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https://www.readbyqxmd.com/read/27911376/rapid-and-efficient-generation-of-recombinant-human-pluripotent-stem-cells-by-recombinase-mediated-cassette-exchange-in-the-aavs1-locus
#1
Laura Ordovás, Ruben Boon, Mariaelena Pistoni, Yemiao Chen, Rangarajan Sambathkumar, Nicky Helsen, Jolien Vanhove, Pieter Berckmans, Qing Cai, Kim Vanuytsel, Susanna Raitano, Catherine M Verfaillie
Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects...
November 20, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27909303/conditional-knockout-of-n-wasp-in-mouse-fibroblast-caused-keratinocyte-hyper-proliferation-and-enhanced-wound-closure
#2
Neeraj Jain, Pazhanichamy Kalailingam, Kai Wei Tan, Hui Bing Tan, Ming Keat Sng, Jeremy Soon Kiat Chan, Nguan Soon Tan, Thirumaran Thanabalu
Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASP(FKO) (N-WASP(fl/fl); Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions...
December 2, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27909096/excitatory-hindbrain-forebrain-communication-is-required-for-cisplatin-induced-anorexia-and-weight-loss
#3
Amber L Alhadeff, Ruby A Holland, Huiyuan Zheng, Linda Rinaman, Harvey J Grill, Bart C De Jonghe
: Cisplatin chemotherapy is commonly used to treat cancer despite severe energy balance side effects. In rats, cisplatin activates nucleus tractus solitarius (NTS) projections to the lateral parabrachial nucleus (lPBN), and calcitonin-gene related peptide (CGRP) projections from the lPBN to the central nucleus of the amygdala (CeA). We previously demonstrated that CeA glutamate receptor signaling mediates cisplatin-induced anorexia and body weight loss. Here, we use neuroanatomical tracing, immunofluorescence and confocal imaging to demonstrate that virtually all NTS→lPBN and lPBN→CeA CGRP projections co-express vesicular glutamate transporter 2 (VGLUT2), providing evidence that excitatory projections mediate cisplatin-induced energy balance dysregulation...
December 1, 2016: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
https://www.readbyqxmd.com/read/27907066/b-cell-based-seamless-engineering-of-antibody-fc-domains
#4
Koji Hashimoto, Kohei Kurosawa, Akiho Murayama, Hidetaka Seo, Kunihiro Ohta
Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody's Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the 'ADLib system')...
2016: PloS One
https://www.readbyqxmd.com/read/27906051/the-lysine-methyltransferase-ehmt2-g9a-is-dispensable-for-skeletal-muscle-development-and-regeneration
#5
Regan-Heng Zhang, Robert N Judson, David Y Liu, Jürgen Kast, Fabio M V Rossi
BACKGROUND: Euchromatic histone-lysine N-methyltransferase 2 (G9a/Ehmt2) is the main enzyme responsible for the apposition of H3K9 di-methylation on histones. Due to its dual role as an epigenetic regulator and in the regulation of non-histone proteins through direct methylation, G9a has been implicated in a number of biological processes relevant to cell fate control. Recent reports employing in vitro cell lines indicate that Ehmt2 methylates MyoD to repress its transcriptional activity and therefore its ability to induce differentiation of activated myogenic cells...
May 27, 2016: Skeletal Muscle
https://www.readbyqxmd.com/read/27893096/chemogenetic-activation-of-iprgcs-drives-changes-in-dark-adapted-scotopic-electroretinogram
#6
Nina Milosavljevic, Annette E Allen, Jasmina Cehajic-Kapetanovic, Robert J Lucas
Purpose: The purpose of this study was to investigate the impact of activating melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) on dark-adapted (scotopic) electroretinograms (ERG). Methods: We used mice (Opn4Cre/+) expressing cre recombinase in melanopsin-expressing cells for a targeted gene delivery of a chemogenetic Gq-coupled receptor, hM3Dq, to ipRGCs. Intraperitoneal injection of clozapine N-oxide (CNO) at 5 mg/kg was used for acute activation of hM3Dq and thus excitation of ipRGCs in darkness...
November 1, 2016: Investigative Ophthalmology & Visual Science
https://www.readbyqxmd.com/read/27881487/osteogenesis-requires-fak-dependent-collagen-synthesis-by-fibroblasts-and-osteoblasts
#7
Dhaarmini Rajshankar, Yongqiang Wang, Christopher A McCulloch
Focal adhesion kinase (FAK) is critical in adhesion-dependent signaling, but its role in osteogenesis in vivo is ill defined. We deleted Fak in fibroblasts and osteoblasts in Floxed-Fak mice bred with those expressing Cre-recombinase driven by 3.6 kb α1(I)-collagen promoter. Compared with wild-type (WT), conditional FAK-knockout (CFKO) mice were shorter (2-fold; P < 0.0001) and had crooked, shorter tails (50%; P < 0.0001). Microcomputed tomography analysis showed reduced bone volume (4-fold in tails; P < 0...
