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Sample records for achieve efficient gene

  1. A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

    PubMed

    Vankayala, Raviraj; Chiang, Chi-Shiun; Chao, Jui-I; Yuan, Chiun-Jye; Lin, Shyr-Yeu; Hwang, Kuo Chu

    2014-09-01

    Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented. PMID:24973297

  2. A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

    PubMed

    Vankayala, Raviraj; Chiang, Chi-Shiun; Chao, Jui-I; Yuan, Chiun-Jye; Lin, Shyr-Yeu; Hwang, Kuo Chu

    2014-09-01

    Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented.

  3. Achieving energy efficiency during collective communications

    SciTech Connect

    Sundriyal, Vaibhav; Sosonkina, Masha; Zhang, Zhao

    2012-09-13

    Energy consumption has become a major design constraint in modern computing systems. With the advent of petaflops architectures, power-efficient software stacks have become imperative for scalability. Techniques such as dynamic voltage and frequency scaling (called DVFS) and CPU clock modulation (called throttling) are often used to reduce the power consumption of the compute nodes. To avoid significant performance losses, these techniques should be used judiciously during parallel application execution. For example, its communication phases may be good candidates to apply the DVFS and CPU throttling without incurring a considerable performance loss. They are often considered as indivisible operations although little attention is being devoted to the energy saving potential of their algorithmic steps. In this work, two important collective communication operations, all-to-all and allgather, are investigated as to their augmentation with energy saving strategies on the per-call basis. The experiments prove the viability of such a fine-grain approach. They also validate a theoretical power consumption estimate for multicore nodes proposed here. While keeping the performance loss low, the obtained energy savings were always significantly higher than those achieved when DVFS or throttling were switched on across the entire application run

  4. Daily energy balance in growth hormone receptor/binding protein (GHR−/−) gene-disrupted mice is achieved through an increase in dark-phase energy efficiency

    PubMed Central

    Longo, Kenneth A.; Berryman, Darlene E.; Kelder, Bruce; Charoenthongtrakul, Soratree; DiStefano, Peter S.; Geddes, Brad J.; Kopchick, John

    2009-01-01

    The goal of this study was to examine factors that contribute to energy balance in female GHR −/− mice. We measured energy intake, energy expenditure (EE), fuel utilization, body mass (Mb) changes and physical activity in 17 month-old female GHR −/− mice and their age-matched wild type littermates. The GHR −/− mice were smaller, consumed more food per unit Mb, had greater EE per unit Mb and had an increase in 24-h EE/Mb that was similar to the increase in their surface-area-to-volume ratio. Locomotor activity (LMA) was reduced in the GHR −/− mice, but the energetic cost associated with their LMA was greater than in wild type controls. Furthermore, Mb and LMA were independent explanatory covariates of most of the variance in EE, and when adjusted for Mb and LMA, the GHR −/− mice had higher EE during both the light and dark phases of the daily cycle. Respiratory quotient was lower in GHR −/− mice during the light phase, which indicated a greater utilization of lipid relative to carbohydrate in these mice. Additionally, GHR −/− mice had higher ratios of caloric intake to EE at several intervals during the dark phase, and this effect was greater and more sustained in the final three hours of the dark phase. Therefore, we conclude that GHR −/− mice are able to overcome the substantial energetic challenges of dwarfism through several mechanisms that promote stable Mb. Relative to wild type mice, the GHR −/− mice consumed more calories per unit Mb, which offset the disproportionate increase in their daily energy expenditure. While GHR −/− mice oxidized a greater proportion of lipid during the light phase in order to meet their energy requirements, they achieved greater energy efficiency and storage during the dark phase through a combination of higher energy consumption and lower LMA. PMID:19747867

  5. Achieving Energy Efficiency Through Real-Time Feedback

    SciTech Connect

    Nesse, Ronald J.

    2011-09-01

    Through the careful implementation of simple behavior change measures, opportunities exist to achieve strategic gains, including greater operational efficiencies, energy cost savings, greater tenant health and ensuing productivity and an improved brand value through sustainability messaging and achievement.

  6. Using the network to achieve energy efficiency

    SciTech Connect

    Giglio, M.

    1995-12-01

    Novell, the third largest software company in the world, has developed Netware Embedded Systems Technology (NEST). NEST will take the network deeper into non-traditional computing environments and will imbed networking into more intelligent devices. Ultimately, this will lead to energy efficiencies in the office. NEST can make point-of-sale terminals, alarm systems, televisions, traffic controls, printers, lights, fax machines, copiers, HVAC controls, PBX machines, etc., either intelligent or more intelligent than they are currently. The mission statement for this particular group is to integrate over 30 million new intelligent devices into the workplace and the home with Novell networks by 1997. Computing trends have progressed from mainframes in the 1960s to keys, security systems, and airplanes in the year 2000. In fact, the new Boeing 777 has NEST in it, and it also has network servers on board. NEST enables the embedded network with the ability to put intelligence into devices. This gives one more control of the devices from wherever one is. For example, the pharmaceutical industry could use NEST to coordinate what the consumer is buying, what is in the warehouse, what the manufacturing plant is tooled for, and so on. Through NEST technology, the pharmaceutical industry now uses a camera that takes pictures of the pills. It can see whether an {open_quotes}overdose{close_quotes} or {open_quotes}underdose{close_quotes} of a particular type of pill is being manufactured. The plant can be shut down and corrections made immediately.

  7. Upside-Down Solar Cell Achieves Record Efficiencies (Fact Sheet)

    SciTech Connect

    Not Available

    2010-12-01

    The inverted metamorphic multijunction (IMM) solar cell is an exercise in efficient innovation - literally, as the technology boasted the highest demonstrated efficiency for converting sunlight into electrical energy at its debut in 2005. Scientists at the National Renewable Energy Laboratory (NREL) inverted the conventional photovoltaic (PV) structure to revolutionary effect, achieving solar conversion efficiencies of 33.8% and 40.8% under one-sun and concentrated conditions, respectively.

  8. The efficient algorithms for achieving Euclidean distance transformation.

    PubMed

    Shih, Frank Y; Wu, Yi-Ta

    2004-08-01

    Euclidean distance transformation (EDT) is used to convert a digital binary image consisting of object (foreground) and nonobject (background) pixels into another image where each pixel has a value of the minimum Euclidean distance from nonobject pixels. In this paper, the improved iterative erosion algorithm is proposed to avoid the redundant calculations in the iterative erosion algorithm. Furthermore, to avoid the iterative operations, the two-scan-based algorithm by a deriving approach is developed for achieving EDT correctly and efficiently in a constant time. Besides, we discover when obstacles appear in the image, many algorithms cannot achieve the correct EDT except our two-scan-based algorithm. Moreover, the two-scan-based algorithm does not require the additional cost of preprocessing or relative-coordinates recording.

  9. Some methods for achieving more efficient performance of fuel assemblies

    NASA Astrophysics Data System (ADS)

    Boltenko, E. A.

    2014-07-01

    More efficient operation of reactor plant fuel assemblies can be achieved through the use of new technical solutions aimed at obtaining more uniform distribution of coolant over the fuel assembly section, more intense heat removal on convex heat-transfer surfaces, and higher values of departure from nucleate boiling ratio (DNBR). Technical solutions using which it is possible to obtain more intense heat removal on convex heat-transfer surfaces and higher DNBR values in reactor plant fuel assemblies are considered. An alternative heat removal arrangement is described using which it is possible to obtain a significantly higher power density in a reactor plant and essentially lower maximal fuel rod temperature.

  10. A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios

    NASA Astrophysics Data System (ADS)

    Wang, Mingming; Liu, Hongmei; Li, Lei; Cheng, Yiyun

    2014-01-01

    Polymers have shown great promise in the design of high efficient and low cytotoxic gene vectors. Here we synthesize fluorinated dendrimers for use as gene vectors. Fluorinated dendrimers achieve excellent gene transfection efficacy in several cell lines (higher than 90% in HEK293 and HeLa cells) at extremely low N/P ratios. These polymers show superior efficacy and biocompatibility compared with several commercial transfection reagents such as Lipofectamine 2000 and SuperFect. Fluorination enhances the cellular uptake of the dendrimer/DNA polyplexes and facilitates their endosomal escape. In addition, the fluorinated dendrimer shows excellent serum resistance and exhibits high gene transfection efficacy even in medium containing 50% FBS. The results suggest that fluorinated dendrimers are a new class of highly efficient gene vectors and fluorination is a promising strategy to design gene vectors without involving sophisticated syntheses.

  11. Carbon nanotubes as vectors for gene therapy: past achievements, present challenges and future goals.

    PubMed

    Bates, Katie; Kostarelos, Kostas

    2013-12-01

    Promising therapeutic and prophylactic effects have been achieved following advances in the gene therapy research arena, giving birth to the new generation of disease-modifying therapeutics. The greatest challenge that gene therapy vectors still face is the ability to deliver sufficient genetic payloads in order to enable efficient gene transfer into target cells. A wide variety of viral and non-viral gene therapy vectors have been developed and explored over the past 10years, including carbon nanotubes. In this review we will address the application of carbon nanotubes as non-viral vectors in gene therapy with the aim to give a perspective on the past achievements, present challenges and future goals. A series of important topics concerning carbon nanotubes as gene therapy vectors will be addressed, including the benefits that carbon nanotubes offer over other non-viral delivery systems. Furthermore, a perspective is given on what the ideal genetic cargo to deliver using carbon nanotubes is and finally the geno-pharmacological impact of carbon nanotube-mediated gene therapy is discussed.

  12. Air Force Achieves Fuel Efficiency through Industry Best Practices

    SciTech Connect

    2012-12-01

    The U.S. Air Force’s Air Mobility Command (AMC) is changing the way it does business. It is saving energy and money through an aircraft fleet fuel-efficiency program inspired by private industry best practices and ideas resulting from the empowered fuel savings culture.

  13. Telescoping Solar Array Concept for Achieving High Packaging Efficiency

    NASA Technical Reports Server (NTRS)

    Mikulas, Martin; Pappa, Richard; Warren, Jay; Rose, Geoff

    2015-01-01

    Lightweight, high-efficiency solar arrays are required for future deep space missions using high-power Solar Electric Propulsion (SEP). Structural performance metrics for state-of-the art 30-50 kW flexible blanket arrays recently demonstrated in ground tests are approximately 40 kW/cu m packaging efficiency, 150 W/kg specific power, 0.1 Hz deployed stiffness, and 0.2 g deployed strength. Much larger arrays with up to a megawatt or more of power and improved packaging and specific power are of interest to mission planners for minimizing launch and life cycle costs of Mars exploration. A new concept referred to as the Compact Telescoping Array (CTA) with 60 kW/cu m packaging efficiency at 1 MW of power is described herein. Performance metrics as a function of array size and corresponding power level are derived analytically and validated by finite element analysis. Feasible CTA packaging and deployment approaches are also described. The CTA was developed, in part, to serve as a NASA reference solar array concept against which other proposed designs of 50-1000 kW arrays for future high-power SEP missions could be compared.

  14. 10 CFR 435.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...-RISE RESIDENTIAL BUILDINGS Mandatory Energy Efficiency Standards for Federal Low-Rise Residential Buildings. § 435.7 Water used to achieve energy efficiency. ... 10 Energy 3 2011-01-01 2011-01-01 false Water used to achieve energy efficiency. 435.7 Section...

  15. 10 CFR 433.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 3 2011-01-01 2011-01-01 false Water used to achieve energy efficiency. 433.7 Section 433.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR THE DESIGN AND... achieve energy efficiency....

  16. 10 CFR 433.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 3 2014-01-01 2014-01-01 false Water used to achieve energy efficiency. 433.7 Section 433.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR NEW FEDERAL COMMERCIAL AND MULTI-FAMILY HIGH-RISE RESIDENTIAL BUILDINGS § 433.7 Water used to achieve energy efficiency....

  17. 10 CFR 433.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Water used to achieve energy efficiency. 433.7 Section 433.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR THE DESIGN AND... achieve energy efficiency....

  18. 10 CFR 433.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 3 2013-01-01 2013-01-01 false Water used to achieve energy efficiency. 433.7 Section 433.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR NEW FEDERAL COMMERCIAL AND MULTI-FAMILY HIGH-RISE RESIDENTIAL BUILDINGS § 433.7 Water used to achieve energy efficiency....

  19. 10 CFR 433.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 false Water used to achieve energy efficiency. 433.7 Section 433.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR NEW FEDERAL COMMERCIAL AND MULTI-FAMILY HIGH-RISE RESIDENTIAL BUILDINGS § 433.7 Water used to achieve energy efficiency....

  20. Barriers to Achieving Textbook Multigrid Efficiency (TME) in CFD

    NASA Technical Reports Server (NTRS)

    Brandt, Achi

    1998-01-01

    As a guide to attaining this optimal performance for general CFD problems, the table below lists every foreseen kind of computational difficulty for achieving that goal, together with the possible ways for resolving that difficulty, their current state of development, and references. Included in the table are staggered and nonstaggered, conservative and nonconservative discretizations of viscous and inviscid, incompressible and compressible flows at various Mach numbers, as well as a simple (algebraic) turbulence model and comments on chemically reacting flows. The listing of associated computational barriers involves: non-alignment of streamlines or sonic characteristics with the grids; recirculating flows; stagnation points; discretization and relaxation on and near shocks and boundaries; far-field artificial boundary conditions; small-scale singularities (meaning important features, such as the complete airplane, which are not visible on some of the coarse grids); large grid aspect ratios; boundary layer resolution; and grid adaption.

  1. Laboratory 2000--the challenge of achieving efficiency and compliance.

    PubMed

    Potter, J A

    2001-01-01

    Significant advances within the field of laboratory automation and instrumentation have greatly benefited the pharmaceutical industry in its quest to discover, develop and monitor the quality of its products. Necessitated by the need for efficiency and greater productivity, faster and more cost-effective means of analyses exist in the form of devices made up of complex electromechanical components, all logically controlled and most with the capability to interface with sophisticated information systems. This benefit does come with a price, a greater responsibility to ensure data quality while complying with increased regulatory requirements. Commitment to this responsibility presents a substantial challenge to scientists and managers throughout the industry. Due diligence must be demonstrated. A comprehensive evaluation of every laboratory system utilized, a solid plan of action for correcting any known deficiencies including upgrades or complete replacement, and an accurate monitoring procedure with the ability to measure progress are all absolute necessities to ensure success. Crossfunctional team effort and communication must transpire with full managerial support. Vendors need to be audited, made aware of any functional or quality inadequacies they possess as well as the pharmaceutical industry's expectation for these shortcomings to be rapidly corrected. Suppliers of these systems should also be encouraged to provide complete 'off-the-shelf solutions' to eliminate the need for in-house customization. The requirements for regulatory compliance in today's electronic environment have been well publicized. The players involved are not only listening, but also taking the necessary steps to retain and improve efficiency without sacrificing quality. With the proper measures, planning and action, a highly automated, cost-effective and compliant laboratory operation can become a reality.

  2. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    PubMed

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

  3. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    PubMed Central

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  4. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    PubMed

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  5. 10 CFR 435.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 3 2013-01-01 2013-01-01 false Water used to achieve energy efficiency. 435.7 Section 435.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR NEW FEDERAL LOW-RISE RESIDENTIAL BUILDINGS Mandatory Energy Efficiency Standards for Federal Low-Rise...

  6. 10 CFR 435.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 3 2014-01-01 2014-01-01 false Water used to achieve energy efficiency. 435.7 Section 435.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR NEW FEDERAL LOW-RISE RESIDENTIAL BUILDINGS Mandatory Energy Efficiency Standards for Federal Low-Rise...

  7. 10 CFR 435.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 false Water used to achieve energy efficiency. 435.7 Section 435.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR NEW FEDERAL LOW-RISE RESIDENTIAL BUILDINGS Mandatory Energy Efficiency Standards for Federal Low-Rise...

  8. 10 CFR 435.7 - Water used to achieve energy efficiency. [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Water used to achieve energy efficiency. 435.7 Section 435.7 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY STANDARDS FOR NEW FEDERAL LOW-RISE RESIDENTIAL BUILDINGS Mandatory Energy Efficiency Standards for Federal Low-Rise...

  9. A visible, targeted high-efficiency gene delivery and transfection strategy

    PubMed Central

    2011-01-01

    Background To enhance myocardial angiogenic gene expression, a novel gene delivery strategy was tested. Direct intramyocardial injection of an angiogenic gene with microbubbles and insonation were applied in a dog animal model. Dogs received one of the four different treatments in conjunction with either the enhanced green fluorescence protein (EGFP) gene or the hepatocyte growth factor (HGF) gene: gene with microbubbles (MB) and ultrasound (US); gene with US; gene with MB; or the gene alone. Results Distribution of MB and the gene in the myocardium was visualized during the experiment. Compared with the EGFP gene group, an average 14.7-fold enhancement in gene expression was achieved in the EGFP+MB/US group (P < 0.01). Compared with the HGF gene group, an average 10.7-fold enhancement in gene expression was achieved in the HGF+MB/US group (P < 0.01). In addition, capillary density increased from 20.8 ± 3.4/mm2 in the HGF gene group to 146.7 ± 31.4/mm2 in HGF+MB/US group (P < 0.01). Conclusions Thus, direct intramyocardial injection of an angiogenic gene in conjunction with microbubbles plus insonation synergistically enhances angiogenesis. This method offers an observable gene delivery procedure with enhanced expression efficiency of the delivered gene. PMID:21600027

  10. Improvement of efficiency and viability in plasma gene transfection by plasma minimization and optimization electrode configuration

    NASA Astrophysics Data System (ADS)

    Jinno, Masafumi; Tachibana, Kunihide; Motomura, Hideki; Saeki, Noboru; Satoh, Susumu

    2016-07-01

    Plasma gene transfection is expected as a safe and useful method of gene transfection. However, in this method, there is difficulty in keeping both high transfection efficiency and less cell damage simultaneously. The authors have evaluated transfection efficiency and cell viability using four different plasma sources, such as arc discharge, plasma jet, dielectric barrier discharge (DBD), and microplasma. A high transfection efficiency was achieved by discharge forms in which the electric current flows via the cells. This suggested that an electric current plays an important role in plasma gene transfection. The total volume of gas flow must be small or zero and the area in which the cells are directly irradiated by plasma must be small in order to achieve a higher cell viability. The microplasma that satisfies these conditions achieved both the highest transfection efficiency and the highest cell viability simultaneously.

  11. Thermodynamic and achievable efficiencies for solar-driven electrochemical reduction of carbon dioxide to transportation fuels.

    PubMed

    Singh, Meenesh R; Clark, Ezra L; Bell, Alexis T

    2015-11-10

    Thermodynamic, achievable, and realistic efficiency limits of solar-driven electrochemical conversion of water and carbon dioxide to fuels are investigated as functions of light-absorber composition and configuration, and catalyst composition. The maximum thermodynamic efficiency at 1-sun illumination for adiabatic electrochemical synthesis of various solar fuels is in the range of 32-42%. Single-, double-, and triple-junction light absorbers are found to be optimal for electrochemical load ranges of 0-0.9 V, 0.9-1.95 V, and 1.95-3.5 V, respectively. Achievable solar-to-fuel (STF) efficiencies are determined using ideal double- and triple-junction light absorbers and the electrochemical load curves for CO2 reduction on silver and copper cathodes, and water oxidation kinetics over iridium oxide. The maximum achievable STF efficiencies for synthesis gas (H2 and CO) and Hythane (H2 and CH4) are 18.4% and 20.3%, respectively. Whereas the realistic STF efficiency of photoelectrochemical cells (PECs) can be as low as 0.8%, tandem PECs and photovoltaic (PV)-electrolyzers can operate at 7.2% under identical operating conditions. We show that the composition and energy content of solar fuels can also be adjusted by tuning the band-gaps of triple-junction light absorbers and/or the ratio of catalyst-to-PV area, and that the synthesis of liquid products and C2H4 have high profitability indices.

  12. Thermodynamic and achievable efficiencies for solar-driven electrochemical reduction of carbon dioxide to transportation fuels

    PubMed Central

    Singh, Meenesh R.; Clark, Ezra L.; Bell, Alexis T.

    2015-01-01

    Thermodynamic, achievable, and realistic efficiency limits of solar-driven electrochemical conversion of water and carbon dioxide to fuels are investigated as functions of light-absorber composition and configuration, and catalyst composition. The maximum thermodynamic efficiency at 1-sun illumination for adiabatic electrochemical synthesis of various solar fuels is in the range of 32–42%. Single-, double-, and triple-junction light absorbers are found to be optimal for electrochemical load ranges of 0–0.9 V, 0.9–1.95 V, and 1.95–3.5 V, respectively. Achievable solar-to-fuel (STF) efficiencies are determined using ideal double- and triple-junction light absorbers and the electrochemical load curves for CO2 reduction on silver and copper cathodes, and water oxidation kinetics over iridium oxide. The maximum achievable STF efficiencies for synthesis gas (H2 and CO) and Hythane (H2 and CH4) are 18.4% and 20.3%, respectively. Whereas the realistic STF efficiency of photoelectrochemical cells (PECs) can be as low as 0.8%, tandem PECs and photovoltaic (PV)-electrolyzers can operate at 7.2% under identical operating conditions. We show that the composition and energy content of solar fuels can also be adjusted by tuning the band-gaps of triple-junction light absorbers and/or the ratio of catalyst-to-PV area, and that the synthesis of liquid products and C2H4 have high profitability indices. PMID:26504215

  13. Thermodynamic and achievable efficiencies for solar-driven electrochemical reduction of carbon dioxide to transportation fuels

    NASA Astrophysics Data System (ADS)

    Singh, Meenesh R.; Clark, Ezra L.; Bell, Alexis T.

    2015-11-01

    Thermodynamic, achievable, and realistic efficiency limits of solar-driven electrochemical conversion of water and carbon dioxide to fuels are investigated as functions of light-absorber composition and configuration, and catalyst composition. The maximum thermodynamic efficiency at 1-sun illumination for adiabatic electrochemical synthesis of various solar fuels is in the range of 32-42%. Single-, double-, and triple-junction light absorbers are found to be optimal for electrochemical load ranges of 0-0.9 V, 0.9-1.95 V, and 1.95-3.5 V, respectively. Achievable solar-to-fuel (STF) efficiencies are determined using ideal double- and triple-junction light absorbers and the electrochemical load curves for CO2 reduction on silver and copper cathodes, and water oxidation kinetics over iridium oxide. The maximum achievable STF efficiencies for synthesis gas (H2 and CO) and Hythane (H2 and CH4) are 18.4% and 20.3%, respectively. Whereas the realistic STF efficiency of photoelectrochemical cells (PECs) can be as low as 0.8%, tandem PECs and photovoltaic (PV)-electrolyzers can operate at 7.2% under identical operating conditions. We show that the composition and energy content of solar fuels can also be adjusted by tuning the band-gaps of triple-junction light absorbers and/or the ratio of catalyst-to-PV area, and that the synthesis of liquid products and C2H4 have high profitability indices.

  14. Thermodynamic and achievable efficiencies for solar-driven electrochemical reduction of carbon dioxide to transportation fuels.

    PubMed

    Singh, Meenesh R; Clark, Ezra L; Bell, Alexis T

    2015-11-10

    Thermodynamic, achievable, and realistic efficiency limits of solar-driven electrochemical conversion of water and carbon dioxide to fuels are investigated as functions of light-absorber composition and configuration, and catalyst composition. The maximum thermodynamic efficiency at 1-sun illumination for adiabatic electrochemical synthesis of various solar fuels is in the range of 32-42%. Single-, double-, and triple-junction light absorbers are found to be optimal for electrochemical load ranges of 0-0.9 V, 0.9-1.95 V, and 1.95-3.5 V, respectively. Achievable solar-to-fuel (STF) efficiencies are determined using ideal double- and triple-junction light absorbers and the electrochemical load curves for CO2 reduction on silver and copper cathodes, and water oxidation kinetics over iridium oxide. The maximum achievable STF efficiencies for synthesis gas (H2 and CO) and Hythane (H2 and CH4) are 18.4% and 20.3%, respectively. Whereas the realistic STF efficiency of photoelectrochemical cells (PECs) can be as low as 0.8%, tandem PECs and photovoltaic (PV)-electrolyzers can operate at 7.2% under identical operating conditions. We show that the composition and energy content of solar fuels can also be adjusted by tuning the band-gaps of triple-junction light absorbers and/or the ratio of catalyst-to-PV area, and that the synthesis of liquid products and C2H4 have high profitability indices. PMID:26504215

  15. New Construct Approaches for Efficient Gene Silencing in Plants

    PubMed Central

    Yan, Hua; Chretien, Robert; Ye, Jingsong; Rommens, Caius M.

    2006-01-01

    An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts. PMID:16766670

  16. Efficient exploration of the space of reconciled gene trees.

    PubMed

    Szöllõsi, Gergely J; Rosikiewicz, Wojciech; Boussau, Bastien; Tannier, Eric; Daubin, Vincent

    2013-11-01

    Gene trees record the combination of gene-level events, such as duplication, transfer and loss (DTL), and species-level events, such as speciation and extinction. Gene tree-species tree reconciliation methods model these processes by drawing gene trees into the species tree using a series of gene and species-level events. The reconstruction of gene trees based on sequence alone almost always involves choosing between statistically equivalent or weakly distinguishable relationships that could be much better resolved based on a putative species tree. To exploit this potential for accurate reconstruction of gene trees, the space of reconciled gene trees must be explored according to a joint model of sequence evolution and gene tree-species tree reconciliation. Here we present amalgamated likelihood estimation (ALE), a probabilistic approach to exhaustively explore all reconciled gene trees that can be amalgamated as a combination of clades observed in a sample of gene trees. We implement the ALE approach in the context of a reconciliation model (Szöllősi et al. 2013), which allows for the DTL of genes. We use ALE to efficiently approximate the sum of the joint likelihood over amalgamations and to find the reconciled gene tree that maximizes the joint likelihood among all such trees. We demonstrate using simulations that gene trees reconstructed using the joint likelihood are substantially more accurate than those reconstructed using sequence alone. Using realistic gene tree topologies, branch lengths, and alignment sizes, we demonstrate that ALE produces more accurate gene trees even if the model of sequence evolution is greatly simplified. Finally, examining 1099 gene families from 36 cyanobacterial genomes we find that joint likelihood-based inference results in a striking reduction in apparent phylogenetic discord, with respectively. 24%, 59%, and 46% reductions in the mean numbers of duplications, transfers, and losses per gene family. The open source

  17. Intellectual interest mediates gene × socioeconomic status interaction on adolescent academic achievement.

    PubMed

    Tucker-Drob, Elliot M; Harden, K Paige

    2012-01-01

    Recent studies have demonstrated that genetic influences on cognitive ability and academic achievement are larger for children raised in higher socioeconomic status (SES) homes. However, little work has been done to document the psychosocial processes that underlie this Gene × Environment interaction. One process may involve the conversion of intellectual interest into academic achievement. Analyses of data from 777 pairs of 17-year-old twins indicated that Gene × SES effects on achievement scores can be accounted for by stronger influences of genes for intellectual interest on achievement at higher levels of SES. These findings are consistent with the hypothesis that higher SES affords greater opportunity for children to seek out and benefit from learning experiences that are congruent with their genetically influenced intellectual interests.

  18. Intellectual Interest Mediates Gene-by-SES Interaction on Adolescent Academic Achievement

    PubMed Central

    Tucker-Drob, Elliot M.; Harden, K. Paige

    2011-01-01

    Recent studies have demonstrated that genetic influences on cognitive ability and academic achievement are larger for children raised in higher socioeconomic status (SES) homes. However, little work has been done to document the psychosocial processes that underlie this gene-by-environment interaction. One process may involve the conversion of intellectual interest into academic achievement. Analyses of data from 777 pairs of 17-year-old twins indicated that gene-by-SES effects on achievement scores can be accounted for by stronger influences of genes for intellectual interest on achievement at higher levels of SES. These findings are consistent with the hypothesis that higher SES affords greater opportunity for children to seek out and benefit from learning experiences that are congruent with their genetically influenced intellectual interests. PMID:22288554

  19. Controlling surface enrichment in polymeric hole extraction layers to achieve high-efficiency organic photovoltaic cells.

    PubMed

    Kim, Dong-Hun; Lim, Kyung-Geun; Park, Jong Hyeok; Lee, Tae-Woo

    2012-10-01

    Hole extraction in organic photovoltaic cells (OPVs) can be modulated by a surface-enriched layer formed on top of the conducting polymer-based hole extraction layer (HEL). This tunes the surface work function of the HEL to better align with the ionization potential of the polymeric photoactive layer. Results show noticeable improvement in device power conversion efficiencies (PCEs) in OPVs. We achieved a 6.1 % PCE from the OPV by optimizing the surface-enriched layer.

  20. Chemical modification of chitosan for efficient gene therapy.

    PubMed

    Jiang, Hu-Lin; Cui, Peng-Fei; Xie, Rong-Lin; Cho, Chong-Su

    2014-01-01

    Gene therapy involves the introduction of foreign genetic material into cells in order to exert a therapeutic effect. Successful gene therapy relies on effective vector system. Viral vectors are highly efficient in transfecting cells, but the undesirable complications limit their therapeutic applications. As a natural biopolymer, chitosan has been considered to be a good gene carrier candidate due to its ideal character which combines biocompatibility, low toxicity with high cationic density together. However, the low cell specificity and low transfection efficiency of chitosan as a gene carrier need to be overcome before undertaking clinical trials. This chapter is principally on those endeavors such as chemical modifications using cell-specific ligands and stimuli-response groups as well as penetrating modifications that have been done to increase the performances of chitosan in gene therapy.

  1. Device engineering of perovskite solar cells to achieve near ideal efficiency

    SciTech Connect

    Agarwal, Sumanshu E-mail: prnair@ee.iitb.ac.in; Nair, Pradeep R. E-mail: prnair@ee.iitb.ac.in

    2015-09-21

    Despite the exciting recent research on perovskite based solar cells, the design space for further optimization and the practical limits of efficiency are not well known in the community. In this letter, we address these aspects through theoretical calculations and detailed numerical simulations. Here, we first provide the detailed balance limit efficiency in the presence of radiative and Auger recombination. Then, using coupled optical and carrier transport simulations, we identify the physical mechanisms that contribute towards bias dependent carrier collection, and hence low fill factors of current perovskite based solar cells. Our detailed simulations indicate that it is indeed possible to achieve efficiencies and fill factors greater than 25% and 85%, respectively, with near ideal super-position characteristics even in the presence of Auger recombination.

  2. Tuning charge balance in PHOLEDs with ambipolar host materials to achieve high efficiency

    SciTech Connect

    Padmaperuma, Asanga B.; Koech, Phillip K.; Cosimbescu, Lelia; Polikarpov, Evgueni; Swensen, James S.; Chopra, Neetu; So, Franky; Sapochak, Linda S.; Gaspar, Daniel J.

    2009-08-27

    The efficiency and stability of blue organic light emitting devices (OLEDs) continue to be a primary roadblock to developing organic solid state white lighting. For OLEDs to meet the high power conversion efficiency goal, they will require both close to 100% internal quantum efficiency and low operating voltage in a white light emitting device.1 It is generally accepted that such high quantum efficiency, can only be achieved with the use of organometallic phosphor doped OLEDs. Blue OLEDs are particularly important for solid state lighting. The simplest (and therefore likely the lowest cost) method of generating white light is to down convert part of the emission from a blue light source with a system of external phosphors.2 A second method of generating white light requires the superposition of the light from red, green and blue OLEDs in the correct ratio. Either of these two methods (and indeed any method of generating white light with a high color rendering index) critically depends on a high efficiency blue light component.3 A simple OLED generally consists of a hole-injecting anode, a preferentially hole transporting organic layer (HTL), an emissive layer that contains the recombination zone and ideally transports both holes and electrons, a preferentially electron-transporting layer (ETL) and an electron-injecting cathode. Color in state-of-the-art OLEDs is generated by an organometallic phosphor incorporated by co-sublimation into the emissive layer (EML).4 New materials functioning as hosts, emitters, charge transporting, and charge blocking layers have been developed along with device architectures leading to electrophosphorescent based OLEDs with high quantum efficiencies near the theoretical limit. However, the layers added to the device architecture to enable high quantum efficiencies lead to higher operating voltages and correspondingly lower power efficiencies. Achievement of target luminance power efficiencies will require new strategies for lowering

  3. Energy efficiency enhancements for semiconductors, communications, sensors and software achieved in cool silicon cluster project

    NASA Astrophysics Data System (ADS)

    Ellinger, Frank; Mikolajick, Thomas; Fettweis, Gerhard; Hentschel, Dieter; Kolodinski, Sabine; Warnecke, Helmut; Reppe, Thomas; Tzschoppe, Christoph; Dohl, Jan; Carta, Corrado; Fritsche, David; Tretter, Gregor; Wiatr, Maciej; Detlef Kronholz, Stefan; Mikalo, Ricardo Pablo; Heinrich, Harald; Paulo, Robert; Wolf, Robert; Hübner, Johannes; Waltsgott, Johannes; Meißner, Klaus; Richter, Robert; Michler, Oliver; Bausinger, Markus; Mehlich, Heiko; Hahmann, Martin; Möller, Henning; Wiemer, Maik; Holland, Hans-Jürgen; Gärtner, Roberto; Schubert, Stefan; Richter, Alexander; Strobel, Axel; Fehske, Albrecht; Cech, Sebastian; Aßmann, Uwe; Pawlak, Andreas; Schröter, Michael; Finger, Wolfgang; Schumann, Stefan; Höppner, Sebastian; Walter, Dennis; Eisenreich, Holger; Schüffny, René

    2013-07-01

    An overview about the German cluster project Cool Silicon aiming at increasing the energy efficiency for semiconductors, communications, sensors and software is presented. Examples for achievements are: 1000 times reduced gate leakage in transistors using high-fc (HKMG) materials compared to conventional poly-gate (SiON) devices at the same technology node; 700 V transistors integrated in standard 0.35 μm CMOS; solar cell efficiencies above 19% at < 200 W/m2 irradiation; 0.99 power factor, 87% efficiency and 0.088 distortion factor for dc supplies; 1 ns synchronization resolution via Ethernet; database accelerators allowing 85% energy savings for servers; adaptive software yielding energy reduction of 73% for e-Commerce applications; processors and corresponding data links with 40% and 70% energy savings, respectively, by adaption of clock frequency and supply voltage in less than 20 ns; clock generator chip with tunable frequency from 83-666 MHz and 0.62-1.6 mW dc power; 90 Gb/s on-chip link over 6 mm and efficiency of 174 fJ/mm; dynamic biasing system doubling efficiency in power amplifiers; 60 GHz BiCMOS frontends with dc power to bandwidth ratio of 0.17 mW/MHz; driver assistance systems reducing energy consumption by 10% in cars Contribution to the Topical Issue “International Semiconductor Conference Dresden-Grenoble - ISCDG 2012”, Edited by Gérard Ghibaudo, Francis Balestra and Simon Deleonibus.

  4. Efficient Exploration of the Space of Reconciled Gene Trees

    PubMed Central

    Szöllősi, Gergely J.; Rosikiewicz, Wojciech; Boussau, Bastien; Tannier, Eric; Daubin, Vincent

    2013-01-01

    Gene trees record the combination of gene-level events, such as duplication, transfer and loss (DTL), and species-level events, such as speciation and extinction. Gene tree–species tree reconciliation methods model these processes by drawing gene trees into the species tree using a series of gene and species-level events. The reconstruction of gene trees based on sequence alone almost always involves choosing between statistically equivalent or weakly distinguishable relationships that could be much better resolved based on a putative species tree. To exploit this potential for accurate reconstruction of gene trees, the space of reconciled gene trees must be explored according to a joint model of sequence evolution and gene tree–species tree reconciliation. Here we present amalgamated likelihood estimation (ALE), a probabilistic approach to exhaustively explore all reconciled gene trees that can be amalgamated as a combination of clades observed in a sample of gene trees. We implement the ALE approach in the context of a reconciliation model (Szöllősi et al. 2013), which allows for the DTL of genes. We use ALE to efficiently approximate the sum of the joint likelihood over amalgamations and to find the reconciled gene tree that maximizes the joint likelihood among all such trees. We demonstrate using simulations that gene trees reconstructed using the joint likelihood are substantially more accurate than those reconstructed using sequence alone. Using realistic gene tree topologies, branch lengths, and alignment sizes, we demonstrate that ALE produces more accurate gene trees even if the model of sequence evolution is greatly simplified. Finally, examining 1099 gene families from 36 cyanobacterial genomes we find that joint likelihood-based inference results in a striking reduction in apparent phylogenetic discord, with respectively. 24%, 59%, and 46% reductions in the mean numbers of duplications, transfers, and losses per gene family. The open source

  5. Achievement-Relevant Personality: Relations with the Big Five and Validation of an Efficient Instrument.

    PubMed

    Briley, Daniel A; Domiteaux, Matthew; Tucker-Drob, Elliot M

    2014-05-01

    Many achievement-relevant personality measures (APMs) have been developed, but the interrelations among APMs or associations with the broader personality landscape are not well-known. In Study 1, 214 participants were measured on 36 APMs and a measure of the Big Five. Factor analytic results supported the convergent and discriminant validity of five latent dimensions: performance, mastery, self-doubt, effort, and intellectual investment. Conscientiousness, neuroticism, and openness to experience had the most consistent associations with APMs. We constructed a more efficient scale- the Multidimensional Achievement-Relevant Personality Scale (MAPS). In Study 2, we replicated the factor structure and external correlates of the MAPS in a sample of 359 individuals. Finally, we validated the MAPS with four indicators of academic performance and demonstrated incremental validity.

  6. Achievement-Relevant Personality: Relations with the Big Five and Validation of an Efficient Instrument

    PubMed Central

    Briley, Daniel A.; Domiteaux, Matthew; Tucker-Drob, Elliot M.

    2014-01-01

    Many achievement-relevant personality measures (APMs) have been developed, but the interrelations among APMs or associations with the broader personality landscape are not well-known. In Study 1, 214 participants were measured on 36 APMs and a measure of the Big Five. Factor analytic results supported the convergent and discriminant validity of five latent dimensions: performance, mastery, self-doubt, effort, and intellectual investment. Conscientiousness, neuroticism, and openness to experience had the most consistent associations with APMs. We constructed a more efficient scale– the Multidimensional Achievement-Relevant Personality Scale (MAPS). In Study 2, we replicated the factor structure and external correlates of the MAPS in a sample of 359 individuals. Finally, we validated the MAPS with four indicators of academic performance and demonstrated incremental validity. PMID:24839374

  7. Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

    PubMed Central

    2009-01-01

    Background Trypanosoma cruzi, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial in silico studies of the T. cruzi genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in T. cruzi are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems. Results While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes. Conclusion Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of T. cruzi. This methodology should greatly facilitate reverse genetic studies in T. cruzi. PMID:19432966

  8. Learning Motivation Mediates Gene-by-Socioeconomic Status Interaction on Mathematics Achievement in Early Childhood

    ERIC Educational Resources Information Center

    Tucker-Drob, Elliot M.; Harden, K. Paige

    2012-01-01

    There is accumulating evidence that genetic influences on achievement are more pronounced among children living in higher socioeconomic status homes, and that these gene-by-environment interactions occur prior to children's entry into formal schooling. We hypothesized that one pathway through which socioeconomic status promotes genetic influences…

  9. Intellectual Interest Mediates Gene x Socioeconomic Status Interaction on Adolescent Academic Achievement

    ERIC Educational Resources Information Center

    Tucker-Drob, Elliot M.; Harden, K. Paige

    2012-01-01

    Recent studies have demonstrated that genetic influences on cognitive ability and academic achievement are larger for children raised in higher socioeconomic status (SES) homes. However, little work has been done to document the psychosocial processes that underlie this Gene x Environment interaction. One process may involve the conversion of…

  10. FEMP's O & M Best Practices Guide: A Guide to Achieving Operational Efficiency

    SciTech Connect

    Sullivan, Gregory P. ); Melendez, Aldo P. ); Pugh, Ray )

    2002-10-01

    FEMP's O & M Best Practices Guide (O & M BPG) highlights O & M programs targeting energy efficiency that are estimated to save between 5% and 20% on energy bills without a significant capital investment. Depending on the Federal site, these savings can represent thousands to hundreds-of-thousands of dollars each year, and many can be achieved with minimal cash outlays. In addition to energy/resource savings, a well-run O & M program will (1)increase the safety of all staff because properly maintained equipment is safer equipment; (2)ensure the comfort, health and safety of building occupants through properly functioning equipment providing a healthy indoor environment; (3)confirm the design life expectancy of equipment is achieved; and (4)facilitate the compliance with Federal legislation such as the Clean Air Act and the Clean Water Act. The focus of this guide is to provide the Federal O & M/Energy manager and practitioner with information and actions aimed at achieving these savings and benefits. The O & M BPG was developed under the direction of the Department of Energy's Federal Energy Management Program (FEMP).

