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https://www.readbyqxmd.com/read/28937836/optimizing-a-3d-model-system-for-molecular-manipulation-of-tenogenesis
#1
Chun Chien, Brian Pryce, Sara F Tufa, Douglas R Keene, Alice H Huang
PURPOSE: Tendon injuries are clinically challenging due to poor healing. A better understanding of the molecular events that regulate tendon differentiation would improve current strategies for repair. The mouse model system has been instrumental to tendon studies and several key molecules were initially established in mouse. However, the study of gene function has been limited by the absence of a standard in vitro tendon system for efficiently testing multiple mutations, physical manipulations, and mis-expression...
September 22, 2017: Connective Tissue Research
https://www.readbyqxmd.com/read/28934476/crystal-structure-of-an-engineered-hiv-specific-recombinase-for-removal-of-integrated-proviral-dna
#2
Gretchen Meinke, Janet Karpinski, Frank Buchholz, Andrew Bohm
As part of the HIV infection cycle, viral DNA inserts into the genome of host cells such that the integrated DNA encoding the viral proteins is flanked by long terminal repeat (LTR) regions from the retrovirus. In an effort to develop novel genome editing techniques that safely excise HIV provirus from cells, Tre, an engineered version of Cre recombinase, was designed to target a 34-bp sequence within the HIV-1 LTR (loxLTR). The sequence targeted by Tre lacks the symmetry present in loxP, the natural DNA substrate for Cre...
September 19, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28934293/generation-of-a-pancreatic-cancer-model-using-a-pdx1-flp-recombinase-knock-in-allele
#3
Jinghai Wu, Xin Liu, Sunayana G Nayak, Jason R Pitarresi, Maria C Cuitiño, Lianbo Yu, Blake E Hildreth, Katie A Thies, Daniel J Schilling, Soledad A Fernandez, Gustavo Leone, Michael C Ostrowski
The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the in vivo investigation of stromal genetics in tumor initiation and progression...
2017: PloS One
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
#4
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/28892037/induction-and-micro-ct-imaging-of-cerebral-cavernous-malformations-in-mouse-model
#5
Jaesung P Choi, Xi Yang, Matthew Foley, Xian Wang, Xiangjian Zheng
Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains...
September 4, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28886748/erbb3-drives-mammary-epithelial-survival-and-differentiation-during-pregnancy-and-lactation
#6
Michelle M Williams, David B Vaught, Meghan Morrison Joly, Donna J Hicks, Violeta Sanchez, Philip Owens, Bushra Rahman, David L Elion, Justin M Balko, Rebecca S Cook
BACKGROUND: During pregnancy, as the mammary gland prepares for synthesis and delivery of milk to newborns, a luminal mammary epithelial cell (MEC) subpopulation proliferates rapidly in response to systemic hormonal cues that activate STAT5A. While the receptor tyrosine kinase ErbB4 is required for STAT5A activation in MECs during pregnancy, it is unclear how ErbB3, a heterodimeric partner of ErbB4 and activator of phosphatidyl inositol-3 kinase (PI3K) signaling, contributes to lactogenic expansion of the mammary gland...
September 8, 2017: Breast Cancer Research: BCR
https://www.readbyqxmd.com/read/28884467/cre-expressing-neurons-in-visual-cortex-of-ntsr1-cre-gn220-mice-are-corticothalamic-and-are-depolarized-by-acetylcholine
#7
Sofie Charlotte Sundberg, Sarah Helen Lindström, Gonzalo Manuel Sanchez, Björn Granseth
The Ntsr1-Cre GN220 mouse expresses Cre-recombinase in corticothalamic (CT) neurons in neocortical layer 6. It is not known if the other major types of pyramidal neurons in this layer also express this enzyme. By electrophysiological recordings in slices and histological analysis of the uptake of retrogradely transported beads we show that Cre-positive neurons are corticothalamic and not corticocortical or cortico-claustral types. Furthermore, we show that Ntsr1-Cre-positive cells are immuno-positive for the nuclear transcription factor Forkhead box protein P2 (FoxP2)...
September 7, 2017: Journal of Comparative Neurology
https://www.readbyqxmd.com/read/28875587/a-new-mouse-line-for-cell-ablation-by-diphtheria-toxin-subunit-a-controlled-by-a-cre-dependent-flex-switch
#8
Nicholas W Plummer, Erica K Ungewitter, Kathleen G Smith, Humphrey H-C Yao, Patricia Jensen
Recombinase responsive mouse lines expressing diphtheria toxin subunit A (DTA) are well established tools for targeted ablation of genetically defined cell populations. Here we describe a new knock-in allele at the Gt(Rosa)26Sor locus that retains the best features of previously described DTA alleles-including a CAG promoter, attenuated mutant DTA cDNA, and ubiquitous EGFP labeling-with the addition of a Cre-dependent FLEx switch for tight control of expression. The FLEx switch consists of two pairs of antiparallel lox sites requiring Cre-mediated recombination for inversion of the DTA to the proper orientation for transcription...
