
#14
Yoshiko Hosoe-Nagai, Teruo Hidaka, Ayano Sonoda, Yu Sasaki, Kanae Yamamoto-Nonaka, Takuto Seki, Rin Asao, Eriko Tanaka, Juan Alejandro Oliva Trejo, Fumiko Kodama, Miyuki Takagi, Nobuhiro Tada, Takashi Ueno, Ryuichi Nishinakamura, Yasuhiko Tomino, Katsuhiko Asanuma
The highly conserved spalt (sal) gene family members encode proteins characterized by multiple double zinc finger motifs of the C2H2 type. Humans and mice each have four known Sal-like genes (SALL1-4 in humans and Sall1-4 in mice). Sall1 is known to have a crucial role in kidney development. To explore the significance of Sall1 in differentiated podocytes, we investigated podocyte-specific Sall1-deficient mice (Sall1 KO(p)°(d)°(/p)°(d)°) using a podocin-Cre/loxP system and siRNA Sall1 knockdown (Sall1 KD) podocytes...
July 31, 2017: Laboratory Investigation; a Journal of Technical Methods and Pathology
https://www.readbyqxmd.com/read/28751573/sox10-cells-contribute-to-vascular-development-in-multiple-organs
#15
Dong Wang, Fan Wu, Haoyong Yuan, Aijun Wang, Gyeong Jin Kang, Tan Truong, Lu Chen, Andrew S McCallion, Xiaohua Gong, Song Li
OBJECTIVE: Previous genetic lineage tracing studies showed that Sox10+ cells differentiate into vascular mural cells, limited to neural crest-derived blood vessels in craniofacial tissues, aortic arch, pulmonary arch arteries, brachiocephalic, carotid arteries, and thymus. The purpose of this study was to investigate the contribution of Sox10+ cells to the vascular development in other tissues and organs and their relationship with neural crest. APPROACH AND RESULTS: Using genetic lineage tracing technique based on Cre/LoxP system, we examined blood vessels of the adult organs of Sox10-Cre/Rosa-LoxP-red fluorescent protein and Wnt1-Cre/Rosa-LoxP-red fluorescent protein mice by immunohistological analysis...
July 27, 2017: Arteriosclerosis, Thrombosis, and Vascular Biology
https://www.readbyqxmd.com/read/28722653/directing-visceral-white-adipocyte-precursors-to-a-thermogenic-adipocyte-fate-improves-insulin-sensitivity-in-obese-mice
#16
Chelsea Hepler, Mengle Shao, Jonathan Y Xia, Alexandra L Ghaben, Mackenzie J Pearson, Lavanya Vishvanath, Ankit X Sharma, Thomas S Morley, William L Holland, Rana K Gupta
Visceral adiposity confers significant risk for developing metabolic disease in obesity whereas preferential expansion of subcutaneous white adipose tissue (WAT) appears protective. Unlike subcutaneous WAT, visceral WAT is resistant to adopting a protective thermogenic phenotype characterized by the accumulation of Ucp1(+) beige/BRITE adipocytes (termed "browning"). In this study, we investigated the physiological consequences of browning murine visceral WAT by selective genetic ablation of Zfp423, a transcriptional suppressor of the adipocyte thermogenic program...
July 19, 2017: ELife
https://www.readbyqxmd.com/read/28715425/elimination-of-huntingtin-in-the-adult-mouse-leads-to-progressive-behavioral-deficits-bilateral-thalamic-calcification-and-altered-brain-iron-homeostasis
#17
Paula Dietrich, Irudayam Maria Johnson, Shanta Alli, Ioannis Dragatsis
Huntington's Disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by cognitive, behavioral and motor dysfunctions. HD is caused by a CAG repeat expansion in exon 1 of the HD gene that is translated into an expanded polyglutamine tract in the encoded protein, huntingtin (HTT). While the most significant neuropathology of HD occurs in the striatum, other brain regions are also affected and play an important role in HD pathology. To date there is no cure for HD, and recently strategies aiming at silencing HTT expression have been initiated as possible therapeutics for HD...
July 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28707700/deleting-the-arntl-clock-gene-in-the-granular-layer-of-the-mouse-cerebellum-impact-on-the-molecular-circadian-clockwork
#18
Tenna Bering, Mikkel Bloss Carstensen, Martin Fredensborg Rath
The suprachiasmatic nucleus houses the central circadian clock and is characterized by the timely regulated expression of clock genes. However, neurons of the cerebellar cortex also contain a circadian oscillator with circadian expression of clock genes being controlled by the suprachiasmatic nucleus. It has been suggested that the cerebellar circadian oscillator is involved in food anticipation, but direct molecular evidence of the role of the circadian oscillator of the cerebellar cortex is currently unavailable...
July 14, 2017: Journal of Neurochemistry
https://www.readbyqxmd.com/read/28705795/selective-deletion-of-leptin-signaling-in-endothelial-cells-enhances-neointima-formation-and-phenocopies-the-vascular-effects-of-diet-induced-obesity-in-mice
#19
Astrid Hubert, Magdalena L Bochenek, Eva Schütz, Rajinikanth Gogiraju, Thomas Münzel, Katrin Schäfer
OBJECTIVE: Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. APPROACH AND RESULTS: Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter...
July 13, 2017: Arteriosclerosis, Thrombosis, and Vascular Biology
https://www.readbyqxmd.com/read/28702330/neonatal-glp1r-activation-limits-adult-adiposity-by-durably-altering-hypothalamic-architecture
#20
Andrea V Rozo, Daniella A Babu, PoMan A Suen, David N Groff, Randy J Seeley, Rebecca A Simmons, Patrick Seale, Rexford S Ahima, Doris A Stoffers
OBJECTIVE: Adult obesity risk is influenced by alterations to fetal and neonatal environments. Modifying neonatal gut or neurohormone signaling pathways can have negative metabolic consequences in adulthood. Here we characterize the effect of neonatal activation of glucagon like peptide-1 (GLP-1) receptor (GLP1R) signaling on adult adiposity and metabolism. METHODS: Wild type C57BL/6 mice were injected with 1 nmol/kg Exendin-4 (Ex-4), a GLP1R agonist, for 6 consecutive days after birth...
July 2017: Molecular Metabolism
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