  11. Alternative Formats to Achieve More Efficient Energy Codes for Commercial Buildings

    SciTech Connect

    Conover, David R.; Rosenberg, Michael I.; Halverson, Mark A.; Taylor, Zachary T.; Makela, Eric J.

    2013-01-26

    This paper identifies and examines several formats or structures that could be used to create the next generation of more efficient energy codes and standards for commercial buildings. Pacific Northwest National Laboratory (PNNL) is funded by the U.S. Department of Energy’s Building Energy Codes Program (BECP) to provide technical support to the development of ANSI/ASHRAE/IES Standard 90.1. While the majority of PNNL’s ASHRAE Standard 90.1 support focuses on developing and evaluating new requirements, a portion of its work involves consideration of the format of energy standards. In its current working plan, the ASHRAE 90.1 committee has approved an energy goal of 50% improvement in Standard 90.1-2013 relative to Standard 90.1-2004, and will likely be considering higher improvement targets for future versions of the standard. To cost-effectively achieve the 50% goal in manner that can gain stakeholder consensus, formats other than prescriptive must be considered. Alternative formats that include reducing the reliance on prescriptive requirements may make it easier to achieve these aggressive efficiency levels in new codes and standards. The focus on energy code and standard formats is meant to explore approaches to presenting the criteria that will foster compliance, enhance verification, and stimulate innovation while saving energy in buildings. New formats may also make it easier for building designers and owners to design and build the levels of efficiency called for in the new codes and standards. This paper examines a number of potential formats and structures, including prescriptive, performance-based (with sub-formats of performance equivalency and performance targets), capacity constraint-based, and outcome-based. The paper also discusses the pros and cons of each format from the viewpoint of code users and of code enforcers.

  12. Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery.

    PubMed

    Nam, Kihoon; Jung, Simhyun; Nam, Joung-Pyo; Kim, Sung Wan

    2015-12-28

    Branched poly(ethylenimine) (PEI) 25 kDa is an efficient gene delivery vector with outstanding gene condensation ability and great endosome escape activity. However, it also induces higher cytotoxicity. Transfection efficiency and toxicity of PEI are highly dependent upon their molecular weight and structure. We developed a bioreducible poly(ethylenimine) (PEI (-s-s-)) derived from low molecular weight PEI (1.8 kDa) for efficient gene delivery. Bioreducible core molecule is expected to increase molecular weight and reduce the cytotoxicity of the copolymer. PEI (-s-s-) polyplexes showed higher transfection efficiency and lower cytotoxicity compared to branched PEI 25 kDa, Lipofectamine® 2000 and, FuGENE® 6. In addition, PEI (-s-s-) derivative (16 kDa) formed stable polyplexes with a zeta-potential value of +34 mV and polyplex size of 61 nm. PEI (-s-s-) derivative (16 kDa) showed excellent transfection efficiency: 3.6 times higher than branched PEI 25 kDa in HeLa cells and 7.4 times higher than Lipofectamine® 2000 in H9C2 cell. The derivatives also showed lower cytotoxicity compared with Lipofectamine® 2000 and PEI 25 kDa in various cell types. In addition, newly synthesized PEI (-s-s-) derivatives have high reproducibility. PMID:26551343

  13. Pyramiding B genes in cotton achieves broader but not always higher resistance to bacterial blight.

    PubMed

    Essenberg, Margaret; Bayles, Melanie B; Pierce, Margaret L; Verhalen, Laval M

    2014-10-01

    Near-isogenic lines of upland cotton (Gossypium hirsutum) carrying single, race-specific genes B4, BIn, and b7 for resistance to bacterial blight were used to develop a pyramid of lines with all possible combinations of two and three genes to learn whether the pyramid could achieve broad and high resistance approaching that of L. A. Brinkerhoff's exceptional line Im216. Isogenic strains of Xanthomonas axonopodis pv. malvacearum carrying single avirulence (avr) genes were used to identify plants carrying specific resistance (B) genes. Under field conditions in north-central Oklahoma, pyramid lines exhibited broader resistance to individual races and, consequently, higher resistance to a race mixture. It was predicted that lines carrying two or three B genes would also exhibit higher resistance to race 1, which possesses many avr genes. Although some enhancements were observed, they did not approach the level of resistance of Im216. In a growth chamber, bacterial populations attained by race 1 in and on leaves of the pyramid lines decreased significantly with increasing number of B genes in only one of four experiments. The older lines, Im216 and AcHR, exhibited considerably lower bacterial populations than any of the one-, two-, or three-B-gene lines. A spreading collapse of spray-inoculated AcBIn and AcBInb7 leaves appears to be a defense response (conditioned by BIn) that is out of control. PMID:24655289

  14. Efficient transformation and expression of gfp gene in Valsa mali var. mali.

    PubMed

    Chen, Liang; Sun, Gengwu; Wu, Shujing; Liu, Huixiang; Wang, Hongkai

    2015-01-01

    Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species. PMID:25423905

  15. The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection.

    PubMed

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2010-10-15

    An unsaturated hydrocarbon chain in phospholipid was reported to affect a phase transition and a fusogenic activity after mixing membranes, and consequently to achieve a high DNA transfection efficiency. We previously showed that a biosurfactant mannosylerythritol lipid-A (MEL-A) enhances the gene transfection efficiency of cationic liposomes. Here, we have studied the effects of unsaturated fatty acid ratio of MEL-A on the physicochemical properties and gene delivery into cells of cationic liposomes using MEL-A with three different unsaturated fatty acid ratios (9.1%, 21.5%, and 46.3%). The gene transfer efficiency of cationic liposomes containing MEL-A (21.5%) was much higher than that of those containing MEL-A (9.1%) and MEL-A (46.3%). MEL-A (21.5%)-containing cationic liposomes induced highly efficient membrane fusion after addition of anionic liposomes and led to subsequent DNA release. Imaging analysis revealed that MEL-A (21.5%)-containing liposomes fused with the plasma membrane and delivered DNA into the nucleus of NIH-3T3 cells, MEL-A (46.3%)-containing liposomes fused with the plasma membrane did not deliver DNA into the nucleus, and MEL-A (9.1%)-containing liposomes neither fused with the plasma membrane nor delivered DNA into the nucleus. Thus, it is understandable that the unsaturated fatty acid ratio of MEL-A strongly influences the gene transfection efficiency of cationic liposomes. PMID:20674726

  16. Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae.

    PubMed Central

    Stewart, P; Whitwam, R E; Kersten, P J; Cullen, D; Tien, M

    1996-01-01

    A manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3' untranslated region of the glucoamylase gene of Aspergillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies. PMID:8975615

  17. Starch synthesis in Arabidopsis is achieved by spatial cotranscription of core starch metabolism genes.

    PubMed

    Tsai, Huang-Lung; Lue, Wei-Ling; Lu, Kuan-Jen; Hsieh, Ming-Hsiun; Wang, Shue-Mei; Chen, Jychian

    2009-11-01

    Starch synthesis and degradation require the participation of many enzymes, occur in both photosynthetic and nonphotosynthetic tissues, and are subject to environmental and developmental regulation. We examine the distribution of starch in vegetative tissues of Arabidopsis (Arabidopsis thaliana) and the expression of genes encoding core enzymes for starch synthesis. Starch is accumulated in plastids of epidermal, mesophyll, vascular, and root cap cells but not in root proper cells. We also identify cells that can synthesize starch heterotrophically in albino mutants. Starch synthesis in leaves is regulated by developmental stage and light. Expression of gene promoter-beta-glucuronidase fusion constructs in transgenic seedlings shows that starch synthesis genes are transcriptionally active in cells with starch synthesis and are inactive in root proper cells except the plastidial phosphoglucose isomerase. In addition, ADG2 (for ADPG PYROPHOSPHORYLASE2) is not required for starch synthesis in root cap cells. Expression profile analysis reveals that starch metabolism genes can be clustered into two sets based on their tissue-specific expression patterns. Starch distribution and expression pattern of core starch synthesis genes are common in Arabidopsis and rice (Oryza sativa), suggesting that the regulatory mechanism for starch metabolism genes may be conserved evolutionarily. We conclude that starch synthesis in Arabidopsis is achieved by spatial coexpression of core starch metabolism genes regulated by their promoter activities and is fine-tuned by cell-specific endogenous and environmental controls.

  18. Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system

    PubMed Central

    Singh, Atul K.; Carette, Xavier; Potluri, Lakshmi-Prasad; Sharp, Jared D.; Xu, Ranfei; Prisic, Sladjana; Husson, Robert N.

    2016-01-01

    Despite many methodological advances that have facilitated investigation of Mycobacterium tuberculosis pathogenesis, analysis of essential gene function in this slow-growing pathogen remains difficult. Here, we describe an optimized CRISPR-based method to inhibit expression of essential genes based on the inducible expression of an enzymatically inactive Cas9 protein together with gene-specific guide RNAs (CRISPR interference). Using this system to target several essential genes of M. tuberculosis, we achieved marked inhibition of gene expression resulting in growth inhibition, changes in susceptibility to small molecule inhibitors and disruption of normal cell morphology. Analysis of expression of genes containing sequences similar to those targeted by individual guide RNAs did not reveal significant off-target effects. Advantages of this approach include the ability to compare inhibited gene expression to native levels of expression, lack of the need to alter the M. tuberculosis chromosome, the potential to titrate the extent of transcription inhibition, and the ability to avoid off-target effects. Based on the consistent inhibition of transcription and the simple cloning strategy described in this work, CRISPR interference provides an efficient approach to investigate essential gene function that may be particularly useful in characterizing genes of unknown function and potential targets for novel small molecule inhibitors. PMID:27407107

  19. Calibration of STUD+ parameters to achieve optimally efficient broadband adiabatic decoupling in a single transient

    PubMed

    Bendall; Skinner

    1998-10-01

    for a single sech/tanh pulse. Residual splitting of the centerband, normally associated with incomplete or inefficient decoupling, is not seen in sech/tanh decoupling and therefore cannot be used as a measure of adiabatic decoupling efficiency. The calibrated experimental performance levels achieved in this study are within 20% of theoretical performance levels derived previously for ideal sech/tanh decoupling at high power, indicating a small scope for further improvement at practical RF power levels. The optimization procedures employed here will be generally applicable to any good combination of adiabatic inversion pulse and phase cycle. Copyright 1998 Academic Press. PMID:9761708

  20. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  1. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  2. Modification of nanostructured calcium carbonate for efficient gene delivery.

    PubMed

    Zhao, Dong; Wang, Chao-Qun; Zhuo, Ren-Xi; Cheng, Si-Xue

    2014-06-01

    In this study, a facile method to modify nanostructured calcium carbonate (CaCO3) gene delivery systems by adding calcium phosphate (CaP) component was developed. CaCO3/CaP/DNA nanoparticles were prepared by the co-precipitation of Ca(2+) ions with plasmid DNA in the presence of carbonate and phosphate ions. For comparison, CaCO3/DNA nanoparticles and CaP/DNA co-precipitates were also prepared. The effects of carbonate ion/phosphate ion (CO3(2-)/PO4(3-)) ratio on the particle size and gene delivery efficiency were investigated. With an appropriate CO3(2-)/PO4(3-) ratio, the co-existence of carbonate and phosphate ions could control the size of co-precipitates effectively, and CaCO3/CaP/DNA nanoparticles with a decreased size and improved stability could be obtained. The in vitro gene transfections mediated by different nanoparticles in 293T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. The gene transfection efficiency of CaCO3/CaP/DNA nanoparticles could be significantly improved as compared with CaCO3/DNA nanoparticles and CaP/DNA co-precipitates. The confocal microscopy study indicated that the cellular uptake and nuclear localization of CaCO3/CaP/DNA nanoparticles were significantly enhanced as compared with unmodified CaCO3/DNA nanoparticles.

  3. A novel promoterless gene targeting vector to efficiently disrupt PRNP gene in cattle.

    PubMed

    Wang, Shaohua; Zhang, Kun; Ding, Fangrong; Zhao, Rui; Li, Song; Li, Rong; Xu, Lingling; Song, Chi; Dai, Yunping; Li, Ning

    2013-02-20

    The PRNP gene encodes a cellular protein named prion, whose misfolded form has been implicated in a number of neuropathic diseases in mammals such as the Bovine Spongiform Encephalopathy (BSE) in cattle. BSE has brought devastating impact on the world economy and human health. Recently, several groups have performed the gene targeting strategy to disrupt the PRNP gene in bovine fibroblast cells and produce BSE-resistant cattle by somatic cell nuclear transfer (SCNT). However, the enrichment efficiency of the gene targeting vector was low. Here, we constructed a novel promoterless gene targeting vector to sequentially disrupt the PRNP gene in bovine fibroblast cells and generate gene targeted cattle by SCNT. The enrichment efficiency of the novel vector was 100% and 60%, respectively. After nuclear transfer, no significant difference was found in the rate of cleavage and blastocyst formation between the knockout and wild type cloned embryos. One PRNP⁺/⁻ calf was born with no obvious abnormal development by now. Fusion RT-PCR and real-time PCR showed one allele of the PRNP gene was functionally disrupted, and the mRNA expression reduced dramatically in the PRNP⁺/⁻ cattle. The reconstituted PRNP⁻/⁻ embryos showed double alleles disruption, and no difference in the rate of cleavage and blastocyst formation.

  4. Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex.

    PubMed Central

    Cristiano, R J; Smith, L C; Kay, M A; Brinkley, B R; Woo, S L

    1993-01-01

    Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies. Images Fig. 2 Fig. 4 PMID:8265587

  5. Achieving Highly Efficient, Selective, and Stable CO2 Reduction on Nitrogen-Doped Carbon Nanotubes.

    PubMed

    Wu, Jingjie; Yadav, Ram Manohar; Liu, Mingjie; Sharma, Pranav P; Tiwary, Chandra Sekhar; Ma, Lulu; Zou, Xiaolong; Zhou, Xiao-Dong; Yakobson, Boris I; Lou, Jun; Ajayan, Pulickel M

    2015-05-26

    The challenge in the electrosynthesis of fuels from CO2 is to achieve durable and active performance with cost-effective catalysts. Here, we report that carbon nanotubes (CNTs), doped with nitrogen to form resident electron-rich defects, can act as highly efficient and, more importantly, stable catalysts for the conversion of CO2 to CO. The unprecedented overpotential (-0.18 V) and selectivity (80%) observed on nitrogen-doped CNTs (NCNTs) are attributed to their unique features to facilitate the reaction, including (i) high electrical conductivity, (ii) preferable catalytic sites (pyridinic N defects), and (iii) low free energy for CO2 activation and high barrier for hydrogen evolution. Indeed, DFT calculations show a low free energy barrier for the potential-limiting step to form key intermediate COOH as well as strong binding energy of adsorbed COOH and weak binding energy for the adsorbed CO. The highest selective site toward CO production is pyridinic N, and the NCNT-based electrodes exhibit no degradation over 10 h of continuous operation, suggesting the structural stability of the electrode.

  6. Targeted and highly efficient gene transfer into CD4+ cells by a recombinant human immunodeficiency virus retroviral vector.

    PubMed Central

    Shimada, T; Fujii, H; Mitsuya, H; Nienhuis, A W

    1991-01-01

    We have established a recombinant HIV gene transfer system based on transient expression of the HIV packaging functions and a recombinant vector genome in monkey kidney Cos cells. The recombinant HIV retroviral vector introduced the neoR gene into CD4+ cells with high efficiency, comparable to that achieved with the highest titer amphotropic murine recombinant retrovirus. Vector preparations were devoid of replication competent, infectious HIV. Gene transfer was dependent on CD4 expression, as shown by expression of the CD4 gene in HeLa cells, and could be inhibited by soluble CD4. This specific and efficient gene transfer system may be useful for development of gene therapy for which T cells are the desired targets. Images PMID:1885765

  7. Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum.

    PubMed

    Levy, M; Erental, A; Yarden, O

    2008-09-01

    Homologous recombination is required for gene-targeted procedures such as gene disruption and gene replacement. Ku80 is part of the non-homologous end-joining DNA repair mechanism in many organisms. We identified and disrupted the Ku80 homologue in Sclerotinia sclerotiorum and generated heterokaryon mutants enriched with Ku80-deficient nuclei (ssku80). Sclerotial formation and pathogenicity of ssku80 mutants were normal on tomato fruits. The frequencies of homologous recombination in these strains were much higher than those of the wild type when transformed with a cna1 (encoding calcineurin) replacement construct. We coupled the increase in homologous recombination with a direct BIM-LAB-mediated transformation procedure, which utilizes compressed air to assist the transforming DNA in penetrating fungal hyphae of S. sclerotiorum. We found this method to be efficient and reproducible, and it did not alter the fitness of the mutants. We also demonstrated the first case of direct transformation of sclerotia. Nourseothricin was introduced as a selectable marker in S. sclerotiorum. The tools and procedures described will improve our ability to study gene function in S. sclerotiorum and are most likely to be adaptable for use in other plant pathogens. PMID:19019000

  8. Dendrimer type bio-reducible polymer for efficient gene delivery.

    PubMed

    Nam, Hye Yeong; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan; Bull, David A

    2012-06-28

    Arginine-grafted bio-reducible poly(disulfide amine) (ABP) was incorporated into the poly(amido amine) (PAMAM) dendrimer, creating a high molecular weight bio-reducible polymer, PAM-ABP, to overcome the limitations of the low molecular weight ABP. The newly synthesized PAM-ABP was studied to determine its efficacy as a gene delivery carrier. The PAM-ABP demonstrated superior condensing ability for plasmid DNA through the formation of compact nanosized polyplexes. These compact polyplexes enhanced cellular uptake and were less susceptible to reducing agents, resulting in greater transfection efficiency compared to ABP alone. Based on these results, this newly developed PAM-ABP polyplex is a promising delivery system for clinical gene therapy. PMID:22546681

  9. Strategies for achieving high-level expression of genes in Escherichia coli.

    PubMed Central

    Makrides, S C

    1996-01-01

    Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli. PMID:8840785

  10. Identification of Nitrogen Use Efficiency Genes in Barley: Searching for QTLs Controlling Complex Physiological Traits

    PubMed Central

    Han, Mei; Wong, Julia; Su, Tao; Beatty, Perrin H.; Good, Allen G.

    2016-01-01

    Over the past half century, the use of nitrogen (N) fertilizers has markedly increased crop yields, but with considerable negative effects on the environment and human health. Consequently, there has been a strong push to reduce the amount of N fertilizer used by maximizing the nitrogen use efficiency (NUE) of crops. One approach would be to use classical genetics to improve the NUE of a crop plant. This involves both conventional breeding and quantitative trait loci (QTL) mapping in combination with marker-assisted selection (MAS) to track key regions of the chromosome that segregate for NUE. To achieve this goal, one of initial steps is to characterize the NUE-associated genes, then use the profiles of specific genes to combine plant physiology and genetics to improve plant performance. In this study, on the basis of genetic homology and expression analysis, barley candidate genes from a variety of families that exhibited potential roles in enhancing NUE were identified and mapped. We then performed an analysis of QTLs associated with NUE in field trials and further analyzed their map-location data to narrow the search for these candidate genes. These results provide a novel insight on the identification of NUE genes and for the future prospects, will lead to a more thorough understanding of physiological significances of the diverse gene families that may be associated with NUE in barley.

  11. Efficient intranuclear gene delivery by CdSe aqueous quantum dots electrostatically-coated with polyethyleneimine

    NASA Astrophysics Data System (ADS)

    Au, Giang H. T.; Y Shih, Wan; Shih, Wei-Heng

    2015-01-01

    Quantum dots (QDs) are semiconducting nanoparticles with photoluminescence properties that do not photobleach. Due to these advantages, using QDs for non-viral gene delivery has the additional benefit of being able to track the delivery of the genes in real time as it happens. We investigate the efficacy of mercaptopropionic acid (MPA)-capped CdSe aqueous quantum dots (AQDs) electrostatically complexed with branched polyethyleneimine (PEI) both as a non-viral gene delivery vector and as a fluorescent probe for tracking the delivery of genes into nuclei. The MPA-capped CdSe AQDs that were completely synthesized in water were the model AQDs. A nominal MPA:Cd:Se = 4:3:1 was chosen for optimal photoluminescence and zeta potential. The gene delivery study was carried out in vitro using a human colon cancer cell line, HT29 (ATCC). The model gene was a plasmid DNA (pDNA) that can express red fluorescent protein (RFP). Positively charged branched PEI was employed to provide a proton buffer to the AQDs to allow for endosomal escape. It is shown that by using a PEI-AQD complex with a PEI/AQD molar ratio of 300 and a nominal pDNA/PEI-AQD ratio of 6, we can achieve 75 ± 2.6% RFP expression efficiency with cell vitality remaining at 78 ± 4% of the control.

  12. A simple, universal, efficient PCR-based gene synthesis method: sequential OE-PCR gene synthesis.

    PubMed

    Zhang, Pingping; Ding, Yingying; Liao, Wenting; Chen, Qiuli; Zhang, Huaqun; Qi, Peipei; He, Ting; Wang, Jinhong; Deng, Songhua; Pan, Tianyue; Ren, Hao; Pan, Wei

    2013-07-25

    Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience. PMID:23597923

  13. Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae

    SciTech Connect

    Stewart, P.; Whitwam, R.E.; Tien, Ming

    1996-03-01

    A manganese peroxidase (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3{prime} untranslated region of the glucoamylase gene of Asperigillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies. 34 refs., 7 figs., 1 tab.

  14. Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9

    PubMed Central

    Wang, Longxiang; Wang, Longlong; Tan, Qian; Fan, Qiuling; Zhu, Hui; Hong, Zonglie; Zhang, Zhongming; Duanmu, Deqiang

    2016-01-01

    The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.

  15. Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9

    PubMed Central

    Wang, Longxiang; Wang, Longlong; Tan, Qian; Fan, Qiuling; Zhu, Hui; Hong, Zonglie; Zhang, Zhongming; Duanmu, Deqiang

    2016-01-01

    The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus. PMID:27630657

  16. Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9.

    PubMed

    Wang, Longxiang; Wang, Longlong; Tan, Qian; Fan, Qiuling; Zhu, Hui; Hong, Zonglie; Zhang, Zhongming; Duanmu, Deqiang

    2016-01-01

    The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus. PMID:27630657

  17. Expert Meeting Report: Achieving the Best Installed Performance from High-Efficiency Residential Gas Furnaces

    SciTech Connect

    Brand, L.

    2012-03-01

    This report describes a Building America expert meeting hosted on July 28, 2011, by the Partnership for Advanced Residential Retrofit team. The purpose of this meeting was to identify installation practices that provide the best installed efficiency for residential gas furnaces, explain how AFUE and field efficiency can differ, and investigate the impact of installation practices on the efficiency and long-term durability of the furnace.

  18. Expert Meeting Report: Achieving the Best Installed Performance from High-Efficiency Residential Gas Furnaces

    SciTech Connect

    Brand, Larry

    2012-03-01

    This report describes a Building America expert meeting hosted on July 28, 2011, by the Partnership for Advanced Residential Retrofit (PARR) team. The purpose of this meeting was to identify installation practices that provide the best installed efficiency for residential gas furnaces, explain how AFUE and field efficiency can differ, and investigate the impact of installation practices on the efficiency and long-term durability of the furnace.

  19. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    PubMed

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells. PMID:27386153

  20. High-Efficiency Rooftop Air Conditioners: Innovative Procurement to Achieve Advances in Technology

    SciTech Connect

    Hollomon, Brad

    2003-08-01

    The U.S. Department of Energy, Defense Logistics Agency, and Pacific Northwest National Laboratory recently conducted a technology procurement to increase the availability of energy-efficient, packaged unitary ''rooftop'' air conditioners. The procurement encouraged air conditioner manufacturers to produce equipment that exceeded US energy efficiency standards by at least 25% at a lower life-cycle cost. An outgrowth of the project, a web-based cost estimator tool is now available to help consumers determine the cost-effectiveness of purchasing energy-efficient air conditioners based on climate conditions and other factors at their own locations.

  1. The Effect of Curriculum for Developing Efficient Studying Skills on Academic Achievements and Studying Skills of Learners

    ERIC Educational Resources Information Center

    Demir, Semra; Kilinc, Mehmet; Dogan, Ali

    2012-01-01

    Purpose of this study is to examine the effect of "Development of Efficient Studying Skills Curriculum" on academic achievements and studying skills of 7th grade primary school students. In this study, pre-test post-test from experiment models and semi-experimental model with control group were preferred. The reason for the preference is…

  2. A nanoparticle-based epigenetic modulator for efficient gene modulation

    NASA Astrophysics Data System (ADS)

    Pongkulapa, Thanapat

    Modulation of gene expression through chromatin remodeling involves epigenetic mechanisms, such as histone acetylation. Acetylation is tightly regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Molecules that can regulate these enzymes by altering (activating or inhibiting) their functions have become a valuable tool for understanding cell development and diseases. HAT activators, i.e. N-(4-Chloro-(3-trifluoromethyl)phenyl)-2-ethoxybenzamide (CTB), have shown a therapeutic potential for many diseases, including cancer and neurodegeneration. However, these compounds encounter a solubility and a membrane permeability issue, which restricts their full potential for practical usage, especially for in vivo applications. To address this issue, in this work, we developed a nanoparticle-based HAT activator CTB, named Au-CTB, by incorporating a new CTB analogue onto gold nanoparticles (AuNPs) along with a poly(ethylene glycol) moiety and a nuclear localization signal (NLS) peptide to assist with solubility and membrane permeability. We found that our new CTB analogue and Au-CTB could activate HAT activity. Significantly, an increase in potency to activate HAT activity by Au-CTB proved the effectiveness of using the nanoparticle delivery platform. In addition, the versatility of Au-CTB platform permits the attachment of multiple ligands with tunable ratios on the nanoparticle surface via facile surface functionalization of gold nanoparticles. Due to its high delivery efficiency and versatility, Au-CTB can be a powerful platform for applications in epigenetic regulation of gene expression.

  3. Efficient TALEN-mediated gene knockout in livestock.

    PubMed

    Carlson, Daniel F; Tan, Wenfang; Lillico, Simon G; Stverakova, Dana; Proudfoot, Chris; Christian, Michelle; Voytas, Daniel F; Long, Charles R; Whitelaw, C Bruce A; Fahrenkrug, Scott C

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

  4. O&M Best Practices - A Guide to Achieving Operational Efficiency (Release 2.0)

    SciTech Connect

    Sullivan, Gregory P.; Pugh, Ray; Melendez, Aldo P.; Hunt, W. D.

    2004-07-31

    This guide, sponsored by DOE's Federal Energy Management Program, highlights operations and maintenance (O&M) programs targeting energy efficiency that are estimated to save 5% to 20% on energy bills without a significant capital investment. The purpose of this guide is to provide the federal O&M energy manager and practitioner with useful information about O&M management, technologies, energy efficiency and cost-reduction approaches.

  5. Efficient Immunoglobulin Gene Disruption and Targeted Replacement in Rabbit Using Zinc Finger Nucleases

    PubMed Central

    Offner, Sonja; Ros, Francesca; Lifke, Valeria; Zeitler, Bryan; Rottmann, Oswald; Vincent, Anna; Zhang, Lei; Jenkins, Shirin; Niersbach, Helmut; Kind, Alexander J.; Gregory, Philip D.; Schnieke, Angelika E.; Platzer, Josef

    2011-01-01

    Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM+ and IgG+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields. PMID:21695153

  6. Rapid and Efficient Stable Gene Transfer to Mesenchymal Stromal Cells Using a Modified Foamy Virus Vector

    PubMed Central

    Sweeney, Nathan Paul; Regan, Cathy; Liu, Jiahui; Galleu, Antonio; Dazzi, Francesco; Lindemann, Dirk; Rupar, Charles Anthony; McClure, Myra Olga

    2016-01-01

    Mesenchymal stromal cells (MSCs) hold great promise for regenerative medicine. Stable ex vivo gene transfer to MSCs could improve the outcome and scope of MSC therapy, but current vectors require multiple rounds of transduction, involve genotoxic viral promoters and/or the addition of cytotoxic cationic polymers in order to achieve efficient transduction. We describe a self-inactivating foamy virus vector (FVV), incorporating the simian macaque foamy virus envelope and using physiological promoters, which efficiently transduces murine MSCs (mMSCs) in a single-round. High and sustained expression of the transgene, whether GFP or the lysosomal enzyme, arylsulphatase A (ARSA), was achieved. Defining MSC characteristics (surface marker expression and differentiation potential), as well as long-term engraftment and distribution in the murine brain following intracerebroventricular delivery, are unaffected by FVV transduction. Similarly, greater than 95% of human MSCs (hMSCs) were stably transduced using the same vector, facilitating human application. This work describes the best stable gene transfer vector available for mMSCs and hMSCs. PMID:27133965

  7. Swine herds achieve high performance by culling low lifetime efficiency sows in early parity.

    PubMed

    Takanashi, Ariko; McTaggart, Iain; Koketsu, Yuzo

    2011-11-01

    Sow lifetime performance and by-parity performance were analyzed using a 3 by 3 factorial design, comprising 3 herd productivity groups and 3 sow efficiency groups. Data was obtained from 101 Japanese herds, totaling 173,526 parity records of 34,929 sows, for the years 2001 to 2006. Sows were categorized into 3 groups based on the lower and upper 25th percentiles of the annualized lifetime pigs born alive: low lifetime efficiency sows (LE sows), intermediate lifetime efficiency sows or high lifetime efficiency sows. Herds were grouped on the basis of the upper and lower 25th percentiles of pigs weaned per mated female per year, averaged over 6 years: high-, intermediate- or low-performing herds. Mixed-effects models were used for comparisons. LE sows in high-performing herds had 57.8 fewer lifetime nonproductive days and 0.5 earlier parity at removal than those in low-performing herds (P<0.05). The number of pigs born alive of LE sows continuously decreased from parity 1 to 5, whereas those of high lifetime efficiency sows gradually increased from parity 1 to 4 before decreasing up to parity ≥ 6 (P<0.05). In conclusion, the LE sows have a performance pattern of decreasing number of pigs born alive across parity. The present study also indicates that high-performing herds culled potential LE sows earlier than the other herds.

  8. Student Achievement and Efficiency in Missouri Schools and the No Child Left Behind Act

    ERIC Educational Resources Information Center

    Primont, Diane F.; Domazlicky, Bruce

    2006-01-01

    The 2001 No Child Left Behind Act requires that schools make ''annual yearly progress'' in raising student achievement, or face possible sanctions. The No Child Left Behind Act places added emphasis on test scores, such as scores from the Missouri Assessment Program (MAP), to evaluate the performance of schools. In this paper, we investigate…

  9. A Thorough and Efficient Education: School Funding, Student Achievement and Productivity

    ERIC Educational Resources Information Center

    Ahlgrim, Richard W.

    2010-01-01

    Many school districts are facing stagnant or reduced funding (input) concurrent with demands for improved student achievement (output). In other words, there is pressure for all schools, even those schools with student populations of low socioeconomic status, to improve academic results (accountability for output) without a directly proportionate…

  10. A Strategy to Achieve High-Efficiency Organolead Trihalide Perovskite Solar Cells

    NASA Astrophysics Data System (ADS)

    Andalibi, Shabnam; Rostami, Ali; Darvish, Ghafar; Moravvej-Farshi, Mohammad Kazem

    2016-11-01

    Recent theoretical and experimental reports have shown that organometal lead halide perovskite solar cells have attracted attention as a low-cost photovoltaic technology offering high power conversion efficiency. However, the photovoltaic efficiency of these materials is still limited by poor chemical and structural stability in the case of methylammonium lead triiodide and by large bandgap in the case of methylammonium lead tribromide or trichloride. To obtain high-performance devices, we have investigated the computationally optimal efficiency for these materials using the detailed-balance method and present optimal intermediate-band perovskite solar cells with high open-circuit voltage. We model different halide perovskites using density function theory calculations and study their bandgap and absorption coefficient. Based on calculation results, surprisingly Hg doping in different halide perovskites introduces a narrow partially filled intermediate band in the forbidden bandgap. We investigate electrical and optical properties of MAPb0.97Hg0.03I3, MAPb0.96Hg0.04Br3, and MAPb0.96Hg0.04Cl3 and calculate the high absorption efficiency of the different perovskite structures to create thin films suitable for photovoltaic devices.

  11. A Strategy to Achieve High-Efficiency Organolead Trihalide Perovskite Solar Cells

    NASA Astrophysics Data System (ADS)

    Andalibi, Shabnam; Rostami, Ali; Darvish, Gafar; Moravvej-Farshi, Mohammad Kazem

    2016-07-01

    Recent theoretical and experimental reports have shown that organometal lead halide perovskite solar cells have attracted attention as a low-cost photovoltaic technology offering high power conversion efficiency. However, the photovoltaic efficiency of these materials is still limited by poor chemical and structural stability in the case of methylammonium lead triiodide and by large bandgap in the case of methylammonium lead tribromide or trichloride. To obtain high-performance devices, we have investigated the computationally optimal efficiency for these materials using the detailed-balance method and present optimal intermediate-band perovskite solar cells with high open-circuit voltage. We model different halide perovskites using density function theory calculations and study their bandgap and absorption coefficient. Based on calculation results, surprisingly Hg doping in different halide perovskites introduces a narrow partially filled intermediate band in the forbidden bandgap. We investigate electrical and optical properties of MAPb0.97Hg0.03I3, MAPb0.96Hg0.04Br3, and MAPb0.96Hg0.04Cl3 and calculate the high absorption efficiency of the different perovskite structures to create thin films suitable for photovoltaic devices.

  12. Achieving strategic cost advantages by focusing on back-office efficiency.

    PubMed

    McDowell, Jim

    2010-06-01

    A study of more than 270 hospitals over a four-year period highlighted a number of investments that can reduce hospitals' costs and improve efficiency, including the following: E-procurement systems. Electronic exchange of invoices and payments (and electronic receipt of payments). Human resources IT systems that reduce the need for manual entry of data. Shared services deployment.

  13. Combination of molecular, morphological, and interfacial engineering to achieve highly efficient and stable plastic solar cells.

    PubMed

    Chang, Chih-Yu; Cheng, Yen-Ju; Hung, Shih-Hsiu; Wu, Jhong-Sian; Kao, Wei-Shun; Lee, Chia-Hao; Hsu, Chain-Shu

    2012-01-24

    A flexible solar device showing exceptional air and mechanical stability is produced by simultaneously optimizing molecular structure, active layer morphology, and interface characteristics. The PFDCTBT-C8-based devices with inverted architecture exhibited excellent power conversion efficiencies of 7.0% and 6.0% on glass and flexible substrates, respectively.

  14. Achieving high performance polymer optoelectronic devices for high efficiency, long lifetime and low fabrication cost

    NASA Astrophysics Data System (ADS)

    Huang, Jinsong

    This thesis described three types of organic optoelectronic devices: polymer light emitting diodes (PLED), polymer photovoltaic solar cell, and organic photo detector. The research in this work focuses improving their performance including device efficiency, operation lifetime simplifying fabrication process. With further understanding in PLED device physics, we come up new device operation model and improved device architecture design. This new method is closely related to understanding of the science and physics at organic/metal oxide and metal oxide/metal interface. In our new device design, both material and interface are considered in order to confine and balance all injected carriers, which has been demonstrated very be successful in increasing device efficiency. We created two world records in device efficiency: 18 lm/W for white emission fluorescence PLED, 22 lm/W for red emission phosphorescence PLED. Slow solvent drying process has been demonstrated to significantly increase device efficiency in poly(3-hexylthiophene) (P3HT) and [6,6]-phenyl C 61-butyric acid methyl ester (PCBM) mixture polymer solar cell. From the mobility study by time of flight, the increase of efficiency can be well correlated to the improved carrier transport property due to P3HT crystallization during slow solvent drying. And it is found that, similar to PLED, balanced carrier mobility is essential in high efficient polymer solar cell. There is also a revolution in our device fabrication method. A unique device fabrication method is presented by an electronic glue based lamination process combined with interface modification as a one-step polymer solar cell fabrication process. It can completely skip the thermal evaporation process, and benefit device lifetime by several merits: no air reactive. The device obtained is metal free, semi-transparent, flexible, self-encapsulated, and comparable efficiency with that by regular method. We found the photomultiplication (PM) phenomenon in C

  15. Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

    PubMed

    Khan, Zahidul; Knecht, Wolfgang; Willer, Mette; Rozpedowska, Elzbieta; Kristoffersen, Peter; Clausen, Anders Ranegaard; Munch-Petersen, Birgitte; Almqvist, Per M; Gojkovic, Zoran; Piskur, Jure; Ekström, Tomas J

    2010-06-01

    The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate, but also to AZT diphosphate, with excellent kinetics. The efficiency of the toTK1/AZT system was confirmed when toTK1-transduced human glioblastoma (GBM) cells displayed a 500-fold increased sensitivity to AZT compared with wild-type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT suicide gene therapy system in combination with stem cell-mediated gene delivery promises new treatment of malignant gliomas. PMID:20154339

  16. Identification of Energy Efficiency Opportunities through Building Data Analysis and Achieving Energy Savings through Improved Controls

    SciTech Connect

    Katipamula, Srinivas; Taasevigen, Danny J.; Koran, Bill

    2014-09-04

    This chapter will highlight analysis techniques to identify energy efficiency opportunities to improve operations and controls. A free tool, Energy Charting and Metrics (ECAM), will be used to assist in the analysis of whole-building, sub-metered, and/or data from the building automation system (BAS). Appendix A describes the features of ECAM in more depth, and also provide instructions for downloading ECAM and all resources pertaining to using ECAM.

  17. Efficient shRNA-mediated inhibition of gene expression in zebrafish.

    PubMed

    De Rienzo, Gianluca; Gutzman, Jennifer H; Sive, Hazel

    2012-09-01

    Despite the broad repertoire of loss of function (LOF) tools available for use in the zebrafish, there remains a need for a simple and rapid method that can inhibit expression of genes at later stages. RNAi would fulfill that role, and a previous report (Dong et al. 2009) provided encouraging data. The goal of this study was to further address the ability of expressed shRNAs to inhibit gene expression. This included quantifying RNA knockdown, testing specificity of shRNA effects, and determining whether tissue-specific LOF could be achieved. Using an F0 transgenic approach, this report demonstrates that for two genes, wnt5b and zDisc1, each with described mutant and morphant phenotypes, shRNAs efficiently decrease endogenous RNA levels. Phenotypes elicited by shRNA resemble those of mutants and morphants, and are reversed by expression of cognate RNA, further demonstrating specificity. Tissue-specific expression of zDisc1 shRNAs in F0 transgenics demonstrates that conditional LOF can be readily obtained. These results suggest that shRNA expression presents a viable approach for rapid inhibition of zebrafish gene expression.

  18. Self-assembled Messenger RNA Nanoparticles (mRNA-NPs) for Efficient Gene Expression

    PubMed Central

    Kim, Hyejin; Park, Yongkuk; Lee, Jong Bum

    2015-01-01

    Although mRNA has several advantages over plasmid DNA when delivered into cells for gene expression, mRNA transfection is a very rare occurrence in gene delivery. This is mainly because of the labile nature of RNA, resulting in a low expression level of the desired protein. In this study, self-assembled mRNA nanoparticles (mRNA-NPs) packed with multiple repeats of mRNA were synthesized to achieve efficient gene expression. This approach required only a one-step process to synthesize particles with a minimal amount of plasmid DNA to produce the RNA transcripts via rolling circle transcription. Moreover, there are no concerns for cytotoxicity which can be caused by chemical condensates because mRNA-NPs are made entirely of mRNA. An examination of the cells transfected with the mRNA-NPs encoding the green fluorescence protein (GFP) confirmed that the mRNA-NPs can be used as a novel platform for effective gene delivery. PMID:26235529

  19. Ternary polyplex micelles with PEG shells and intermediate barrier to complexed DNA cores for efficient systemic gene delivery.

