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Cre/loxp

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https://www.readbyqxmd.com/read/28446024/enhanced-vector-design-for-cancer-gene-therapy-with-hierarchical-enhancement-of-therapeutic-transgene-expression
#1
Maria B Kostina, Alexader V Sass, Elena A Stukacheva, Igor V Korobko, Eugeny D Sverdlov
A set of vectors for Cre recombinase-dependent expression of hybrid suicidal FCU1 transgene was constructed which included two-plasmid system where FCU1 and Cre transgenes reside in separate vectors, and single-plasmid variants when a single plasmid bears both transgenes. To improve safety profile and specificity in cancer gene therapy applications, as well as to assure stable propagation of plasmids in bacterial cells, Cre/LoxP system components were optimized. Bicistronic vector with Cre expression cassette placed between LoxP sites unidirectionally with FCU1 cDNA resulted in higher therapeutic efficiency compared to double-plasmid system in enzyme-prodrug suicide cancer gene therapy scheme...
April 27, 2017: Human Gene Therapy Methods
https://www.readbyqxmd.com/read/28433771/a-versatile-tumor-gene-deletion-system-reveals-a-crucial-role-for-fgfr1-in-breast-cancer-metastasis
#2
Wei Wang, Yanling Meng, Bingning Dong, Jie Dong, Michael M Ittmann, Chad J Creighton, Lu Yang, Hong Zhang, Tao Shen, Jianghua Wang, David R Rowley, Yi Li, Fengju Chen, David D Moore, Feng Yang
RCAS avian viruses have been used to deliver oncogene expression and induce tumors in transgenic mice expressing the virus receptor TVA. Here we report the generation and characterization of a novel RCAS-Cre-IRES-PyMT (RCI-PyMT) virus designed to specifically knockout genes of interest in tumors generated in appropriate mutant mouse hosts. FGF receptor 1 (FGFR1) is a gene that is amplified in human breast cancer, but there have been no definitive studies on its function in mammary tumorigenesis, progression, and metastasis in vivo in spontaneous tumors in mice...
April 20, 2017: Neoplasia: An International Journal for Oncology Research
https://www.readbyqxmd.com/read/28420716/a-men1-pancreatic-neuroendocrine-tumour-mouse-model-under-temporal-control
#3
Kate E Lines, Roeland P Vas Nunes, Morten Frost, Christopher J Yates, Mark Stevenson, Rajesh Thakker
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by occurrence of parathyroid tumours, and neuroendocrine tumours (NETs) of the pancreatic islets and anterior pituitary. The MEN1 gene, encoding menin, is a tumour suppressor, but its precise role in initiating in vivo tumourigenesis remains to be elucidated. The availability of a temporally controlled conditional MEN1 mouse model would greatly facilitate the study of such early tumourigenic events, and overcome the limitations of other MEN1 knockout models, in which menin is lost from conception, or tumour development occurs asynchronously...
April 18, 2017: Endocrine Connections
https://www.readbyqxmd.com/read/28416649/optic-nerve-astrocyte-reactivity-protects-function-in-experimental-glaucoma-and-other-nerve-injuries
#4
Daniel Sun, Sara Moore, Tatjana C Jakobs
Reactive remodeling of optic nerve head astrocytes is consistently observed in glaucoma and other optic nerve injuries. However, it is unknown whether this reactivity is beneficial or harmful for visual function. In this study, we used the Cre recombinase (Cre)-loxP system under regulation of the mouse glial fibrillary acidic protein promoter to knock out the transcription factor signal transducer and activator of transcription 3 (STAT3) from astrocytes and test the effect this has on reactive remodeling, ganglion cell survival, and visual function after experimental glaucoma and nerve crush...
