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Sleeping beauty transposon

Yasunori Sasakura
Enhancer trap is a famous application of transposons. This method is useful for the creation of marker transgenic lines that express a reporter gene in tissue- or organ-specific manner, characterization of enhancers in the genome, finding novel patterns of gene expression, and mutagenesis. In Ciona intestinalis, efficient enhancer traps with Minos and Sleeping beauty transposons have been reported. With the enhancer trap lines, the intronic enhancers regulating the expression of the Musashi gene, the compartment in the digestive tube, the presence of enhancers sensitive to the orientation of the gene that they regulate, and the functions of the Hox1 gene have been revealed...
2018: Advances in Experimental Medicine and Biology
Marta Holstein, Cristina Mesa-Nuñez, Csaba Miskey, Elena Almarza, Valentina Poletti, Marco Schmeer, Esther Grueso, Juan Carlos Ordóñez Flores, Dennis Kobelt, Wolfgang Walther, Manish K Aneja, Johannes Geiger, Halvard B Bonig, Zsuzsanna Izsvák, Martin Schleef, Carsten Rudolph, Fulvio Mavilio, Juan A Bueren, Guillermo Guenechea, Zoltán Ivics
The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size...
January 31, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Bo Gao, Wei Wang, Han Wu, Cai Chen, Dan Shen, Saisai Wang, Wei Chen, Li Zhang, Shuheng Chan, Chengyi Song
Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5'- and 3'-regulatory regions of porcine skeletal α-actin gene...
February 22, 2018: Biochemical Genetics
Yabin Guo, Yin Zhang, Kaishun Hu
Background: Sleeping Beauty transposon (SB) has become an increasingly important genetic tool for generating mutations in vertebrate cells. It is widely thought that SB exclusively integrates into TA dinucleotides. However, this strict TA-preference has not been rigorously tested in large numbers of insertion sites that now can be detected with next generation sequencing. Li et al. found 71 SB insertions in non-TA dinucleotides in 2013, suggesting that TA dinucleotides are not the only sites of SB integration, yet further studies on this topic have not been carried out...
2018: Mobile DNA
Katie Weihbrecht, Wesley A Goar, Calvin S Carter, Val C Sheffield, Seongjin Seo
Nephronophthisis-related ciliopathies (NPHP-RC) are a group of disorders that present with end-stage renal failure in childhood/adolescence, kidney cysts, retinal degeneration, and cerebellar hypoplasia. One disorder that shares clinical features with NPHP-RC is Bardet-Biedl Syndrome (BBS). Serologically defined colon cancer antigen 8 (SDCCAG8; also known as NPHP10 and BBS16) is an NPHP gene that is also associated with BBS. To better understand the patho-mechanisms of NPHP and BBS caused by loss of SDCCAG8 function, we characterized an SDCCAG8 mouse model (Sdccag8Tn(sb-Tyr)2161B...
2018: PloS One
Eric K Hubner, Christian Lechler, Thomas N Rösner, Birgit Kohnke-Ertel, Roland M Schmid, Ursula Ehmer
In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems...
February 2, 2018: Journal of Visualized Experiments: JoVE
Min Wu, Jin Li, Lei Yue, Lu Bai, Yaming Li, Jieliang Chen, Xiaonan Zhang, Zhenghong Yuan
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP)...
February 9, 2018: Antiviral Research
Gergő Vőfély, Tünde Berecz, Eszter Szabó, Kornélia Szebényi, Edit Hathy, Tamás I Orbán, Balázs Sarkadi, László Homolya, Maria C Marchetto, János M Réthelyi, Ágota Apáti
Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons...
February 6, 2018: Molecular and Cellular Neurosciences
Na Zhan, Adeola Adebayo Michael, Kaiyuan Wu, Gang Zeng, Aaron Bell, Junyan Tao, Satdarshan P Monga
Simultaneous mutations in CTNNB1 and activation of c-MET occurs in 9-12.5% of patients with hepatocellular cancer (HCC). Co-expression of c-MET-V5 and mutant-β-catenin-Myc in mouse liver by sleeping beauty transposon/transposase and hydrodynamic tail vein injection (SB-HTVI) led to development of HCC with 70% molecular identity to the clinical subset. Using this model, we investigated the effect of EMD1214063, a highly selective c-MET inhibitor. Five weeks after SB-HTVI when tumors were established, EMD1214063 (10mg/kg) was administered by gastric lavage as a single-agent on 5-days-on/3-days-off schedule, compared to vehicle only control...
February 6, 2018: Gene Expression
Daniela Benati, Fabienne Cocchiarella, Alessandra Recchia
The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a "cut and paste" SB mechanism...
January 12, 2018: Journal of Visualized Experiments: JoVE
Yu Pan, Jing Lv, Donghui Pan, Min Yang, Huijun Ju, Jinxin Zhou, Liying Zhu, Yifan Zhang
Reporter gene imaging is widely used for non-invasively detecting tumorigenesis, trafficking therapeutic cells, and monitoring treatment effect. Baculoviral vectors (BVs) have been utilized as transgenic vectors in the reporter gene imaging systems in recent years. However, BV-mediated report gene imaging can only provide short-term investigation due to its transient transgene expression, which is incompetent for the long-term applications. In the current study, we reconstructed a series of hybrid BVs with several elements, to investigate the feasibility of this hybrid BV-mediated long-term reporter gene imaging in vivo...