    PubMed

    Li, Junjie; Chen, Qixian; Zha, Zengshi; Li, Hui; Toh, Kazuko; Dirisala, Anjaneyulu; Matsumoto, Yu; Osada, Kensuke; Kataoka, Kazunori; Ge, Zhishen

    2015-07-10

    Simultaneous achievement of prolonged retention in blood circulation and efficient gene transfection activity in target tissues has always been a major challenge hindering in vivo applications of nonviral gene vectors via systemic administration. Herein, we constructed novel rod-shaped ternary polyplex micelles (TPMs) via complexation between the mixed block copolymers of poly(ethylene glycol)-b-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-PAsp(DET)) and poly(N-isopropylacrylamide)-b-PAsp(DET) (PNIPAM-b-PAsp(DET)) and plasmid DNA (pDNA) at room temperature, exhibiting distinct temperature-responsive formation of a hydrophobic intermediate layer between PEG shells and pDNA cores through facile temperature increase from room temperature to body temperature (~37 °C). As compared with binary polyplex micelles of PEG-b-PAsp(DET) (BPMs), TPMs were confirmed to condense pDNA into a more compact structure, which achieved enhanced tolerability to nuclease digestion and strong counter polyanion exchange. In vitro gene transfection results demonstrated TPMs exhibiting enhanced gene transfection efficiency due to efficient cellular uptake and endosomal escape. Moreover, in vivo performance evaluation after intravenous injection confirmed that TPMs achieved significantly prolonged blood circulation, high tumor accumulation, and promoted gene expression in tumor tissue. Moreover, TPMs loading therapeutic pDNA encoding an anti-angiogenic protein remarkably suppressed tumor growth following intravenous injection into H22 tumor-bearing mice. These results suggest TPMs with PEG shells and facilely engineered intermediate barrier to inner complexed pDNA have great potentials as systemic nonviral gene vectors for cancer gene therapy. PMID:25912408

  20. [Bariatric surgery is more efficient than medical treatment in achieving remission in diabetes mellitus type 2].

    PubMed

    Klein, Mads; Rosenberg, Jacob; Gögenur, Ismail

    2013-04-01

    Observational studies have shown that bariatric surgery can lead to remission of diabetes mellitus type 2 (DMII), but randomized controlled trials have been lacking. Recently, randomized controlled trials comparing bariatric surgery with optimal medical treatment in patients suffering from poorly controlled DMII, have been performed. These trials show that bariatric surgery in general, and the malabsorptive procedures in particular, are more effective than medical treatment in achieving remission of DMII. These procedures should therefore be considered in the treatment of patients with DMII and obesity.

  1. Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency.

    PubMed

    Ji, Yewei; Nie, Bin; Li, Ping; Xu, Xiaoyu; Zhou, Yuanguo

    2007-07-01

    The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β (Hsp90β) gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection. Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and BglII. Recombinant plasmids were transformed into Escherichia coli, DH5a, and the colonies were picked and grown in the Amp-agarose. The presence of positive clones was checked by the means of endodigestion and sequencing. Three cell strains, HepG2, Human umbilicus vein endothelium cells (HUVEC) and HeK293, were cultured. Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine. The transfection efficiency was measured by detection of enhanced green fluorescence protein (EGFP). The presence of positive recombinant clones were verified by double-digestion and sequencing. The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1, pSuper-Hsp90β2 and pSuper-Hsp90β3. After optimizing the ratio of plasmid to Lipofectamine, we achieved high transfection efficiency in HeK293 cells. Transfection efficiency was still low in the HepG2 cells. In conclusion, the si-RNA-synthesizing plasmids targeting Hsp90β were constructed and transfected into cells with different transfection efficiency.

  2. Optimal thickness of silicon membranes to achieve maximum thermoelectric efficiency: A first principles study

    NASA Astrophysics Data System (ADS)

    Mangold, Claudia; Neogi, Sanghamitra; Donadio, Davide

    2016-08-01

    Silicon nanostructures with reduced dimensionality, such as nanowires, membranes, and thin films, are promising thermoelectric materials, as they exhibit considerably reduced thermal conductivity. Here, we utilize density functional theory and Boltzmann transport equation to compute the electronic properties of ultra-thin crystalline silicon membranes with thickness between 1 and 12 nm. We predict that an optimal thickness of ˜7 nm maximizes the thermoelectric figure of merit of membranes with native oxide surface layers. Further thinning of the membranes, although attainable in experiments, reduces the electrical conductivity and worsens the thermoelectric efficiency.

  3. Advanced liquid cooling in HCPVT systems to achieve higher energy efficiencies

    NASA Astrophysics Data System (ADS)

    Zimmermann, S.; Helmers, H.; Tiwari, M. K.; Escher, W.; Paredes, S.; Neves, P.; Poulikakos, D.; Wiesenfarth, M.; Bett, A. W.; Michel, B.

    2013-09-01

    The benefits of advanced thermal packaging are demonstrated through a receiver package consisting of a monolithic interconnected module (MIM) which is directly attached to a high performance microchannel heat sink. Those packages can be applied in high-concentration photovoltaic systems and the generated heat can be used in addition to the electrical power output (CPVT systems). Thus, the total energy efficiency of the system increases significantly. A detailed exergy analysis of the receiver power output underscores the advantages of the new cooling approach.

  4. Enhancement of p53 gene transfer efficiency in hepatic tumor mediated by transferrin receptor through trans-arterial delivery.

    PubMed

    Lu, Qin; Teng, Gao-Jun; Zhang, Yue; Niu, Huan-Zhang; Zhu, Guang-Yu; An, Yan-Li; Yu, Hui; Li, Guo-Zhao; Qiu, Ding-Hong; Wu, Chuan-Ging

    2008-02-01

    Transferrin-DNA complex mediated by transferrin receptor in combination with interventional trans-arterial injection into a target organ may be a duel-target-oriented delivery means to achieve an efficient gene therapy. In this study, transferrin receptor expression in normal human hepatocyte and two hepatocellular-carcinoma cells (Huh7/SK-Hep1) was determined. p53-LipofectAMINE with different amounts of transferrin was transfected into the cells and the gene transfection efficiency was evaluated. After VX2 rabbit hepatocarcinoma model was established, the transferrin-p53-LipofectAMINE complex was delivered into the hepatic artery via interventional techniques to analyze the therapeutic p53 gene transfer efficiency in vivo by Western blot, immunohistochemical/immunofluorescence staining analysis and survival time. The results were transferrin receptor expression in Huh7 and SK-Hep1 cells was higher than in normal hepatocyte. Transfection efficiency of p53 was increased in vitro in both Huh7 and SK-Hep1 cells with increasing transferrin in a dose-dependent manner. As compared to intravenous administration, interventional injection of p53-gene complex into hepatic tumor mediated by transferrin-receptor, could enhance the gene transfer efficiency in vivo as evaluated by Western blot, immunohistochemical/immunofluorenscence staining analyses and improved animal survival (H = 12.567, p = 0.0019). These findings show the transferrin-transferrin receptor system combined with interventional techniques enhanced p53-gene transfer to hepatic tumor and the duel-target-oriented gene delivery may be an effective approach for gene therapy. PMID:18347429

  5. Efficient Method of Achieving Agreements between Individuals and Organizations about RFID Privacy

    NASA Astrophysics Data System (ADS)

    Cha, Shi-Cho

    This work presents novel technical and legal approaches that address privacy concerns for personal data in RFID systems. In recent years, to minimize the conflict between convenience and the privacy risk of RFID systems, organizations have been requested to disclose their policies regarding RFID activities, obtain customer consent, and adopt appropriate mechanisms to enforce these policies. However, current research on RFID typically focuses on enforcement mechanisms to protect personal data stored in RFID tags and prevent organizations from tracking user activity through information emitted by specific RFID tags. A missing piece is how organizations can obtain customers' consent efficiently and flexibly. This study recommends that organizations obtain licenses automatically or semi-automatically before collecting personal data via RFID technologies rather than deal with written consents. Such digitalized and standard licenses can be checked automatically to ensure that collection and use of personal data is based on user consent. While individuals can easily control who has licenses and license content, the proposed framework provides an efficient and flexible way to overcome the deficiencies in current privacy protection technologies for RFID systems.

  6. Achieving Selective and Efficient Electrocatalytic Activity for CO2 Reduction Using Immobilized Silver Nanoparticles.

    PubMed

    Kim, Cheonghee; Jeon, Hyo Sang; Eom, Taedaehyeong; Jee, Michael Shincheon; Kim, Hyungjun; Friend, Cynthia M; Min, Byoung Koun; Hwang, Yun Jeong

    2015-11-01

    Selective electrochemical reduction of CO2 is one of the most sought-after processes because of the potential to convert a harmful greenhouse gas to a useful chemical. We have discovered that immobilized Ag nanoparticles supported on carbon exhibit enhanced Faradaic efficiency and a lower overpotential for selective reduction of CO2 to CO. These electrocatalysts were synthesized directly on the carbon support by a facile one-pot method using a cysteamine anchoring agent resulting in controlled monodispersed particle sizes. These synthesized Ag/C electrodes showed improved activities, specifically decrease of the overpotential by 300 mV at 1 mA/cm(2), and 4-fold enhanced CO Faradaic efficiency at -0.75 V vs RHE with the optimal particle size of 5 nm compared to polycrystalline Ag foil. DFT calculations enlightened that the specific interaction between Ag nanoparticle and the anchoring agents modified the catalyst surface to have a selectively higher affinity to the intermediate COOH over CO, which effectively lowers the overpotential. PMID:26447349

  7. Achieving Internet-based efficiencies in a rural IDS: a case study.

    PubMed

    Bacus, R; Zunke, R

    2001-09-01

    After suffering payment cuts resulting from the Balanced Budget Act of 1997, Colorado-Fayette Medical Center (CFMC), a not-for-profit, rural integrated delivery system in Texas, wanted to reduce costs by gaining systemwide Internet access for its internal information system at a reasonable price. An application service provider affiliated with the Texas Hospital Association, helped CFMC achieve its goals for the project by performing a needs assessment, installing a wide-area network (WAN) with Internet access, and training staff. The new WAN enabled CFMC to improve its Web presence, allow radiologic image viewing at all sites, negotiate more favorable prices from vendors, implement electronic communication for staff members, and take advantage of on-line education opportunities. CFMC has found that the monthly fee paid to THN is offset by savings on long-distance calls, Internet service provider fees, and marketing and advertising costs. PMID:11552587

  8. Metering Best Practices, A Guide to Achieving Utility Resource Efficiency, Release 2.0

    SciTech Connect

    Sullivan, Greg; Hunt, W. D.; Pugh, Ray; Sandusky, William F.; Koehler, Theresa M.; Boyd, Brian K.

    2011-08-31

    This release is an update and expansion of the information provided in Release 1.0 of the Metering Best Practice Guide that was issued in October 2007. This release, as was the previous release, was developed under the direction of the U.S. Department of Energy's Federal Energy Management Program (FEMP). The mission of FEMP is to facilitate the Federal Government's implementation of sound cost-effective energy management and investment practices to enhance the nation's energy security and environmental stewardship. Each of these activities is directly related to achieving requirements set forth in the Energy Policy Acts of 1992 and 2005, the Energy Independence and Security Act (EISA) of 2007, and the goals that have been established in Executive Orders 13423 and 13514 - and also those practices that are inherent in sound management of Federal financial and personnel resources.

  9. Efficient Gene Induction and Endogenous Gene Repression Systems for the Filamentous Cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Higo, Akiyoshi; Isu, Atsuko; Fukaya, Yuki; Hisabori, Toru

    2016-02-01

    In the last decade, many studies have been conducted to employ genetically engineered cyanobacteria in the production of various metabolites. However, the lack of a strict gene regulation system in cyanobacteria has hampered these attempts. The filamentous cyanobacterium Anabaena sp. PCC 7120 performs both nitrogen and carbon fixation and is, therefore, a good candidate organism for such production. To employ Anabaena cells for this purpose, we intended to develop artificial gene regulation systems to alter the cell metabolic pathways efficiently. We introduced into Anabaena a transcriptional repressor TetR, widely used in diverse organisms, and green fluorescent protein (GFP) as a reporter. We found that anhydrotetracycline (aTc) substantially induced GFP fluorescence in a concentration-dependent manner. By expressing tetR under the nitrate-specific promoter nirA, we successfully reduced the concentration of aTc required for the induction of gfp under nitrogen fixation conditions (to 10% of the concentration needed under nitrate-replete conditions). Further, we succeeded in the overexpression of GFP by depletion of nitrate without the inducer by means of promoter engineering of the nirA promoter. Moreover, we applied these gene regulation systems to a metabolic enzyme in Anabaena and successfully repressed glnA, the gene encoding glutamine synthetase that is essential for nitrogen assimilation in cyanobacteria, by expressing the small antisense RNA for glnA. Consequently, the ammonium production of an ammonium-excreting Anabaena mutant was significantly enhanced. We therefore conclude that the gene regulation systems developed in this study are useful tools for the regulation of metabolic enzymes and will help to increase the production of desired substances in Anabaena. PMID:26684202

  10. Efficient Gene Induction and Endogenous Gene Repression Systems for the Filamentous Cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Higo, Akiyoshi; Isu, Atsuko; Fukaya, Yuki; Hisabori, Toru

    2016-02-01

    In the last decade, many studies have been conducted to employ genetically engineered cyanobacteria in the production of various metabolites. However, the lack of a strict gene regulation system in cyanobacteria has hampered these attempts. The filamentous cyanobacterium Anabaena sp. PCC 7120 performs both nitrogen and carbon fixation and is, therefore, a good candidate organism for such production. To employ Anabaena cells for this purpose, we intended to develop artificial gene regulation systems to alter the cell metabolic pathways efficiently. We introduced into Anabaena a transcriptional repressor TetR, widely used in diverse organisms, and green fluorescent protein (GFP) as a reporter. We found that anhydrotetracycline (aTc) substantially induced GFP fluorescence in a concentration-dependent manner. By expressing tetR under the nitrate-specific promoter nirA, we successfully reduced the concentration of aTc required for the induction of gfp under nitrogen fixation conditions (to 10% of the concentration needed under nitrate-replete conditions). Further, we succeeded in the overexpression of GFP by depletion of nitrate without the inducer by means of promoter engineering of the nirA promoter. Moreover, we applied these gene regulation systems to a metabolic enzyme in Anabaena and successfully repressed glnA, the gene encoding glutamine synthetase that is essential for nitrogen assimilation in cyanobacteria, by expressing the small antisense RNA for glnA. Consequently, the ammonium production of an ammonium-excreting Anabaena mutant was significantly enhanced. We therefore conclude that the gene regulation systems developed in this study are useful tools for the regulation of metabolic enzymes and will help to increase the production of desired substances in Anabaena.

  11. Progress toward achieving high power and high efficiency semipolar LEDs and their characterization

    NASA Astrophysics Data System (ADS)

    Zhong, Hong

    Performance of current commercially available wurtzite nitride based light-emitting diodes (LEDs), grown along the polar (0001) c-plane orientation, is limited by the presence of polarization-related electric fields inside multi-quantum wells (MQWs). The discontinuities in both spontaneous and piezoelectric polarization at the heterointerfaces result in internal electric fields in the quantum wells. These electric fields cause carrier separation [quantum confined Stark effect (QCSE)] and reduce the radiative recombination rate within the quantum wells. One approach to reduce and possibly eliminate the polarization-related effects is to grow III-nitride devices on crystal planes that are inclined with respect to the c-axis, i.e., on semipolar planes. In this dissertation, metalorganic chemical vapor deposition (MOCVD) has been employed for the homoepitaxial growth of GaN based LEDs on semipolar orientations. As a consequence of growing on high-quality bulk GaN substrates, the LEDs have significantly reduced threading dislocation and stacking fault densities, resulting in remarkable improvements in EQE and output power. High efficiency semipolar (1011) violet-blue and blue LEDs have been demonstrated without any intentional effort to enhance the light extraction from those devices. Optimizations of epitaxial structures have led to increased output power and external quantum efficiency. A silicone encapsulated single quantum well blue LED with peak wavelength of 444 nm with output power of 24.3 mW, external quantum efficiency of 43% and luminous efficacy of 75 lm/W (with phosphorescent coating) at 20 mA has been demonstrated. Polarization fields in strained (1011) and (112¯2) InGaN quantum wells have been experimentally determined through bias-dependent optical studies. Our results show that the polarization field flips its direction in semipolar InGaN quantum wells with large inclination angles (i.e. around 60°). This suggests that there exists a polarization

  12. Dye-sensitized solar cells with 13% efficiency achieved through the molecular engineering of porphyrin sensitizers.

    PubMed

    Mathew, Simon; Yella, Aswani; Gao, Peng; Humphry-Baker, Robin; Curchod, Basile F E; Ashari-Astani, Negar; Tavernelli, Ivano; Rothlisberger, Ursula; Nazeeruddin, Md Khaja; Grätzel, Michael

    2014-03-01

    Dye-sensitized solar cells have gained widespread attention in recent years because of their low production costs, ease of fabrication and tunable optical properties, such as colour and transparency. Here, we report a molecularly engineered porphyrin dye, coded SM315, which features the prototypical structure of a donor-π-bridge-acceptor and both maximizes electrolyte compatibility and improves light-harvesting properties. Linear-response, time-dependent density functional theory was used to investigate the perturbations in the electronic structure that lead to improved light harvesting. Using SM315 with the cobalt(II/III) redox shuttle resulted in dye-sensitized solar cells that exhibit a high open-circuit voltage VOC of 0.91 V, short-circuit current density JSC of 18.1 mA cm(-2), fill factor of 0.78 and a power conversion efficiency of 13%.

  13. Dye-sensitized solar cells with 13% efficiency achieved through the molecular engineering of porphyrin sensitizers

    NASA Astrophysics Data System (ADS)

    Mathew, Simon; Yella, Aswani; Gao, Peng; Humphry-Baker, Robin; Curchod, Basile F. E.; Ashari-Astani, Negar; Tavernelli, Ivano; Rothlisberger, Ursula; Nazeeruddin, Md. Khaja; Grätzel, Michael

    2014-03-01

    Dye-sensitized solar cells have gained widespread attention in recent years because of their low production costs, ease of fabrication and tunable optical properties, such as colour and transparency. Here, we report a molecularly engineered porphyrin dye, coded SM315, which features the prototypical structure of a donor-π-bridge-acceptor and both maximizes electrolyte compatibility and improves light-harvesting properties. Linear-response, time-dependent density functional theory was used to investigate the perturbations in the electronic structure that lead to improved light harvesting. Using SM315 with the cobalt(II/III) redox shuttle resulted in dye-sensitized solar cells that exhibit a high open-circuit voltage VOC of 0.91 V, short-circuit current density JSC of 18.1 mA cm-2, fill factor of 0.78 and a power conversion efficiency of 13%.

  14. Operations & Maintenance Best Practices - A Guide to Achieving Operational Efficiency (Release 3)

    SciTech Connect

    Sullivan, Greg; Pugh, Ray; Melendez, Aldo P.; Hunt, W. D.

    2010-08-04

    This guide highlights operations and maintenance programs targeting energy and water efficiency that are estimated to save 5% to 20% on energy bills without a significant capital investment. The purpose of this guide is to provide you, the Operations and Maintenance (O&M)/Energy manager and practitioner, with useful information about O&M management, technologies, energy and water efficiency, and cost-reduction approaches. To make this guide useful and to reflect your needs and concerns, the authors met with O&M and Energy managers via Federal Energy Management Program (FEMP) workshops. In addition, the authors conducted extensive literature searches and contacted numerous vendors and industry experts. The information and case studies that appear in this guide resulted from these activities. It needs to be stated at the outset that this guide is designed to provide information on effective O&M as it applies to systems and equipment typically found at Federal facilities. This guide is not designed to provide the reader with step-by-step procedures for performing O&M on any specific piece of equipment. Rather, this guide first directs the user to the manufacturer's specifications and recommendations. In no way should the recommendations in this guide be used in place of manufacturer's recommendations. The recommendations in this guide are designed to supplement those of the manufacturer, or, as is all too often the case, provide guidance for systems and equipment for which all technical documentation has been lost. As a rule, this guide will first defer to the manufacturer's recommendations on equipment operation and maintenance.

  15. Understanding the DNA damage response in order to achieve desired gene editing outcomes in mosquitoes.

    PubMed

    Overcash, Justin M; Aryan, Azadeh; Myles, Kevin M; Adelman, Zach N

    2015-02-01

    Mosquitoes are high-impact disease vectors with the capacity to transmit pathogenic agents that cause diseases such as malaria, yellow fever, chikungunya, and dengue. Continued growth in knowledge of genetic, molecular, and physiological pathways in mosquitoes allows for the development of novel control methods and for the continued optimization of existing ones. The emergence of site-specific nucleases as genomic engineering tools promises to expedite research of crucial biological pathways in these disease vectors. The utilization of these nucleases in a more precise and efficient manner is dependent upon knowledge and manipulation of the DNA repair pathways utilized by the mosquito. While progress has been made in deciphering DNA repair pathways in some model systems, research into the nature of the hierarchy of mosquito DNA repair pathways, as well as in mechanistic differences that may exist, is needed. In this review, we will describe progress in the use of site-specific nucleases in mosquitoes, along with the hierarchy of DNA repair in the context of mosquito chromosomal organization and structure, and how this knowledge may be manipulated to achieve precise chromosomal engineering in mosquitoes. PMID:25596822

  16. Understanding the DNA damage response in order to achieve desired gene editing outcomes in mosquitoes.

    PubMed

    Overcash, Justin M; Aryan, Azadeh; Myles, Kevin M; Adelman, Zach N

    2015-02-01

    Mosquitoes are high-impact disease vectors with the capacity to transmit pathogenic agents that cause diseases such as malaria, yellow fever, chikungunya, and dengue. Continued growth in knowledge of genetic, molecular, and physiological pathways in mosquitoes allows for the development of novel control methods and for the continued optimization of existing ones. The emergence of site-specific nucleases as genomic engineering tools promises to expedite research of crucial biological pathways in these disease vectors. The utilization of these nucleases in a more precise and efficient manner is dependent upon knowledge and manipulation of the DNA repair pathways utilized by the mosquito. While progress has been made in deciphering DNA repair pathways in some model systems, research into the nature of the hierarchy of mosquito DNA repair pathways, as well as in mechanistic differences that may exist, is needed. In this review, we will describe progress in the use of site-specific nucleases in mosquitoes, along with the hierarchy of DNA repair in the context of mosquito chromosomal organization and structure, and how this knowledge may be manipulated to achieve precise chromosomal engineering in mosquitoes.

  17. Progresses towards safe and efficient gene therapy vectors.

    PubMed

    Chira, Sergiu; Jackson, Carlo S; Oprea, Iulian; Ozturk, Ferhat; Pepper, Michael S; Diaconu, Iulia; Braicu, Cornelia; Raduly, Lajos-Zsolt; Calin, George A; Berindan-Neagoe, Ioana

    2015-10-13

    The emergence of genetic engineering at the beginning of the 1970's opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

  18. The use of ECDIS equipment to achieve an optimum value for energy efficiency operation index

    NASA Astrophysics Data System (ADS)

    Acomi, N.; Acomi, O. C.; Stanca, C.

    2015-11-01

    To reduce air pollution produced by ships, the International Maritime Organization has developed a set of technical, operational and management measures. The subject of our research addresses the operational measures for minimizing CO2 air emissions and the way how the emission value could be influenced by external factors regardless of ship-owners’ will. This study aims to analyse the air emissions for a loaded voyage leg performed by an oil tanker. The formula that allows us to calculate the predicted Energy Efficiency Operational Index involves the estimation of distance and fuel consumption, while the quantity of cargo is known. The electronic chart display and information system, ECDIS Simulation Software, will be used for adjusting the passage plan in real time, given the predicted severe environmental conditions. The distance will be determined using ECDIS, while the prediction of the fuel consumption will consider the sea trial and the vessel experience records. That way it will be possible to compare the estimated EEOI value in the case of great circle navigation in adverse weather condition with the estimated EEOI value for weather navigation.

  19. Depth Filters Containing Diatomite Achieve More Efficient Particle Retention than Filters Solely Containing Cellulose Fibers

    PubMed Central

    Buyel, Johannes F.; Gruchow, Hannah M.; Fischer, Rainer

    2015-01-01

    The clarification of biological feed stocks during the production of biopharmaceutical proteins is challenging when large quantities of particles must be removed, e.g., when processing crude plant extracts. Single-use depth filters are often preferred for clarification because they are simple to integrate and have a good safety profile. However, the combination of filter layers must be optimized in terms of nominal retention ratings to account for the unique particle size distribution in each feed stock. We have recently shown that predictive models can facilitate filter screening and the selection of appropriate filter layers. Here we expand our previous study by testing several filters with different retention ratings. The filters typically contain diatomite to facilitate the removal of fine particles. However, diatomite can interfere with the recovery of large biopharmaceutical molecules such as virus-like particles and aggregated proteins. Therefore, we also tested filtration devices composed solely of cellulose fibers and cohesive resin. The capacities of both filter types varied from 10 to 50 L m−2 when challenged with tobacco leaf extracts, but the filtrate turbidity was ~500-fold lower (~3.5 NTU) when diatomite filters were used. We also tested pre–coat filtration with dispersed diatomite, which achieved capacities of up to 120 L m−2 with turbidities of ~100 NTU using bulk plant extracts, and in contrast to the other depth filters did not require an upstream bag filter. Single pre-coat filtration devices can thus replace combinations of bag and depth filters to simplify the processing of plant extracts, potentially saving on time, labor and consumables. The protein concentrations of TSP, DsRed and antibody 2G12 were not affected by pre-coat filtration, indicating its general applicability during the manufacture of plant-derived biopharmaceutical proteins. PMID:26734037

  20. Depth Filters Containing Diatomite Achieve More Efficient Particle Retention than Filters Solely Containing Cellulose Fibers.

    PubMed

    Buyel, Johannes F; Gruchow, Hannah M; Fischer, Rainer

    2015-01-01

    The clarification of biological feed stocks during the production of biopharmaceutical proteins is challenging when large quantities of particles must be removed, e.g., when processing crude plant extracts. Single-use depth filters are often preferred for clarification because they are simple to integrate and have a good safety profile. However, the combination of filter layers must be optimized in terms of nominal retention ratings to account for the unique particle size distribution in each feed stock. We have recently shown that predictive models can facilitate filter screening and the selection of appropriate filter layers. Here we expand our previous study by testing several filters with different retention ratings. The filters typically contain diatomite to facilitate the removal of fine particles. However, diatomite can interfere with the recovery of large biopharmaceutical molecules such as virus-like particles and aggregated proteins. Therefore, we also tested filtration devices composed solely of cellulose fibers and cohesive resin. The capacities of both filter types varied from 10 to 50 L m(-2) when challenged with tobacco leaf extracts, but the filtrate turbidity was ~500-fold lower (~3.5 NTU) when diatomite filters were used. We also tested pre-coat filtration with dispersed diatomite, which achieved capacities of up to 120 L m(-2) with turbidities of ~100 NTU using bulk plant extracts, and in contrast to the other depth filters did not require an upstream bag filter. Single pre-coat filtration devices can thus replace combinations of bag and depth filters to simplify the processing of plant extracts, potentially saving on time, labor and consumables. The protein concentrations of TSP, DsRed and antibody 2G12 were not affected by pre-coat filtration, indicating its general applicability during the manufacture of plant-derived biopharmaceutical proteins.

  1. Bacterial magnetic particles as a novel and efficient gene vaccine delivery system

    PubMed Central

    Tang, Y-S; Wang, D; Zhou, C; Ma, W; Zhang, Y-Q; Liu, B; Zhang, S

    2012-01-01

    DNA vaccination is an attractive approach for eliciting antigen-specific immunity. In this study, we used magnetosomes (bacterial magnetic particles, BMPs) as carriers of a recombinant DNA composed of a secondary lymphoid tissue chemokine, human papillomavirus type E7 (HPV-E7) and Ig-Fc fragment (pSLC-E7-Fc) to generate a gene vaccine (BMP-V) for tumour immunotherapy. The results indicate that BMPs linked to DNA more efficiently in phosphate-buffered saline (pH=4–5) than in physiological saline. Efficient transfection of BMP-V in vitro and in vivo was achieved when a 600-mT static magnetic field was applied for 10 min. In a mouse tumour model, subcutaneous injection of BMP-V (5 μg, × 3 at 4-day intervals) plus magnetic exposure elicited systemic HPV-E7-specific immunity leading to significant tumour inhibition. The treated mice tolerated BMP-V immunisation well with no toxic side effects, as shown by histopathological examinations of major internal organs. Taken together, these results suggest that BMP can be used as a gene carrier to elicit a systemic immune response. PMID:22170341

  2. Overexpression of β-expansin gene GmEXPB2 improves phosphorus efficiency in soybean.

    PubMed

    Zhou, Jia; Xie, Jianna; Liao, Hong; Wang, Xiurong

    2014-02-01

    Soybean (Glycine max) is an important oil crop in agricultural production, but low phosphorus (P) availability limits soybean growth and production. Expansin is a family of plant cell wall proteins and involved in a variety of physiological processes, including cell division and enlargement, root growth and leaf development. To test the potential effects of expansins on crop production, we have developed soybean transgenic plants overexpressing a soybean β-expansin gene GmEXPB2, which was significantly induced by phosphate (Pi) starvation. The results indicated that constitutive overexpression of GmEXPB2 promoted leaf expansion, sequentially stimulated root growth and consequently resulted in improved P efficiency in the transgenic plants under P-limited conditions in hydroponics. In particular, when tested in calcareous (CS) and acid soils (AS), the two GmEXPB2 transgenic soybean lines showed above 25 and 40% increases in plant dry weight and P content, respectively to wild-type plants in low-P CS, but not in AS. To our knowledge, this is the first report in which improvement of P efficiency could be achieved through constitutive overexpression of an endogenous EXPB gene in soybean. These findings suggest that genetic modification of root and leaf traits might be a suitable strategy for improving crop production in low-P soils.

  3. Molecular Assemblies, Genes and Genomics Integrated Efficiently (MAGGIE)

    SciTech Connect

    Baliga, Nitin S

    2011-05-26

    Final report on MAGGIE. We set ambitious goals to model the functions of individual organisms and their community from molecular to systems scale. These scientific goals are driving the development of sophisticated algorithms to analyze large amounts of experimental measurements made using high throughput technologies to explain and predict how the environment influences biological function at multiple scales and how the microbial systems in turn modify the environment. By experimentally evaluating predictions made using these models we will test the degree to which our quantitative multiscale understanding wilt help to rationally steer individual microbes and their communities towards specific tasks. Towards this end we have made substantial progress towards understanding evolution of gene families, transcriptional structures, detailed structures of keystone molecular assemblies (proteins and complexes), protein interactions, biological networks, microbial interactions, and community structure. Using comparative analysis we have tracked the evolutionary history of gene functions to understand how novel functions evolve. One level up, we have used proteomics data, high-resolution genome tiling microarrays, and 5' RNA sequencing to revise genome annotations, discover new genes including ncRNAs, and map dynamically changing operon structures of five model organisms: For Desulfovibrio vulgaris Hildenborough, Pyrococcus furiosis, Sulfolobus solfataricus, Methanococcus maripaludis and Haiobacterium salinarum NROL We have developed machine learning algorithms to accurately identify protein interactions at a near-zero false positive rate from noisy data generated using tagfess complex purification, TAP purification, and analysis of membrane complexes. Combining other genome-scale datasets produced by ENIGMA (in particular, microarray data) and available from literature we have been able to achieve a true positive rate as high as 65% at almost zero false positives when

  4. Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA

    PubMed Central

    Wang, Yong; Du, Yinan; Shen, Bin; Zhou, Xiaoyang; Li, Jian; Liu, Yu; Wang, Jianying; Zhou, Jiankui; Hu, Bian; Kang, Nannan; Gao, Jimin; Yu, Liqing; Huang, Xingxu; Wei, Hong

    2015-01-01

    Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs. PMID:25653176

  5. Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA.

    PubMed

    Wang, Yong; Du, Yinan; Shen, Bin; Zhou, Xiaoyang; Li, Jian; Liu, Yu; Wang, Jianying; Zhou, Jiankui; Hu, Bian; Kang, Nannan; Gao, Jimin; Yu, Liqing; Huang, Xingxu; Wei, Hong

    2015-01-01

    Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs. PMID:25653176

  6. Highly efficient method for gene delivery into mouse dorsal root ganglia neurons.

    PubMed

    Yu, Lingli; Reynaud, Florie; Falk, Julien; Spencer, Ambre; Ding, Yin-Di; Baumlé, Véronique; Lu, Ruisheng; Castellani, Valérie; Yuan, Chonggang; Rudkin, Brian B

    2015-01-01

    The development of gene transfection technologies has greatly advanced our understanding of life sciences. While use of viral vectors has clear efficacy, it requires specific expertise and biological containment conditions. Electroporation has become an effective and commonly used method for introducing DNA into neurons and in intact brain tissue. The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5-16. This cell type has been particularly recalcitrant and refractory to physical or chemical methods for introduction of DNA. By optimizing the culture condition and parameters including voltage and duration for this specific electroporation system, high efficiency (60-80%) and low toxicity (>60% survival) were achieved with robust differentiation in response to Nerve growth factor (NGF). Moreover, 3-50 times fewer cells are needed (6 × 10(4)) compared with other traditional electroporation methods. This approach underlines the efficacy of this type of electroporation, particularly when only limited amount of cells can be obtained, and is expected to greatly facilitate the study of gene function in dorsal root ganglia neuron cultures. PMID:25698920

  7. Efficient and anonymous two-factor user authentication in wireless sensor networks: achieving user anonymity with lightweight sensor computation.

    PubMed

    Nam, Junghyun; Choo, Kim-Kwang Raymond; Han, Sangchul; Kim, Moonseong; Paik, Juryon; Won, Dongho

    2015-01-01

    A smart-card-based user authentication scheme for wireless sensor networks (hereafter referred to as a SCA-WSN scheme) is designed to ensure that only users who possess both a smart card and the corresponding password are allowed to gain access to sensor data and their transmissions. Despite many research efforts in recent years, it remains a challenging task to design an efficient SCA-WSN scheme that achieves user anonymity. The majority of published SCA-WSN schemes use only lightweight cryptographic techniques (rather than public-key cryptographic techniques) for the sake of efficiency, and have been demonstrated to suffer from the inability to provide user anonymity. Some schemes employ elliptic curve cryptography for better security but require sensors with strict resource constraints to perform computationally expensive scalar-point multiplications; despite the increased computational requirements, these schemes do not provide user anonymity. In this paper, we present a new SCA-WSN scheme that not only achieves user anonymity but also is efficient in terms of the computation loads for sensors. Our scheme employs elliptic curve cryptography but restricts its use only to anonymous user-to-gateway authentication, thereby allowing sensors to perform only lightweight cryptographic operations. Our scheme also enjoys provable security in a formal model extended from the widely accepted Bellare-Pointcheval-Rogaway (2000) model to capture the user anonymity property and various SCA-WSN specific attacks (e.g., stolen smart card attacks, node capture attacks, privileged insider attacks, and stolen verifier attacks).

  8. Efficient and anonymous two-factor user authentication in wireless sensor networks: achieving user anonymity with lightweight sensor computation.

    PubMed

    Nam, Junghyun; Choo, Kim-Kwang Raymond; Han, Sangchul; Kim, Moonseong; Paik, Juryon; Won, Dongho

    2015-01-01

    A smart-card-based user authentication scheme for wireless sensor networks (hereafter referred to as a SCA-WSN scheme) is designed to ensure that only users who possess both a smart card and the corresponding password are allowed to gain access to sensor data and their transmissions. Despite many research efforts in recent years, it remains a challenging task to design an efficient SCA-WSN scheme that achieves user anonymity. The majority of published SCA-WSN schemes use only lightweight cryptographic techniques (rather than public-key cryptographic techniques) for the sake of efficiency, and have been demonstrated to suffer from the inability to provide user anonymity. Some schemes employ elliptic curve cryptography for better security but require sensors with strict resource constraints to perform computationally expensive scalar-point multiplications; despite the increased computational requirements, these schemes do not provide user anonymity. In this paper, we present a new SCA-WSN scheme that not only achieves user anonymity but also is efficient in terms of the computation loads for sensors. Our scheme employs elliptic curve cryptography but restricts its use only to anonymous user-to-gateway authentication, thereby allowing sensors to perform only lightweight cryptographic operations. Our scheme also enjoys provable security in a formal model extended from the widely accepted Bellare-Pointcheval-Rogaway (2000) model to capture the user anonymity property and various SCA-WSN specific attacks (e.g., stolen smart card attacks, node capture attacks, privileged insider attacks, and stolen verifier attacks). PMID:25849359

  9. Efficient and Anonymous Two-Factor User Authentication in Wireless Sensor Networks: Achieving User Anonymity with Lightweight Sensor Computation

    PubMed Central

    Nam, Junghyun; Choo, Kim-Kwang Raymond; Han, Sangchul; Kim, Moonseong; Paik, Juryon; Won, Dongho

    2015-01-01

    A smart-card-based user authentication scheme for wireless sensor networks (hereafter referred to as a SCA-WSN scheme) is designed to ensure that only users who possess both a smart card and the corresponding password are allowed to gain access to sensor data and their transmissions. Despite many research efforts in recent years, it remains a challenging task to design an efficient SCA-WSN scheme that achieves user anonymity. The majority of published SCA-WSN schemes use only lightweight cryptographic techniques (rather than public-key cryptographic techniques) for the sake of efficiency, and have been demonstrated to suffer from the inability to provide user anonymity. Some schemes employ elliptic curve cryptography for better security but require sensors with strict resource constraints to perform computationally expensive scalar-point multiplications; despite the increased computational requirements, these schemes do not provide user anonymity. In this paper, we present a new SCA-WSN scheme that not only achieves user anonymity but also is efficient in terms of the computation loads for sensors. Our scheme employs elliptic curve cryptography but restricts its use only to anonymous user-to-gateway authentication, thereby allowing sensors to perform only lightweight cryptographic operations. Our scheme also enjoys provable security in a formal model extended from the widely accepted Bellare-Pointcheval-Rogaway (2000) model to capture the user anonymity property and various SCA-WSN specific attacks (e.g., stolen smart card attacks, node capture attacks, privileged insider attacks, and stolen verifier attacks). PMID:25849359

  10. Split vector systems for ultra-targeted gene delivery: a contrivance to achieve ethical assurance of somatic gene therapy in vivo.

    PubMed

    Tolmachov, Oleg E

    2014-08-01

    Tightly controlled spatial localisation of therapeutic gene delivery is essential to maximize the benefits of somatic gene therapy in vivo and to reduce its undesired effects on the 'bystander' cell populations, most importantly germline cells. Indeed, complete ethical assurance of somatic gene therapy can only be achieved with ultra-targeted gene delivery, which excludes the risk of inadvertent germline gene transfer. Thus, it is desired to supplement existing strategies of physical focusing and biological (cell-specific) targeting of gene delivery with an additional principle for the rigid control over spread of gene transfer within the body. In this paper I advance the concept of 'combinatorial' targeting of therapeutic gene transfer in vivo. I hypothesize that it is possible to engineer complex gene delivery vector systems consisting of several components, each one of them capable of independent spread within the human body but incapable of independent facilitation of gene transfer. As the gene delivery augmented by such split vector systems would be reliant on the simultaneous availability of all the vector system components at a predetermined body site, it is envisaged that higher order reaction kinetics required for the assembly of the functional gene transfer configuration would sharpen spatial localisation of gene transfer via curtailing the blurring effect of the vector spread within the body. A particular implementation of such split vector system could be obtained through supplementing a viral therapeutic gene vector with a separate auxiliary vector carrying a non-integrative and non-replicative form of a gene (e.g., mRNA) coding for a cellular receptor of the therapeutic vector component. Gene-transfer-enabling components of the vector system, which would be delivered separately from the vector component loaded with the therapeutic gene cargo, could also be cell-membrane-insertion-proficient receptors, elements of artificial transmembrane channels

  11. Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

    PubMed

    Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

    2014-03-21

    Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor.