April 17, 2017: Journal of Experimental Medicine
https://www.readbyqxmd.com/read/28407687/cytokeratin-19-promoter-directs-the-expression-of-cre-recombinase-in-various-epithelia-of-transgenic-mice
#5
Gui-Feng Zhao, Shuang Zhao, Jia-Jie Liu, Ji-Cheng Wu, Hao-Yu He, Xiao-Qing Ding, Xue-Wen Yu, Ke-Qiang Huang, Zhi-Jie Li, Hua-Chuan Zheng
Cytokeratin 19 (K19) is expressed in various differentiated cells, including gastric, intestinal and bronchial epithelial cells, and liver duct cells. Here, we generated a transgenic mouse line, K19-Cre, in which the expression of Cre recombinase was controlled by the promoter of K19. To test the tissue distribution and excision activity of Cre recombinase, K19-Cre transgenic mice were bred with Rosa26 reporter strain and a mouse strain that carries PTEN conditional alleles (PTENLoxp/Loxp). At mRNA level, Cre was strongly expressed in the stomach, lung and intestine, while in stomach, lung, and liver at protein level...
March 14, 2017: Oncotarget
https://www.readbyqxmd.com/read/28401685/characterization-of-new-bone-morphogenetic-protein-bmp-2-regulatory-alleles
#6
Tapan A Shah, Youhua Zhu, Nadia N Shaikh, Marie A Harris, Stephen E Harris, Melissa B Rogers
Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell proliferation, differentiation, and apoptosis. Key events requiring precise Bmp2 regulation include heart specification and morphogenesis and neural development. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence pattern formation and organogenesis, the mechanisms that regulate Bmp2 are crucial...
April 12, 2017: Genesis: the Journal of Genetics and Development
https://www.readbyqxmd.com/read/28401157/engineering-of-systematic-elimination-of-a-targeted-chromosome-in-human-cells
#7
Hiroshi Sato, Hiroki Kato, Haruyoshi Yamaza, Keiji Masuda, Huong Thi Nguyen Nguyen, Thanh Thi Mai Pham, Xu Han, Yuta Hirofuji, Kazuaki Nonaka
Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28389020/seeds-in-the-liver
#8
REVIEW
Hongjie Ji, Yanrong Lu, Yujun Shi
The liver is a crucial organ for homeostasis and has a tremendous self-renewal and regenerative capacity. It has long been believed that the self-renewal and repair of the liver within a given physiological condition or its repopulation in chronic liver diseases, when hepatocyte proliferation is impaired, will primarily be conducted by the proliferating duct cells, termed "oval cells" or hepatic progenitor cells (HPCs). In addition, numerous studies have revealed that HPCs are the initial tumor cells of liver cancer under certain micro-environments...
April 4, 2017: Acta Histochemica
https://www.readbyqxmd.com/read/28387239/survival-of-human-embryonic-stem-cells-implanted-in-the-guinea-pig-auditory-epithelium
#9
Min Young Lee, Sandra Hackelberg, Kari L Green, Kelly G Lunghamer, Takaomi Kurioka, Benjamin R Loomis, Donald L Swiderski, R Keith Duncan, Yehoash Raphael
Hair cells in the mature cochlea cannot spontaneously regenerate. One potential approach for restoring hair cells is stem cell therapy. However, when cells are transplanted into scala media (SM) of the cochlea, they promptly die due to the high potassium concentration. We previously described a method for conditioning the SM to make it more hospitable to implanted cells and showed that HeLa cells could survive for up to a week using this method. Here, we evaluated the survival of human embryonic stem cells (hESC) constitutively expressing GFP (H9 Cre-LoxP) in deaf guinea pig cochleae that were pre-conditioned to reduce potassium levels...
April 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28384353/generation-and-characterization-of-epoxide-hydrolase-3-ephx3-deficient-mice
#10
Samantha L Hoopes, Artiom Gruzdev, Matthew L Edin, Joan P Graves, J Alyce Bradbury, Gordon P Flake, Fred B Lih, Laura M DeGraff, Darryl C Zeldin
Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH) and soluble epoxide hydrolase (EPHX2, sEH) were identified >30 years ago and are capable of hydrolyzing EETs to DHETs...
2017: PloS One
https://www.readbyqxmd.com/read/28379340/liver-nf-%C3%AE%C2%BAb-inducing-kinase-nik-promotes-liver-steatosis-and-glucose-counterregulation-in-male-mice-with-obesity
#11
Yan Liu, Liang Sheng, Yi Xiong, Hong Shen, Yong Liu, Liangyou Rui
Obesity is associated with chronic inflammation and liver steatoses. Numerous proinflammatory cytokines have been reported to regulate liver glucose and lipid metabolism, thus contributing to the pathogenesis of liver steatosis and/or metabolic dysfunction. NIK is stimulated by many cytokines and mediates activation of the noncanonical NF-κB pathway. We previously reported that liver NIK is aberrantly activated in obesity; inactivation of NIK by overexpressing dominant negative NIK(KA) suppresses hepatic glucose production...