November 23, 2016: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/27880796/foxn1-cre-expression-in-the-male-germline
#8
Jianjun Shi, Irina Getun, Bivian Torres, Howard T Petrie
Foxn1 (forkhead box N1), also known as the nude gene or winged-helix nude (Whn), is a forkhead transcription factor thought to be restricted to keratinocytes in the skin and thymus. Consistent with this tissue distribution, spontaneous or targeted mutation of Foxn1 results in the absence of both hair and a thymus. Genetic manipulation of the Foxn1 locus thus represents a powerful tool for tissue specific gene control in the skin and thymus, and tools such as Cre recombinase under control of the Foxn1 locus are widely used for this purpose...
2016: PloS One
https://www.readbyqxmd.com/read/27879338/functional-interplay-between-murine-leukemia-virus-glycogag-serinc5-and-surface-glycoprotein-governs-virus-entry-with-opposite-effects-on-gammaretroviral-and-ebolavirus-glycoproteins
#9
Yadvinder S Ahi, Shu Zhang, Yashna Thappeta, Audrey Denman, Amin Feizpour, Suryaram Gummuluru, Bjoern Reinhard, Delphine Muriaux, Matthew J Fivash, Alan Rein
: Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called "glycogag" (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others...
November 22, 2016: MBio
https://www.readbyqxmd.com/read/27878291/adenovirus-mediated-truncated-bid-overexpression-induced-by-the-cre-loxp-system-promotes-the-cell-apoptosis-of-cd133-ovarian-cancer-stem-cells
#10
Qifang Long, Ru Yang, Weixian Lu, Weipei Zhu, Jundong Zhou, Cui Zheng, Dongmei Zhou, Ling Yu, Jinchang Wu
Cancer stem cells are a small subset of cancer cells that contribute to cancer progression, metastasis, chemoresistance and recurrence. CD133-positive (CD133+) ovarian cancer cells have been identified as ovarian cancer stem cells. Adenovirus-mediated gene therapy is an innovative therapeutic method for cancer treatment. In the present study, we aimed to develop a new gene therapy to specifically eliminate CD133+ ovarian cancer stem cells by targeting CD133. We used the Cre/LoxP system to augment the selective expression of the truncated Bid (tBid) gene as suicide gene therapy in CD133+ ovarian cancer stem cells...
November 21, 2016: Oncology Reports
https://www.readbyqxmd.com/read/27867498/bone-muscle-crosstalk-targets-muscle-regeneration-pathway-regulated-by-core-circadian-transcriptional-repressors-dec1-and-dec2
#11
REVIEW
Jeffrey P Gorski, Jeffrey L Price
Deletion of proprotein convertase Mbtps1 in bone osteocytes leads to a significant postnatal increase in skeletal muscle size and contractile function, while causing only a 25% increase in stiffness in long bones. Concerns about leakiness in skeletal muscle were discounted since Cre recombinase expression does not account for our findings, and, Mbtps1 protein and mRNA is not deleted. Interestingly, the response of normal skeletal muscle to exercise and the regenerative response of skeletal muscle to the deletion of Mbtps1 in bone share some key regulatory features including a preference for slow twitch muscle fibers...
2016: BoneKEy Reports
https://www.readbyqxmd.com/read/27866960/ethanol-seeking-behavior-is-expressed-directly-through-an-extended-amygdala-to-midbrain-neural-circuit
#12
Melanie M Pina, Christopher L Cunningham
Abstinent alcohol-dependent individuals experience an enduring sensitivity to cue-induced craving and relapse to drinking. There is considerable evidence indicating that structures within the midbrain and extended amygdala are involved in this process. Individually, the ventral tegmental area (VTA) and the bed nucleus of the stria terminalis (BNST) have been shown to modulate cue-induced ethanol-seeking behavior. It is hypothesized that cue-induced seeking is communicated through a direct projection from the BNST to VTA...
November 17, 2016: Neurobiology of Learning and Memory
https://www.readbyqxmd.com/read/27861152/rad9a-is-required-for-spermatogonia-differentiation-in-mice
#13
Lin Huang, Zhen-Bo Wang, Shu-Tao Qi, Xue-Shan Ma, Qiu-Xia Liang, Guo Lei, Tie-Gang Meng, Li-Feng Liang, Ye-Xin Xian, Yi Hou, Xiao-Fang Sun, Yong Zhao, Wei-Hua Wang, Qing-Yuan Sun
Spermatogenesis in testes requires precise spermatogonia differentiation. Spermatocytes lacking the Rad9a gene are arrested in pachytene prophase, implying a possible role for RAD9A in spermatogonia differentiation. However, numerous RAD9A-positive pachytene spermatocytes are still observed in mouse testes following Rad9a excision using the Stra8-Cre system, and it is unclear whether Rad9a deletion in spermatogonia interrupts differentiation. Here, we generated a mouse model in which Rad9a was specifically deleted in spermatogonial stem cells (SSCs) using Cre recombinase expression driven by the germ cell-specific Vasa promoter...