  12. Titanate cathodes with enhanced electrical properties achieved via growing surface Ni particles toward efficient carbon dioxide electrolysis.

    PubMed

    Gan, Lizhen; Ye, Lingting; Tao, Shanwen; Xie, Kui

    2016-01-28

    Ionic conduction in perovskite oxide is commonly tailored by element doping in lattices to create charge carriers, while few studies have been focused on ionic conduction enhancement through tailoring microstructures. In this work, remarkable enhancement of ionic conduction in titanate has been achieved via in situ growing active nickel nanoparticles on an oxide surface by controlling the oxide material nonstoichiometry. The combined use of XRD, SEM, XPS and EDS indicates that the exsolution/dissolution of the nickel nanoparticles is completely reversible in redox cycles. With the synergetic effect of enhanced ionic conduction of titanate and the presence of catalytic active Ni nanocatalysts, significant improvement of electrocatalytic performances of the titanate cathode is demonstrated. A current density of 0.3 A cm(-2) with a Faradic efficiency of 90% has been achieved for direct carbon dioxide electrolysis in a 2 mm-thick YSZ-supported solid oxide electrolyzer with the modified titanate cathode at 2 V and 1073 K. PMID:26743799

  13. Benefits of Hybrid-Electric Propulsion to Achieve 4x Increase in Cruise Efficiency for a VTOL Aircraft

    NASA Technical Reports Server (NTRS)

    Fredericks, William J.; Moore, Mark D.; Busan, Ronald C.

    2013-01-01

    Electric propulsion enables radical new vehicle concepts, particularly for Vertical Takeoff and Landing (VTOL) aircraft because of their significant mismatch between takeoff and cruise power conditions. However, electric propulsion does not merely provide the ability to normalize the power required across the phases of flight, in the way that automobiles also use hybrid electric technologies. The ability to distribute the thrust across the airframe, without mechanical complexity and with a scale-free propulsion system, is a new degree of freedom for aircraft designers. Electric propulsion is scale-free in terms of being able to achieve highly similar levels of motor power to weight and efficiency across a dramatic scaling range. Applying these combined principles of electric propulsion across a VTOL aircraft permits an improvement in aerodynamic efficiency that is approximately four times the state of the art of conventional helicopter configurations. Helicopters typically achieve a lift to drag ratio (L/D) of between 4 and 5, while the VTOL aircraft designed and developed in this research were designed to achieve an L/D of approximately 20. Fundamentally, the ability to eliminate the problem of advancing and retreating rotor blades is shown, without resorting to unacceptable prior solutions such as tail-sitters. This combination of concept and technology also enables a four times increase in range and endurance while maintaining the full VTOL and hover capability provided by a helicopter. Also important is the ability to achieve low disc-loading for low ground impingement velocities, low noise and hover power minimization (thus reducing energy consumption in VTOL phases). This combination of low noise and electric propulsion (i.e. zero emissions) will produce a much more community-friendly class of vehicles. This research provides a review of the concept brainstorming, configuration aerodynamic and mission analysis, as well as subscale prototype construction and

  14. Efficient Gene Tree Correction Guided by Genome Evolution

    PubMed Central

    Lafond, Manuel; Seguin, Jonathan; Boussau, Bastien; Guéguen, Laurent; El-Mabrouk, Nadia; Tannier, Eric

    2016-01-01

    Motivations Gene trees inferred solely from multiple alignments of homologous sequences often contain weakly supported and uncertain branches. Information for their full resolution may lie in the dependency between gene families and their genomic context. Integrative methods, using species tree information in addition to sequence information, often rely on a computationally intensive tree space search which forecloses an application to large genomic databases. Results We propose a new method, called ProfileNJ, that takes a gene tree with statistical supports on its branches, and corrects its weakly supported parts by using a combination of information from a species tree and a distance matrix. Its low running time enabled us to use it on the whole Ensembl Compara database, for which we propose an alternative, arguably more plausible set of gene trees. This allowed us to perform a genome-wide analysis of duplication and loss patterns on the history of 63 eukaryote species, and predict ancestral gene content and order for all ancestors along the phylogeny. Availability A web interface called RefineTree, including ProfileNJ as well as a other gene tree correction methods, which we also test on the Ensembl gene families, is available at: http://www-ens.iro.umontreal.ca/~adbit/polytomysolver.html. The code of ProfileNJ as well as the set of gene trees corrected by ProfileNJ from Ensembl Compara version 73 families are also made available. PMID:27513924

  15. Progresses towards safe and efficient gene therapy vectors

    PubMed Central

    Chira, Sergiu; Jackson, Carlo S.; Oprea, Iulian; Ozturk, Ferhat; Pepper, Michael S.; Diaconu, Iulia; Braicu, Cornelia; Raduly, Lajos-Zsolt; Calin, George A.; Berindan-Neagoe, Ioana

    2015-01-01

    The emergence of genetic engineering at the beginning of the 1970′s opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors. PMID:26362400

  16. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    NASA Astrophysics Data System (ADS)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  17. Efficient Reverse-Engineering of a Developmental Gene Regulatory Network

    PubMed Central

    Cicin-Sain, Damjan; Ashyraliyev, Maksat; Jaeger, Johannes

    2012-01-01

    Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology. Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data. This approach is called reverse engineering. It has been successfully applied to many gene networks in various biological systems. However, to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge. Here, we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster. A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data. We have developed a simplified data processing pipeline that considerably increases the throughput of the method, but results in data of reduced accuracy compared to those previously used for gap gene network inference. We demonstrate that we can infer the correct network structure using our reduced data set, and investigate minimal data requirements for successful reverse engineering. Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data, while it is less important to precisely measure expression levels. Based on this, we define minimal data requirements for gap gene network inference. Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort. This enables more widespread use of the method in different developmental contexts and organisms. Such systematic application of data-driven models to real-world networks has enormous potential. Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to

  18. An efficient method for in vitro gene delivery via regulation of cellular endocytosis pathway

    PubMed Central

    Luo, Jing; Li, Caixia; Chen, Jianlin; Wang, Gang; Gao, Rong; Gu, Zhongwei

    2015-01-01

    Transfection efficiency was the primary goal for in vitro gene delivery mediated by nonviral gene carriers. Here, we report a modified gene transfection method that could greatly increase the efficiency of, and accelerate the process mediated by, 25 kDa branched polyethyleneimine and Lipofectamine™ 2000 in a broad range of cell strains, including tumor, normal, primary, and embryonic stem cells. In this method, the combination of transfection procedure with optimized complexation volume had a determinant effect on gene delivery result. The superiorities of the method were found to be related to the change of cellular endocytosis pathway and decrease of particle size. The efficient and simple method established in this study can be widely used for in vitro gene delivery into cultured cells. We think it may also be applicable for many more nonviral gene delivery materials than polyethyleneimine and liposome. PMID:25767387

  19. Design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo.

    PubMed

    Li, Yong-Fu; Morcos, Paul A

    2008-07-01

    Safe and efficient in vivo delivery of Morpholino antisense oligos was probably the last and most difficult challenge for the broad application of antisense in animal research and therapeutics. Several arginine-rich peptides effective for in vivo delivery of Morpholino antisense oligos require rather complex and expensive procedures for synthesis and conjugation. This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffold, functional group multiplication, orthogonal protecting group manipulation, solid phase conjugation, and off-resin perguanidinylation of the transporter structure are all orchestrated for efficient assembly. We utilized triazine as a core to provide a site for on-column conjugation to the Morpholino oligo and to anchor functional side arms which, after extension, multiplication, and deprotection, are subsequently converted from primary amines to the eight guanidinium headgroups that serve for transport across cell membranes. Intravenous administration of the delivery-enabled Morpholino into a splice-reporter strain of transgenic living mice results in de novo expression of splice-corrected green fluorescent protein in a broad range of tissues and organs in those treated mice. This rigorously demonstrates that this new dendritic transporter achieves effective delivery of a Morpholino oligo into the cytosol/nuclear compartment of cells systemically in vivo. The practical conjugation process may overcome any availability limitation for routine use by the scientific community, and the efficient delivery ability of this transporter may advance the application of Morpholino antisense technology in animals.

  20. Analyzing the possibility of achieving more efficient cooling of water in the evaporative cooling towers of the Armenian NPP

    NASA Astrophysics Data System (ADS)

    Petrosyan, V. G.; Yeghoyan, E. A.

    2015-10-01

    The specific features of the service cooling water system used at the Armenian NPP and modifications made in the arrangement for supplying water to the water coolers in order to achieve more efficient cooling are presented. The mathematical model applied in carrying out the analyses is described, the use of which makes it possible to investigate the operation of parallel-connected cooling towers having different hydraulic and thermal loads. When the third standby cooling tower is put into operation (with the same flow rate of water supplied to the water coolers), the cooled water temperature is decreased by around 2-3°C in the range of atmospheric air temperatures 0-35°C. However, the introduced water distribution arrangement with a decreased spraying density has limitation on its use at negative outdoor air temperatures due to the hazard intense freezing of the fill in the cooling tower peripheral zone. The availability of standby cooling towers in the shutdown Armenian NPP power unit along with the planned full replacement of the cooling tower process equipment create good possibilities for achieving a deeper water cooling extent and better efficiency of the NPP. The present work was carried out with the aim of achieving maximally efficient use of existing possibilities and for elaborating the optimal cooling tower modernization version. Individual specific heat-andmass transfer processes in the chimney-type evaporative cooling towers are analyzed. An improved arrangement for distributing cooled water over the cooling tower spraying area (during its operation with a decreased flow rate) is proposed with the aim of cooling water to a deeper extent and preserving the possibility of using the cooling towers in winter. The main idea behind improving the existing arrangement is to exclude certain zones of the cooling tower featuring inefficient cooling from operation. The effectiveness of introducing the proposed design is proven by calculations (taking as an

  1. AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

    PubMed Central

    2014-01-01

    Background Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous

  2. A novel cationic liposome formulation for efficient gene delivery via a pulmonary route

    NASA Astrophysics Data System (ADS)

    Li, Peng; Liu, Donghua; Sun, Xiaoli; Liu, Chunxi; Liu, Yongjun; Zhang, Na

    2011-06-01

    The clinical success of gene therapy for lung cancer is not only dependent on efficient gene carriers but also on a suitable delivery route. A pulmonary delivery route can directly deliver gene vectors to the lung which is more efficient than a systemic delivery route. For gene carriers, cationic liposomes have recently emerged as leading non-viral vectors in worldwide gene therapy clinical trials. However, cytotoxic effects or apoptosis are often observed which is mostly dependent on the cationic lipid used. Therefore, an efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), previously synthesized by our group was first used to prepare cationic liposomes. Physicochemical and biological properties of LHLN-liposomes were investigated. LHLN-liposome/DNA complexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA binding capability. Compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited not only lower cytotoxicity (P < 0.05) but also similar transfection efficiency in A549 and HepG2 lung cancer cells for in vitro tests. When administered by intratracheal instillation into rat lungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higher pulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P < 0.05). These results suggested that LHLN-liposomes may have great potential for efficient pulmonary gene delivery.

  3. Synergistic effect of a biosurfactant and protamine on gene transfection efficiency.

    PubMed

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2013-04-11

    Several barriers need to be overcome to ensure successful gene transfection, including passing of the foreign gene through the plasma membrane, escape of this material from lysosomal degradation, and its translocation into the nucleus. We previously showed that the biosurfactant mannosylerythritol lipid-A (MEL-A) enhanced the efficiency of gene transfection mediated by cationic liposomes by facilitating rapid delivery of foreign genes into target cells through membrane fusion between liposomes and the plasma membrane. Moreover, using MEL-A-containing cationic liposomes, the foreign gene was efficiently delivered into the nucleus because it was released directly into the cytosol and thus escaped lysosomal degradation. Here we investigated the effect of pre-condensation of plasmid DNA by a cationic polymer, protamine, on gene transfection. We found that the efficiency of pre-condensed DNA transfection mediated by MEL-A-containing OH liposomes was >10 times higher than that of non-condensed DNA transfection. In contrast, the efficiency of pre-condensed DNA transfection mediated by OH liposomes was only 1.5 times higher than that of non-condensed DNA transfection. MEL-A did not influence plasmid DNA encapsulation by cationic liposomes, but it greatly accelerated the nuclear delivery of pre-condensed plasmid DNA. Our findings indicate that MEL-A and protamine synergistically accelerate the nuclear delivery of foreign gene and consequently promote gene transfection efficiency. PMID:23422688

  4. New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

    DOE PAGES

    Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; Kulkkarni, Gargi; Metcalf, William W.

    2008-01-01

    A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline inmore » strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

  5. New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

    PubMed Central

    Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; Kulkkarni, Gargi; Metcalf, William W.

    2008-01-01

    A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion. PMID:19054746

  6. SpeedyGenes: Exploiting an Improved Gene Synthesis Method for the Efficient Production of Synthetic Protein Libraries for Directed Evolution.

    PubMed

    Currin, Andrew; Swainston, Neil; Day, Philip J; Kell, Douglas B

    2017-01-01

    Gene synthesis is a fundamental technology underpinning much research in the life sciences. In particular, synthetic biology and biotechnology utilize gene synthesis to assemble any desired DNA sequence, which can then be incorporated into novel parts and pathways. Here, we describe SpeedyGenes, a gene synthesis method that can assemble DNA sequences with greater fidelity (fewer errors) than existing methods, but that can also be used to encode extensive, statistically designed sequence variation at any position in the sequence to create diverse (but accurate) variant libraries. We summarize the integrated use of GeneGenie to design DNA and oligonucleotide sequences, followed by the procedure for assembling these accurately and efficiently using SpeedyGenes.

  7. SpeedyGenes: Exploiting an Improved Gene Synthesis Method for the Efficient Production of Synthetic Protein Libraries for Directed Evolution.

    PubMed

    Currin, Andrew; Swainston, Neil; Day, Philip J; Kell, Douglas B

    2017-01-01

    Gene synthesis is a fundamental technology underpinning much research in the life sciences. In particular, synthetic biology and biotechnology utilize gene synthesis to assemble any desired DNA sequence, which can then be incorporated into novel parts and pathways. Here, we describe SpeedyGenes, a gene synthesis method that can assemble DNA sequences with greater fidelity (fewer errors) than existing methods, but that can also be used to encode extensive, statistically designed sequence variation at any position in the sequence to create diverse (but accurate) variant libraries. We summarize the integrated use of GeneGenie to design DNA and oligonucleotide sequences, followed by the procedure for assembling these accurately and efficiently using SpeedyGenes. PMID:27671932

  8. Babesia and its hosts: adaptation to long-lasting interactions as a way to achieve efficient transmission

    PubMed Central

    Chauvin, Alain; Moreau, Emmanuelle; Bonnet, Sarah; Plantard, Olivier; Malandrin, Laurence

    2009-01-01

    Babesia, the causal agent of babesiosis, are tick-borne apicomplexan protozoa. True babesiae (Babesia genus sensu stricto) are biologically characterized by direct development in erythrocytes and by transovarial transmission in the tick. A large number of true Babesia species have been described in various vertebrate and tick hosts. This review presents the genus then discusses specific adaptations of Babesia spp. to their hosts to achieve efficient transmission. The main adaptations lead to long-lasting interactions which result in the induction of two reservoirs: in the vertebrate host during low long-term parasitemia and throughout the life cycle of the tick host as a result of transovarial and transstadial transmission. The molecular bases of these adaptations in vertebrate hosts are partially known but few of the tick-host interaction mechanisms have been elucidated. PMID:19379662

  9. From here to efficiency : time lags between the introduction of new technology and the achievement of fuel savings.

    SciTech Connect

    Mintz, M.; Vyas, A.; Wang, M.; Stodolsky, F.; Cuenca, R.; Gaines, L.

    1999-12-03

    In this paper, the energy savings of new technology offering significant improvements in fuel efficiency are tracked for over 20 years as vehicles incorporating that technology enter the fleet and replace conventional light-duty vehicles. Two separate analyses are discussed: a life-cycle analysis of aluminum-intensive vehicles and a fuel-cycle analysis of the energy and greenhouse gas emissions of double vs. triple fuel-economy vehicles. In both efforts, market-penetration modeling is used to simulate the rate at which new technology enters the new fleet, and stock-adjustment modeling is used to capture the inertia in turnover of new and existing current-technology vehicles. Together, these two effects--slowed market penetration and delayed vehicle replacement--increase the time lag between market introduction and the achievement of substantial energy savings. In both cases, 15-20 years elapse, before savings approach these levels.

  10. The potential of gene therapy approaches for the treatment of hemoglobinopathies: achievements and challenges

    PubMed Central

    Goodman, Michael A.; Malik, Punam

    2016-01-01

    Hemoglobinopathies, including β-thalassemia and sickle cell disease (SCD), are a heterogeneous group of commonly inherited disorders affecting the function or levels of hemoglobin. Disease phenotype can be severe with substantial morbidity and mortality. Bone marrow transplantation is curative, but limited to those patients with an appropriately matched donor. Genetic therapy, which utilizes a patient’s own cells, is thus an attractive therapeutic option. Numerous therapies are currently in clinical trials or in development, including therapies utilizing gene replacement therapy using lentiviruses and the latest gene editing techniques. In addition, methods are being developed that may be able to expand gene therapies to those with poor access to medical care, potentially significantly decreasing the global burden of disease. PMID:27695619

  11. Folate-associated lipoplexes mediate efficient gene delivery and potent antitumoral activity in vitro and in vivo.

    PubMed

    Duarte, Sónia; Faneca, Henrique; Lima, Maria C Pedroso de

    2012-02-28

    The lack of suitable vectors for efficient nucleic acid delivery into target cells represents a major hurdle for the successful application of gene therapy. Cationic liposomes exhibit attractive features for gene delivery, but their efficacy is still unsatisfactory, particularly for in vivo applications, which justifies the drive to further improve their performance by developing novel and efficient formulations. In the present study, we generated a new formulation of lipoplexes through electrostatic association of folate (FA) to 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC):cholesterol (Chol) liposomes, prepared at different lipid/DNA charge ratios, and explored their potential to mediate gene delivery. The optimal FA-lipoplex formulation was evaluated for its efficacy to mediate antitumoral activity upon application of HSV-tk suicide gene therapy, both in vitro and in an animal model of oral cancer. Our results demonstrate that FA-EPOPC:Chol/DNA lipoplexes were able to promote a great enhancement of transfection and high in vitro antitumoral activity compared to plain lipoplexes in two different cancer cell lines. Most importantly, a considerable reduction of tumor growth was achieved with the developed FA-lipoplexes as compared to that observed for control FA-lipoplexes or plain lipoplexes. Overall, our study shows that FA-EPOPC:Chol/DNA lipoplexes constitute a promising system for the successful application of suicide gene therapy aiming at treating solid tumors. PMID:22209825

  12. Biocleavable Polycationic Micelles as Highly Efficient Gene Delivery Vectors

    NASA Astrophysics Data System (ADS)

    Zhang, Min; Xue, Ya-Nan; Liu, Min; Zhuo, Ren-Xi; Huang, Shi-Wen

    2010-11-01

    An amphiphilic disulfide-containing polyamidoamine was synthesized by Michael-type polyaddition reaction of piperazine to equimolar N, N'-bis(acryloyl)cystamine with 90% yield. The polycationic micelles (198 nm, 32.5 mV), prepared from the amphiphilic polyamidoamine by dialysis method, can condense foreign plasmid DNA to form nanosized polycationic micelles/DNA polyelectrolyte complexes with positive charges, which transfected 293T cells with high efficiency. Under optimized conditions, the transfection efficiencies of polycationic micelles/DNA complexes are comparable to, or even higher than that of commercially available branched PEI (Mw 25 kDa).

  13. Discovering gene re-ranking efficiency and conserved gene-gene relationships derived from gene co-expression network analysis on breast cancer data.

    PubMed

    Bourdakou, Marilena M; Athanasiadis, Emmanouil I; Spyrou, George M

    2016-01-01

    Systemic approaches are essential in the discovery of disease-specific genes, offering a different perspective and new tools on the analysis of several types of molecular relationships, such as gene co-expression or protein-protein interactions. However, due to lack of experimental information, this analysis is not fully applicable. The aim of this study is to reveal the multi-potent contribution of statistical network inference methods in highlighting significant genes and interactions. We have investigated the ability of statistical co-expression networks to highlight and prioritize genes for breast cancer subtypes and stages in terms of: (i) classification efficiency, (ii) gene network pattern conservation, (iii) indication of involved molecular mechanisms and (iv) systems level momentum to drug repurposing pipelines. We have found that statistical network inference methods are advantageous in gene prioritization, are capable to contribute to meaningful network signature discovery, give insights regarding the disease-related mechanisms and boost drug discovery pipelines from a systems point of view. PMID:26892392

  14. Discovering gene re-ranking efficiency and conserved gene-gene relationships derived from gene co-expression network analysis on breast cancer data

    PubMed Central

    Bourdakou, Marilena M.; Athanasiadis, Emmanouil I.; Spyrou, George M.

    2016-01-01

    Systemic approaches are essential in the discovery of disease-specific genes, offering a different perspective and new tools on the analysis of several types of molecular relationships, such as gene co-expression or protein-protein interactions. However, due to lack of experimental information, this analysis is not fully applicable. The aim of this study is to reveal the multi-potent contribution of statistical network inference methods in highlighting significant genes and interactions. We have investigated the ability of statistical co-expression networks to highlight and prioritize genes for breast cancer subtypes and stages in terms of: (i) classification efficiency, (ii) gene network pattern conservation, (iii) indication of involved molecular mechanisms and (iv) systems level momentum to drug repurposing pipelines. We have found that statistical network inference methods are advantageous in gene prioritization, are capable to contribute to meaningful network signature discovery, give insights regarding the disease-related mechanisms and boost drug discovery pipelines from a systems point of view. PMID:26892392

  15. Lipid Encapsulation Provides Insufficient Total-Tract Digestibility to Achieve an Optimal Transfer Efficiency of Fatty Acids to Milk Fat

    PubMed Central

    Bainbridge, Melissa; Kraft, Jana

    2016-01-01

    Transfer efficiencies of rumen-protected n-3 fatty acids (FA) to milk are low, thus we hypothesized that rumen-protection technologies allow for biohydrogenation and excretion of n-3 FA. The objectives of this study were to i) investigate the ruminal protection and post-ruminal release of the FA derived from the lipid-encapsulated echium oil (EEO), and ii) assess the bioavailability and metabolism of the EEO-derived FA through measuring the FA content in plasma lipid fractions, feces, and milk. The EEO was tested for rumen stability using the in situ nylon bag technique, then the apparent total-tract digestibility was assessed in vivo using six Holstein dairy cattle. Diets consisted of a control (no EEO); 1.5% of dry matter (DM) as EEO and 1.5% DM as encapsulation matrix; and 3% DM as EEO. The EEO was rumen-stable and had no effect on animal production. EEO-derived FA were incorporated into all plasma lipid fractions, with the highest proportion of n-3 FA observed in cholesterol esters. Fecal excretion of EEO-derived FA ranged from 7–14%. Biohydrogenation products increased in milk, plasma, and feces with EEO supplementation. In conclusion, lipid-encapsulation provides inadequate digestibility to achieve an optimal transfer efficiency of n-3 FA to milk. PMID:27741299

  16. Links between Transcription, Environmental Adaptation and Gene Variability in Escherichia coli: Correlations between Gene Expression and Gene Variability Reflect Growth Efficiencies.

    PubMed

    Feugeas, Jean-Paul; Tourret, Jerome; Launay, Adrien; Bouvet, Odile; Hoede, Claire; Denamur, Erick; Tenaillon, Olivier

    2016-10-01

    Gene expression is known to be the principle factor explaining how fast genes evolve. Highly transcribed genes evolve slowly because any negative impact caused by a particular mutation is magnified by protein abundance. However, gene expression is a phenotype that depends both on the environment and on the strains or species. We studied this phenotypic plasticity by analyzing the transcriptome profiles of four Escherichia coli strains grown in three different culture media, and explored how expression variability was linked to gene allelic diversity. Genes whose expression changed according to the media and not to the strains were less polymorphic than other genes. Genes for which transcription depended predominantly on the strain were more polymorphic than other genes and were involved in sensing and responding to environmental changes, with an overrepresentation of two-component system genes. Surprisingly, we found that the correlation between transcription and gene diversity was highly variable among growth conditions and could be used to quantify growth efficiency of a strain in a medium. Genetic variability was found to increase with gene expression in poor growth conditions. As such conditions are also characterized by down-regulation of all DNA repair systems, including transcription-coupled repair, we suggest that gene expression under stressful conditions may be mutagenic and thus leads to a variability in mutation rate among genes in the genome which contributes to the pattern of protein evolution.

  17. Application of an Efficient Gene Targeting System Linking Secondary Metabolites to their Biosynthetic Genes in Aspergillus terreus

    SciTech Connect

    Guo, Chun-Jun; Knox, Benjamin P.; Sanchez, James F.; Chiang, Yi-Ming; Bruno, Kenneth S.; Wang, Clay C.

    2013-07-19

    Nonribosomal peptides (NRPs) are natural products biosynthesized by NRP synthetases. A kusA-, pyrG- mutant strain of Aspergillusterreus NIH 2624 was developed that greatly facilitated the gene targeting efficiency in this organism. Application of this tool allowed us to link four major types of NRP related secondary metabolites to their responsible genes in A. terreus. In addition, an NRP related melanin synthetase was also identified in this species.

  18. CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.

    PubMed

    Mandegar, Mohammad A; Huebsch, Nathaniel; Frolov, Ekaterina B; Shin, Edward; Truong, Annie; Olvera, Michael P; Chan, Amanda H; Miyaoka, Yuichiro; Holmes, Kristin; Spencer, C Ian; Judge, Luke M; Gordon, David E; Eskildsen, Tilde V; Villalta, Jacqueline E; Horlbeck, Max A; Gilbert, Luke A; Krogan, Nevan J; Sheikh, Søren P; Weissman, Jonathan S; Qi, Lei S; So, Po-Lin; Conklin, Bruce R

    2016-04-01

    Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease. PMID:26971820

  19. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase

    PubMed Central

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-01

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1–2.5 µg) conferring optimal levels of long-term expression (>1011 photons/second/cm2). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>1010 photons/second/cm2) was achieved at a transposon dose of 5–125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver. PMID:26784638

  20. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase.

    PubMed

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-19

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1-2.5 µg) conferring optimal levels of long-term expression (>10(11) photons/second/cm(2)). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>10(10) photons/second/cm(2)) was achieved at a transposon dose of 5-125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver.

  1. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase.

    PubMed

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-01

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1-2.5 µg) conferring optimal levels of long-term expression (>10(11) photons/second/cm(2)). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>10(10) photons/second/cm(2)) was achieved at a transposon dose of 5-125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver. PMID:26784638

  2. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

    PubMed

    Michielse, C B; Arentshorst, M; Ram, A F J; van den Hondel, C A M J J

    2005-01-01

    In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.

  3. Differential expression of genes in the jejunum of steers with feed efficiency phenotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The small intestine is an important site of digestion and absorption of nutrients in cattle, and has the potential to significantly impact feed efficiency. We hypothesized that the differences in feed efficiency phenotypes of beef cattle can be partially explained by the differences in gene expressi...

  4. Hands-On, Demonstration, and Videotape Laboratories for Non-Science Majors in a Food Science Course: Achievement, Attitude, and Efficiency

    ERIC Educational Resources Information Center

    Johnson, H. L.; Trout, B. L.; Brekke, C. J.; Luedecke, L. O.

    2004-01-01

    Student achievement, attitude, and instructional efficiency were determined for hands-on and for live and videotape demonstration laboratories for nonscience majors. Each of 3 laboratory sections experienced 3 different teaching methods for one 4-wk unit. No significant difference in achievement was found among the laboratory methods. An attitude…

  5. A 12%-efficient upgraded metallurgical grade silicon-organic heterojunction solar cell achieved by a self-purifying process.

    PubMed

    Zhang, Jie; Song, Tao; Shen, Xinlei; Yu, Xuegong; Lee, Shuit-Tong; Sun, Baoquan

    2014-11-25

    Low-quality silicon such as upgraded metallurgical-grade (UMG) silicon promises to reduce the material requirements for high-performance cost-effective photovoltaics. So far, however, UMG silicon currently exhibits the short diffusion length and serious charge recombination associated with high impurity levels, which hinders the performance of solar cells. Here, we used a metal-assisted chemical etching (MACE) method to partially upgrade the UMG silicon surface. The silicon was etched into a nanostructured one by the MACE process, associated with removing impurities on the surface. Meanwhile, nanostructured forms of UMG silicon can benefit improved light harvesting with thin substrates, which can relax the requirement of material purity for high photovoltaic performance. In order to suppress the large surface recombination due to increased surface area of nanostructured UMG silicon, a post chemical treatment was used to decrease the surface area. A solution-processed conjugated polymer of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) was deposited on UMG silicon at low temperature (<150 °C) to form a heterojunction to avoid any impurity diffusion in the silicon substrate. By optimizing the thickness of silicon and suppressing the charge recombination at the interface between thin UMG silicon/PEDOT:PSS, we are able to achieve 12.0%-efficient organic-inorganic hybrid solar cells, which are higher than analogous UMG silicon devices. We show that the modified UMG silicon surface can increase the minority carrier lifetime because of reduced impurity and surface area. Our results suggest a design rule for an efficient silicon solar cell with low-quality silicon absorbers.

  6. Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs

    PubMed Central

    Cade, Lindsay; Reyon, Deepak; Hwang, Woong Y.; Tsai, Shengdar Q.; Patel, Samir; Khayter, Cyd; Joung, J. Keith; Sander, Jeffry D.; Peterson, Randall T.; Yeh, Jing-Ruey Joanna

    2012-01-01

    Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish. PMID:22684503

  7. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

    PubMed Central

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  8. Efficient Consistency Achievement of Federated Identity and Access Management Based on a Novel Self-Adaptable Approach

    NASA Astrophysics Data System (ADS)

    Cha, Shi-Cho; Chang, Hsiang-Meng

    Federated identity and access management (FIAM) systems enable a user to access services provided by various organizations seamlessly. In FIAM systems, service providers normally stipulate that their users show assertions issued by allied parties to use their services as well as determine user privileges based on attributes in the assertions. However, the integrity of the attributes is important under certain circumstances. In such a circumstance, all released assertions should reflect modifications made to user attributes. Despite the ability to adopt conventional certification revocation technologies, including CRL or OCSP, to revoke an assertion and request the corresponding user to obtain a new assertion, re-issuing an entirely new assertion if only one attribute, such as user location or other environmental information, is changed would be inefficient. Therefore, this work presents a self-adaptive framework to achieve consistency in federated identity and access management systems (SAFIAM). In SAFIAM, an identity provider (IdP), which authenticates users and provides user attributes, should monitor access probabilities according to user attributes. The IdP can then adopt the most efficient means of ensuring data integrity of attributes based on related access probabilities. While Internet-based services emerge daily that have various access probabilities with respect to their user attributes, the proposed self-adaptive framework significantly contributes to efforts to streamline the use of FIAM systems.

  9. Novel benzimidazole derivatives as electron-transporting type host to achieve highly efficient sky-blue phosphorescent organic light-emitting diode (PHOLED) device.

    PubMed

    Huang, Jau-Jiun; Leung, Man-Kit; Chiu, Tien-Lung; Chuang, Ya-Ting; Chou, Pi-Tai; Hung, Yu-Hsiang

    2014-10-17

    The development of benzimidazole substituted biphenyls as electron-transporting hosts for bis[2-(4,6-difluorophenyl)pyridinato-C(2),N](picolinato)iridium(III) is reported. Under the optimized conditions, the organic light-emitting diode (OLED) achieves the maximum current efficiency of 57.2 cd/A, power efficiency of 50.4 lm/W, and external quantum efficiency 25.7%. PMID:25296531

  10. A pH-sensitive multifunctional gene carrier assembled via layer-by-layer technique for efficient gene delivery

    PubMed Central

    Li, Peng; Liu, Donghua; Miao, Lei; Liu, Chunxi; Sun, Xiaoli; Liu, Yongjun; Zhang, Na

    2012-01-01

    Background The success of gene therapy asks for the development of multifunctional vectors that could overcome various gene delivery barriers, such as the cell membrane, endosomal membrane, and nuclear membrane. Layer-by-layer technique is an efficient method with easy operation which can be used for the assembly of multifunctional gene carriers. This work describes a pH-sensitive multifunctional gene vector that offered long circulation property but avoided the inhibition of tumor cellular uptake of gene carriers associated with the use of polyethylene glycol. Methods Deoxyribonucleic acid (DNA) was firstly condensed with protamine into a cationic core which was used as assembly template. Then, additional layers of anionic DNA, cationic liposomes, and o-carboxymethyl-chitosan (CMCS) were alternately adsorbed onto the template via layer-by-layer technique and finally the multifunctional vector called CMCS-cationic liposome-coated DNA/protamine/DNA complexes (CLDPD) was constructed. For in vitro test, the cytotoxicity and transfection investigation was carried out on HepG2 cell line. For in vivo evaluation, CMCS-CLDPD was intratumorally injected into tumor-bearing mice and the tumor cells were isolated for fluorescence determination of transfection efficiency. Results CMCS-CLDPD had ellipsoidal shapes and showed “core-shell” structure which showed stabilization property in serum and effective protection of DNA from nuclease degradation. In vitro and in vivo transfection results demonstrated that CMCS-CLDPD had pH-sensitivity and the outermost layer of CMCS fell off in the tumor tissue, which could not only protect CMCS- CLDPD from serum interaction but also enhance gene transfection efficiency. Conclusion These results demonstrated that multifunctional CMCS-CLDPD had pH- sensitivity, which may provide a new approach for the antitumor gene delivery. PMID:22393290

  11. An efficient method for mining cross-timepoint gene regulation sequential patterns from time course gene expression datasets

    PubMed Central

    2013-01-01

    Background Observation of gene expression changes implying gene regulations using a repetitive experiment in time course has become more and more important. However, there is no effective method which can handle such kind of data. For instance, in a clinical/biological progression like inflammatory response or cancer formation, a great number of differentially expressed genes at different time points could be identified through a large-scale microarray approach. For each repetitive experiment with different samples, converting the microarray datasets into transactional databases with significant singleton genes at each time point would allow sequential patterns implying gene regulations to be identified. Although traditional sequential pattern mining methods have been successfully proposed and widely used in different interesting topics, like mining customer purchasing sequences from a transactional database, to our knowledge, the methods are not suitable for such biological dataset because every transaction in the converted database may contain too many items/genes. Results In this paper, we propose a new algorithm called CTGR-Span (Cross-Timepoint Gene Regulation Sequential pattern) to efficiently mine CTGR-SPs (Cross-Timepoint Gene Regulation Sequential Patterns) even on larger datasets where traditional algorithms are infeasible. The CTGR-Span includes several biologically designed parameters based on the characteristics of gene regulation. We perform an optimal parameter tuning process using a GO enrichment analysis to yield CTGR-SPs more meaningful biologically. The proposed method was evaluated with two publicly available human time course microarray datasets and it was shown that it outperformed the traditional methods in terms of execution efficiency. After evaluating with previous literature, the resulting patterns also strongly correlated with the experimental backgrounds of the datasets used in this study. Conclusions We propose an efficient CTGR

  12. Cell wall, lignin and fatty acid-related transcriptome in soybean: Achieving gene expression patterns for bioenergy legume

    PubMed Central

    Pestana-Calsa, Maria Clara; Pacheco, Cinthya Mirella; de Castro, Renata Cruz; de Almeida, Renata Rodrigues; de Lira, Nayara Patrícia Vieira; Junior, Tercilio Calsa

    2012-01-01

    Increasing efforts to preserve environmental resources have included the development of more efficient technologies to produce energy from renewable sources such as plant biomass, notably through biofuels and cellulosic residues. The relevance of the soybean industry is due mostly to oil and protein production which, although interdependent, results from coordinated gene expression in primary metabolism. Concerning biomass and biodiesel, a comprehensive analysis of gene regulation associated with cell wall components (as polysaccharides and lignin) and fatty acid metabolism may be very useful for finding new strategies in soybean breeding for the expanding bioenergy industry. Searching the Genosoja transcriptional database for enzymes and proteins directly involved in cell wall, lignin and fatty acid metabolism provides gene expression datasets with frequency distribution and specific regulation that is shared among several cultivars and organs, and also in response to different biotic/abiotic stress treatments. These results may be useful as a starting point to depict the Genosoja database regarding gene expression directly associated with potential applications of soybean biomass and/or residues for bioenergy-producing technologies. PMID:22802717

  13. Functional and biodegradable dendritic macromolecules with controlled architectures as nontoxic and efficient nanoscale gene vectors.

    PubMed

    Luo, Kui; He, Bin; Wu, Yao; Shen, Youqing; Gu, Zhongwei

    2014-01-01

    Gene therapy has provided great potential to revolutionize the treatment of many diseases. This therapy is strongly relied on whether a delivery vector efficiently and safely directs the therapeutic genes into the target tissue/cells. Nonviral gene delivery vectors have been emerging as a realistic alternative to the use of viral analogs with the potential of a clinically relevant output. Dendritic polymers were employed as nonviral vectors due to their branched and layered architectures, globular shape and multivalent groups on their surface, showing promise in gene delivery. In the present review, we try to bring out the recent trend of studies on functional and biodegradable dendritic polymers as nontoxic and efficient gene delivery vectors. By regulating dendritic polymer design and preparation, together with recent progress in the design of biodegradable polymers, it is possible to precisely manipulate their architectures, molecular weight and chemical composition, resulting in predictable tuning of their biocompatibility as well as gene transfection activities. The multifunctional and biodegradable dendritic polymers possessing the desirable characteristics are expected to overcome extra- and intracellular obstacles, and as efficient and nontoxic gene delivery vectors to move into the clinical arena.

  14. Stable and Efficient Gene Transfer into the Retina Using an HIV-Based Lentiviral Vector

    NASA Astrophysics Data System (ADS)

    Miyoshi, Hiroyuki; Takahashi, Masayo; Gage, Fred H.; Verma, Inder M.

    1997-09-01

    The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

  15. Advances in the Development of Gene-Targeting Vectors to Increase the Efficiency of Genetic Modification.

    PubMed

    Saito, Shinta; Adachi, Noritaka

    2016-01-01

    Gene targeting via homologous recombination, albeit highly inefficient in human cells, is considered a powerful tool for analyzing gene functions. Despite recent progress in the application of artificial nucleases for genome editing, safety issues remain a concern, particularly when genetic modification is used for therapeutic purposes. Therefore, the development of gene-targeting vectors is necessary for safe and sophisticated genetic modification. In this paper, we describe the effect of vector structure on random integration, which is a major obstacle in efficient gene targeting. In addition, we focus on the features of exon-trapping-type gene-targeting vectors, and discuss a novel strategy for negative selection to enhance gene targeting in human cells.

  16. Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes

    PubMed Central

    Yu, Yan-Hua; Lu, Ye; He, Yong-Qiang; Huang, Sheng; Tang, Ji-Liang

    2015-01-01

    Xanthomonas TALE transcriptional activators act as virulence or avirulence factors by activating host disease susceptibility or resistance genes. Their specificity is determined by a tandem repeat domain. Some Xanthomonas pathogens contain 10–30 TALEs per strain. Although TALEs play critical roles in pathogenesis, their studies have so far been limited to a few examples, due to their highly repetitive gene structure and extreme similarity among different members, which constrict sequencing and assembling. To facilitate TALE studies, we developed an efficient and rapid pipeline for genome-wide cloning of tal genes as many as possible from a strain. Here, we report the pipeline and its use to identify all 18 tal genes from a newly isolated strain of the rice pathogen Xathomonas oryzae. Target prediction revealed a number of potential rice targets including several notable genes such as genes encoding SWEET, WRKY, Hen1, and BAK1 proteins, which provide candidates for further experimental functional analysis of the TALEs. PMID:26271455

  17. Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes.