April 3, 2017: Endocrinology
https://www.readbyqxmd.com/read/28378505/prolactin-receptors-in-rip-cre-cells-but-not-in-agrp-neurons-are-involved-in-energy-homeostasis
#12
S R Ladyman, M A MacLeod, Z Khant Aung, P Knowles, H R Phillipps, R S E Brown, D R Grattan
Among its many functions, prolactin has been implicated in energy homeostasis, particularly during pregnancy and lactation. The arcuate nucleus is a key site in the regulation of energy balance. The aim of this study was to examine whether arcuate nucleus neuronal populations involved in energy homeostasis are prolactin responsive and if they can mediate the effects of prolactin on energy homeostasis. To determine if Agrp neurons or Rip-Cre neurons are prolactin responsive, transgenic mice expressing the reporter td-tomato in Agrp neurons (td-tomato/AgRP-Cre) or Rip-Cre neurons (td-tomato/Rip-Cre) were treated with prolactin and perfused 45 minutes later...
April 4, 2017: Journal of Neuroendocrinology
https://www.readbyqxmd.com/read/28373488/popping-out-mx-cassettes-from-saccharomyces-cerevisiae
#13
John H McCusker
MX cassettes are frequently used to generate knockout (KO) mutations in Saccharomyces cerevisiae. The recycling or "popping out" of an MX cassette flanked by direct repeats allows the same cassette to be reused in a strain to generate additional KO mutations. Popping out MX cassettes also eliminates MX homology in a strain, which facilitates the subsequent generation of additional KO mutations with other MX cassettes. MX cassettes can be recycled or "popped out" of the genome by spontaneous recombination between large, cassette-borne MX3 or PR direct repeats and by Cre-mediated, site-specific recombination between small, cassette-borne loxP direct repeats...
April 3, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28371069/specific-and-spatial-labeling-of-p0-cre-versus-wnt1-cre-in-cranial-neural-crest-in-early-mouse-embryos
#14
Guiqian Chen, Mohamed Ishan, Jingwen Yang, Satoshi Kishigami, Tomokazu Fukuda, Greg Scott, Manas K Ray, Chenming Sun, Shi-You Chen, Yoshihiro Komatsu, Yuji Mishina, Hong-Xiang Liu
P0-Cre and Wnt1-Cre mouse lines have been widely used in combination with loxP-flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1-Cre has been regarded as the gold standard and there have been concerns about the specificity of P0-Cre because it is not clear about the timing and spatial distribution of the P0-Cre transgene in labeling NC cells at early embryonic stages. We re-visited P0-Cre and Wnt1-Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC...
March 29, 2017: Genesis: the Journal of Genetics and Development
https://www.readbyqxmd.com/read/28364122/a-convenient-cas9-based-conditional-knockout-strategy-for-simultaneously-targeting-multiple-genes-in-mouse
#15
Jiang Chen, Yinan Du, Xueyan He, Xingxu Huang, Yun S Shi
The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding...
March 31, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28363520/sequence-segregation-improves-non-covalent-protein-delivery
#16
Federica Sgolastra, Coralie M Backlund, E Ilker Ozay, Brittany M deRonde, Lisa M Minter, Gregory N Tew
The impermeability of the plasma membrane towards large, hydrophilic biomolecules is a major obstacle in their use and development against intracellular targets. To overcome such limitations, protein transduction domains (PTDs) have been used as protein carriers, however they often require covalent fusion to the protein for efficient delivery. In an effort to develop more efficient and versatile biological vehicles, a series of PTD-inspired polyoxanorbornene-based synthetic mimics with identical chemical compositions but different hydrophobic/hydrophilic segregation were used to investigate the role of sequence segregation on protein binding and uptake into Jurkat T cells and HEK293Ts...