January 22, 2018: Applied Microbiology and Biotechnology
Irina Portier, Karen Vanhoorelbeke, Sebastien Verhenne, Inge Pareyn, Nele Vandeputte, Hans Deckmyn, Daniel S Goldenberg, Himanshu B Samal, Manvendra Singh, Zoltán Ivics, Zsuzsanna Izsvák, Simon F De Meyer
BACKGROUND: Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short-term solution. Gene therapy offers the potential for a long-term treatment for VWD. OBJECTIVES: To develop an integrative Sleeping Beauty (SB) transposon-mediated VWF gene transfer approach in a preclinical mouse model of severe VWD. METHODS: We established a robust platform for sustained transgene murine (m)VWF expression in the liver of Vwf-/- mice by combining a liver-specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery...
December 29, 2017: Journal of Thrombosis and Haemostasis: JTH
Laura Garcia-Garcia, Sergio Recalde, Maria Hernandez, Jaione Bezunartea, Juan Roberto Rodriguez-Madoz, Sandra Johnen, Sabine Diarra, Corinne Marie, Zsuzsanna Izsvák, Zoltán Ivics, Daniel Scherman, Martina Kropp, Gabriele Thumann, Felipe Prosper, Patricia Fernandez-Robredo, Alfredo Garcia-Layana
Pigment epithelium derived factor (PEDF) is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF) in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids...
December 15, 2017: Molecular Therapy. Nucleic Acids
Jaitip Tipanee, Yoke Chin Chai, Thierry VandenDriessche, Marinee K Chuah
Transposons derived from Sleeping Beauty (SB), piggyBac (PB), or Tol2 typically require cotransfection of transposon DNA with a transposase either as an expression plasmid or mRNA. Consequently, this results in genomic integration of the potentially therapeutic gene into chromosomes of the desired target cells, and thus conferring stable expression. Non-viral transfection methods are typically preferred to deliver the transposon components into the target cells. However, these methods do not match the efficacy typically attained with viral vectors and are sometimes associated with cellular toxicity evoked by the DNA itself...
December 22, 2017: Bioscience Reports
Andrea Kerekes, Orsolya Ivett Hoffmann, Gergely Iski, Nándor Lipták, Elen Gócza, Wilfried A Kues, Zsuzsanna Bősze, László Hiripi
Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter...
2017: PloS One
Justin Y Newberg, Karen M Mann, Michael B Mann, Nancy A Jenkins, Neal G Copeland
Large-scale oncogenomic studies have identified few frequently mutated cancer drivers and hundreds of infrequently mutated drivers. Defining the biological context for rare driving events is fundamentally important to increasing our understanding of the druggable pathways in cancer. Sleeping Beauty (SB) insertional mutagenesis is a powerful gene discovery tool used to model human cancers in mice. Our lab and others have published a number of studies that identify cancer drivers from these models using various statistical and computational approaches...
October 20, 2017: Nucleic Acids Research
Barbara R Tschida, Nuri A Temiz, Timothy P Kuka, Lindsey A Lee, Jesse D Riordan, Carlos A Tierrablanca, Robert Hullsiek, Sandra Wagner, Wendy A Hudson, Michael A Linden, Khalid Amin, Pauline J Beckmann, Rachel A Heuer, Aaron L Sarver, Ju Dong Yang, Lewis R Roberts, Joseph H Nadeau, Adam J Dupuy, Vincent W Keng, David A Largaespada
Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways...
December 1, 2017: Cancer Research
Partow Kebriaei, Zsuzsanna Izsvák, Suneel A Narayanavari, Harjeet Singh, Zoltán Ivics
The widespread clinical implementation of gene therapy requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective, and economical manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient nonviral gene delivery approaches that are prevalent in ongoing clinical trials. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use...
November 2017: Trends in Genetics: TIG
Jesse D Riordan, Charlotte R Feddersen, Barbara R Tschida, Pauline Jackson, Vincent W Keng, Michael A Linden, Khalid Amin, Christopher S Stipp, David A Largaespada, Adam J Dupuy
Most hepatocellular carcinomas (HCCs) develop in a chronically injured liver, yet the extent to which this microenvironment promotes neoplastic transformation or influences selective pressures for genetic drivers of HCC remains unclear. We sought to determine the impact of hepatic injury in an established mouse model of HCC induced by Sleeping Beauty transposon mutagenesis. Chemically-induced chronic liver injury dramatically increased tumor penetrance and significantly altered driver mutation profiles, likely reflecting distinct selective pressures...
September 29, 2017: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
Evangelia K Siska, Itamar Weisman, Jacob Romano, Zoltán Ivics, Zsuzsanna Izsvák, Uriel Barkai, Spyros Petrakis, George Koliakos
Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations...
2017: PloS One
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