    PubMed

    Yu, Yan-Hua; Lu, Ye; He, Yong-Qiang; Huang, Sheng; Tang, Ji-Liang

    2015-01-01

    Xanthomonas TALE transcriptional activators act as virulence or avirulence factors by activating host disease susceptibility or resistance genes. Their specificity is determined by a tandem repeat domain. Some Xanthomonas pathogens contain 10-30 TALEs per strain. Although TALEs play critical roles in pathogenesis, their studies have so far been limited to a few examples, due to their highly repetitive gene structure and extreme similarity among different members, which constrict sequencing and assembling. To facilitate TALE studies, we developed an efficient and rapid pipeline for genome-wide cloning of tal genes as many as possible from a strain. Here, we report the pipeline and its use to identify all 18 tal genes from a newly isolated strain of the rice pathogen Xathomonas oryzae. Target prediction revealed a number of potential rice targets including several notable genes such as genes encoding SWEET, WRKY, Hen1, and BAK1 proteins, which provide candidates for further experimental functional analysis of the TALEs. PMID:26271455

  18. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

    PubMed

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2014-06-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

  19. Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

    PubMed Central

    Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E.; Ha, Un-Hwan; Wu, Donghai

    2015-01-01

    The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies. Significance The present study describes a novel and powerful tool for the delivery of the genome editing enzyme transcription activator-like effector nuclease (TALEN) directly into pluripotent stem cells (PSCs), achieving desired base changes on the genomes of PSCs with high efficiency. This novel approach uses bacteria as a protein delivery

  20. An efficient platform for genetic selection and screening of gene switches in Escherichia coli.

    PubMed

    Muranaka, Norihito; Sharma, Vandana; Nomura, Yoko; Yokobayashi, Yohei

    2009-04-01

    Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay. PMID:19190095

  1. Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Xue, Chunsheng; Wu, Dongliang; Condon, Bradford J; Bi, Qing; Wang, Weiwei; Turgeon, B Gillian

    2013-06-01

    Setosphaeria turcica, a hemibiotrophic pathogenic dothideomycete, is the causal agent of Northern Leaf Blight of maize, which periodically causes significant yield losses worldwide. To explore molecular mechanisms of fungal pathogenicity and virulence to the host, an efficient targeted gene knockout transformation system using Agrobacterium tumefaciens was established with field collected strains. The starting materials, incubation time, induction medium type, Agrobacterium cell density, and method of co-incubation were optimized for deletion of 1,3,8-trihydroxynaphthalene reductase, a gene in the melanin biosynthesis pathway, as a test case. Four additional genes were deleted in two different S. turcica field isolates to confirm robustness of the method. One of these mutant strains was reduced in virulence compared with the wild-type strain when inoculated on susceptible maize. Transformation efficiency was ≈20 ± 3 transformants per 1× 10(6) germlings and homologous recombination efficiency was 33.3 to 100%. PMID:23384859

  2. Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

    PubMed Central

    Kamau, Sarah W.; Hassa, Paul O.; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hofmann-Amtenbrink, Margarethe; von Rechenberg, Brigitte; Hottiger, Michael O.

    2006-01-01

    New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs. PMID:16540591

  3. Identification of Candidate Genes Underlying an Iron Efficiency Quantitative Trait Locus in Soybean1

    PubMed Central

    Peiffer, Gregory A.; King, Keith E.; Severin, Andrew J.; May, Gregory D.; Cianzio, Silvia R.; Lin, Shun Fu; Lauter, Nicholas C.; Shoemaker, Randy C.

    2012-01-01

    Prevalent on calcareous soils in the United States and abroad, iron deficiency is among the most common and severe nutritional stresses in plants. In soybean (Glycine max) commercial plantings, the identification and use of iron-efficient genotypes has proven to be the best form of managing this soil-related plant stress. Previous studies conducted in soybean identified a significant iron efficiency quantitative trait locus (QTL) explaining more than 70% of the phenotypic variation for the trait. In this research, we identified candidate genes underlying this QTL through molecular breeding, mapping, and transcriptome sequencing. Introgression mapping was performed using two related near-isogenic lines in which a region located on soybean chromosome 3 required for iron efficiency was identified. The region corresponds to the previously reported iron efficiency QTL. The location was further confirmed through QTL mapping conducted in this study. Transcriptome sequencing and quantitative real-time-polymerase chain reaction identified two genes encoding transcription factors within the region that were significantly induced in soybean roots under iron stress. The two induced transcription factors were identified as homologs of the subgroup lb basic helix-loop-helix (bHLH) genes that are known to regulate the strategy I response in Arabidopsis (Arabidopsis thaliana). Resequencing of these differentially expressed genes unveiled a significant deletion within a predicted dimerization domain. We hypothesize that this deletion disrupts the Fe-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)/bHLH heterodimer that has been shown to induce known iron acquisition genes. PMID:22319075

  4. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system.

    PubMed

    Song, Yuning; Yuan, Lin; Wang, Yong; Chen, Mao; Deng, Jichao; Lv, Qingyan; Sui, Tingting; Li, Zhanjun; Lai, Liangxue

    2016-08-01

    The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been extensively used to edit the genome of several organisms. However, most mutations reported to date have been are indels, resulting in multiple mutations and numerous alleles in targeted genes. In the present study, a large deletion of 105 kb in the TYR (tyrosinase) gene was generated in rabbit via a dual sgRNA-directed CRISPR/Cas9 system. The typical symptoms of albinism accompanied significantly decreased expression of TYR in the TYR knockout rabbits. Furthermore, the same genotype and albinism phenotype were found in the F1 generation, suggesting that large-fragment deletions can be efficiently transmitted to the germline and stably inherited in offspring. Taken together, our data demonstrate that mono and biallelic large deletions can be achieved using the dual sgRNA-directed CRISPR/Cas9 system. This system produces no mosaic mutations or off-target effects, making it an efficient tool for large-fragment deletions in rabbit and other organisms. PMID:26817461

  5. Histone H2A-mediated transient cytokine gene delivery induces efficient antitumor responses in murine neuroblastoma.

    PubMed

    Balicki, D; Reisfeld, R A; Pertl, U; Beutler, E; Lode, H N

    2000-10-10

    A major goal of cancer immunotherapy is the induction of a cell-mediated antitumor response in poorly immunogenic malignancies. We tested the hypothesis that this can be achieved by cytokine gene therapy with a novel histone H2A-based transient transfection procedure. This was tested by using cytokine genes encoding for IL-2 and a single chain IL-12 (scIL-12) fusion protein in a recently developed murine neuroblastoma model. Here, we demonstrate that cytokine gene transfer of IL-2 and scIL-12 with histone H2A results in the induction of an antitumor immune response that is superior in some respects to gene transfer with Superfect, a commercially available activated dendrimer commonly used to effect transfection with plasmids. Three lines of evidence support this contention. First, histone H2A-mediated transfection of IL-2 induces a natural killer cell-induced rejection of primary tumors in contrast to Superfect, which produces only a partial reduction in primary tumor growth. Second, the induction of a T cell-mediated protective tumor immunity following gene transfer of scIL-12 is more efficient with the histone H2A-mediated gene transfer because rejection of a lethal wild-type tumor cell challenge is accompanied by the greatest degree of MHC class I-restricted tumor cell killing in vitro. Third, histone H2A-mediated scIL-12 gene therapy induces the greatest release of mIFN-gamma from splenocytes of vaccinated animals in contrast to Superfect and other controls.

  6. Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

    PubMed

    Shigeta, Mitsuki; Sakane, Yuto; Iida, Midori; Suzuki, Miyuki; Kashiwagi, Keiko; Kashiwagi, Akihiko; Fujii, Satoshi; Yamamoto, Takashi; Suzuki, Ken-Ichi T

    2016-07-01

    Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders.

  7. Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9.

    PubMed

    Zhang, Xiya; Liang, Puping; Ding, Chenhui; Zhang, Zhen; Zhou, Jianwen; Xie, Xiaowei; Huang, Rui; Sun, Ying; Sun, Hongwei; Zhang, Jinran; Xu, Yanwen; Songyang, Zhou; Huang, Junjiu

    2016-01-01

    The CRISPR/Cas system is an efficient genome-editing tool to modify genes in mouse zygotes. However, only the Streptococcus pyogenes Cas9 (SpCas9) has been systematically tested for generating gene-modified mice. The protospacer adjacent motif (PAM, 5'-NGG-3') recognized by SpCas9 limits the number of potential target sites for this system. Staphylococcus aureus Cas9 (SaCas9), with its smaller size and unique PAM (5'-NNGRRT-3') preferences, presents an alternative for genome editing in zygotes. Here, we showed that SaCas9 could efficiently and specifically edit the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes. SaCas9-mediated disruption of the tyrosinase (Tyr) gene led to C57BL/6J mice with mosaic coat color. Furthermore, multiplex targeting proved efficient multiple genes disruption when we co-injected gRNAs targeting Slx2, Zp1, and Tyr together with SaCas9 mRNA. We were also able to insert a Flag tag at the C-terminus of histone H1c, when a Flag-encoding single-stranded DNA oligo was co-introduced into mouse zygotes with SaCas9 mRNA and the gRNA. These results indicate that SaCas9 can specifically cleave the target gene locus, leading to successful gene knock-out and precise knock-in in mouse zygotes, and highlight the potential of using SaCas9 for genome editing in preimplantation embryos and producing gene-modified animal models. PMID:27586692

  8. Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9

    PubMed Central

    Zhang, Xiya; Liang, Puping; Ding, Chenhui; Zhang, Zhen; Zhou, Jianwen; Xie, Xiaowei; Huang, Rui; Sun, Ying; Sun, Hongwei; Zhang, Jinran; Xu, Yanwen; Songyang, Zhou; Huang, Junjiu

    2016-01-01

    The CRISPR/Cas system is an efficient genome-editing tool to modify genes in mouse zygotes. However, only the Streptococcus pyogenes Cas9 (SpCas9) has been systematically tested for generating gene-modified mice. The protospacer adjacent motif (PAM, 5′-NGG-3′) recognized by SpCas9 limits the number of potential target sites for this system. Staphylococcus aureus Cas9 (SaCas9), with its smaller size and unique PAM (5′-NNGRRT-3′) preferences, presents an alternative for genome editing in zygotes. Here, we showed that SaCas9 could efficiently and specifically edit the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes. SaCas9-mediated disruption of the tyrosinase (Tyr) gene led to C57BL/6J mice with mosaic coat color. Furthermore, multiplex targeting proved efficient multiple genes disruption when we co-injected gRNAs targeting Slx2, Zp1, and Tyr together with SaCas9 mRNA. We were also able to insert a Flag tag at the C-terminus of histone H1c, when a Flag-encoding single-stranded DNA oligo was co-introduced into mouse zygotes with SaCas9 mRNA and the gRNA. These results indicate that SaCas9 can specifically cleave the target gene locus, leading to successful gene knock-out and precise knock-in in mouse zygotes, and highlight the potential of using SaCas9 for genome editing in preimplantation embryos and producing gene-modified animal models. PMID:27586692

  9. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  10. Efficient PRNP gene targeting in bovine fibroblasts by adeno-associated virus vectors.

    PubMed

    Hirata, Roli K; Xu, Cong; Dong, Rong; Miller, Daniel G; Ferguson, Stacy; Russell, David W

    2004-01-01

    Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathy.

  11. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation.

    PubMed

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  12. Using interlayer step-wise triplet transfer to achieve an efficient white organic light-emitting diode with high color-stability

    SciTech Connect

    Wang, Qi; Ma, Dongge Ding, Junqiao; Wang, Lixiang; Leo, Karl; Qiao, Qiquan; Jia, Huiping; Gnade, Bruce E.

    2014-05-12

    An efficient phosphorescent white organic light emitting-diode with a red-green-blue tri-emitting-layer structure is reported. The host of the red dopant possesses a lower triplet-energy than the green dye. An interlayer step-wise triplet transfer via blue dye → green dye → red host → red dye is achieved. This mechanism allows an efficient triplet harvesting by the three dopants, thus maintaining a balanced white light and reducing energy loss. Moreover, the color stability of the device is improved significantly. The white device not only achieves a peak external quantum efficiency of 21.1 ± 0.8% and power efficiency of 37.5 ± 1.4 lm/W but shows no color shift over a wide range of voltages.

  13. Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells.

    PubMed

    Matthews, K E; Mills, G B; Horsfall, W; Hack, N; Skorecki, K; Keating, A

    1993-05-01

    We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.

  14. Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents.

    PubMed

    Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

    2015-12-01

    The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. PMID:26670247

  15. Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

    PubMed Central

    Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

    2015-01-01

    The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. PMID:26670247

  16. Efficient Gene Knockout in Goats Using CRISPR/Cas9 System

    PubMed Central

    Ni, Wei; Qiao, Jun; Hu, Shengwei; Zhao, Xinxia; Regouski, Misha; Yang, Min; Polejaeva, Irina A.; Chen, Chuangfu

    2014-01-01

    The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications. PMID:25188313

  17. Characterization of biosurfactant-containing liposomes and their efficiency for gene transfection.

    PubMed

    Ueno, Yoshinobu; Hirashima, Naohide; Inoh, Yoshikazu; Furuno, Tadahide; Nakanishi, Mamoru

    2007-01-01

    Recently we showed significance of biosurfactants in the field of non-viral vectors for gene transfection. There, a biosurfactant, mannosylerythritol lipid A (MEL-A), especially increased the efficiency of gene transfection mediated with cationic liposomes. However, the molecular mechanism has not been well-understood yet. Here, through the examination of the ability of cationic liposomes containing an MEL (MEL-A, MEL-B or MEL-C) for important transfectional processes of the DNA capsulation and the membrane fusion with anionic liposomes, we found that MEL-A-containing liposomes increased both processes, but that MEL-B and MEL-C-containing liposomes just increased either of them. The results indicated that these kinds of the physicochemical properties in MEL-A-containing liposomes are able to increase the efficiency of liposome-mediated gene transfection. PMID:17202680

  18. Achieving greenhouse gas emission reductions in developing countries through energy efficient lighting projects in the Clean Development Mechanism (CDM)

    SciTech Connect

    Figueres, C.; Bosi, M.

    2006-11-15

    Energy efficiency can help address the challenge of increasing access to modern energy services, reduce the need for capital-intensive supply investments as well as mitigating climate change. Efficient lighting is a promising sector for improving the adequacy and reliability of power systems and reducing emissions in developing countries. However, these measures are hardly represented in the CDM portfolio. The COP/MOP decision to include programs of activities in the CDM could open the door to the implementation of a large number of energy efficiency projects in developing countries. Since GHG reductions are essentially the emission equivalent of energy savings, the CDM can benefit from long established energy efficiency methodologies for quantifying energy savings and fulfilling CDM methodological requirements. The integration of the CDM into energy efficiency programs could help spur a necessary transformation in the lighting market.

  19. Avidin-biotin interaction mediated peptide assemblies as efficient gene delivery vectors for cancer therapy.

    PubMed

    Qu, Wei; Chen, Wei-Hai; Kuang, Ying; Zeng, Xuan; Cheng, Si-Xue; Zhou, Xiang; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-01-01

    Gene therapy offers a bright future for the treatment of cancers. One of the research highlights focuses on smart gene delivery vectors with good biocompatibility and tumor-targeting ability. Here, a novel gene vector self-assembled through avidin-biotin interaction with optimized targeting functionality, biotinylated tumor-targeting peptide/avidin/biotinylated cell-penetrating peptide (TAC), was designed and prepared to mediate the in vitro and in vivo delivery of p53 gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53 complexes showed higher transfection efficiency and expression amount of p53 protein in MCF-7 cells as compared with 293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7 cells. Additionally, by in situ administration of TAC/p53 complexes into tumor-bearing mice, the expression of p53 gene was obviously upregulated in tumor cells, and the tumor growth was significantly suppressed. This study provides an alternative and unique strategy to assemble functionalized peptides, and the novel self-assembled vector TAC developed is a promising gene vector for cancer therapy.

  20. A novel potential biocompatible hyperbranched polyspermine for efficient lung cancer gene therapy.

    PubMed

    Xie, Rong-Lin; Jang, Yoon-Jeong; Xing, Lei; Zhang, Bing-Feng; Wang, Feng-Zhen; Cui, Peng-Fei; Cho, Myung-Haing; Jiang, Hu-Lin

    2015-01-15

    The clinical successful application of gene therapy critically depends upon the development of non-toxic and efficient delivery system. Although polycationic non-viral vectors hold great promise in nanomedicine, the exploring of application in clinics still remains a big challenge. To develop a non-toxic and efficient non-viral gene delivery system, two kinds of endogenous substance, citric acid (CA) and spermine (SPE), were used to prepare a new low charge density hyperbranched polyspermine (HPSPE) by one-pot polymerization. The biocompatibility evaluated by hemolytic activity and red blood cell (RBC) aggregation indicated that HPSPE was highly biocompatible without causing hemolysis and RBC aggregation compared with PEI as well as SPE. The MTS assay also demonstrated that the cell viability of HPSPE was above 90% even at 200 μg/mL at different time (24 and 72 h), which much higher than PEI 25K. Besides, HPSPE showed high transfection efficiency without any toxic effect after aerosol delivery to the mice. Moreover, aerosol delivery of HPSPE/Akt1 shRNA significantly reduced tumor size and numbers and efficiently suppressed lung tumorigenesis ultimately in K-ras(LA1) lung cancer model mice. These results suggest that low charge density as well as endogenous substance skeleton endow HPSPE with great potential for toxicity-free and efficient gene therapy.

  1. A novel potential biocompatible hyperbranched polyspermine for efficient lung cancer gene therapy.

    PubMed

    Xie, Rong-Lin; Jang, Yoon-Jeong; Xing, Lei; Zhang, Bing-Feng; Wang, Feng-Zhen; Cui, Peng-Fei; Cho, Myung-Haing; Jiang, Hu-Lin

    2015-01-15

    The clinical successful application of gene therapy critically depends upon the development of non-toxic and efficient delivery system. Although polycationic non-viral vectors hold great promise in nanomedicine, the exploring of application in clinics still remains a big challenge. To develop a non-toxic and efficient non-viral gene delivery system, two kinds of endogenous substance, citric acid (CA) and spermine (SPE), were used to prepare a new low charge density hyperbranched polyspermine (HPSPE) by one-pot polymerization. The biocompatibility evaluated by hemolytic activity and red blood cell (RBC) aggregation indicated that HPSPE was highly biocompatible without causing hemolysis and RBC aggregation compared with PEI as well as SPE. The MTS assay also demonstrated that the cell viability of HPSPE was above 90% even at 200 μg/mL at different time (24 and 72 h), which much higher than PEI 25K. Besides, HPSPE showed high transfection efficiency without any toxic effect after aerosol delivery to the mice. Moreover, aerosol delivery of HPSPE/Akt1 shRNA significantly reduced tumor size and numbers and efficiently suppressed lung tumorigenesis ultimately in K-ras(LA1) lung cancer model mice. These results suggest that low charge density as well as endogenous substance skeleton endow HPSPE with great potential for toxicity-free and efficient gene therapy. PMID:25448566

  2. Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides.

    PubMed

    McKenzie, D L; Collard, W T; Rice, K G

    1999-10-01

    In a previous report (M.S. Wadhwa et al. (1997) Bioconjugate Chem. 8, 81-88), we synthesized a panel of polylysine-containing peptides and determined that a minimal repeating lysine chain of 18 residues followed by a tryptophan and an alkylated cysteine residue (AlkCWK18) resulted in the formation of optimal size (78 nm diameter) plasmid DNA condensates that mediated efficient in vitro gene transfer. Shorter polylysine chains produced larger DNA condensates and mediated much lower gene expression while longer lysine chains were equivalent to AlkCWK18. Surprisingly, AlkCWK18 (molecular weight 2672) was a much better gene transfer agent than commercially available low molecular weight polylysine (molecular weight 1000-4000), despite its similar molecular weight. Possible explanations were that the cysteine or tryptophan residue in AlkCWK18 contributed to the DNA binding and the formation of small condensates or that the homogeneity of AlkCWK18 relative to low molecular weight polylysine facilitated optimal condensation. To test these hypotheses, the present study prepared AlkCYK18 and K20 and used these to form DNA condensates and conduct in vitro gene transfer. The results established that DNA condensates prepared with either AlkCYK18 or K20 possessed identical particle size and mediated in vitro gene transfer efficiencies that were indistinguishable from AlkCWK18 DNA condensates, eliminating the possibility of contributions from cysteine or tryptophan. However, a detailed chromatographic and electrospray mass spectrometry analysis of low molecular weight polylysine revealed it to possess a much lower than anticipated average chain length of dp 6. Thus, the short chain length of low molecular weight polylysine explains its inability to form small DNA condensates and mediate efficient gene transfer relative to AlkCWK18 DNA condensates. These experiments further emphasize the need to develop homogenous low molecular weight carrier molecules for nonviral gene delivery.

  3. Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9

    PubMed Central

    Li, Jinhuan; Shou, Jia; Guo, Ya; Tang, Yuanxiao; Wu, Yonghu; Jia, Zhilian; Zhai, Yanan; Chen, Zhifeng; Xu, Quan; Wu, Qiang

    2015-01-01

    The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters. PMID:25757625

  4. Efficient synthesis and cell-transfection properties of a new multivalent cationic lipid for nonviral gene delivery.

    PubMed

    Ewert, Kai; Ahmad, Ayesha; Evans, Heather M; Schmidt, Hans-Werner; Safinya, Cyrus R

    2002-11-01

    Lipid-mediated delivery of DNA into cells holds great promise both for gene therapy and basic research applications. This paper describes the efficient and facile synthesis and the characterization of a new multivalent cationic lipid with a double-branched headgroup structure for gene delivery applications. The synthetic scheme can be extended to give cationic lipids of different charge, spacer, or lipid chain length. The chemical and physical properties of self-assembled complexes of the cationic liposomes (CLs) with DNA give indications of why multivalent cationic lipids possess superior transfection properties. The lipid bears a headgroup with five charges in the fully protonated state, which is attached to an unsaturated double-chain hydrophobic moiety based on 3,4-dihydroxybenzoic acid. Liposomes consisting of the new multivalent lipid and the neutral lipid 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) were used to prepare complexes with DNA. Investigations of the structures of these complexes by optical microscopy and small-angle X-ray scattering reveal a lamellar L(alpha)(C) phase of CL-DNA complexes with the DNA molecules sandwiched between bilayers of the lipids. Experiments using plasmid DNA containing the firefly luciferase reporter gene show that these complexes efficiently transfect mammalian cells. When compared to the monovalent cationic lipid 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP), the higher charge density of the membranes of CL-DNA complexes achievable with the new multivalent lipid greatly increases transfection efficiency in the regime of small molar ratios of cationic to neutral lipid. This is desired to minimize the known toxicity effects of cationic lipids.

  5. High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes.

    PubMed

    Remacle, Claire; Cardol, Pierre; Coosemans, Nadine; Gaisne, Mauricette; Bonnefoy, Nathalie

    2006-03-21

    Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Deltand4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I. PMID:16537419

  6. Photosynthesis and chloroplast genes are involved in water-use efficiency in common bean.

    PubMed

    Ruiz-Nieto, Jorge E; Aguirre-Mancilla, César L; Acosta-Gallegos, Jorge A; Raya-Pérez, Juan C; Piedra-Ibarra, Elías; Vázquez-Medrano, Josefina; Montero-Tavera, Victor

    2015-01-01

    A recent proposal to mitigate the effects of climatic change and reduce water consumption in agriculture is to develop cultivars with high water-use efficiency. The aims of this study were to characterize this trait as a differential response mechanism to water-limitation in two bean cultivars contrasting in their water stress tolerance, to isolate and identify gene fragments related to this response in a model cultivar, as well as to evaluate transcription levels of genes previously identified. Keeping CO2 assimilation through a high photosynthesis rate under limited conditions was the physiological response which allowed the cultivar model to maintain its growth and seed production with less water. Chloroplast genes stood out among identified genetic elements, which confirmed the importance of photosynthesis in such response. ndhK, rpoC2, rps19, rrn16, ycf1 and ycf2 genes were expressed only in response to limited water availability.

  7. Specific Destruction of HIV Proviral p17 Gene in T Lymphoid Cells Achieved by the Genome Editing Technology

    PubMed Central

    Kishida, Tsunao; Ejima, Akika; Mazda, Osam

    2016-01-01

    Recent development in genome editing technologies has enabled site-directed deprivation of a nucleotide sequence in the chromosome in mammalian cells. Human immunodeficiency (HIV) infection causes integration of proviral DNA into the chromosome, which potentially leads to re-emergence of the virus, but conventional treatment cannot delete the proviral DNA sequence from the cells infected with HIV. In the present study, the transcription activator-like effector nucleases (TALENs) specific for the HIV p17 gene were constructed, and their activities to destroy the target sequence were evaluated. SSA assay showed a high activity of a pair of p17-specific TALENs. A human T lymphoid cell line, Jurkat, was infected with a lentivirus vector followed by transfection with the TALEN–HIV by electroporation. The target sequence was destructed in approximately 10–95% of the p17 polymerase chain reaction clones, and the efficiencies depended on the Jurkat–HIV clones. Because p17 plays essential roles for assembly and budding of HIV, and this gene has relatively low nucleotide sequence diversity, genome editing procedures targeting p17 may provide a therapeutic benefit for HIV infection. PMID:27446041

  8. Hyaluronidase and collagenase increase the transfection efficiency of gene electrotransfer in various murine tumors.

    PubMed

    Cemazar, Maja; Golzio, Muriel; Sersa, Gregor; Escoffre, Jean-Michel; Coer, Andrej; Vidic, Suzana; Teissie, Justin

    2012-01-01

    One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy. PMID:21797718

  9. Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing.

    PubMed

    Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin

    2014-04-01

    RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

  10. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    PubMed Central

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium. PMID:25520733

  11. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

    PubMed

    Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

    2015-04-01

    Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

  12. Efficient Gene Delivery of Primary Human Cells Using Peptide Linked Polyethylenimine Polymer Hybrid

    PubMed Central

    Dey, Devaveena; Inayathullah, Mohammed; Lee, Andrew S; Limiuex, Melbes; Zhang, Xuexiang; Wu, Yi; Nag, Divya; De Almeida, Patricia Eliza; Han, Leng; Rajadas, Jayakumar; Wu, Joseph C

    2011-01-01

    Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nano gels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of ‘reactive oxygen species’ (ROS) in presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress. PMID:21477858

  13. Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors.

    SciTech Connect

    Filichkin, Sergei A; DiFazio, Steven P; Brunner, Amy M; Davis, John M; Yang, Zamin Koo; Kalluri, Udaya C; Arias, Renee S; Etherington, Elizabeth; Tuskan, Gerald A; Strauss, S

    2007-01-01

    We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.

  14. High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation.

    PubMed

    Lavertu, Marc; Méthot, Stephane; Tran-Khanh, Nicolas; Buschmann, Michael D

    2006-09-01

    Chitosan is a biodegradable natural polysaccharide that has shown potential for gene delivery, although the ideal molecular weight (MW) and degree of deacetylation (DDA) for this application have not been elucidated. To examine the influence of these parameters on gene transfer, we produced chitosans with different DDAs (98%, 92%, 80% and 72%) and depolymerized them with nitrous acid to obtain different MWs (150, 80, 40 and 10 kDa). We produced 64 formulations of chitosan/pDNA complexes (16 chitosans, 2 amine-to-phosphate (N:P) ratios of 5:1 and 10:1 and 2 transfection media pH of 6.5 and 7.1), characterized them for size and surface charge, and tested them for gene transfection in HEK 293 cells in vitro. Several formulations produced high levels of transgene expression while two conditions, 92-10-5 and 80-10-10 [DDA-MW-N:P ratio] at pH 6.5, showed equivalence to our best positive control. The results also revealed an important coupling between DDA and MW of chitosan in determining transgene expression. Maximum expression was obtained with a certain combination of DDA and MW that depended on N:P ratio and the pH, but similar expression levels could be achieved by simultaneously lowering MW and increasing DDA or lowering DDA and increasing MW, suggesting a predominant role of particle stability, through co-operative electrostatic binding, in determining transfection efficiency.

  15. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing.

    PubMed

    Kam, Nadine Wong Shi; Liu, Zhuang; Dai, Hongjie

    2005-09-14

    We present a novel functionalization scheme for single-walled carbon nanotubes (SWNTs) to afford nanotube-biomolecule conjugates with the incorporation of cleavable bonds to enable controlled molecular releasing from nanotube surfaces, thus creating "smart" nanomaterials with high potential for chemical and biological applications. With this versatile functionalization, we demonstrate transporting, enzymatic cleaving and releasing of DNA from SWNT transporters, and subsequent nuclear translocation of DNA oligonucleotides in mammalian cells. We further show highly efficient delivery of siRNA by SWNTs and achieving more potent RNAi functionality than a widely used conventional transfection agent. Thus, the novel functionalization of SWNTs with cleavable bonds is highly promising for a wide range of applications including gene and protein therapy.

  16. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    SciTech Connect

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  17. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    PubMed

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  18. Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells.

    PubMed

    Choi, WooJae; Kim, Eunji; Yum, Soo-Young; Lee, ChoongIl; Lee, JiHyun; Moon, JoonHo; Ramachandra, Sisitha; Malaweera, Buddika Oshadi; Cho, JongKi; Kim, Jin-Soo; Kim, SeokJoong; Jang, Goo

    2015-01-01

    Even though prion (encoded by the PRNP gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with Bmi-1and hTert. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle. PMID:26217959

  19. Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing.

    PubMed

    Fischer, Konrad; Kraner-Scheiber, Simone; Petersen, Björn; Rieblinger, Beate; Buermann, Anna; Flisikowska, Tatiana; Flisikowski, Krzysztof; Christan, Susanne; Edlinger, Marlene; Baars, Wiebke; Kurome, Mayuko; Zakhartchenko, Valeri; Kessler, Barbara; Plotzki, Elena; Szczerbal, Izabela; Switonski, Marek; Denner, Joachim; Wolf, Eckhard; Schwinzer, Reinhard; Niemann, Heiner; Kind, Alexander; Schnieke, Angelika

    2016-01-01

    Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - 'gene stacking', and cointegration of multiple engineered large vectors - 'combineering', to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age. PMID:27353424

  20. ACHIEVING PERCEPTUAL-MOTOR EFFICIENCY, A SPACE-ORIENTED APPROACH TO LEARNING. PERCEPTUAL MOTOR CURRICULUM, VOLUME I.

    ERIC Educational Resources Information Center

    BARSCH, RAY H.

    THE FIRST OF A 3-VOLUME PERCEPTUAL MOTOR CURRICULUM, THE BOOK DESCRIBES A PROGRAM BASED ON A THEORY OF MOVEMENT WHICH THE AUTHOR LABELS MOVIGENICS (THE STUDY OF THE ORIGIN AND DEVELOPMENT OF PATTERNS OF MOVEMENT IN MAN AND THE RELATIONSHIP OF THESE MOVEMENTS TO HIS LEARNING EFFICIENCY). TEN BASIC CONSTRUCTS OF MOVIGENICS ARE OUTLINED, AND THE…

  1. Efficient gene silencing in mesenchymal stem cells by substrate-mediated RNA interference.

    PubMed

    Hsu, Shan-Hui; Huang, Guo-Shiang; Ho, Tung-Tso; Feng, Fuh

    2014-11-01

    We described a novel substrate-mediated RNA interference (RNAi) technology to investigate the effect of neural crest marker expression on the multipotency of human gingival fibroblasts (HGFs). HGFs showed significantly higher neural and chondrogenic differentiation potentials compared with adult bone-marrow-derived mesenchymal stem cells and stem cells from human exfoliated deciduous teeth. By sending target-specific RNAi agents with the conventional vehicle (PolyFect), we observed that the multipotency of HGFs was closely associated with the expression of neural crest marker gene Forkhead box D3 (FoxD3). Using the novel chitosan substrate-mediated method, we successfully delivered short-hairpin RNA constructs to HGFs grown on chitosan without the use of conventional vehicles. The delivery efficiency measured by flow cytometry showed a 10-fold increase for HGFs on chitosan versus those on culture dish, and the cell viability was >95%. Moreover, HGFs with FoxD3 gene knockdown did not form spheroids on chitosan. Based on this working principle, we further selected the gene-silenced population from HGFs. The nonsilenced HGFs showed much higher neural differentiation ability with the nestin expression 40-fold greater than FoxD3-silenced population after induction, suggesting the feasibility of the method to silence genes. The new substrate-mediated gene silencing platform that combines the use of substrate and RNAi can be used to clarify the functions of important genes without suffering the toxicity. PMID:24624901

  2. Solar-to-hydrogen efficiency exceeding 2.5% achieved for overall water splitting with an all earth-abundant dual-photoelectrode.

    PubMed

    Ding, Chunmei; Qin, Wei; Wang, Nan; Liu, Guiji; Wang, Zhiliang; Yan, Pengli; Shi, Jingying; Li, Can

    2014-08-01

    The solar-to-hydrogen (STH) efficiency of a traditional mono-photoelectrode photoelectrochemical water splitting system has long been limited as large external bias is required. Herein, overall water splitting with STH efficiency exceeding 2.5% was achieved using a self-biased photoelectrochemical-photovoltaic coupled system consisting of an all earth-abundant photoanode and a Si-solar-cell-based photocathode connected in series under parallel illumination. We found that parallel irradiation mode shows higher efficiency than tandem illumination especially for photoanodes with a wide light absorption range, probably as the driving force for water splitting reaction is larger and the photovoltage loss is smaller in the former. This work essentially takes advantage of a tandem solar cell which can enhance the solar-to-electricity efficiency from another point of view.

  3. Solar-to-hydrogen efficiency exceeding 2.5% achieved for overall water splitting with an all earth-abundant dual-photoelectrode.

    PubMed

    Ding, Chunmei; Qin, Wei; Wang, Nan; Liu, Guiji; Wang, Zhiliang; Yan, Pengli; Shi, Jingying; Li, Can

    2014-08-01

    The solar-to-hydrogen (STH) efficiency of a traditional mono-photoelectrode photoelectrochemical water splitting system has long been limited as large external bias is required. Herein, overall water splitting with STH efficiency exceeding 2.5% was achieved using a self-biased photoelectrochemical-photovoltaic coupled system consisting of an all earth-abundant photoanode and a Si-solar-cell-based photocathode connected in series under parallel illumination. We found that parallel irradiation mode shows higher efficiency than tandem illumination especially for photoanodes with a wide light absorption range, probably as the driving force for water splitting reaction is larger and the photovoltage loss is smaller in the former. This work essentially takes advantage of a tandem solar cell which can enhance the solar-to-electricity efficiency from another point of view. PMID:24956231

  4. Tailor-made TALEN system for highly efficient targeted gene replacement in the rice blast fungus.

    PubMed

    Arazoe, Takayuki; Ogawa, Tetsuo; Miyoshi, Kennosuke; Yamato, Tohru; Ohsato, Shuichi; Sakuma, Tetsushi; Yamamoto, Takashi; Arie, Tsutomu; Kuwata, Shigeru

    2015-07-01

    Genetic manipulation is key to unraveling gene functions and creating genetically modified strains of microbial organisms. Recently, engineered nucleases that can generate DNA double-strand breaks (DSBs) at a specific site in the desired locus within genome are utilized in a rapidly developing genome editing technology via DSBs repair. However, the use of engineered nucleases in filamentous fungi has not been validated. In this study, we demonstrated that tailor-made transcriptional activator-like effector nucleases (TALENs) system, Platinum-Fungal TALENs (PtFg TALENs), could improve the efficiency of homologous recombination-mediated targeted gene replacement by up to 100% in the rice blast fungus Pyricularia oryzae. This high-efficiency PtFg TALEN has great potential for basic and applied biological applications in filamentous fungi. PMID:25683503

  5. Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.

    PubMed

    Murphy, Andrew J; Macdonald, Lynn E; Stevens, Sean; Karow, Margaret; Dore, Anthony T; Pobursky, Kevin; Huang, Tammy T; Poueymirou, William T; Esau, Lakeisha; Meola, Melissa; Mikulka, Warren; Krueger, Pamela; Fairhurst, Jeanette; Valenzuela, David M; Papadopoulos, Nicholas; Yancopoulos, George D

    2014-04-01

    Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

  6. Carrots and Sticks: A Comprehensive Business Model for the Successful Achievement of Energy Efficiency Resource Standards Environmental Energy Technologies DivisionMarch 2011

    SciTech Connect

    Satchwell, Andrew; Cappers, Peter; Goldman, Charles

    2011-03-22

    Energy efficiency resource standards (EERS) are a prominent strategy to potentially achieve rapid and aggressive energy savings goals in the U.S. As of December 2010, twenty-six U.S. states had some form of an EERS with savings goals applicable to energy efficiency (EE) programs paid for by utility customers. The European Union has initiated a similar type of savings goal, the Energy End-use Efficiency and Energy Services Directive, where it is being implemented in some countries through direct partnership with regulated electric utilities. U.S. utilities face significant financial disincentives under traditional regulation which affects the interest of shareholders and managers in aggressively pursuing cost-effective energy efficiency. Regulators are considering some combination of mandated goals ('sticks') and alternative utility business model components ('carrots' such as performance incentives) to align the utility's business and financial interests with state and federal energy efficiency public policy goals. European countries that have directed their utilities to administer EE programs have generally relied on non-binding mandates and targets; in the U.S., most state regulators have increasingly viewed 'carrots' as a necessary condition for successful achievement of energy efficiency goals and targets. In this paper, we analyze the financial impacts of an EERS on a large electric utility in the State of Arizona using a pro-forma utility financial model, including impacts on utility earnings, customer bills and rates. We demonstrate how a viable business model can be designed to improve the business case while retaining sizable ratepayer benefits. Quantifying these concerns and identifying ways they can be addressed are crucial steps in gaining the support of major stakeholder groups - lessons that can apply to other countries looking to significantly increase savings targets that can be achieved from their own utility-administered EE programs.

  7. Pullulan-protamine as efficient haemocompatible gene delivery vector: synthesis and in vitro characterization.

    PubMed

    Priya, S S; Rekha, M R; Sharma, Chandra P

    2014-02-15

    Biodegradable non-viral vectors with good transfection efficiency is essential for successful gene delivery. The purpose of this study was to design a non-viral vector by conjugating protamine to pullulan and elucidate the potential use of pullulan protamine conjugate (PPA) as an effective, non toxic and haemocompatible gene delivery system. The particle size and surface charge were measured using Nanosizer. Derivatization was confirmed by NMR, FTIR and DSC analyses. Acid base titration revealed the buffering behaviour of the conjugate. The protection of DNA from nuclease enzyme and interaction of plasma components on the stability of nanoplexes were also analysed. The uptake studies confirmed the plasmid delivery into the nucleus and the inhibitor studies determined the uptake mechanism. Transfection experiments revealed the capability of PPA to cellular uptake in C6 cells and facilitate high gene expression. Thus, PPA proves to be a promising non-viral vector.

  8. Codon-optimized antibiotic resistance gene improves efficiency of transient transformation in Frankia.

    PubMed

    Kucho, Ken-Ichi; Kakoi, Kentaro; Yamaura, Masatoshi; Iwashita, Mari; Abe, Mikiko; Uchiumi, Toshiki

    2013-11-01

    Frankia is a unique actinobacterium having abilities to fix atmospheric dinitrogen and to establish endosymbiosis with trees, but molecular bases underlying these interesting characteristics are poorly understood because of a lack of stable transformation system. Extremely high GC content of Frankia genome (more than 70 percent) can be a hindrance to successful transformation. We generated a synthetic gentamicin resistance gene whose codon usage is optimized to Frankia (fgmR) and evaluated its usefulness as a selection marker using a transient transformation system. Success rate of transient transformation and cell growth in selective culture were significantly increased by use of fgmR instead of a native gentamicin resistance gene, suggesting that codon optimization improved translation efficiency of the marker gene and increased antibiotic resistance. Our result shows that similarity in codon usage pattern is an important factor to be taken into account when exogenous transgenes are expressed in Frankia cells. PMID:24287650

  9. An efficient approach of attractor calculation for large-scale Boolean gene regulatory networks.

    PubMed

    He, Qinbin; Xia, Zhile; Lin, Bin

    2016-11-01

    Boolean network models provide an efficient way for studying gene regulatory networks. The main dynamics of a Boolean network is determined by its attractors. Attractor calculation plays a key role for analyzing Boolean gene regulatory networks. An approach of attractor calculation was proposed in this study, which improved the predecessor-based approach. Furthermore, the proposed approach combined with the identification of constant nodes and simplified Boolean networks to accelerate attractor calculation. The proposed algorithm is effective to calculate all attractors for large-scale Boolean gene regulatory networks. If the average degree of the network is not too large, the algorithm can get all attractors of a Boolean network with dozens or even hundreds of nodes.