November 16, 2016: Oncotarget
https://www.readbyqxmd.com/read/27859428/low-p16-ink4a-expression-in-early-passage-human-prostate-basal-epithelial-cells-enables-immortalization-by-telomerase-expression-alone
#14
Mindy Kim Graham, Lorenzo Principessa, Lizamma Antony, Alan K Meeker, John T Isaacs
BACKGROUND: There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16(INK4a) /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten...
November 8, 2016: Prostate
https://www.readbyqxmd.com/read/27858314/genetically-dissecting-p2rx7-expression-within-the-central-nervous-system-using-conditional-humanized-mice
#15
Michael W Metzger, Sandra M Walser, Fernando Aprile-Garcia, Nina Dedic, Alon Chen, Florian Holsboer, Eduardo Arzt, Wolfgang Wurst, Jan M Deussing
The purinergic P2X7 receptor (P2X7R) has attracted considerable interest as a potential target for various central nervous system (CNS) pathologies including affective and neurodegenerative disorders. To date, the distribution and cellular localization of the P2X7R in the brain are not fully resolved and a matter of debate mainly due to the limitations of existing tools. However, this knowledge should be a prerequisite for understanding the contribution of the P2X7R to brain disease. Here, we generated a genetic mouse model by humanizing the P2X7R in the mouse as mammalian model organism...
November 17, 2016: Purinergic Signalling
https://www.readbyqxmd.com/read/27858140/adipocyte-stat5-deficiency-promotes-adiposity-and-impairs-lipid-mobilisation-in-mice
#16
Doris Kaltenecker, Kristina M Mueller, Pia Benedikt, Ursula Feiler, Madeleine Themanns, Michaela Schlederer, Lukas Kenner, Martina Schweiger, Guenter Haemmerle, Richard Moriggl
AIMS/HYPOTHESIS: Dysfunction of lipid metabolism in white adipose tissue can substantially interfere with health and quality of life, for example in obesity and associated metabolic diseases. Therefore, it is important to characterise pathways that regulate lipid handling in adipocytes and determine how they affect metabolic homeostasis. Components of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway are involved in adipocyte physiology and pathophysiology...
November 17, 2016: Diabetologia
https://www.readbyqxmd.com/read/27857685/conditional-knockout-of-cav2-1-disrupts-the-accuracy-of-spatial-recognition-of-ca1-place-cells-and-spatial-contextual-recognition-behavior
#17
Dahee Jung, Yu J Hwang, Hoon Ryu, Masanobu Kano, Kenji Sakimura, Jeiwon Cho
Hippocampal pyramidal neurons play an essential role in processing spatial information as implicated with its place-dependent firing. Although, previous slice physiology studies have reported that voltage gated calcium channels contribute to spike shapes and corresponding firing rate in the hippocampus, the roles of P/Q type calcium channels (Cav2.1) underlying neural activity in behaving mice have not been well-investigated. To determine physiological and behavioral roles of Cav2.1, we conducted place cell recordings in CA1 and hippocampus dependent learning/memory tasks using mice lacking Cav2...
2016: Frontiers in Behavioral Neuroscience
https://www.readbyqxmd.com/read/27856232/improved-recombinant-antibody-production-by-cho-cells-using-a-production-enhancer-dna-element-with-repeated-transgene-integration-at-a-predetermined-chromosomal-site
#18
Yoshinori Kawabe, Takanori Inao, Shodai Komatsu, Guan Huang, Akira Ito, Takeshi Omasa, Masamichi Kamihira
Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome...
November 14, 2016: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/27822501/inhibition-of-ikk%C3%AE-reduces-ethanol-consumption-in-c57bl-6j-mice
#19
Jay M Truitt, Yuri A Blednov, Jillian M Benavidez, Mendy Black, Olga Ponomareva, Jade Law, Morgan Merriman, Sami Horani, Kelly Jameson, Amy W Lasek, R Adron Harris, R Dayne Mayfield
Proinflammatory pathways in neuronal and non-neuronal cells are implicated in the acute and chronic effects of alcohol exposure in animal models and humans. The nuclear factor-κB (NF-κB) family of DNA transcription factors plays important roles in inflammatory diseases. The kinase IKKβ mediates the phosphorylation and subsequent proteasomal degradation of cytosolic protein inhibitors of NF-κB, leading to activation of NF-κB. The role of IKKβ as a potential regulator of excessive alcohol drinking had not previously been investigated...
September 2016: ENeuro
https://www.readbyqxmd.com/read/27821018/geminin-is-essential-for-pluripotent-cell-viability-during-teratoma-formation-but-not-for-differentiated-cell-viability-during-teratoma-expansion
#20
Diane C Adler-Wailes, Joshua A Kramer, Melvin DePamphilis
Pluripotent embryonic stem cells (ESCs) are unusual in that geminin has been reported to be essential either to prevent differentiation by maintaining expression of pluripotency genes, or to prevent DNA re-replication dependent apoptosis. To distinguish between these two incompatible hypotheses, immune-compromised mice were inoculated subcutaneously with ESCs harboring conditional Gmnn alleles alone or together with a tamoxifen dependent Cre recombinase gene. Mice were then injected with tamoxifen at various times during which the ESCs proliferated and differentiated into a teratoma...
November 7, 2016: Stem Cells and Development
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