March 29, 2017: Journal of Controlled Release: Official Journal of the Controlled Release Society
https://www.readbyqxmd.com/read/28361238/a-novel-genetic-tool-for-metabolic-optimization-of-corynebacterium-glutamicum-efficient-and-repetitive-chromosomal-integration-of-synthetic-promoter-driven-expression-libraries
#17
Jing Shen, Jun Chen, Peter Ruhdal Jensen, Christian Solem
Fine-tuning the expression level of multiple genes is usually pivotal for metabolic optimization. We have developed a tool for this purpose for the important industrial workhorse Corynebacterium glutamicum that allows for the introduction of synthetic promoter-driven expression libraries of arbitrary genes. We first devised a method for introducing genetic elements into the chromosome repeatedly, relying on site-specific recombinases and the vector pJS31 serving as the carrier. The pJS31 vector contains a synthetic cassette including a phage attachment site attP for integration, a bacterial attachment site attB for subsequent integration, a multiple cloning site, and two modified loxP sites to facilitate easy removal of undesirable vector elements...
March 30, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28357594/knockdown-of-ggps1-in-chondrocyte-expedites-fracture-healing-by-accelerating-the-progression-of-endochondral-ossification-in-mice
#18
Bingyang Dai, Qiangqiang Li, Xiaoxiao Song, Yuxiang Ge, Jing Wu, Kaijia Zhang, Chao Wang, Yifeng Zhang, Huajian Teng, Chaojun Li, Qing Jiang
Bone fracture healing is achieved through the proliferation and differentiation of stem cells, while bone marrow stem cells (BMSCs) contribute to endochondral ossification. During fracture healing, mesenchymal progenitor cells first form a cartilaginous blastema that becomes vascularized to recruit precursor cells of osteoblasts through the bone morphogenetic protein 2 (Bmp2)/Smad-dependent Runx2 pathway. Statins deplete geranylgeranyl diphosphate (GGPP), which participates in the regulation of BMSCs differentiation, through the inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, leading to impaired protein geranylgeranylation, which strongly impacts the bone synthesis induced by Bmp2...
March 29, 2017: Journal of Bone and Mineral Metabolism
https://www.readbyqxmd.com/read/28352551/an-easy-method-for-preparation-of-cre-loxp-regulated-fluorescent-adenoviral-expression-vectors-and-its-application-for-direct-reprogramming-into-hepatocytes
#19
Chitose Kurihara, Koji Nakade, Jianzhi Pan, Jing Huang, Bohdan Wasylyk, Yuichi Obata
The recombinant adenoviral gene expression system is a powerful tool for gene delivery. However, it is difficult to obtain high titers of infectious virus, principally due to the toxicity of the expressed gene which affects on virus replication in the host HEK293 cells. To avoid these problems, we generated a Cre-loxP-regulated fluorescent universal vector (termed pAxCALRL). This vector produces recombinant adenoviruses that express the red fluorescent protein (RFP) instead of the inserted gene during proliferation, which limits toxicity and can be used to monitor viral replication...
December 2016: Biotechnology Reports
https://www.readbyqxmd.com/read/28335584/atrap-expression-in-brown-adipose-tissue-does-not-influence-the-development-of-diet-induced-metabolic-disorders-in-mice
#20
Kohji Ohki, Hiromichi Wakui, Kengo Azushima, Kazushi Uneda, Sona Haku, Ryu Kobayashi, Kotaro Haruhara, Sho Kinguchi, Miyuki Matsuda, Masato Ohsawa, Akinobu Maeda, Shintaro Minegishi, Tomoaki Ishigami, Yoshiyuki Toya, Akio Yamashita, Satoshi Umemura, Kouichi Tamura
Activation of tissue renin-angiotensin system (RAS), mainly mediated by an angiotensin II (Ang II) type 1 receptor (AT1R), plays an important role in the development of obesity-related metabolic disorders. We have shown that AT1R-associated protein (ATRAP), a specific binding protein of AT1R, functions as an endogenous inhibitor to prevent excessive activation of tissue RAS. In the present study, we newly generated ATRAP/Agtrap-floxed (ATRAP(fl/fl)) mice and adipose tissue-specific ATRAP downregulated (ATRAP(adipoq)) mice by the Cre/loxP system using Adipoq-Cre...
March 21, 2017: International Journal of Molecular Sciences
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