  10. Simple and efficient recycling of fungal selectable marker genes with the Cre-loxP recombination system via anastomosis

    PubMed Central

    Zhang, Dong-Xiu; Lu, Hsiao-Ling; Liao, Xinggang; St. Leger, Raymond J.; Nuss, Donald L.

    2013-01-01

    Reverse-genetics analysis has played a significant role in advancing fungal biology, but is limited by the number of available selectable marker genes (SMGs). The Cre-loxP recombination system has been adapted for use in filamentous fungi to overcome this limitation. Expression of the Cre recombinase results in excision of an integrated SMG that is flanked by loxP sites, allowing a subsequent round of transformation with the same SMG. However, current protocols for regulated expression or presentation of Cre require multiple time-consuming steps. During efforts to disrupt four different RNA-dependent RNA polymerase genes in a single strain of the chestnut blight fungus Cryphonectria parasitica, we tested whether Cre could successfully excise loxP-flanked SMGs when provided in trans via anastomosis. Stable Cre-producing donor strains were constructed by transformation of wild-type C. parasitica strain EP155 with the Cre-coding domain under the control of a constitutive promoter. Excision of multiple loxP-flanked SMGs was efficiently achieved by simply pairing the Cre-donor strain and the loxP-flanked SMGs-transformed recipient strain and recovering mycelia from the margin of the recipient colony near the anastomosis zone. This method was shown to be as efficient as and much less time consuming than excision by transformation-mediated expression of Cre. It also allows unlimited recycling of loxP-flanked SMGs and the generation of disruption mutant strains that are free of any foreign gene. The successful application of this method to Metarhizium robertsii suggests potential use for optimizing reverse-genetics analysis in a broad range of filamentous fungi. PMID:24007936

  11. Gene delivery efficiency and cytotoxicity of heterocyclic amine-modified PAMAM and PPI dendrimers.

    PubMed

    Hashemi, Maryam; Tabatabai, Seyed Meghdad; Parhiz, Hamideh; Milanizadeh, Soroush; Amel Farzad, Sara; Abnous, Khalil; Ramezani, Mohammad

    2016-04-01

    Poly-(amidoamine) (PAMAM) and poly-(propylenimine) (PPI) are the two most widely investigated dendrimers for drug and gene delivery. In order to enhance DNA transfection activity of these dendrimers, generation 3 and 4 PAMAM and generation 4 and 5 PPI were modified by partial substitution of surface primary amines with histidine, pyridine, and piperazine, which have buffering capacity properties. It was shown that higher dendrimer generations and higher grafting percentages (30% and 50% of primary amines) were associated with higher transfection activity. Pyridine was the most effective substituent for PPI, while piperazine-modified PAMAM dendrimers showed the best transfection efficiency among PAMAM-based vectors in murine neuroblastoma (Neuro-2a) cells. None of the modified carriers showed remarkable cytotoxicity in vitro. Pretreatment of cells with bafilomycin A indicated that endosomal buffering capacity is the main mechanism of endosomal escape. In conclusion, PAMAM and PPI may exhibit different gene delivery efficiency and cytotoxicity profiles with the same chemical modifications. These modified dendrimers could be considered as efficient and safe gene carriers in neuroblastoma cells in vitro. PMID:26838910

  12. An efficient virus-induced gene silencing vector for maize functional genomics research.

    PubMed

    Wang, Rong; Yang, Xinxin; Wang, Nian; Liu, Xuedong; Nelson, Richard S; Li, Weimin; Fan, Zaifeng; Zhou, Tao

    2016-04-01

    Maize is a major crop whose rich genetic diversity provides an advanced resource for genetic research. However, a tool for rapid transient gene function analysis in maize that may be utilized in most maize cultivars has been lacking, resulting in reliance on time-consuming stable transformation and mutation studies to obtain answers. We developed an efficient virus-induced gene silencing (VIGS) vector for maize based on a naturally maize-infecting cucumber mosaic virus (CMV) strain, ZMBJ-CMV. An infectious clone of ZMBJ-CMV was constructed, and a vascular puncture inoculation method utilizing Agrobacterium was optimized to improve its utility for CMV infection of maize. ZMBJ-CMV was then modified to function as a VIGS vector. The ZMBJ-CMV vector induced mild to moderate symptoms in many maize lines, making it useful for gene function studies in critically important maize cultivars, such as the sequenced reference inbred line B73. Using this CMV VIGS system, expression of two endogenous genes, ZmPDS and ZmIspH, was found to be decreased by 75% and 78%, respectively, compared with non-silenced tissue. Inserts with lengths of 100-300 bp produced the most complete transcriptional and visual silencing phenotypes. Moreover, genes related to autophagy, ZmATG3 and ZmATG8a, were also silenced, and it was found that they function in leaf starch degradation. These results indicate that our ZMBJ-CMV VIGS vector provides a tool for rapid and efficient gene function studies in maize. PMID:26921244

  13. Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

    PubMed Central

    Bartlett, J S; Sethna, M; Ramamurthy, L; Gowen, S A; Samulski, R J; Marzluff, W F

    1996-01-01

    Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8799116

  14. Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum.

    PubMed

    Gilles, Anna F; Schinko, Johannes B; Averof, Michalis

    2015-08-15

    Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs. PMID:26160901

  15. Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum.

    PubMed

    Gilles, Anna F; Schinko, Johannes B; Averof, Michalis

    2015-08-15

    Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs.

  16. Construction of simple and efficient siRNA validation systems for screening and identification of effective RNAi-targeted sequences from mammalian genes.

    PubMed

    Tsai, Wen-Hui; Chang, Wen-Tsan

    2014-01-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing induced by double-stranded RNAs (dsRNAs). Among the widely used dsRNAs, small interfering RNAs (siRNAs) and short hairpin RNAs have evolved as extremely powerful and the most popular gene silencing reagents. The key challenge to achieving efficient gene silencing especially for the purpose of therapeutics is mainly dependent on the effectiveness and specificity of the selected RNAi-targeted sequences. Practically, only a small number of dsRNAs are capable of inducing highly effective and sequence-specific gene silencing via RNAi mechanism. In addition, the efficiency of gene silencing induced by dsRNAs can only be experimentally examined based on inhibition of the target gene expression. Therefore, it is essential to develop a fully robust and comparative validation system for measuring the efficacy of designed dsRNAs. In this chapter, we focus our discussion on a reliable and quantitative reporter-based siRNA validation system that has been previously established in our laboratory. The system consists of a short synthetic DNA fragment containing an RNAi-targeted sequence of interest and two expression vectors for targeting reporter and triggering siRNA expressions. The efficiency of siRNAs is determined by their abilities to inhibit expression of the targeting reporters with easily quantified readouts including enhanced green fluorescence protein and firefly luciferase. Since only a readily available short synthetic DNA fragment is needed for constructing this reliable and efficient reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective RNAi-targeted sequences from mammalian genes but also implicates the use of RNAi-based dsRNA reagents for reverse functional genomics and molecular therapeutics.

  17. Strategies to enhance transductional efficiency of adenoviral-based gene transfer to primary human fibroblasts and keratinocytes as a platform in dermal wounds

    PubMed Central

    Stoff, Alexander; Rivera, Angel A.; Banerjee, N. S.; Mathis, J. Michael; Espinosa-de-los-Monteros, Antonio; Le, Long P.; De la Torre, Jorge I.; Vasconez, Luis O.; Broker, Thomas R.; Richter, Dirk F.; Stoff-Khalili, Mariam A.; Curiel, David T.

    2007-01-01

    Genetically modified keratinocytes and fibroblasts are suitable for delivery of therapeutic genes capable of modifying the wound healing process. However, efficient gene delivery is a prerequisite for successful gene therapy of wounds. Whereas adenoviral vectors (Ads) exhibit superior levels of in vivo gene transfer, their transductional efficiency to cells resident within wounds may nonetheless be suboptimal, due to deficiency of the primary adenovirus receptor, coxsackie-adenovirus receptor (CAR). We explored CAR-independent transduction to fibroblasts and keratinocytes using a panel of CAR-independent fiber-modified Ads to determine enhancement of infectivity. These fiber-modified adenoviral vectors included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7). To evaluate whether transduction efficiencies of the fiber-modified adenoviral vectors correlated with the expression of their putative receptors on keratinocytes and fibroblasts, we analyzed the mRNA levels of CAR, αυ integrin, syndecan-1, and glypican-1 using quantitative polymerase chain reaction. Analysis of luciferase and green fluorescent protein transgene expression showed superior transduction efficiency of Ad5.pK7 in keratinocytes and Ad5.RGD.pK7 in fibroblasts. mRNA expression of αυ integrin, syndecan-1 and glypican-1 was significantly higher in primary fibroblasts than CAR. In keratinocytes, syndecan-1 expression was significantly higher than all the other receptors tested. Significant infectivity enhancement was achieved in keratinocytes and fibroblasts using fiber-modified adenoviral vectors. These strategies to enhance infectivity may help to achieve higher clinical efficacy of wound gene therapy. PMID:17014674

  18. Phylo-MCOA: a fast and efficient method to detect outlier genes and species in phylogenomics using multiple co-inertia analysis.

    PubMed

    de Vienne, Damien M; Ollier, Sébastien; Aguileta, Gabriela

    2012-06-01

    Full genome data sets are currently being explored on a regular basis to infer phylogenetic trees, but there are often discordances among the trees produced by different genes. An important goal in phylogenomics is to identify which individual gene and species produce the same phylogenetic tree and are thus likely to share the same evolutionary history. On the other hand, it is also essential to identify which genes and species produce discordant topologies and therefore evolve in a different way or represent noise in the data. The latter are outlier genes or species and they can provide a wealth of information on potentially interesting biological processes, such as incomplete lineage sorting, hybridization, and horizontal gene transfers. Here, we propose a new method to explore the genomic tree space and detect outlier genes and species based on multiple co-inertia analysis (MCOA), which efficiently captures and compares the similarities in the phylogenetic topologies produced by individual genes. Our method allows the rapid identification of outlier genes and species by extracting the similarities and discrepancies, in terms of the pairwise distances, between all the species in all the trees, simultaneously. This is achieved by using MCOA, which finds successive decomposition axes from individual ordinations (i.e., derived from distance matrices) that maximize a covariance function. The method is freely available as a set of R functions. The source code and tutorial can be found online at http://phylomcoa.cgenomics.org.

  19. Low-cost and no-cost practice to achieve energy efficiency of government office buildings: A case study in federal territory of Malaysia

    NASA Astrophysics Data System (ADS)

    Tahir, Mohamad Zamhari; Nawi, Mohd Nasrun Mohd; Ibrahim, Amlus

    2016-08-01

    This paper presents the findings of a case study to achieve energy-efficient performance of conventional office buildings in Malaysia. Two multi-storey office buildings in Federal Territory of Malaysia have been selected. The aim is to study building energy saving potential then to highlight the appropriate measures that can be implemented. Data was collected using benchmarking method by comparing the measured consumption to other similar office buildings and a series of preliminary audit which involves interviews, a brief review of utility and operating data as well as a walkthrough in the buildings. Additionally, in order to get a better understanding of major energy consumption in the selected buildings, general audit have been conducted to collect more detailed information about building operation. In the end, this study emphasized low-cost and no-cost practice to achieve energy efficiency with significant results in some cases.

  20. Limits on Achievable Dimensional and Photon Efficiencies with Intensity-Modulation and Photon-Counting Due to Non-Ideal Photon-Counter Behavior

    NASA Technical Reports Server (NTRS)

    Moision, Bruce; Erkmen, Baris I.; Farr, William; Dolinar, Samuel J.; Birnbaum, Kevin M.

    2012-01-01

    An ideal intensity-modulated photon-counting channel can achieve unbounded photon information efficiencies (PIEs). However, a number of limitations of a physical system limit the practically achievable PIE. In this paper, we discuss several of these limitations and illustrate their impact on the channel. We show that, for the Poisson channel, noise does not strictly bound PIE, although there is an effective limit, as the dimensional information efficiency goes as e[overline] e PIE beyond a threshold PIE. Since the Holevo limit is bounded in the presence of noise, this illustrates that the Poisson approximation is invalid at large PIE for any number of noise modes. We show that a finite transmitter extinction ratio bounds the achievable PIE to a maximum that is logarithmic in the extinction ratio. We show how detector jitter limits the ability to mitigate noise in the PPM signaling framework. We illustrate a method to model detector blocking when the number of detectors is large, and illustrate mitigation of blocking with spatial spreading and altering. Finally, we illustrate the design of a high photon efficiency system using state-of-the-art photo-detectors and taking all these effects into account.

  1. Endosomal pH responsive polymers for efficient cancer targeted gene therapy.

    PubMed

    Shi, Bingyang; Zhang, Hu; Bi, Jingxiu; Dai, Sheng

    2014-07-01

    Treatment of human diseases at gene level is always limited by effective gene delivery vectors. In this study, we designed and developed an endosomal pH sensitive targeted gene delivery system, folic acid functionalized Schiff-base linked imidazole chitosan (FA-SLICS), for cancer therapy. The FA-SLICS is able to self-assemble plasmid DNA (pDNA) into nano-scaled polyplexes under a neutral condition and to release the loaded pDNA in the endosomal microenvironment due to the presence of pH sensitive Schiff-base moieties along chitosan backbones. The FA-SLICS has negligible cytotoxicity to normal cells (CHO), but displays slight toxicity to cancer cells (HeLa and HepG2). In addition, FA-SLICS can selectively and efficiently transfect FR (folate receptor) positive cells (HeLa cells) as a gene carrier. Therefore, the FA-SLICS should be a promising delivery vector in cancer gene therapy based on its cell targeting capability and intracellular microenvironment controlled delivery mechanism.

  2. Dextran-protamine polycation: an efficient nonviral and haemocompatible gene delivery system.

    PubMed

    Thomas, Jane Joy; Rekha, M R; Sharma, Chandra P

    2010-11-01

    Despite the remarkable progress in the field of gene therapy with viral vectors, nonviral vectors have attracted great interests due to their unique properties. Imparting desired characteristics to nonviral gene delivery systems requires the development of cationic polymers. The purpose of this work was to design a cationic derivative (Dex-P) of dextran using protamine in order to assert target specific cellular binding. Our objective was to elucidate the potential use of Dex-P as a haemocompatible, nontoxic and efficient nonviral candidate for gene therapy. Nanoplexes were prepared with calf thymus DNA and Dex-P. Derivatization was confirmed by FTIR, gel permeation chromatography and TNBS assay. Dynamic light scattering and TEM studies determined the size and morphology of the nanoplex. The buffering behaviour was assessed by acid base titration. Complexation stability was evaluated using agarose gel electrophoresis and EtBr displacement assay. The protection of ctDNA from nuclear digestion and the effect of plasma components towards stability of the nanoplexes were also analyzed. Various haemocompatible studies were performed to check haemolysis, aggregation, clotting time, and complement activation. Transfection and cytotoxicity experiments were performed in vitro. The nanosize, spherical shape and stability of nanoplexes were affirmed. Various experiments conducted confirmed Dex-P to be nontoxic and haemocompatible. Transfection experiments revealed the capability of Dex-P to facilitate high gene expression and cellular uptake in HepG2 cells. With the improved physicochemical, biological and transfection properties, Dex-P seems to be a promising gene delivery system.

  3. Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing.

    PubMed

    Chen, Chao; Posocco, Paola; Liu, Xiaoxuan; Cheng, Qiang; Laurini, Erik; Zhou, Jiehua; Liu, Cheng; Wang, Yang; Tang, Jingjie; Col, Valentina Dal; Yu, Tianzhu; Giorgio, Suzanne; Fermeglia, Maurizio; Qu, Fanqi; Liang, Zicai; Rossi, John J; Liu, Minghua; Rocchi, Palma; Pricl, Sabrina; Peng, Ling

    2016-07-01

    Self-assembly is a fundamental concept and a powerful approach in molecular science. However, creating functional materials with the desired properties through self-assembly remains challenging. In this work, through a combination of experimental and computational approaches, the self-assembly of small amphiphilic dendrons into nanosized supramolecular dendrimer micelles with a degree of structural definition similar to traditional covalent high-generation dendrimers is reported. It is demonstrated that, with the optimal balance of hydrophobicity and hydrophilicity, one of the self-assembled nanomicellar systems, totally devoid of toxic side effects, is able to deliver small interfering RNA and achieve effective gene silencing both in cells - including the highly refractory human hematopoietic CD34(+) stem cells - and in vivo, thus paving the way for future biomedical implementation. This work presents a case study of the concept of generating functional supramolecular dendrimers via self-assembly. The ability of carefully designed and gauged building blocks to assemble into supramolecular structures opens new perspectives on the design of self-assembling nanosystems for complex and functional applications. PMID:27244195

  4. Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing.

    PubMed

    Chen, Chao; Posocco, Paola; Liu, Xiaoxuan; Cheng, Qiang; Laurini, Erik; Zhou, Jiehua; Liu, Cheng; Wang, Yang; Tang, Jingjie; Col, Valentina Dal; Yu, Tianzhu; Giorgio, Suzanne; Fermeglia, Maurizio; Qu, Fanqi; Liang, Zicai; Rossi, John J; Liu, Minghua; Rocchi, Palma; Pricl, Sabrina; Peng, Ling

    2016-07-01

    Self-assembly is a fundamental concept and a powerful approach in molecular science. However, creating functional materials with the desired properties through self-assembly remains challenging. In this work, through a combination of experimental and computational approaches, the self-assembly of small amphiphilic dendrons into nanosized supramolecular dendrimer micelles with a degree of structural definition similar to traditional covalent high-generation dendrimers is reported. It is demonstrated that, with the optimal balance of hydrophobicity and hydrophilicity, one of the self-assembled nanomicellar systems, totally devoid of toxic side effects, is able to deliver small interfering RNA and achieve effective gene silencing both in cells - including the highly refractory human hematopoietic CD34(+) stem cells - and in vivo, thus paving the way for future biomedical implementation. This work presents a case study of the concept of generating functional supramolecular dendrimers via self-assembly. The ability of carefully designed and gauged building blocks to assemble into supramolecular structures opens new perspectives on the design of self-assembling nanosystems for complex and functional applications.

  5. Self-assembled supramolecular nano vesicles for safe and highly efficient gene delivery to solid tumors

    PubMed Central

    Li, Wei; Li, Huafei; Li, Jinfeng; Wang, Huajing; Zhao, He; Zhang, Li; Xia, Yu; Ye, Zengwei; Gao, Jie; Dai, Jianxin; Wang, Hao; Guo, Yajun

    2012-01-01

    The main obstacles for cationic polyplexes in gene delivery are in vivo instability and low solid-tumor accumulation. Safe vectors with high transfection efficiency and in vivo tumor accumulation are therefore highly desirable. In this study, the amphiphilic block copolymer poly(n-butyl methacrylate)-b-poly(N-acryloylmorpholine) was synthesized by reversible addition–fragmentation chain-transfer (RAFT) radical polymerization. The corresponding well-defined vesicles with narrow size distribution were tailored by finely regulating the packing parameter (β) of copolymer (1/2 < β < 1). Compared with traditional “gold-standard” polycation (polyethylenimine, 25 kDa), plasmid DNA condensing efficiency, DNase I degradation protection, and cellular uptake were improved by the supramolecular nano vesicles. In addition, the plasmid DNA transferring efficiency in 10% fetal bovine serum medium was enlarged five times to that of polyethylenimine in renal tubular epithelial and human hepatocellular carcinoma cell lines. This improved in vitro transfection was mainly attributed to the densely packed bilayer. This stealth polyplex showed high serum stability via entropic repulsion, which further protected the polyplex from being destroyed during sterilization. As indicated by the IVIS® Lumina II Imaging System (Caliper Life Sciences, Hopkinton, MA) 24 hours post-intravenous administration, intra-tumor accumulation of the stealth polyplex was clearly promoted. This study successfully circumvented the traditional dilemma of efficient gene transfection at a high nitrogen-from-polyethylenimine to phosphate-from-DNA ratio that is accompanied with site cytotoxicity and low stability. As such, these simply tailored noncytotoxic nano vesicles show significant potential for use in practical gene therapy. PMID:22977303

  6. Efficient gene delivery to human umbilical cord mesenchymal stem cells by cationized Porphyra yezoensis polysaccharide nanoparticles

    PubMed Central

    Yu, Qingtong; Cao, Jin; Chen, Baoding; Deng, Wenwen; Cao, Xia; Chen, Jingjing; Wang, Yan; Wang, Shicheng; Yu, Jiangnan; Xu, Ximing; Gao, Xiangdong

    2015-01-01

    This study centered on an innovative application of Porphyra yezoensis polysaccharide (PPS) with cationic modification as a safe and efficient nonviral gene vector to deliver a plasmid encoding human Wnt3a (pWnt3a) into human umbilical cord mesenchymal stem cells (HUMSCs). After modification with branched low-molecular-weight (1,200 Da) polyethylenimine, the cationized PPS (CPPS) was combined with pWnt3a to form spherical nanoscale particles (CPPS-pWnt3a nanoparticles). Particle size and distribution indicated that the CPPS-pWnt3a nanoparticles at a CPPS:pWnt3a weight ratio of 40:1 might be a potential candidate for DNA plasmid transfection. A cytotoxicity assay demonstrated that the nanoparticles prepared at a CPPS:pWnt3a weight ratio of 40:1 were nontoxic to HUMSCs compared to those of Lipofectamine 2000 and polyethylenimine (25 kDa). These nanoparticles were further transfected to HUMSCs. Western blotting demonstrated that the nanoparticles (CPPS:pWnt3a weight ratio 40:1) had the greatest transfection efficiency in HUMSCs, which was significantly higher than that of Lipofectamine 2000; however, when the CPPS:pWnt3a weight ratio was increased to 80:1, the nanoparticle-treated group showed no obvious improvement in translation efficiency over Lipofectamine 2000. Therefore, CPPS, a novel cationic polysaccharide derived from P. yezoensis, could be developed into a safe, efficient, nonviral gene vector in a gene-delivery system. PMID:26604758

  7. Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells.

    PubMed

    Tsuchiya, Megumi; Ogawa, Hidesato; Koujin, Takako; Kobayashi, Shouhei; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

    2016-08-01

    Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here, we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery. PMID:27317902

  8. Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations.

    PubMed

    Jiang, WenZhi; Yang, Bing; Weeks, Donald P

    2014-01-01

    The newly developed CRISPR/Cas9 system for targeted gene knockout or editing has recently been shown to function in plants in both transient expression systems as well as in primary T1 transgenic plants. However, stable transmission of genes modified by the Cas9/single guide RNA (sgRNA) system to the T2 generation and beyond has not been demonstrated. Here we provide extensive data demonstrating the efficiency of Cas9/sgRNA in causing modification of a chromosomally integrated target reporter gene during early development of transgenic Arabidopsis plants and inheritance of the modified gene in T2 and T3 progeny. Efficient conversion of a nonfunctional, out-of-frame GFP gene to a functional GFP gene was confirmed in T1 plants by the observation of green fluorescent signals in leaf tissues as well as the presence of mutagenized DNA sequences at the sgRNA target site within the GFP gene. All GFP-positive T1 transgenic plants and nearly all GFP-negative plants examined contained mutagenized GFP genes. Analyses of 42 individual T2 generation plants derived from 6 different T1 progenitor plants showed that 50% of T2 plants inherited a single T-DNA insert. The efficiency of the Cas9/sgRNA system and stable inheritance of edited genes point to the promise of this system for facile editing of plant genes.

  9. Absorber and emitter for solar thermo-photovoltaic systems to achieve efficiency exceeding the Shockley-Queisser limit.

    PubMed

    Rephaeli, Eden; Fan, Shanhui

    2009-08-17

    We present theoretical considerations as well as detailed numerical design of absorber and emitter for Solar Thermophotovoltaics (STPV) applications. The absorber, consisting of an array of tungsten pyramids, was designed to provide near-unity absorptivity over all solar wavelengths for a wide angular range, enabling it to absorb light effectively from solar sources regardless of concentration. The emitter, a tungsten slab with Si/SiO(2) multilayer stack, provides a sharp emissivity peak at the solar cell band-gap while suppressing emission at lower frequencies. We show that, under a suitable light concentration condition, and with a reasonable area ratio between the emitter and absorber, a STPV system employing such absorber-emitter pair and a single-junction solar cell can attain efficiency that exceeds the Shockley-Queisser limit.

  10. Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing

    NASA Astrophysics Data System (ADS)

    Mok, Hyejung; Lee, Soo Hyeon; Park, Ji Won; Park, Tae Gwan

    2010-03-01

    Small interfering RNA (siRNA) with 19-21 base pairs has been recently recognized as a new therapeutic agent for effectively silencing a specific gene on a post-transcription level. For siRNA therapeutics, safe and efficient delivery issues are significant hurdles to clinical applications. Here we present a new class of biologically active siRNA structure based on chemically self-crosslinked and multimerized siRNA through cleavable disulphide linkages. The multimerized siRNA can produce more stable and compact polyelectrolyte complexes with less cytotoxic cationic carriers than naked siRNA because of substantially increased charge densities and the presence of flexible chemical linkers in the backbone. The cleavable and multimerized siRNA shows greatly enhanced gene-silencing efficiencies in vitro and in vivo through a target-messenger-RNA-specific RNA interference processing without significantly eliciting immune induction. This study demonstrates that the multimerized siRNA structure complexed with selected cationic condensing agents can serve as potential gene-silencing therapeutics for treating various diseases.

  11. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9

    PubMed Central

    Mandal, Pankaj K.; Ferreira, Leonardo M. R.; Collins, Ryan; Meissner, Torsten B.; Boutwell, Christian L.; Friesen, Max; Vrbanac, Vladimir; Garrison, Brian S.; Stortchevoi, Alexei; Bryder, David; Musunuru, Kiran; Brand, Harrison; Tager, Andrew M.; Allen, Todd M.; Talkowski, Michael E.; Rossi, Derrick J.; Cowan, Chad A.

    2014-01-01

    SUMMARY Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9 mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multi-lineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy. PMID:25517468

  12. Water soluble cationic dextran derivatives containing poly(amidoamine) dendrons for efficient gene delivery.

    PubMed

    Mai, Kaijin; Zhang, Shanshan; Liang, Bing; Gao, Cong; Du, Wenjun; Zhang, Li-Ming

    2015-06-01

    To develop new dextran derivatives for efficient gene delivery, the conjugation of poly(amidoamine) dendrons onto biocompatible dextran was carried out by a Cu(I)-catalyzed azide-alkyne cycloaddition, as confirmed by FTIR and (1)H NMR analyses. For resultant dextran conjugates with various generations of poly(amidoamine) dendrons, their buffering capacity and in vitro cytotoxicity were evaluated by acid-base titration and MTT tests, respectively. In particular, their physicochemical characteristics for the complexation with plasmid DNA were investigated by the combined analyses from agarose gel electrophoresis, zeta potential, particle size, transmission electron microscopy and fluorescence emission spectra. Moreover, their complexes with plasmid DNA were studied with respect to their transfection efficiency in human embryonic kidney (HEK293) cell lines. In the case of a higher generation of poly(amidoamine) dendrons, such a dextran conjugate was found to have much lower cytotoxicity and better gene delivery capability when compared to branched polyethylenimine (bPEI, 25kDa), a commonly used gene vector. PMID:25843855

  13. Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.

    PubMed

    Fan, Zhiqiang; Li, Wei; Lee, Sang R; Meng, Qinggang; Shi, Bi; Bunch, Thomas D; White, Kenneth L; Kong, Il-Keun; Wang, Zhongde

    2014-01-01

    The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)--three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C--and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.

  14. How endogenous plant cell-wall degradation mechanisms can help achieve higher efficiency in saccharification of biomass.

    PubMed

    Tavares, Eveline Q P; De Souza, Amanda P; Buckeridge, Marcos S

    2015-07-01

    Cell-wall recalcitrance to hydrolysis still represents one of the major bottlenecks for second-generation bioethanol production. This occurs despite the development of pre-treatments, the prospect of new enzymes, and the production of transgenic plants with less-recalcitrant cell walls. Recalcitrance, which is the intrinsic resistance to breakdown imposed by polymer assembly, is the result of inherent limitations in its three domains. These consist of: (i) porosity, associated with a pectin matrix impairing trafficking through the wall; (ii) the glycomic code, which refers to the fine-structural emergent complexity of cell-wall polymers that are unique to cells, tissues, and species; and (iii) cellulose crystallinity, which refers to the organization in micro- and/or macrofibrils. One way to circumvent recalcitrance could be by following cell-wall hydrolysis strategies underlying plant endogenous mechanisms that are optimized to precisely modify cell walls in planta. Thus, the cell-wall degradation that occurs during fruit ripening, abscission, storage cell-wall mobilization, and aerenchyma formation are reviewed in order to highlight how plants deal with recalcitrance and which are the routes to couple prospective enzymes and cocktail designs with cell-wall features. The manipulation of key enzyme levels in planta can help achieving biologically pre-treated walls (i.e. less recalcitrant) before plants are harvested for bioethanol production. This may be helpful in decreasing the costs associated with producing bioethanol from biomass.

  15. Exciplex-triplet energy transfer: A new method to achieve extremely efficient organic light-emitting diode with external quantum efficiency over 30% and drive voltage below 3 V

    NASA Astrophysics Data System (ADS)

    Seo, Satoshi; Shitagaki, Satoko; Ohsawa, Nobuharu; Inoue, Hideko; Suzuki, Kunihiko; Nowatari, Hiromi; Yamazaki, Shunpei

    2014-04-01

    A novel approach to enhance the power efficiency of an organic light-emitting diode (OLED) by employing energy transfer from an exciplex to a phosphorescent emitter is reported. It was found that excitation energy of an exciplex formed between an electron-transporting material with a π-deficient quinoxaline moiety and a hole-transporting material with aromatic amine structure can be effectively transferred to a phosphorescent iridium complex in an emission layer of a phosphorescent OLED. Moreover, such an exciplex formation increases quantum efficiency and reduces drive voltage. A highly efficient, low-voltage, and long-life OLED based on this energy transfer is also demonstrated. This OLED device exhibited extremely high external quantum efficiency of 31% even without any attempt to enhance light outcoupling and also achieved a low drive voltage of 2.8 V and a long lifetime of approximately 1,000,000 h at a luminance of 1,000 cd/m2.

  16. A Simple Zn2+ Complex-Based Composite System for Efficient Gene Delivery.

    PubMed

    Zhang, Zhe; Zhao, Yanjie; Meng, Xianggao; Zhao, Dan; Zhang, Dan; Wang, Li; Liu, Changlin

    2016-01-01

    Metal complexes might become a new type of promising gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, and appropriate density and distribution of positive charges. In this study, Zn2+ complexes (1-10) formed with a series of ligands contained benzimidazole(bzim)were prepared and characterized. They were observed to have different affinities for DNA, dependent on their numbers of positive charges, bzim groups, and coordination structures around Zn2+. The binding induced DNA to condensate into spherical nanoparticles with ~ 50 nm in diameter. The cell transfection efficiency of the DNA nanoparticles was poor, although they were low toxic. The sequential addition of the cell-penetrating peptide (CPP) TAT(48-60) and polyethylene glycol (PEG) resulted in the large DNA condensates (~ 100 nm in diameter) and the increased cellular uptake. The clathrin-mediated endocytosis was found to be a key cellular uptake pathway of the nanoparticles formed with or without TAT(48-60) or/and PEG. The DNA nanoparticles with TAT(48-60) and PEG was found to have the cell transfection efficiency up to 20% of the commercial carrier Lipofect. These results indicated that a simple Zn2+-bzim complex-based composite system can be developed for efficient and low toxic gene delivery through the combination with PEG and CPPs such as TAT. PMID:27433798

  17. A Simple Zn2+ Complex-Based Composite System for Efficient Gene Delivery

    PubMed Central

    Zhang, Zhe; Zhao, Yanjie; Meng, Xianggao; Zhao, Dan; Zhang, Dan; Wang, Li; Liu, Changlin

    2016-01-01

    Metal complexes might become a new type of promising gene delivery systems because of their low cytotoxicity, structural diversity, controllable aqua- and lipo-solubility, and appropriate density and distribution of positive charges. In this study, Zn2+ complexes (1–10) formed with a series of ligands contained benzimidazole(bzim)were prepared and characterized. They were observed to have different affinities for DNA, dependent on their numbers of positive charges, bzim groups, and coordination structures around Zn2+. The binding induced DNA to condensate into spherical nanoparticles with ~ 50 nm in diameter. The cell transfection efficiency of the DNA nanoparticles was poor, although they were low toxic. The sequential addition of the cell-penetrating peptide (CPP) TAT(48–60) and polyethylene glycol (PEG) resulted in the large DNA condensates (~ 100 nm in diameter) and the increased cellular uptake. The clathrin-mediated endocytosis was found to be a key cellular uptake pathway of the nanoparticles formed with or without TAT(48–60) or/and PEG. The DNA nanoparticles with TAT(48–60) and PEG was found to have the cell transfection efficiency up to 20% of the commercial carrier Lipofect. These results indicated that a simple Zn2+-bzim complex-based composite system can be developed for efficient and low toxic gene delivery through the combination with PEG and CPPs such as TAT. PMID:27433798

  18. Enhanced Conversion Efficiencies in Dye-Sensitized Solar Cells Achieved through Self-Assembled Platinum(II) Metallacages

    NASA Astrophysics Data System (ADS)

    He, Zuoli; Hou, Zhiqiang; Xing, Yonglei; Liu, Xiaobin; Yin, Xingtian; Que, Meidan; Shao, Jinyou; Que, Wenxiu; Stang, Peter J.

    2016-07-01

    Two-component self-assembly supramolecular coordination complexes with particular photo-physical property, wherein unique donors are combined with a single metal acceptor, can be utilized for many applications including in photo-devices. In this communication, we described the synthesis and characterization of two-component self-assembly supramolecular coordination complexes (SCCs) bearing triazine and porphyrin faces with promising light-harvesting properties. These complexes were obtained from the self-assembly of a 90° Pt(II) acceptor with 2,4,6-tris(4-pyridyl)-1,3,5-triazine (TPyT) or 5,10,15,20-Tetra(4-pyridyl)-21H,23H-porphine (TPyP). The greatly improved conversion efficiencies of the dye-sensitized TiO2 solar cells were 6.79 and 6.08 respectively, while these SCCs were introduced into the TiO2 nanoparticle film photoanodes. In addition, the open circuit voltage (Voc) of dye-sensitized solar cells was also increased to 0.769 and 0.768 V, which could be ascribed to the inhibited interfacial charge recombination due to the addition of SCCs.

  19. Achieving High Efficiency and Eliminating Degradation in Solid Oxide Electrochemical Cells Using High Oxygen-Capacity Perovskite.

    PubMed

    Jun, Areum; Kim, Junyoung; Shin, Jeeyoung; Kim, Guntae

    2016-09-26

    Recently, there have been efforts to use clean and renewable energy because of finite fossil fuels and environmental problems. Owing to the site-specific and weather-dependent characteristics of the renewable energy supply, solid oxide electrolysis cells (SOECs) have received considerable attention to store energy as hydrogen. Conventional SOECs use Ni-YSZ (yttria-stabilized zirconia) and LSM (strontium-doped lanthanum manganites)-YSZ as electrodes. These electrodes, however, suffer from redox-instability and coarsening of the Ni electrode along with delamination of the LSM electrode during steam electrolysis. In this study, we successfully design and fabricate highly efficient SOECs using layered perovskites, PrBaMn2 O5+δ (PBM) and PrBa0.5 Sr0.5 Co1.5 Fe0.5 O5+δ (PBSCF50), as both electrodes for the first time. The SOEC with layered perovskites as both-side electrodes shows outstanding performance, reversible cycling, and remarkable stability over 600 hours. PMID:27604172

  20. Achieving High Efficiency and Eliminating Degradation in Solid Oxide Electrochemical Cells Using High Oxygen-Capacity Perovskite.

    PubMed

    Jun, Areum; Kim, Junyoung; Shin, Jeeyoung; Kim, Guntae

    2016-09-26

    Recently, there have been efforts to use clean and renewable energy because of finite fossil fuels and environmental problems. Owing to the site-specific and weather-dependent characteristics of the renewable energy supply, solid oxide electrolysis cells (SOECs) have received considerable attention to store energy as hydrogen. Conventional SOECs use Ni-YSZ (yttria-stabilized zirconia) and LSM (strontium-doped lanthanum manganites)-YSZ as electrodes. These electrodes, however, suffer from redox-instability and coarsening of the Ni electrode along with delamination of the LSM electrode during steam electrolysis. In this study, we successfully design and fabricate highly efficient SOECs using layered perovskites, PrBaMn2 O5+δ (PBM) and PrBa0.5 Sr0.5 Co1.5 Fe0.5 O5+δ (PBSCF50), as both electrodes for the first time. The SOEC with layered perovskites as both-side electrodes shows outstanding performance, reversible cycling, and remarkable stability over 600 hours.

  1. An approach for an advanced anode interfacial layer with electron-blocking ability to achieve high-efficiency organic photovoltaics.

    PubMed

    Yeo, Jun-Seok; Yun, Jin-Mun; Kang, Minji; Khim, Dongyoon; Lee, Seung-Hoon; Kim, Seok-Soon; Na, Seok-In; Kim, Dong-Yu

    2014-11-26

    The interfacial properties of PEDOT:PSS, pristine r-GO, and r-GO with sulfonic acid (SR-GO) in organic photovoltaic are investigated to elucidate electron-blocking property of PEDOT:PSS anode interfacial layer (AIL), and to explore the possibility of r-GO as electron-blocking layers. The SR-GO results in an optimized power conversion efficiency of 7.54% for PTB7-th:PC71BM and 5.64% for P3HT:IC61BA systems. By combining analyses of capacitance-voltage and photovoltaic-parameters dependence on light intensity, it is found that recombination process at SR-GO/active film is minimized. In contrast, the devices using r-GO without sulfonic acid show trap-assisted recombination. The enhanced electron-blocking properties in PEDOT:PSS and SR-GO AILs can be attributed to surface dipoles at AIL/acceptor. Thus, for electron-blocking, the AIL/acceptor interface should be importantly considered in OPVs. Also, by simply introducing sulfonic acid unit on r-GO, excellent contact selectivity can be realized in OPVs.

  2. Enhanced Conversion Efficiencies in Dye-Sensitized Solar Cells Achieved through Self-Assembled Platinum(II) Metallacages

    PubMed Central

    He, Zuoli; Hou, Zhiqiang; Xing, Yonglei; Liu, Xiaobin; Yin, Xingtian; Que, Meidan; Shao, Jinyou; Que, Wenxiu; Stang, Peter J.