September 5, 2017: Genesis: the Journal of Genetics and Development
https://www.readbyqxmd.com/read/28874756/foxo1-deletion-in-keratinocytes-improves-diabetic-wound-healing-through-mmp9-regulation
#9
Chenying Zhang, Jason Lim, Hyeran Helen Jeon, Fanxing Xu, Chen Tian, Fang Miao, Alhassan Hameedaldeen, Dana T Graves
Keratinocyte migration is a key aspect of re-epithelialization during wound healing. Matrix metalloproteinase 9 (MMP9) contributes to this process and deficiencies in the MMP9 lead to impaired healing. Inappropriate expression of MMP9 also contributes to impaired re-epithelialization. Previously we demonstrated that FOXO1 was activated in wound healing but to higher levels in diabetic wounds. To address mechanisms of impaired re-epithelialization we examined MMP9 expression in vivo in full thickness dermal scalp wounds created in experimental K14...
September 5, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28874450/hypothalamic-tuberomammillary-nucleus-neurons-electrophysiological-diversity-and-essential-role-in-arousal-stability
#10
Akie Fujita, Patricia Bonnavion, Miryam H Wilson, Laura E Mickelsen, Julien Bloit, Luis de Lecea, Alexander C Jackson
Histaminergic (HA) neurons, found in the posterior hypothalamic tuberomammillary nucleus (TMN), extend fibers throughout the brain and exert modulatory influence over numerous physiological systems. Multiple lines of evidence suggest that the activity of HA neurons is important in the regulation of vigilance despite the lack of direct, causal evidence demonstrating its requirement for the maintenance of arousal during wakefulness. Given the strong correlation between HA neuron excitability and behavioral arousal, we investigated both the electrophysiological diversity of HA neurons in brain slices and the effect of their acute silencing in vivo in male mice...
September 5, 2017: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
https://www.readbyqxmd.com/read/28872120/dissection-of-the-mouse-pancreas-for-histological-analysis-and-metabolic-profiling
#11
Michelle J Veite-Schmahl, Daniel P Regan, Adam C Rivers, Joseph F Nowatzke, Michael A Kennedy
We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1a(cre/+);LSL-Kras(G12D/+)) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression...
August 19, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28867552/connexin-43-mediated-astroglial-metabolic-networks-contribute-to-the-regulation-of-the-sleep-wake-cycle
#12
Jerome Clasadonte, Eliana Scemes, Zhongya Wang, Detlev Boison, Philip G Haydon
Astrocytes produce and supply metabolic substrates to neurons through gap junction-mediated astroglial networks. However, the role of astroglial metabolic networks in behavior is unclear. Here, we demonstrate that perturbation of astroglial networks impairs the sleep-wake cycle. Using a conditional Cre-Lox system in mice, we show that knockout of the gap junction subunit connexin 43 in astrocytes throughout the brain causes excessive sleepiness and fragmented wakefulness during the nocturnal active phase. This astrocyte-specific genetic manipulation silenced the wake-promoting orexin neurons located in the lateral hypothalamic area (LHA) by impairing glucose and lactate trafficking through astrocytic networks...
September 13, 2017: Neuron
https://www.readbyqxmd.com/read/28855574/genetic-studies-in-mice-directly-link-oocytes-produced-during-adulthood-to-ovarian-function-and-natural-fertility
#13
Ning Wang, Chonthicha Satirapod, Yasuyo Ohguchi, Eun-Sil Park, Dori C Woods, Jonathan L Tilly
Multiple labs have reported that mammalian ovaries contain oogonial stem cells (OSCs), which can differentiate into oocytes that fertilize to produce offspring. However, the physiological relevance of these observations to adult ovarian function is unknown. Here we performed targeted and reversible ablation of premeiotic germ cells undergoing differentiation into oocytes in transgenic mice expressing the suicide gene, herpes simplex virus thymidine kinase (HSVtk), driven by the promoter of stimulated by retinoic acid gene 8 (Stra8), a germ cell-specific gene activated during meiotic commitment...
August 30, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28842412/imaging-voltage-in-genetically-defined-neuronal-subpopulations-with-a-cre-recombinase-targeted-hybrid-voltage-sensor
#14
Peter O Bayguinov, Yihe Ma, Yu Gao, Xinyu Zhao, Meyer B Jackson
Genetically-encoded voltage indicators (GEVIs) create an opportunity to monitor electrical activity in defined sets of neurons as they participate in the complex patterns of coordinated electrical activity that underlie nervous system function. Taking full advantage of GEVIs requires a generalized strategy for targeting the probe to genetically-defined populations of cells. To this end we have generated a mouse line with an optimized hybrid voltage sensor (hVOS) probe within a locus designed for efficient Cre recombinase dependent expression...