    2016-01-01

    Two-component self-assembly supramolecular coordination complexes with particular photo-physical property, wherein unique donors are combined with a single metal acceptor, can be utilized for many applications including in photo-devices. In this communication, we described the synthesis and characterization of two-component self-assembly supramolecular coordination complexes (SCCs) bearing triazine and porphyrin faces with promising light-harvesting properties. These complexes were obtained from the self-assembly of a 90° Pt(II) acceptor with 2,4,6-tris(4-pyridyl)-1,3,5-triazine (TPyT) or 5,10,15,20-Tetra(4-pyridyl)-21H,23H-porphine (TPyP). The greatly improved conversion efficiencies of the dye-sensitized TiO2 solar cells were 6.79 and 6.08 respectively, while these SCCs were introduced into the TiO2 nanoparticle film photoanodes. In addition, the open circuit voltage (Voc) of dye-sensitized solar cells was also increased to 0.769 and 0.768 V, which could be ascribed to the inhibited interfacial charge recombination due to the addition of SCCs. PMID:27404912

  3. A new efficient statistical test for detecting variability in the gene expression data.

    PubMed

    Mathur, Sunil; Dolo, Samuel

    2008-08-01

    DNA microarray technology allows researchers to monitor the expressions of thousands of genes under different conditions. The detection of differential gene expression under two different conditions is very important in microarray studies. Microarray experiments are multi-step procedures and each step is a potential source of variance. This makes the measurement of variability difficult because approach based on gene-by-gene estimation of variance will have few degrees of freedom. It is highly possible that the assumption of equal variance for all the expression levels may not hold. Also, the assumption of normality of gene expressions may not hold. Thus it is essential to have a statistical procedure which is not based on the normality assumption and also it can detect genes with differential variance efficiently. The detection of differential gene expression variance will allow us to identify experimental variables that affect different biological processes and accuracy of DNA microarray measurements.In this article, a new nonparametric test for scale is developed based on the arctangent of the ratio of two expression levels. Most of the tests available in literature require the assumption of normal distribution, which makes them inapplicable in many situations, and it is also hard to verify the suitability of the normal distribution assumption for the given data set. The proposed test does not require the assumption of the distribution for the underlying population and hence makes it more practical and widely applicable. The asymptotic relative efficiency is calculated under different distributions, which show that the proposed test is very powerful when the assumption of normality breaks down. Monte Carlo simulation studies are performed to compare the power of the proposed test with some of the existing procedures. It is found that the proposed test is more powerful than commonly used tests under almost all the distributions considered in the study. A

  4. Achieving Extreme Utilization of Excitons by an Efficient Sandwich-Type Emissive Layer Architecture for Reduced Efficiency Roll-Off and Improved Operational Stability in Organic Light-Emitting Diodes.

    PubMed

    Wu, Zhongbin; Sun, Ning; Zhu, Liping; Sun, Hengda; Wang, Jiaxiu; Yang, Dezhi; Qiao, Xianfeng; Chen, Jiangshan; Alshehri, Saad M; Ahamad, Tansir; Ma, Dongge

    2016-02-10

    It has been demonstrated that the efficiency roll-off is generally caused by the accumulation of excitons or charge carriers, which is intimately related to the emissive layer (EML) architecture in organic light-emitting diodes (OLEDs). In this article, an efficient sandwich-type EML structure with a mixed-host EML sandwiched between two single-host EMLs was designed to eliminate this accumulation, thus simultaneously achieving high efficiency, low efficiency roll-off and good operational stability in the resulting OLEDs. The devices show excellent electroluminescence performances, realizing a maximum external quantum efficiency (EQE) of 24.6% with a maximum power efficiency of 105.6 lm W(-1) and a maximum current efficiency of 93.5 cd A(-1). At the high brightness of 5,000 cd m(-2), they still remain as high as 23.3%, 71.1 lm W(-1), and 88.3 cd A(-1), respectively. And, the device lifetime is up to 2000 h at initial luminance of 1000 cd m(-2), which is significantly higher than that of compared devices with conventional EML structures. The improvement mechanism is systematically studied by the dependence of the exciton distribution in EML and the exciton quenching processes. It can be seen that the utilization of the efficient sandwich-type EML broadens the recombination zone width, thus greatly reducing the exciton quenching and increasing the probability of the exciton recombination. It is believed that the design concept provides a new avenue for us to achieve high-performance OLEDs.

  5. SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution.

    PubMed

    Currin, Andrew; Swainston, Neil; Day, Philip J; Kell, Douglas B

    2014-09-01

    The de novo synthesis of genes is becoming increasingly common in synthetic biology studies. However, the inherent error rate (introduced by errors incurred during oligonucleotide synthesis) limits its use in synthesising protein libraries to only short genes. Here we introduce SpeedyGenes, a PCR-based method for the synthesis of diverse protein libraries that includes an error-correction procedure, enabling the efficient synthesis of large genes for use directly in functional screening. First, we demonstrate an accurate gene synthesis method by synthesising and directly screening (without pre-selection) a 747 bp gene for green fluorescent protein (yielding 85% fluorescent colonies) and a larger 1518 bp gene (a monoamine oxidase, producing 76% colonies with full catalytic activity, a 4-fold improvement over previous methods). Secondly, we show that SpeedyGenes can accommodate multiple and combinatorial variant sequences while maintaining efficient enzymatic error correction, which is particularly crucial for larger genes. In its first application for directed evolution, we demonstrate the use of SpeedyGenes in the synthesis and screening of large libraries of MAO-N variants. Using this method, libraries are synthesised, transformed and screened within 3 days. Importantly, as each mutation we introduce is controlled by the oligonucleotide sequence, SpeedyGenes enables the synthesis of large, diverse, yet controlled variant sequences for the purposes of directed evolution.

  6. A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells.

    PubMed

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Matsumoto, Yuko; Ohtsuka, Masato; Miura, Hiromi; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2015-01-01

    Isolation of cells harboring exogenous DNA is typically achieved by the introduction of plasmids, but its efficiency remains still low. In this study, we developed a novel strategy to obtain stable transfectants efficiently. Porcine embryonic fibroblasts were transfected with two plasmids: (i) pTransIEnd, which comprises the ubiquitous promoter, the piggyBac (PB) transposase gene, an internal ribosomal entry site, the Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene, and a poly(A) tail and (ii) a PB-based plasmid, termed pT-EGFP, which contains enhanced green fluorescent protein (EGFP) expression unit flanked by PB acceptor sites. The PB transposase can accelerate the chromosomal integration of transposon vectors. EndoGalC expression results in removal of a cell surface α-Gal epitope, which is specifically recognized by Bandeiraea simplicifolia isolectin-B4 (IB4). Four days after transfection, cells were treated with IB4SAP (IB4 conjugated to saporin, which eliminates any α-Gal epitope-expressing cells) for a short period, followed by standard culture for approximately 10 days. Several colonies emerged, most of which were positive for EGFP expression and lacked TransIEnd. These results indicated that the proposed approach is useful and efficient for obtaining stable transfectants without the use of drug-resistance genes, and offers a novel route for gene manipulation in cultured nonhuman mammalian cells. PMID:25345906

  7. A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells.

    PubMed

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Matsumoto, Yuko; Ohtsuka, Masato; Miura, Hiromi; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2015-01-01

    Isolation of cells harboring exogenous DNA is typically achieved by the introduction of plasmids, but its efficiency remains still low. In this study, we developed a novel strategy to obtain stable transfectants efficiently. Porcine embryonic fibroblasts were transfected with two plasmids: (i) pTransIEnd, which comprises the ubiquitous promoter, the piggyBac (PB) transposase gene, an internal ribosomal entry site, the Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene, and a poly(A) tail and (ii) a PB-based plasmid, termed pT-EGFP, which contains enhanced green fluorescent protein (EGFP) expression unit flanked by PB acceptor sites. The PB transposase can accelerate the chromosomal integration of transposon vectors. EndoGalC expression results in removal of a cell surface α-Gal epitope, which is specifically recognized by Bandeiraea simplicifolia isolectin-B4 (IB4). Four days after transfection, cells were treated with IB4SAP (IB4 conjugated to saporin, which eliminates any α-Gal epitope-expressing cells) for a short period, followed by standard culture for approximately 10 days. Several colonies emerged, most of which were positive for EGFP expression and lacked TransIEnd. These results indicated that the proposed approach is useful and efficient for obtaining stable transfectants without the use of drug-resistance genes, and offers a novel route for gene manipulation in cultured nonhuman mammalian cells.

  8. Efficient, Long-term Hepatic Gene Transfer Using Clinically Relevant HDAd Doses by Balloon Occlusion Catheter Delivery in Nonhuman Primates

    PubMed Central

    Brunetti-Pierri, Nicola; Stapleton, Gary E; Law, Mark; Breinholt, John; Palmer, Donna J; Zuo, Yu; Grove, Nathan C; Finegold, Milton J; Rice, Karen; Beaudet, Arthur L; Mullins, Charles E; Ng, Philip

    2008-01-01

    Helper-dependent adenoviral vectors (HDAd) are devoid of all viral coding sequences and are thus an improvement over early generation Ad because they can provide long-term transgene expression in vivo without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic intravenous injection, and this unfortunately results in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver HDAd into the liver of nonhuman primates. Briefly, a balloon occlusion catheter was percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow. HDAd was injected directly into the occluded liver via a percutaneously placed hepatic artery catheter. Compared to systemic vector injection, this approach resulted in substantially higher hepatic transduction efficiency using clinically relevant low vector doses and was accompanied by mild-to-moderate acute but transient toxicities. Transgene expression was sustained for up to 964 days. These results suggest that our minimally invasive method of delivery can significantly improve the vector's therapeutic index and may be a first step toward clinical application of HDAd for liver-directed gene therapy. PMID:19050700

  9. Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles.

    PubMed

    Li, Shyh-Dar; Chono, Sumio; Huang, Leaf

    2008-02-18

    We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and non-targeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the non-targeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed that the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 microg siRNA/kg). This effective formulation also showed very little immunotoxicity.

  10. Efficient Gene Silencing in Metastatic Tumor by siRNA Formulated in Surface-modified Nanoparticles

    PubMed Central

    Li, Shyh-Dar; Chono, Sumio; Huang, Leaf

    2009-01-01

    We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and non-targeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the non-targeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene, in a lung metastasis model. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 μg siRNA/kg). This effective formulation also showed very little immunotoxicity. PMID:18083264

  11. A Sorghum Mutant Resource as an Efficient Platform for Gene Discovery in Grasses[OPEN

    PubMed Central

    Burke, John; Chen, Junping; Wang, Bo; Hayes, Chad; Emendack, Yves

    2016-01-01

    Sorghum (Sorghum bicolor) is a versatile C4 crop and a model for research in family Poaceae. High-quality genome sequence is available for the elite inbred line BTx623, but functional validation of genes remains challenging due to the limited genomic and germplasm resources available for comprehensive analysis of induced mutations. In this study, we generated 6400 pedigreed M4 mutant pools from EMS-mutagenized BTx623 seeds through single-seed descent. Whole-genome sequencing of 256 phenotyped mutant lines revealed >1.8 million canonical EMS-induced mutations, affecting >95% of genes in the sorghum genome. The vast majority (97.5%) of the induced mutations were distinct from natural variations. To demonstrate the utility of the sequenced sorghum mutant resource, we performed reverse genetics to identify eight genes potentially affecting drought tolerance, three of which had allelic mutations and two of which exhibited exact cosegregation with the phenotype of interest. Our results establish that a large-scale resource of sequenced pedigreed mutants provides an efficient platform for functional validation of genes in sorghum, thereby accelerating sorghum breeding. Moreover, findings made in sorghum could be readily translated to other members of the Poaceae via integrated genomics approaches. PMID:27354556

  12. Implementation of a compressive sampling scheme for wireless sensors to achieve energy efficiency in a structural health monitoring system

    NASA Astrophysics Data System (ADS)

    O'Connor, Sean M.; Lynch, Jerome P.; Gilbert, Anna C.

    2013-04-01

    Wireless sensors have emerged to offer low-cost sensors with impressive functionality (e.g., data acquisition, computing, and communication) and modular installations. Such advantages enable higher nodal densities than tethered systems resulting in increased spatial resolution of the monitoring system. However, high nodal density comes at a cost as huge amounts of data are generated, weighing heavy on power sources, transmission bandwidth, and data management requirements, often making data compression necessary. The traditional compression paradigm consists of high rate (>Nyquist) uniform sampling and storage of the entire target signal followed by some desired compression scheme prior to transmission. The recently proposed compressed sensing (CS) framework combines the acquisition and compression stage together, thus removing the need for storage and operation of the full target signal prior to transmission. The effectiveness of the CS approach hinges on the presence of a sparse representation of the target signal in a known basis, similarly exploited by several traditional compressive sensing applications today (e.g., imaging, MRI). Field implementations of CS schemes in wireless SHM systems have been challenging due to the lack of commercially available sensing units capable of sampling methods (e.g., random) consistent with the compressed sensing framework, often moving evaluation of CS techniques to simulation and post-processing. The research presented here describes implementation of a CS sampling scheme to the Narada wireless sensing node and the energy efficiencies observed in the deployed sensors. Of interest in this study is the compressibility of acceleration response signals collected from a multi-girder steel-concrete composite bridge. The study shows the benefit of CS in reducing data requirements while ensuring data analysis on compressed data remain accurate.

  13. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases.

    PubMed

    Sung, Young Hoon; Kim, Jong Min; Kim, Hyun-Taek; Lee, Jaehoon; Jeon, Jisun; Jin, Young; Choi, Jung-Hwa; Ban, Young Ho; Ha, Sang-Jun; Kim, Cheol-Hee; Lee, Han-Woong; Kim, Jin-Soo

    2014-01-01

    RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F₀ and F₁ mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.

  14. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

    PubMed

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H; Yi, Henry; Collarini, Ellen J; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  15. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

    PubMed Central

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H.; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A.

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  16. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.

    PubMed

    Mok, Wendy W K; Li, Yingfu

    2013-04-15

    Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective. PMID:23512843

  17. Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae

    DOEpatents

    Melis, Anastasios; Mitra, Mautusi

    2010-06-29

    The invention provides method and compositions to minimize the chlorophyll antenna size of photosynthesis by decreasing TLA1 gene expression, thereby improving solar conversion efficiencies and photosynthetic productivity in plants, e.g., green microalgae, under bright sunlight conditions.

  18. Development of a successive targeting liposome with multi-ligand for efficient targeting gene delivery

    PubMed Central

    Ma, Kun; Shen, Haijun; Shen, Song; Xie, Men; Mao, Chuanbin; Qiu, Liyan; Jin, Yi

    2012-01-01

    Background A successful gene delivery system needs to breakthrough several barriers to allow efficient transgenic expression. In the present study, successive targeting liposomes (STL) were constructed by integrating various targeting groups into a nanoparticle to address this issue. Methods Polyethylenimine (PEI) 1800-triamcinolone acetonide (TA) with nuclear targeting capability was synthesized by a two-step reaction. Lactobionic acid was connected with cholesterol to obtain a compound of [(2-lactoylamido) ethylamino]formic acid cholesterol ester (CHEDLA) with hepatocyte-targeting capability. The liposome was modified with PEI 1800-TA and CHEDLA to prepare successive targeting liposome (STL). Its physicochemical properties and transfection efficiency were investigated both in vitro and in vivo. Results The diameter of STL was approximately 100 nm with 20 mV of potential. The confocal microscopy observation and potential assay verified that lipid bilayer of STL was decorated with PEI 1800-TA. Cytotoxicity of STL was significantly lower than that of PEI 1800-TA and PEI 25K. The transfection efficiency of 10% CHEDLA STL in HepG2 cells was the higher than of the latter two with serum. Its transfection efficiency was greatly reduced with excessive free galactose, indicating that STL was absorbed via galactose receptor-mediated endocytosis. The in vivo study in mice showed that 10% CHEDLA STL had better transgenic expression in liver than the other carriers. Conclusions STL with multi-ligand was able to overcome the various barriers to target nucleus and special cells and present distinctive transgenic expression. Therefore, it has a great potential for gene therapy as a nonviral carrier. PMID:21574214

  19. Nanosized bioceramic particles could function as efficient gene delivery vehicles with target specificity for the spleen.

    PubMed

    Tan, K; Cheang, P; Ho, I A W; Lam, P Y P; Hui, K M

    2007-05-01

    We have compared the ability of several nanosized bioceramic particles including negatively charged silica (SiO(2)), neutrally charged hydroxyapatite (HA) and positively charged zirconia (ZrO(2)) nanoparticles as non-viral vectors for efficient in vivo gene delivery. A mixture of highly monodispersed aqueous suspension of HA or SiO(2) nanoparticles, coated with protamine sulfate (PS), complexed efficiently with plasmid DNA and significantly enhanced transgene expression in vitro. In comparison, ZrO(2) nanoparticles gave poor transfection efficiency under similar conditions tested. It was also determined that, under the same conditions, PS-SiO(2)-DNA, but not PS-HA-DNA-nanoplexes, were able to mediate efficient transgene expression in vitro in the presence of 50% serum. Intraperitoneal injections of PS-SiO(2)-luciferase DNA nanoplexes targeted the highest level of transgene expression in the spleen of recipient mice that lasted for more than 48 h. Injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes was able to mediate the production of Flt-3L in the sera of recipient mice. Simultaneously, the production of Flt-3L was accompanied by the stimulation of IL-2 and interferon-gamma (IFN-gamma). Most importantly, the injection of PS-SiO(2)-pNGVL-hFLex-MUC-1 nanoplexes could mount potent anti-tumour specific immune responses that led to the subsequent regression of parental tumor cells containing the muc-1 determinant.

  20. An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples.

    PubMed

    Scolnick, Jonathan A; Dimon, Michelle; Wang, I-Ching; Huelga, Stephanie C; Amorese, Douglas A

    2015-01-01

    Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells. PMID:26132974

  1. Functional redundancy of promoter elements ensures efficient transcription of the human 7SK gene in vivo.

    PubMed

    Boyd, D C; Turner, P C; Watkins, N J; Gerster, T; Murphy, S

    1995-11-10

    Deletion and mutation studies of the human 7SK gene transfected into HeLa cells have identified three functional regions of the promoter corresponding to the TATA box at -25, the proximal sequence element (PSE) between -49 and -65 and the distal sequence element (DSE) between -243 and -210. These elements show sequence homology to equivalent regions in other snRNA genes and are functionally analogous. Unlike the DSEs of many snRNA genes however, the 7SK DSE does not contain a consensus binding site for the transcription factor Oct-1 but rather, contains two non-consensus Oct-1 binding sites that can function independently of one another to enhance transcription. Unusually, the 7SK PSE can retain function even after extensive mutation and removal of the conserved TGACC of the PSE has little effect in the context of the whole promoter. However, the same mutation abolishes transcription in the absence of the DSE suggesting that protein/protein interactions between DSE and PSE binding factors can compensate for a mutant PSE. Mutation of the 7SK TATA box allows snRNA type transcription by RNA polymerase II to occur and this is enhanced by the DSE, indicating that both the DSE and the PSE can also function with pol II. In addition, mutation of the TATA box does not abolish pol III dependent transcription, suggesting that other sequence elements may also play a role in the determination of polymerase specificity. Although the human 7SK gene is transcribed efficiently in Xenopus oocytes, analysis of the 7SK wild-type gene and mutants in Xenopus oocytes gives significantly different results from the analysis in HeLa cells indicating that the recognition of functional elements is not the same in the two systems.

  2. Integrase-Deficient Lentiviral Vectors Mediate Efficient Gene Transfer to Human Vascular Smooth Muscle Cells with Minimal Genotoxic Risk

    PubMed Central

    Chick, Helen E.; Nowrouzi, Ali; Fronza, Raffaele; McDonald, Robert A.; Kane, Nicole M.; Alba, Raul; Delles, Christian; Sessa, William C.; Schmidt, Manfred; Thrasher, Adrian J.

    2012-01-01

    Abstract We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer. PMID:22931362

  3. Spermine-alt-poly(ethylene glycol) polyspermine as a safe and efficient aerosol gene carrier for lung cancer therapy.

    PubMed

    Kim, You-Kyoung; Cho, Chong-Su; Cho, Myung-Haing; Jiang, Hu-Lin

    2014-07-01

    The clinical success of gene therapy critically depends upon the safety and efficiency of delivery system used. Although polyethylenimine (PEI) has been commonly used as an efficient cationic polymeric gene carrier due to its high transfection efficiency, its cytotoxicity and nondegradability limit the polymer's therapeutic applications in clinical trials. In this study, biocompatible polyspermine based on spermine (SPE) and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) was synthesized using a Michael-type addition reaction, and its ability as an alternative gene carrier for lung cancer therapy was evaluated. SPE-alt-PEG polyspermine was complexed with plasmid DNA, and the resulting complexes were characterized by particle size and surface charge by dynamic light scattering, complex formation and DNA protection ability by gel retardation, and complex shape by energy-filtering transmission electron microscopy. The SPE-alt-PEG copolymer showed low cytotoxicity, and SPE-alt-PEG/DNA complexes showed efficacious transfection efficiency compared with 25 kDa PEI (PEI 25K). Also SPE-alt-PEG/GFP complexes were efficiently transferred into the lungs after aerosol administration without toxicity, and delivery of Pdcd4 gene as a therapeutic gene with SPE-alt-PEG polyspermine greatly reduced tumor size as well as tumor numbers in K-ras(LA1) lung cancer model mice compared relative to the effect observed for PEI 25K. These results suggest that SPE-alt-PEG has potential as a gene carrier for lung cancer gene therapy. PMID:23929634

  4. Efficient, high-brightness wavelength-beam-combined commercial off-the-shelf diode stacks achieved by use of a wavelength-chirped volume Bragg grating.

    PubMed

    Chann, B; Goyal, A K; Fan, T Y; Sanchez-Rubio, A; Volodin, B L; Ban, V S

    2006-05-01

    We report a method of scaling the spatial brightness from commercial off-the-shelf diode laser stacks through wavelength beam combining, by use of a linearly wavelength-chirped volume Bragg grating (VBG). Using a three-bar commercial stack of broad-area lasers and a VBG, we demonstrate 89.5 W cw of beam-combined output with a beam-combining efficiency of 75%. The output beam has a propagation factor M2 approximately 26 on the slow axis and M2 approximately 21 on the fast axis. This corresponds to a brightness of approximately 20 MW/cm2 sr. To our knowledge, this is the highest brightness broad-area diode laser system. We achieve 81% coupling efficiency into a 100 microm, 0.22 N.A. fiber.

  5. Association analysis between feed efficiency studies and expression of hypothalamic neuropeptide genes in laying ducks.

    PubMed

    Zeng, T; Chen, L; Du, X; Lai, S J; Huang, S P; Liu, Y L; Lu, L Z

    2016-10-01

    Residual feed intake (RFI) is now considered a more reasonable metric to evaluate animal feed efficiency. In this study, the correlation between RFI and other feed efficiency traits was investigated and gene expression within the hypothalamus was determined in low RFI (LRFI) and high RFI (HRFI) ducks. Further, several hypothalamic neuropeptide genes were measured using quantitative real-time PCR. The mean feed intake value was 160 g/day, whereas the egg mass laid (EML) and body weight were approximately 62.4 g/day and 1.46 kg respectively. Estimates for heritability of RFI, feed conversion ratio (FCR) and feed intake were 0.26, 0.18 and 0.23 respectively. RFI is phenotypically positively correlated with feed intake and FCR (P < 0.01). The expression of neuropeptide Y (NPY) and neuropeptide Y receptor Y5 (NPY5R) mRNA was higher in HRFI ducks compared with LRFI ducks (P < 0.05), whereas that of proopiomelanocortin (POMC), melanocortin 4 receptor (MC4R) and cholecystokinin (CCK) was lower (P < 0.05). The mRNA expression of gonadotropin-releasing hormone 1 (luteinizing-releasing hormone) (GNRH1) and prolactin receptor (PRLR) was unchanged between LRFI and HRFI ducks. The results indicate that selection for LRFI could reduce feed intake without significant changes in EML, whereas selection on FCR will increase EML. PMID:27329478

  6. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization.

    PubMed

    Anahtar, Melis N; Bowman, Brittany A; Kwon, Douglas S

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  7. Association analysis between feed efficiency studies and expression of hypothalamic neuropeptide genes in laying ducks.

    PubMed

    Zeng, T; Chen, L; Du, X; Lai, S J; Huang, S P; Liu, Y L; Lu, L Z

    2016-10-01

    Residual feed intake (RFI) is now considered a more reasonable metric to evaluate animal feed efficiency. In this study, the correlation between RFI and other feed efficiency traits was investigated and gene expression within the hypothalamus was determined in low RFI (LRFI) and high RFI (HRFI) ducks. Further, several hypothalamic neuropeptide genes were measured using quantitative real-time PCR. The mean feed intake value was 160 g/day, whereas the egg mass laid (EML) and body weight were approximately 62.4 g/day and 1.46 kg respectively. Estimates for heritability of RFI, feed conversion ratio (FCR) and feed intake were 0.26, 0.18 and 0.23 respectively. RFI is phenotypically positively correlated with feed intake and FCR (P < 0.01). The expression of neuropeptide Y (NPY) and neuropeptide Y receptor Y5 (NPY5R) mRNA was higher in HRFI ducks compared with LRFI ducks (P < 0.05), whereas that of proopiomelanocortin (POMC), melanocortin 4 receptor (MC4R) and cholecystokinin (CCK) was lower (P < 0.05). The mRNA expression of gonadotropin-releasing hormone 1 (luteinizing-releasing hormone) (GNRH1) and prolactin receptor (PRLR) was unchanged between LRFI and HRFI ducks. The results indicate that selection for LRFI could reduce feed intake without significant changes in EML, whereas selection on FCR will increase EML.

  8. Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

    PubMed Central

    Anahtar, Melis N.; Bowman, Brittany A.; Kwon, Douglas S.

    2016-01-01

    There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information. PMID:27168460

  9. Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

    PubMed Central

    Michaud, Edward J; Culiat, Cymbeline T; Klebig, Mitchell L; Barker, Paul E; Cain, KT; Carpenter, Debra J; Easter, Lori L; Foster, Carmen M; Gardner, Alysyn W; Guo, ZY; Houser, Kay J; Hughes, Lori A; Kerley, Marilyn K; Liu, Zhaowei; Olszewski, Robert E; Pinn, Irina; Shaw, Ginger D; Shinpock, Sarah G; Wymore, Ann M; Rinchik, Eugene M; Johnson, Dabney K

    2005-01-01

    Background Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results We produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations. PMID:16300676

  10. Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

    SciTech Connect

    Michaud III, Edward J; Culiat, Cymbeline T; Klebig, Mitch; Barker, Gene; Cain, K T; Carpenter, Debra J S; Easter, Lori L; Foster, Carmen M; Gardner, Alysyn Wallace; Guo, ZY; Houser, Kay J; Hughes, Lori A; Kerley, Marilyn K; Liu, Zhaowei; Olszewski, Robert Edward; Pinn, Irina; Shaw, Ginger D; Shinpock, Sarah G; Wymore, Ann; Rinchik, Eugene M; Johnson, Dabney K

    2005-01-01

    Background: Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results: We produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions: The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

  11. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    SciTech Connect

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  12. Baculoviruses mediate efficient gene expression in a wide range of vertebrate cells.

    PubMed

    Airenne, Kari J; Makkonen, Kaisa-Emilia; Mähönen, Anssi J; Ylä-Herttuala, Seppo

    2011-01-01

    Baculovirus expression vector system (BEVS) is well known as a feasible and safe technology to produce recombinant (re-)proteins in a eukaryotic milieu of insect cells. However, its proven power in gene delivery and gene therapy is still poorly recognized. The basis of BEVS lies in large enveloped DNA viruses derived from insects, the prototype virus being Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Infection of insect cell culture with a virus encoding a desired transgene under powerful baculovirus promoter leads to re-protein production in high quantities. Although the replication of AcMNPV is highly insect specific in nature, it can penetrate and transduce a wide range of cells of other origin. Efficient transduction requires only virus arming with an expression cassette active in the cells under investigation. The inherent safety, ease and speed of virus generation in high quantities, low cytotoxicity and extreme transgene capacity and tropism provides many advantages for gene delivery over the other viral vectors typically derived from human pathogens.

  13. Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes

    PubMed Central

    Hong, Ran; Bai, Weiya; Zhai, Jianwei; Liu, Wei; Li, Xinyan; Zhang, Jiming; Cui, Xiaoxian; Zhao, Xue; Ye, Xiaoli; Deng, Qiang; Tiollais, Pierre; Wen, Yumei

    2013-01-01

    Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection. PMID:23552416

  14. The gene transfection efficiency of a folate-PEI600-cyclodextrin nanopolymer.

    PubMed

    Yao, Hong; Ng, Samuel S; Tucker, Wesley O; Tsang, Yuk-Kai-Tiu; Man, Kwan; Wang, Xiao-Mei; Chow, Billy K C; Kung, Hsiang-Fu; Tang, Gu-Ping; Lin, Marie C

    2009-10-01

    The success of gene therapy relies on a safe and effective gene delivery system. In this communication, we describe the use of folate grafted PEI(600)-CyD (H(1)) as an effective polyplex-forming plasmid delivery agent with low toxicity. The structures of the polymer and polyplex were characterized, and the in vitro transfection efficiency, cytotoxicity, and in vivo transfection of H(1) were examined. We found that folate molecules were successfully grafted to PEI(600)-CyD. At N/P ratios between 5 and 30, the resulting H(1)/DNA polyplexes had diameters less than 120 nm and zeta potentials less than 10 mV. In various tumor cell lines examined (U138, U87, B16, and Lovo), the in vitro transfection efficiency of H(1) was more than 50%, which could be improved by the presence of fetal bovine serum or albumin. The cytotoxicity of H(1) was significantly less than high molecular weight PEI-25 kDa. Importantly, in vivo optical imaging showed that the efficiency of H(1)-mediated transfection (50 microg luciferase plasmid (pLuc), N/P ratio=20/1) was comparable to that of adenovirus-mediated luciferase transduction (1 x 10(9) pfu) in melanoma-bearing mice, and it did not induce any toxicity in the tumor tissue. These results clearly show that H(1) is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA and its application warrants further investigation. PMID:19615741

  15. The combination of a synthetic promoter and a CMV promoter improves foreign gene expression efficiency in myocytes.

    PubMed

    Jianwei, Dai; Qianqian, Zhang; Songcai, Liu; Mingjun, Zhang; Xiaohui, Ren; Linlin, Hao; Qingyan, Jiang; Yongliang, Zhang

    2012-04-15

    Skeletal muscle is becoming an attractive target tissue for gene therapy. Nevertheless, the low level of gene therapeutic expression in this tissue is the major limitation to it becoming an ideal target for gene transfer. The promoter is important element for gene transcription; however, the gene expression efficiencies and specificities of viral promoters and skeletal muscle-specific promotors are in themselves limiting factors. In this study, we established a dual-promoters system in skeletal muscle using a cytomegalovirus (CMV) promoter and a skeletal muscle-specific synthetic promoter. Mouse myoblast cell line C2C12 cells were transfected with the system. We demonstrated that the dual-promoters system could significantly improve exogenous gene expression rate in vitro when compared with a single CMV promoter system and a skeletal muscle-specific synthetic promoter system in C2C12 cell line, by 69.48% and 41.93%, respectively. Next, we evaluated the system efficiency in vivo, the results showed that the dual-promoters system increased gene expression in mice 1.23-fold and 1.60-fold, respectively compared with expression controlled by the two single promoter vectors. Finally, we tested the dual-promoters system in growth hormone-releasing hormone (GHRH) gene therapy, and revealed that when these two promoters co-drove the GHRH gene expression in vivo animal growth was enhanced significantly. All these results indicate that use of the dual-promoter vector was more efficient for gene expression in skeletal muscle tissue than use of the single promoter vectors. These finding could, hopefully, lead to the development of a high efficiency expression system in myocytes and form an ideal approach for gene therapy.

  16. Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure, with Concomitant Bacterial Population Changes

    PubMed Central

    Wang, Hang; Li, Hongyi; Gilbert, Jack A.; Li, Haibo; Wu, Longhua; Liu, Meng; Wang, Liling; Zhou, Qiansheng; Yuan, Junxiang

    2015-01-01

    Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P < 0.05), while those of genes encoding sulfonamide resistance (sul1 and sul2) were increased (P < 0.05) when normalized to 16S rRNA. The abundances of tetracycline resistance genes were correlated (P < 0.05) with the changing concentrations of tetracyclines in the manure. The overall diversity and richness of the bacteria significantly decreased during vermicomposting, accompanied by a 100 times increase in the relative abundance of Flavobacteriaceae spp. Variations in the abundances of ARGs were correlated with the changing microbial community structure and the relative abundances of the family Ruminococcaceae, class Bacilli, or phylum Proteobacteria. Vermicomposting, as a waste management practice, can reduce the overall abundance of ARGs. More research is warranted to assess the use of this waste management practice as a measure to attenuate the dissemination of antimicrobial residues and ARGs from livestock production before vermicompost can be safely used as biofertilizer in agroecosystems. PMID:26296728

  17. Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure, with Concomitant Bacterial Population Changes.

    PubMed

    Wang, Hang; Li, Hongyi; Gilbert, Jack A; Li, Haibo; Wu, Longhua; Liu, Meng; Wang, Liling; Zhou, Qiansheng; Yuan, Junxiang; Zhang, Zhijian

    2015-11-01

    Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P < 0.05), while those of genes encoding sulfonamide resistance (sul1 and sul2) were increased (P < 0.05) when normalized to 16S rRNA. The abundances of tetracycline resistance genes were correlated (P < 0.05) with the changing concentrations of tetracyclines in the manure. The overall diversity and richness of the bacteria significantly decreased during vermicomposting, accompanied by a 100 times increase in the relative abundance of Flavobacteriaceae spp. Variations in the abundances of ARGs were correlated with the changing microbial community structure and the relative abundances of the family Ruminococcaceae, class Bacilli, or phylum Proteobacteria. Vermicomposting, as a waste management practice, can reduce the overall abundance of ARGs. More research is warranted to assess the use of this waste management practice as a measure to attenuate the dissemination of antimicrobial residues and ARGs from livestock production before vermicompost can be safely used as biofertilizer in agroecosystems. PMID:26296728

  18. Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure, with Concomitant Bacterial Population Changes.

    PubMed

    Wang, Hang; Li, Hongyi; Gilbert, Jack A; Li, Haibo; Wu, Longhua; Liu, Meng; Wang, Liling; Zhou, Qiansheng; Yuan, Junxiang; Zhang, Zhijian

    2015-11-01

    Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P < 0.05), while those of genes encoding sulfonamide resistance (sul1 and sul2) were increased (P < 0.05) when normalized to 16S rRNA. The abundances of tetracycline resistance genes were correlated (P < 0.05) with the changing concentrations of tetracyclines in the manure. The overall diversity and richness of the bacteria significantly decreased during vermicomposting, accompanied by a 100 times increase in the relative abundance of Flavobacteriaceae spp. Variations in the abundances of ARGs were correlated with the changing microbial community structure and the relative abundances of the family Ruminococcaceae, class Bacilli, or phylum Proteobacteria. Vermicomposting, as a waste management practice, can reduce the overall abundance of ARGs. More research is warranted to assess the use of this waste management practice as a measure to attenuate the dissemination of antimicrobial residues and ARGs from livestock production before vermicompost can be safely used as biofertilizer in agroecosystems.

  19. Agrobacterium-mediated transformation of Guignardia citricarpa: an efficient tool to gene transfer and random mutagenesis.

    PubMed

    Rodrigues, Maria Beatriz Calderan; Fávaro, Léia Cecília de Lima; Pallu, Ana Paula de Souza; Ferreira, Anderson; Sebastianes, Fernanda de Souza; Rodrigues, Maria Juliana Calderan; Spósito, Marcel Bellato; de Araújo, Welington Luiz; Pizzirani-Kleiner, Aline Aparecida

    2013-01-01

    Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

  20. Efficient gene delivery system mediated by cis-aconitate-modified chitosan-g-stearic acid micelles.

    PubMed

    Yao, Jing-Jing; Du, Yong-Zhong; Yuan, Hong; You, Jian; Hu, Fu-Qiang

    2014-01-01

    Cis-aconitate-modified chitosan-g-stearic acid (CA-CSO-SA) micelles were synthesized in this study to improve the gene transfection efficiency of chitosan-g-stearic acid (CSO-SA). The CA-CSO-SA micelles had a similar size, critical micelle concentration, and morphology, but their zeta potential and cytotoxicity were reduced compared with CSO-SA micelles. After modification with cis-aconitate, the CA-CSO-SA micelles could also compact plasmid DNA (pDNA) to form nanocomplexes. However, the DNA binding ability of CA-CSO-SA was slightly reduced compared with that of CSO-SA. The transfection efficiency mediated by CA-CSO-SA/pDNA against HEK-293 cells reached up to 37%, and was much higher than that of CSO-SA/pDNA (16%). Although the cis-aconitate modification reduced cellular uptake kinetics in the initial stages, the total amount of cellular uptake tended to be the same after 24 hours of incubation. An endocytosis inhibition experiment showed that the internalization mechanism of CA-CSO-SA/pDNA in HEK-293 cells was mainly via clathrin-mediated endocytosis, as well as caveolae-mediated endocytosis and macropinocytosis. Observation of intracellular trafficking indicated that the CSO-SA/pDNA complexes were trapped in endolysosomes, but CA-CSO-SA/pDNA was more widely distributed in the cytosol. This study suggests that modification with cis-aconitate improves the transfection efficiency of CSO-SA/pDNA.

  1. Triolein-based polycation lipid nanocarrier for efficient gene delivery: characteristics and mechanism.

    PubMed

    Zhang, Zhiwen; Fang, Xiaoling; Hao, Junguo; Li, Yajuan; Sha, Xianyi

    2011-01-01

    We proposed to develop a polycation lipid nanocarrier (PLN) with higher transfection efficiency than our previously described polycation nanostrucutred lipid nanocarrier (PNLC). PLN was composed of triolein, cetylated low-molecular-weight polyethylenimine, and dioleoyl phosphatidylethanolamine. The physicochemical properties of PLN and the PLN/DNA complexes (PDC) were characterized. The in vitro transfection was performed in human lung adenocarcinoma (SPC-A1) cells, and the intracellular mechanism was investigated as well. The measurements indicated that PLN and PDC are homogenous nanometer-sized particles with a positive charge. The transfection efficiency of PDC significantly increased with the content of triolein and was higher than that of PNLC and commercial Lipofectamine 2000. In particular, the transfection of PLN in the presence of 10% serum was more effective than that in its absence. With the help of specific inhibitors of chlorpromazine and filipin, the clathrin-dependent endocytosis pathway was determined to be the main contributor to the successful transfection mediated by PLN in SPC-A1 cells. The captured images verified that the fluorescent PDC was localized in the lysosomes and nuclei after endocytosis. Thus, PLN represents a novel efficient nonviral gene delivery vector. PMID:22114487

  2. Establishment of medakafish as a model for stem cell-based gene therapy: efficient gene delivery and potential chromosomal integration by baculoviral vectors.

    PubMed

    Yan, Yan; Du, Juan; Chen, Tiansheng; Yi, Meisheng; Li, Mingyou; Wang, Shu; Li, Chang Ming; Hong, Yunhan

    2009-08-01

    Viral vectors hold promise and challenges in gene therapy. Specifically, we have previously shown that baculoviral (BV) vectors have a high efficiency of gene delivery in human embryonic stem (ES) cells. Here we report the development of a complementary system to further our evaluation by utilizing the laboratory fish medaka that has ES cell lines and tools for experimental analyses in vitro and in vivo. We show that BV vectors can give rise to almost 100% of transient gene delivery in the medaka ES cell line MES1. BV-transduced MES1 cells reproducibly (at approximately 10(-5)) produce GFP-expressing colonies that, upon manual isolation, develop into stable clones during 300 days of culture. Surprisingly, BV transduction can also mediate efficient gene integration in the medaka genome, as fluorescent in situ hybridization revealed the presence of the BV-delivered gfp transgene in multiple locations in nuclei and on various chromosomes of metaphase spreads. We show that BV transduction does not compromise the genome stability and pluripotency of MES1 cells. We conclude that BV can efficiently mediate gene delivery and chromosomal integration in medaka ES cells. Therefore, medaka provides a powerful system for analyzing the potential of BV-mediated gene delivery in stem cells and gene therapy.

  3. Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells.

    PubMed

    Liu, Zhongliang; Hui, Yi; Shi, Lei; Chen, Zhenyu; Xu, Xiangjie; Chi, Liankai; Fan, Beibei; Fang, Yujiang; Liu, Yang; Ma, Lin; Wang, Yiran; Xiao, Lei; Zhang, Quanbin; Jin, Guohua; Liu, Ling; Zhang, Xiaoqing

    2016-09-13

    Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of microRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs. PMID:27594587

  4. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi

    PubMed Central

    Kaulich, Manuel; Lee, Yeon J.; Lönn, Peter; Springer, Aaron D.; Meade, Bryan R.; Dowdy, Steven F.