August 23, 2017: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
https://www.readbyqxmd.com/read/28841687/a-trp53fl-flptenfl-fl-mouse-model-of-undifferentiated-pleomorphic-sarcoma-mediated-by-adeno-cre-injection-and-in-vivo-bioluminescence-imaging
#15
Marisa R Buchakjian, Nicole M Merritt, Devon L Moose, Adam J Dupuy, Munir R Tanas, Michael D Henry
Genetic mouse models of soft tissue sarcoma provide critical insights into disease pathophysiology, which are oftentimes unable to be extracted from human tumor samples or xenograft models. In this study we describe a mouse model of soft tissue sarcoma mediated by adenoviral-Cre recombinase injection into Trp53fl/fl/Ptenfl/fl lox-stop-lox luciferase mice. Injection of adenovirus expressing Cre recombinase, either subcutaneously or intramuscularly in two experimental groups, results in viral infection and gene recombination with 100% penetrance within the first 24 hours following injection...
2017: PloS One
https://www.readbyqxmd.com/read/28835503/murine-olfactory-bulb-interneurons-survive-infection-with-a-neurotropic-coronavirus
#16
D Lori Wheeler, Jeremiah Athmer, David K Meyerholz, Stanley Perlman
Viral infection of the central nervous system is complicated by the mostly irreplaceable nature of neurons, as the loss of neurons has the potential to result in permanent damage to brain function. However, whether neurons or other cells in the CNS sometimes survive infection and the effects of infection on neuronal function are largely unknown. To address this question, we used the rJHM strain (rJ) of mouse hepatitis virus, (MHV), a neurotropic coronavirus, which causes acute encephalitis in susceptible strains of mice...
August 23, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28835125/small-but-increasingly-mighty-latest-advances-in-aav-vector-research-design-and-evolution
#17
Dirk Grimm, Hildegard Büning
Recombinant gene delivery vectors derived from naturally occurring or genetically engineered Adeno-associated viruses (AAV) have taken center stage in human gene therapy, fueled by rapidly accumulating and highly encouraging clinical data. Nonetheless, it has also become evident that the current generation of AAV vectors will require improvements in transduction potency, antibody evasion and cell specificity, to realize their full potential and to widen applicability in larger patient cohorts. Fortunately, in the recent past, the field has seen a flurry of exciting new developments that enhance our understanding of AAV vector biology including virus-host interactions and/or that expand our arsenal of technologies for AAV capsid design and evolution...
August 23, 2017: Human Gene Therapy
https://www.readbyqxmd.com/read/28829806/genome-engineering-in-bacillus-anthracis-using-tyrosine-site-specific-recombinases
#18
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/28818856/deletion-of-nf-%C3%AE%C2%BAb-rela-in-angiotensin-ii-sensitive-mesenchymal-cells-blocks-aortic-vascular-inflammation-and-abdominal-aortic-aneurysm-formation
#19
Talha Ijaz, Hong Sun, Irina V Pinchuk, Dianna M Milewicz, Ronald G Tilton, Allan R Brasier
OBJECTIVE: Infusion of angiotensin II (Ang II) induces extracellular matrix remodeling and inflammation resulting in abdominal aortic aneurysms (AAAs) in normolipidemic mice. Although Ang II activates mesenchymal cells in the media and adventitia to become fibrogenic, the sentinel role of this mesenchymal population in modulating the inflammatory response and aneurysms is not known. We test the hypothesis that these fibrogenic mesenchymal cells play a critical role in Ang II-induced aortic wall vascular inflammation and AAA formation...
August 17, 2017: Arteriosclerosis, Thrombosis, and Vascular Biology
https://www.readbyqxmd.com/read/28815507/using-purified-tyrosine-site-specific-recombinases-in-vitro-to-rapidly-construct-and-diversify-metabolic-pathways
#20
Wei Liu, Laura R Tuck, Jon Marles Wright, Yizhi Cai
The site-specific recombinase Cre was previously reported to have in vitro activity. Here, we describe the method of purifying two new tyrosine site-specific recombinases VCre and Dre along with Cre by nickel affinity chromatography. We proved the in vitro function of the VCre and Dre on their respective conditional recombination sites. We also developed a methodology to one-step construct and optimize the productivity of a biosynthetic pathway through the combinatorial integration of promoters into a plasmid-encoded pathway by simply incubating a DNA mixture with recombinase system at 37 °C in vitro...
2017: Methods in Molecular Biology
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