    2015-01-01

    Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. PMID:25586224

  5. Efficient dye regeneration at low driving force achieved in triphenylamine dye LEG4 and TEMPO redox mediator based dye-sensitized solar cells.

    PubMed

    Yang, Wenxing; Vlachopoulos, Nick; Hao, Yan; Hagfeldt, Anders; Boschloo, Gerrit

    2015-06-28

    Minimizing the driving force required for the regeneration of oxidized dyes using redox mediators in an electrolyte is essential to further improve the open-circuit voltage and efficiency of dye-sensitized solar cells (DSSCs). Appropriate combinations of redox mediators and dye molecules should be explored to achieve this goal. Herein, we present a triphenylamine dye, LEG4, in combination with a TEMPO-based electrolyte in acetonitrile (E(0) = 0.89 V vs. NHE), reaching an efficiency of up to 5.4% under one sun illumination and 40% performance improvement compared to the previously and widely used indoline dye D149. The origin of this improvement was found to be the increased dye regeneration efficiency of LEG4 using the TEMPO redox mediator, which regenerated more than 80% of the oxidized dye with a driving force of only ∼0.2 eV. Detailed mechanistic studies further revealed that in addition to electron recombination to oxidized dyes, recombination of electrons from the conducting substrate and the mesoporous TiO2 film to the TEMPO(+) redox species in the electrolyte accounts for the reduced short circuit current, compared to the state-of-the-art cobalt tris(bipyridine) electrolyte system. The diffusion length of the TEMPO-electrolyte based DSSCs was determined to be ∼0.5 μm, which is smaller than the ∼2.8 μm found for cobalt-electrolyte based DSSCs. These results show the advantages of using LEG4 as a sensitizer, compared to previously record indoline dyes, in combination with a TEMPO-based electrolyte. The low driving force for efficient dye regeneration presented by these results shows the potential to further improve the power conversion efficiency (PCE) of DSSCs by utilizing redox couples and dyes with a minimal need of driving force for high regeneration yields.

  6. Efficient dye regeneration at low driving force achieved in triphenylamine dye LEG4 and TEMPO redox mediator based dye-sensitized solar cells.

    PubMed

    Yang, Wenxing; Vlachopoulos, Nick; Hao, Yan; Hagfeldt, Anders; Boschloo, Gerrit

    2015-06-28

    Minimizing the driving force required for the regeneration of oxidized dyes using redox mediators in an electrolyte is essential to further improve the open-circuit voltage and efficiency of dye-sensitized solar cells (DSSCs). Appropriate combinations of redox mediators and dye molecules should be explored to achieve this goal. Herein, we present a triphenylamine dye, LEG4, in combination with a TEMPO-based electrolyte in acetonitrile (E(0) = 0.89 V vs. NHE), reaching an efficiency of up to 5.4% under one sun illumination and 40% performance improvement compared to the previously and widely used indoline dye D149. The origin of this improvement was found to be the increased dye regeneration efficiency of LEG4 using the TEMPO redox mediator, which regenerated more than 80% of the oxidized dye with a driving force of only ∼0.2 eV. Detailed mechanistic studies further revealed that in addition to electron recombination to oxidized dyes, recombination of electrons from the conducting substrate and the mesoporous TiO2 film to the TEMPO(+) redox species in the electrolyte accounts for the reduced short circuit current, compared to the state-of-the-art cobalt tris(bipyridine) electrolyte system. The diffusion length of the TEMPO-electrolyte based DSSCs was determined to be ∼0.5 μm, which is smaller than the ∼2.8 μm found for cobalt-electrolyte based DSSCs. These results show the advantages of using LEG4 as a sensitizer, compared to previously record indoline dyes, in combination with a TEMPO-based electrolyte. The low driving force for efficient dye regeneration presented by these results shows the potential to further improve the power conversion efficiency (PCE) of DSSCs by utilizing redox couples and dyes with a minimal need of driving force for high regeneration yields. PMID:26016854

  7. Impact of 5-aminolevulinic acid with iron supplementation on exercise efficiency and home-based walking training achievement in older women.

    PubMed

    Masuki, Shizue; Morita, Atsumi; Kamijo, Yoshi-ichiro; Ikegawa, Shigeki; Kataoka, Yufuko; Ogawa, Yu; Sumiyoshi, Eri; Takahashi, Kiwamu; Tanaka, Tohru; Nakajima, Motowo; Nose, Hiroshi

    2016-01-01

    A reduction in exercise efficiency with aging limits daily living activities. We examined whether 5-aminolevulinic acid (ALA) with sodium ferrous citrate (SFC) increased exercise efficiency and voluntary achievement of interval walking training (IWT) in older women. Ten women [65 ± 3(SD) yr] who had performed IWT for >12 mo and were currently performing IWT participated in this study. The study was conducted in a placebo-controlled, double-blind crossover design. All subjects underwent two trials for 7 days each in which they performed IWT with ALA+SFC (100 and 115 mg/day, respectively) or placebo supplement intake (CNT), intermittently with a 2-wk washout period. Before and after each trial, subjects underwent a graded cycling test at 27.0 °C atmospheric temperature and 50% relative humidity, and oxygen consumption rate, carbon dioxide production rate, and lactate concentration in plasma were measured. Furthermore, for the first 6 days of each trial, exercise intensity for IWT was measured by accelerometry. We found that, in the ALA+SFC trial, oxygen consumption rate and carbon dioxide production rate during graded cycling decreased by 12% (P < 0.001) and 11% (P = 0.001) at every workload, respectively, accompanied by a 16% reduction in lactate concentration in plasma (P < 0.001), although all remained unchanged in the CNT trial (P > 0.2). All of the reductions were significantly greater in the ALA+SFC than the CNT trial (P < 0.05). Furthermore, the training days, impulse, and time at fast walking were 42% (P = 0.028), 102% (P = 0.027), and 69% (P = 0.039) higher during the ALA+SFC than the CNT intake period, respectively. Thus ALA+SFC supplementation augmented exercise efficiency and thereby improved IWT achievement in older women.

  8. Impact of 5-aminolevulinic acid with iron supplementation on exercise efficiency and home-based walking training achievement in older women

    PubMed Central

    Masuki, Shizue; Morita, Atsumi; Kamijo, Yoshi-ichiro; Ikegawa, Shigeki; Kataoka, Yufuko; Ogawa, Yu; Sumiyoshi, Eri; Takahashi, Kiwamu; Tanaka, Tohru; Nakajima, Motowo

    2015-01-01

    A reduction in exercise efficiency with aging limits daily living activities. We examined whether 5-aminolevulinic acid (ALA) with sodium ferrous citrate (SFC) increased exercise efficiency and voluntary achievement of interval walking training (IWT) in older women. Ten women [65 ± 3(SD) yr] who had performed IWT for >12 mo and were currently performing IWT participated in this study. The study was conducted in a placebo-controlled, double-blind crossover design. All subjects underwent two trials for 7 days each in which they performed IWT with ALA+SFC (100 and 115 mg/day, respectively) or placebo supplement intake (CNT), intermittently with a 2-wk washout period. Before and after each trial, subjects underwent a graded cycling test at 27.0°C atmospheric temperature and 50% relative humidity, and oxygen consumption rate, carbon dioxide production rate, and lactate concentration in plasma were measured. Furthermore, for the first 6 days of each trial, exercise intensity for IWT was measured by accelerometry. We found that, in the ALA+SFC trial, oxygen consumption rate and carbon dioxide production rate during graded cycling decreased by 12% (P < 0.001) and 11% (P = 0.001) at every workload, respectively, accompanied by a 16% reduction in lactate concentration in plasma (P < 0.001), although all remained unchanged in the CNT trial (P > 0.2). All of the reductions were significantly greater in the ALA+SFC than the CNT trial (P < 0.05). Furthermore, the training days, impulse, and time at fast walking were 42% (P = 0.028), 102% (P = 0.027), and 69% (P = 0.039) higher during the ALA+SFC than the CNT intake period, respectively. Thus ALA+SFC supplementation augmented exercise efficiency and thereby improved IWT achievement in older women. PMID:26514619

  9. Highly Functional TNTs with Superb Photocatalytic, Optical, and Electronic Performance Achieving Record PV Efficiency of 10.1% for 1D-Based DSSCs.

    PubMed

    Qadir, Muhammad Bilal; Li, Yuewen; Sahito, Iftikhar Ali; Arbab, Alvira Ayoub; Sun, Kyung Chul; Mengal, Naveed; Memon, Anam Ali; Jeong, Sung Hoon

    2016-09-01

    Different nanostructures of TiO2 play an important role in the photocatalytic and photoelectronic applications. TiO2 nanotubes (TNTs) have received increasing attention for these applications due to their unique physicochemical properties. Focusing on highly functional TNTs (HF-TNTs) for photocatalytic and photoelectronic applications, this study describes the facile hydrothermal synthesis of HF-TNTs by using commercial and cheaper materials for cost-effective manufacturing. To prove the functionality and applicability, these TNTs are used as scattering structure in dye-sensitized solar cells (DSSCs). Photocatalytic, optical, Brunauer-Emmett-Teller (BET), electrochemical impedance spectrum, incident-photon-to-current efficiency, and intensity-modulated photocurrent spectroscopy/intensity-modulated photovoltage spectroscopy characterizations are proving the functionality of HF-TNTs for DSSCs. HF-TNTs show 50% higher photocatalytic degradation rate and also 68% higher dye loading ability than conventional TNTs (C-TNTs). The DSSCs having HF-TNT and its composite-based multifunctional overlayer show effective light absorption, outstanding light scattering, lower interfacial resistance, longer electron lifetime, rapid electron transfer, and improved diffusion length, and consequently, J SC , quantum efficiency, and record photoconversion efficiency of 10.1% using commercial N-719 dye is achieved, for 1D-based DSSCs. These new and highly functional TNTs will be a concrete fundamental background toward the development of more functional applications in fuel cells, dye-sensitized solar cells, Li-ion batteries, photocatalysis process, ion-exchange/adsorption process, and photoelectrochemical devices. PMID:27432775

  10. Efficient human growth hormone gene expression in the milk of non-transgenic goats.

    PubMed

    Han, Z; Wu, S; Li, Q; Li, J; Gao, D; Li, K; Liu, Z W; Zhao, H

    2009-01-01

    Heterogenous expression of recombinant proteins in milk of livestock at a large scale is very labour-intensive to be achieved with current transgenic animals, and usually seen as time-consuming, expensive and technically most challenging. Here we describe a convenient system for transient production of recombinant human growth hormone and its extensive use in recombinant protein production for therapeutic purposes. In this study, an adenoviral vector containing the GFP gene and hGH gene was constructed for direct infusion into the epithelium of mammary glands of goats via the teat canal during the period of natural lactation. Western-blot analysis of milk samples obtained from all of the viral-treated founders indicated that the recombinant hGH (rhGH) was secreted into the milk of the goats. The concentrations of rhGH in milk ranged from 0.6 to 2.4 mg/ml and lasted for more than 10 days during lactation. These data suggest that it is possible to produce larger amounts of recombinant human growth hormone in the milk of livestock animals by using replication-defective adenoviruses.

  11. The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes.

    PubMed

    Watanabe, Takahiro; Narita, Yohei; Yoshida, Masahiro; Sato, Yoshitaka; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

    2015-10-01

    Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.

  12. Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

    PubMed

    Kimura, Yukiko; Hisano, Yu; Kawahara, Atsuo; Higashijima, Shin-ichi

    2014-01-01

    The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.

  13. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

    PubMed Central

    Wang, Xianlong; Zhou, Jinwei; Cao, Chunwei; Huang, Jiaojiao; Hai, Tang; Wang, Yanfang; Zheng, Qiantao; Zhang, Hongyong; Qin, Guosong; Miao, Xiangnan; Wang, Hongmei; Cao, Suizhong; Zhou, Qi; Zhao, Jianguo

    2015-01-01

    Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs. PMID:26293209

  14. Prediction of microRNA target genes using an efficient genetic algorithm-based decision tree

    PubMed Central

    Rabiee-Ghahfarrokhi, Behzad; Rafiei, Fariba; Niknafs, Ali Akbar; Zamani, Behzad

    2015-01-01

    MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression in almost all plants and animals. They play an important role in key processes, such as proliferation, apoptosis, and pathogen–host interactions. Nevertheless, the mechanisms by which miRNAs act are not fully understood. The first step toward unraveling the function of a particular miRNA is the identification of its direct targets. This step has shown to be quite challenging in animals primarily because of incomplete complementarities between miRNA and target mRNAs. In recent years, the use of machine-learning techniques has greatly increased the prediction of miRNA targets, avoiding the need for costly and time-consuming experiments to achieve miRNA targets experimentally. Among the most important machine-learning algorithms are decision trees, which classify data based on extracted rules. In the present work, we used a genetic algorithm in combination with C4.5 decision tree for prediction of miRNA targets. We applied our proposed method to a validated human datasets. We nearly achieved 93.9% accuracy of classification, which could be related to the selection of best rules. PMID:26649272

  15. Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

    PubMed

    Rao, Shengbin; Fujimura, Tatsuya; Matsunari, Hitomi; Sakuma, Tetsushi; Nakano, Kazuaki; Watanabe, Masahito; Asano, Yoshinori; Kitagawa, Eri; Yamamoto, Takashi; Nagashima, Hiroshi

    2016-01-01

    Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future.

  16. Efficient transformation and artificial miRNA gene silencing in Lemna minor

    PubMed Central

    Cantó-Pastor, Alex; Mollá-Morales, Almudena; Ernst, Evan; Dahl, William; Zhai, Jixian; Yan, Yiheng; Meyers, Blake; Shanklin, John; Martienssen, Robert

    2015-01-01

    Lack of genetic tools in the Lemnaceae (duckweed) has impeded full implementation of this organism as model for biological research, despite its rapid doubling time, simple architecture and unusual metabolic characteristics. Here we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a Magnesium Chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic Lemna minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. PMID:24989135

  17. Polyion complex stability and gene silencing efficiency with a siRNA-grafted polymer delivery system.

    PubMed

    Takemoto, Hiroyasu; Ishii, Atsushi; Miyata, Kanjiro; Nakanishi, Masataka; Oba, Makoto; Ishii, Takehiko; Yamasaki, Yuichi; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2010-11-01

    An siRNA-grafted polymer through disulfide linkage was prepared to improve the physicochemical properties and transfection efficacies of the polyion complexes (PICs) as a nanocarrier of siRNA. The siRNA-grafted polymer formed stable PICs due to its larger numbers and higher density of anionic charges compared with monomeric siRNA, leading to effective internalization by cultured cells. Following the endosomal escape of the PIC, the disulfide linkage of the siRNA-grafted polymer allowed efficient siRNA release from the PIC under intracellular reductive conditions. Consequently, the PIC from the siRNA-grafted polymer showed a potent gene silencing effect without cytotoxicity or immunogenicity, demonstrating a promising feature of the siRNA-grafted polymer to construct the PIC-based nanocarrier for in vivo siRNA delivery.

  18. In the rat liver, Adenoviral gene transfer efficiency is comparable to AAV.

    PubMed

    Montenegro-Miranda, P S; Pichard, V; Aubert, D; Ten Bloemendaal, L; Duijst, S; de Waart, D R; Ferry, N; Bosma, P J

    2014-02-01

    Adenoviral (AdV) and Adenovirus-associated viral (AAV) vectors both are used for in vivo gene therapy of inherited liver disorders, such as Crigler-Najjar syndrome type 1. In a relevant animal model, the Gunn rat, both vectors efficiently correct the severe hyperbilirubinemia characteristic of this liver disorder. Although the clinical use of AAV is more advanced, as demonstrated by the successful phase 1 trial in hemophilia B patients, because of its large cloning capacity AdV remains an attractive option. A direct comparison of the efficacy of these two vectors in the liver in a relevant disease model has not been reported. Aim of this study was to compare the efficiency of clinically applicable doses of both vectors in the Gunn rat. AdV or scAAV (self-complimentary AAV) ferrying identical liver-specific expression cassettes of the therapeutic gene, UGT1A1, were injected into the tail vein. As the titration methods of these two vectors are very different, a comparison based on vector titers is not valid. Therefore, their efficacy was compared by determining the amount of vector genomes delivered to the liver required for therapeutic correction of serum bilirubin. Like AAV, the liver-specific first-generation AdV also provided sustained correction in this relevant disease model. UGT1A1 mRNA expression provided per genome was comparable for both vectors. Flanking the expression cassette in AdV with AAV-ITRs (inverted terminal repeats), increased UGT1A1 mRNA expression eightfold which resulted in a significant improvement of efficacy. Compared with AAV, less AdV genomes were needed for complete correction of hyperbilirubinemia.

  19. Efficient Gene Silencing by Self-Assembled Complexes of siRNA and Symmetrical Fatty Acid Amides of Spermine

    PubMed Central

    Metwally, Abdelkader A.; Pourzand, Charareh; Blagbrough, Ian S.

    2011-01-01

    Gene silencing by siRNA (synthetic dsRNA of 21-25 nucleotides) is a well established biological tool in gene expression studies and has a promising therapeutic potential for difficult-to-treat diseases. Five fatty acids of various chain length and oxidation state (C12:0, C18:0, C18:1, C18:2, C22:1) were conjugated to the naturally occurring polyamine, spermine, and evaluated for siRNA delivery and gene knock-down. siRNA delivery could not be related directly to gene silencing efficiency as N4,N9-dierucoyl spermine resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine. GFP silencing in HeLa cells showed that the unsaturated fatty acid amides are more efficient than saturated fatty acid amides, with N4,N9-dioleoyl spermine resulting in the most efficient gene silencing in the presence of serum. The alamarBlue cell viability assay showed that fatty acid amides of spermine have good viability (75%–85% compared to control) except N4,N9-dilauroyl spermine which resulted in low cell viability. These results prove that unsaturated fatty acid amides of spermine are efficient, non-toxic, non-viral vectors for siRNA mediated gene silencing. PMID:24310492

  20. Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans.

    PubMed

    Peters, Björn; Junker, Anja; Brauer, Katharina; Mühlthaler, Bernadette; Kostner, David; Mientus, Markus; Liebl, Wolfgang; Ehrenreich, Armin

    2013-03-01

    Gluconobacter oxydans, a biotechnologically relevant species which incompletely oxidizes a large variety of carbohydrates, alcohols, and related compounds, contains a gene for pyruvate decarboxylase (PDC). This enzyme is found only in very few species of bacteria where it is normally involved in anaerobic ethanol formation via acetaldehyde. In order to clarify the role of PDC in the strictly oxidative metabolism of acetic acid bacteria, we developed a markerless in-frame deletion system for strain G. oxydans 621H which uses 5-fluorouracil together with a plasmid-encoded uracil phosphoribosyltransferase as counter selection method and used this technique to delete the PDC gene (GOX1081) of G. oxydans 621H. The PDC deletion mutant accumulated large amounts of pyruvate but almost no acetate during growth on D-mannitol, D-fructose or in the presence of L-lactate. This suggested that in G. oxydans acetate formation occurs by decarboxylation of pyruvate and subsequent oxidation of acetaldehyde to acetate. This observation and the efficiency of the markerless deletion system were confirmed by constructing deletion mutants of two acetaldehyde dehydrogenases (GOX1122 and GOX2018) and of the acetyl-CoA-synthetase (GOX0412). Acetate formation during growth of these mutants on mannitol did not differ significantly from the wild-type strain.

  1. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    PubMed

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  2. Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

    PubMed Central

    2012-01-01

    Background Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue. Results Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima). In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days. Conclusions The simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications. PMID:22559320

  3. Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp

    PubMed Central

    Gotic, Ivana; Omidi, Saeed; Fleury-Olela, Fabienne; Molina, Nacho; Naef, Felix; Schibler, Ueli

    2016-01-01

    In mammals, body temperature fluctuates diurnally around a mean value of 36°C–37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells’ circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide “approach to steady-state” kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression. PMID:27633015

  4. Transfection efficiency and structural studies on nonviral gene carriers containing cholesterol and other sterols

    NASA Astrophysics Data System (ADS)

    Evans, Heather

    2005-03-01

    Lipid based nonviral gene delivery currently focuses on cationic liposomes, which typically consist of a mixture of cationic and neutral (helper) lipids. Motivated by the plasma membrane composition of mammalian cells, which contain large amounts of cholesterol, this molecule is often used as a helper lipid. The presented work investigates the effect of cholesterol and structurally related molecules on the transfection efficiency (TE) of cationic lipid-DNA (CL-DNA) complexes in mammalian cells. Previous studies have identified the membrane charge density as a universal parameter, predicting TE for CL-DNA complexes in the lamellar Lα^C phase [1,2]. Addition of cholesterol to low transfecting CL-DNA complexes results in dramatic improvements in TE that significantly deviate from the TE model for lamellar complexes. A model system using negatively charged giant vesicles has been developed to mimic the cell membrane and understand the behavior pattern of CL-DNA complexes containing cholesterol. Funding provided by NIH GM-59288. [1] Lin AJ, Slack NL, Ahmad A, George CX, Samuel CE, Safinya CR, Biophys. J., 2003, V84:3307 [2] Ahmad A, Evans HM, Ewert K, and Safinya CR, J. Gene Med., accepted

  5. Linear cyclen-based polyamine as a novel and efficient reagent in gene delivery.

    PubMed

    Xiang, Yong-Zhe; Feng, Zhi-Hua; Zhang, Ji; Liao, Yi-Le; Yu, Chuan-Jiang; Yi, Wen-Jing; Zhu, Wen; Yu, Xiao-Qi

    2010-02-01

    Linear cyclen-based polyamine (LCPA, M(w) = 7392, M(w)/M(n) = 1.19) as a novel non-viral gene vector was designed and synthesized from 1,7-diprotected 1,4,7,10-tetraazacyclododecane (cyclen), bis(beta-hydroxylethyl)amine and epichlorohydrin. Agarose gel retardation and fluorescent titration using ethidium bromide showed the good DNA-binding ability of LCPA. It could retard pDNA at an N/P ratio of 4 and form polyplexes with sizes around 250-300 nm from an N/P ratio of 10 to 60 and relatively lower zeta-potential values (< +3 mV) even at the N/P ratio of 60. The cytotoxicity of LCPA assayed by MTT is much lower than that of 25 kDa PEI. In vitro transfection against A549 and 293 cells showed that the transfection efficiency of LCPA/DNA polyplexes is close to that of 25 kDa PEI at an N/P ratio of 10-15, indicating that the new material could be a promising non-viral polycationic reagent for gene delivery.

  6. Using the gene pulser MXcell electroporation system to transfect primary cells with high efficiency.

    PubMed

    McCoy, Adam M; Collins, Michelle L; Ugozzoli, Luis A

    2010-01-01

    It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments. PMID:20057352

  7. Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp.

    PubMed

    Gotic, Ivana; Omidi, Saeed; Fleury-Olela, Fabienne; Molina, Nacho; Naef, Felix; Schibler, Ueli

    2016-09-01

    In mammals, body temperature fluctuates diurnally around a mean value of 36°C-37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells' circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide "approach to steady-state" kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression. PMID:27633015

  8. Impact of Pre-Analytical Variables on Cancer Targeted Gene Sequencing Efficiency.

    PubMed

    Araujo, Luiz H; Timmers, Cynthia; Shilo, Konstantin; Zhao, Weiqiang; Zhang, Jianying; Yu, Lianbo; Natarajan, Thanemozhi G; Miller, Clinton J; Yilmaz, Ayse Selen; Liu, Tom; Amann, Joseph; Lapa E Silva, José Roberto; Ferreira, Carlos Gil; Carbone, David P

    2015-01-01

    Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed using custom probes and were paired-end sequenced on a next generation sequencing platform. A PCR-based quality control (QC) assay was utilized to determine DNA quality, and a ratio was generated in comparison to control DNA. We observed that FFPE storage time, PCR/QC ratio, and DNA input in the library preparation were significantly correlated to most parameters of sequencing efficiency including depth of coverage, alignment rate, insert size, and read quality. A combined score using the three parameters was generated and proved highly accurate to predict sequencing metrics. We also showed wide read count variability within the genome, with worse coverage in regions of low GC content like in KRAS. Sample quality and GC content had independent effects on sequencing depth, and the worst results were observed in regions of low GC content in samples with poor quality. Our data confirm that FFPE samples are a reliable source for targeted gene sequencing in cancer, provided adequate sample quality controls are exercised. Tissue quality should be routinely assessed for pre-analytical factors, and sequencing depth may be limited in genomic regions of low GC content if suboptimal samples are utilized. PMID:26605948

  9. A Twin and Adoption Study of Reading Achievement: Exploration of Shared-Environmental and Gene-Environment-Interaction Effects

    ERIC Educational Resources Information Center

    Kirkpatrick, Robert M.; Legrand, Lisa N.; Iacono, William G.; McGue, Matt

    2011-01-01

    Existing behavior-genetic research implicates substantial influence of heredity and modest influence of shared environment on reading achievement and reading disability. Applying DeFries-Fulker analysis to a combined sample of twins and adoptees (N = 4886, including 266 reading-disabled probands), the present study replicates prior findings of…

  10. Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector.

    PubMed

    Shayakhmetov, D M; Papayannopoulou, T; Stamatoyannopoulos, G; Lieber, A

    2000-03-01

    Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.

  11. Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria.

    PubMed

    Dominguez, Wilfredo; O'Sullivan, Daniel J

    2013-02-01

    Bifidobacteria are widely used as probiotics and have attracted increasing research interest worldwide. However, molecular techniques are still very scarce mainly due to the low efficiencies and strain-specific electroporation protocols that have been developed. Bacterial conjugation enables the transfer of genetic material among a relatively wide range of organisms and with virtually no size limitation. A conjugation protocol was developed based on the RP4 conjugative machinery in the Escherichia coli strain WM3064(pBB109). Using this machinery, the newly constructed transmissible E. coli-Bifidobacterium shuttle vector, pDOJHR-WD2, was successfully and consistently transferred into several strains representing four Bifidobacterium species at efficiencies which correlated with the E. coli to bifidobacteria ratios. Higher ratios were found to significantly improve transfer frequency per recipient, with almost 100 % transfer frequency occurring when the ratio was 10(5) : 1. The incompatible resident plasmid, pDOJH10S, in Bifidobacterium longum DJO10A was able to coexist, albeit at lower copy numbers, with the incoming vector pDOJHR-WD2 even though they possess the same ori. In some cases the copy number of this resident plasmid was too low to observe via gel electrophoresis, but it could be detected by Southern hybridization. Plasmid curing resulted in a strain, DJO10A-W3, that had lost both plasmids and this showed a one-log increase in conjugation efficiency due to the lack of plasmid incompatibility. In conclusion, this novel conjugative gene transfer protocol can be used for the introduction of genetic material (without size restriction) into Bifdobacterium species and is particularly useful for strains that are recalcitrant to electroporation.

  12. Efficient gene transfer into normal human B lymphocytes with the chimeric adenoviral vector Ad5/F35.

    PubMed

    Jung, Daniel; Néron, Sonia; Drouin, Mathieu; Jacques, Annie

    2005-09-01

    The failure to efficiently introduce genes into normal cells such as human B lymphocytes limits the characterization of their function on cellular growth, differentiation and survival. Recent studies have shown that a new adenoviral vector Ad5/F35 can efficiently transduce human haematopoietic CD34+ progenitor cells. In this study, we compared the gene transfer efficiencies of the Ad5/F35 vector to that of the parental vector Ad5 in human B lymphocytes. Peripheral blood B cells obtained from healthy individuals were cultured in vitro using CD40-CD154 system. Normal B lymphocytes were infected with replication-defectives Ad5 and Ad5/F35, both containing the GFP reporter gene, and transduction efficiencies were monitored by flow cytometry. Ad5 was highly ineffective, infecting only about 5% of human B lymphocytes. In contrast, Ad5/F35 transduced up to 60% of human B lymphocytes and GFP expression could be detected for up to 5 days post infection. Importantly, physiology of B lymphocytes such as proliferation, viability and antibodies secretion were unaffected following Ad5/F35 transduction. Finally, we observed that memory B lymphocytes were more susceptible to Ad5/F35 infection than naïve B lymphocytes. Thus, our results demonstrate that the adenoviral vector Ad5/F35 is an efficient tool for the functional characterization of genes in B lymphopoiesis.

  13. A new rapid and efficient system with dominant selection developed to inactivate and conditionally express genes in Candida albicans.

    PubMed

    Lai, Wei-Chung; Sun, Hsiao-Fang Sunny; Lin, Pei-Hsuan; Ho Lin, Ho Lin; Shieh, Jia-Ching

    2016-02-01

    Candida albicans is an important human fungal pathogen but its study has been hampered for being a natural diploid that lacks a complete sexual cycle. Gene knock-out and essential gene repression are used to study gene function in C. albicans. To effectively study essential genes in wild-type C. albicans, we took advantage of the compatible effects of the antibiotics hygromycin B and nourseothricin, the recyclable CaSAT1-flipper and the tetracycline-repressible (Tet-off) system. To allow deleting two alleles simultaneously, we created a cassette with a C. albicans HygB resistance gene (CaHygB) flanked with the FLP recombinase target sites that can be operated alongside the CaSAT1-flipper. Additionally, to enable conditionally switching off essential genes, we created a CaHygB-based Tet-off cassette that consisted of the CaTDH3 promoter, which is used for the constitutive expression of the tetracycline-regulated transactivator and a tetracycline response operator. To validate the new systems, all strains were constructed based on the wild-type strain and selected by the two dominant selectable markers, CaHygB and CaSAT1. The C. albicans general transcriptional activator CaGCN4 and its negative regulator CaPCL5 genes were targeted for gene deletion, and the essential cyclin-dependent kinase CaPHO85 gene was placed under the Tet-off system. Cagcn4, Capcl5, the conditional Tet-off CaPHO85 mutants, and mutants bearing two out of the three mutations were generated. By subjecting the mutants to various stress conditions, the functional relationship of the genes was revealed. This new system can efficiently delete genes and conditionally switch off essential genes in wild-type C. albicans to assess functional interaction between genes.

  14. Efficient c-kit Receptor-Targeted Gene Transfer to Primary Human CD34-Selected Hematopoietic Stem Cells

    PubMed Central

    Zhong, Qiu; Oliver, Peter; Huang, Weitao; Good, David; La Russa, Vincent; Zhang, Zili; Cork, John R.; Veith, Robert Woody; Theodossiou, Chris; Kolls, Jay K.; Schwarzenberger, Paul

    2001-01-01

    We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit+ human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer. PMID:11581407

  15. Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters.

    PubMed

    Romdhane, Ines Belhaj-Ben; Frikha, Fakher; Maalej-Achouri, Inès; Gargouri, Ali; Belghith, Hafedh

    2012-02-15

    A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083bp encoding a protein of 269 Aa with an estimated molecular mass of 30kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides. PMID:22178764

  16. Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters.

    PubMed

    Romdhane, Ines Belhaj-Ben; Frikha, Fakher; Maalej-Achouri, Inès; Gargouri, Ali; Belghith, Hafedh

    2012-02-15

    A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083bp encoding a protein of 269 Aa with an estimated molecular mass of 30kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides.

  17. Simple combination of oxidants with zero-valent-iron (ZVI) achieved very rapid and highly efficient removal of heavy metals from water.

    PubMed

    Guo, Xuejun; Yang, Zhe; Dong, Haiyang; Guan, Xiaohong; Ren, Qidong; Lv, Xiaofang; Jin, Xin

    2016-01-01

    This study, for the first time, demonstrated a continuously accelerated Fe(0) corrosion driven by common oxidants (i.e., NaClO, KMnO4 or H2O2) and thereby the rapid and efficient removal of heavy metals (HMs) by zero-valent iron (ZVI) under the experimental conditions of jar tests and column running. ZVI simply coupled with NaClO, KMnO4 or H2O2 (0.5 mM) resulted in almost complete As(V) removal within only 10 min with 1000 μg/L of initial As(V) at initial pH of 7.5(±0.1) and liquid solid ratio of 200:1. Simultaneous removal of 200 μg/L of initial Cd(II) and Hg(II) to 2.4-4.4 μg/L for Cd(II) and to 4.0-5.0 μg/L for Hg(II) were achieved within 30 min. No deterioration of HM removal was observed during the ten recycles of jar tests. The ZVI columns activated by 0.1 mM of oxidants had stably treated 40,200 (NaClO), 20,295 (KMnO4) and 40,200 (H2O2) bed volumes (BV) of HM-contaminated drinking water, but with no any indication of As breakthrough (<10 μg/L) even at short empty bed contact time (EBCT) of 8.0 min. The high efficiency of HMs removal from both the jar tests and column running implied a continuous and stable activation (overcoming of iron passivation) of Fe(0) surface by the oxidants. Via the proper increase in oxidant dosing, the ZVI/oxidant combination was applicable to treat highly As(V)-contaminated wastewater. During Fe(0) surface corrosion accelerated by oxidants, a large amount of fresh and reactive iron oxides and oxyhydroxides were continuously generated, which were responsible for the rapid and efficient removal of HMs through multiple mechanisms including adsorption and co-precipitation. A steady state of Fe(0) surface activation and HM removal enabled this simply coupled system to remove HMs with high speed, efficiency and perdurability. PMID:26575476

  18. Simple combination of oxidants with zero-valent-iron (ZVI) achieved very rapid and highly efficient removal of heavy metals from water.

    PubMed

    Guo, Xuejun; Yang, Zhe; Dong, Haiyang; Guan, Xiaohong; Ren, Qidong; Lv, Xiaofang; Jin, Xin

    2016-01-01

    This study, for the first time, demonstrated a continuously accelerated Fe(0) corrosion driven by common oxidants (i.e., NaClO, KMnO4 or H2O2) and thereby the rapid and efficient removal of heavy metals (HMs) by zero-valent iron (ZVI) under the experimental conditions of jar tests and column running. ZVI simply coupled with NaClO, KMnO4 or H2O2 (0.5 mM) resulted in almost complete As(V) removal within only 10 min with 1000 μg/L of initial As(V) at initial pH of 7.5(±0.1) and liquid solid ratio of 200:1. Simultaneous removal of 200 μg/L of initial Cd(II) and Hg(II) to 2.4-4.4 μg/L for Cd(II) and to 4.0-5.0 μg/L for Hg(II) were achieved within 30 min. No deterioration of HM removal was observed during the ten recycles of jar tests. The ZVI columns activated by 0.1 mM of oxidants had stably treated 40,200 (NaClO), 20,295 (KMnO4) and 40,200 (H2O2) bed volumes (BV) of HM-contaminated drinking water, but with no any indication of As breakthrough (<10 μg/L) even at short empty bed contact time (EBCT) of 8.0 min. The high efficiency of HMs removal from both the jar tests and column running implied a continuous and stable activation (overcoming of iron passivation) of Fe(0) surface by the oxidants. Via the proper increase in oxidant dosing, the ZVI/oxidant combination was applicable to treat highly As(V)-contaminated wastewater. During Fe(0) surface corrosion accelerated by oxidants, a large amount of fresh and reactive iron oxides and oxyhydroxides were continuously generated, which were responsible for the rapid and efficient removal of HMs through multiple mechanisms including adsorption and co-precipitation. A steady state of Fe(0) surface activation and HM removal enabled this simply coupled system to remove HMs with high speed, efficiency and perdurability.

  19. Charge Density and Molecular Weight of Polyphosphoramidate Gene Carrier Are Key Parameters Influencing Its DNA Compaction Ability and Transfection Efficiency

    PubMed Central

    Ren, Yong; Jiang, Xuan; Pan, Deng; Mao, Hai-Quan

    2011-01-01

    A series of polyphosphoramidates (PPA) with different molecular weights (MWs) and charge densities were synthesized and examined for their DNA compaction ability and transfection efficiency. A strong correlation was observed between the transfection efficiency of PPA/DNA nanoparticles and the MW and net positive charge density of the PPA gene carriers in three different cell lines (HeLa, HEK293 and HepG2 cells). An increase in MW and/or net positive charge density of PPA carrier yielded higher DNA compaction capacity, smaller nanoparticles with higher surface charges and higher complex stability against challenges by salt and polyanions. These favorable physicochemical properties of nanoparticles led to enhanced transfection efficiency. PPA/DNA nanoparticles with the highest complex stability showed comparable transfection efficiency as PEI/DNA nanoparticles likely by compensating the low buffering capacity with higher cellular uptake and affording higher level of protection to DNA in endolysosomal compartment. The differences in transfection efficiency were not attributed by any difference in cytotoxicity among the carriers, as all nanoparticles showed minimal level of cytotoxicity under the transfection conditions. Using PPA as a model system, we demonstrated the structural dependence of transfection efficiency of polymer gene carrier. These results offer more insights into nanoparticle engineering for non-viral gene delivery. PMID:21067136

  20. Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency

    PubMed Central

    Zhang, Jian-Ping; Li, Xiao-Lan; Neises, Amanda; Chen, Wanqiu; Hu, Lin-Ping; Ji, Guang-Zhen; Yu, Jun-Yao; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao; Zhang, Xiao-Bing

    2016-01-01

    CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60–70%) and MSCs (65–75%). Furthermore, we observed a decrease of 10–20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable. PMID:27338021

  1. Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency.

    PubMed

    Zhang, Jian-Ping; Li, Xiao-Lan; Neises, Amanda; Chen, Wanqiu; Hu, Lin-Ping; Ji, Guang-Zhen; Yu, Jun-Yao; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao; Zhang, Xiao-Bing

    2016-01-01

    CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60-70%) and MSCs (65-75%). Furthermore, we observed a decrease of 10-20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable. PMID:27338021

  2. Efficient In Planta Detection and Dissection of De Novo Mutation Events in the Arabidopsis thaliana Disease Resistance Gene UNI.

    PubMed

    Ogawa, Tomohiko; Mori, Akiko; Igari, Kadunari; Morita, Miyo Terao; Tasaka, Masao; Uchida, Naoyuki

    2016-06-01

    Plants possess disease resistance (R) proteins encoded by R genes, and each R protein recognizes a specific pathogen factor(s) for immunity. Interestingly, a remarkably high degree of polymorphisms in R genes, which are traces of past mutation events during evolution, suggest the rapid diversification of R genes. However, little is known about molecular aspects that facilitate the rapid change of R genes because of the lack of tools that enable us to monitor de novo R gene mutations efficiently in an experimentally feasible time scale, especially in living plants. Here we introduce a model assay system that enables efficient in planta detection of de novo mutation events in the Arabidopsis thaliana R gene UNI in one generation. The uni-1D mutant harbors a gain-of-function allele of the UNI gene. uni-1D heterozygous individuals originally exhibit dwarfism with abnormally short stems. However, interestingly, morphologically normal stems sometimes emerge spontaneously from the uni-1D plants, and the morphologically reverted tissues carry additional de novo mutations in the UNI gene. Strikingly, under an extreme condition, almost half of the examined population shows the reversion phenomenon. By taking advantage of this phenomenon, we demonstrate that the reversion frequency is remarkably sensitive to a variety of fluctuations in DNA stability, underlying a mutable tendency of the UNI gene. We also reveal that activities of the salicylic acid pathway and DNA damage sensor pathway are involved in the reversion phenomenon. Thus, we provide an experimentally feasible model tool to explore factors and conditions that significantly affect the R gene mutation phenomenon. PMID:27016096

  3. RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

    PubMed

    Kong, Lingdan; Alves, Carla S; Hou, Wenxiu; Qiu, Jieru; Möhwald, Helmuth; Tomás, Helena; Shi, Xiangyang

    2015-03-01

    We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications. PMID:25658033

  4. An efficient RNA isolation procedure and identification of reference genes for normalization of gene expression in blueberry.

    PubMed

    Vashisth, Tripti; Johnson, Lisa Klima; Malladi, Anish

    2011-12-01

    Application of transcriptomics approaches can greatly enhance our understanding of blueberry physiology. The success of transcriptomics approaches is dependent on the extraction of high-quality RNA which is complicated by the abundance of polyphenolics and polysaccharides in blueberry. Additionally, transcriptomics requires the accurate quantification of transcript abundance. Quantitative real-time polymerase chain reaction (qRT-PCR) is a robust method to determine transcript abundance. Normalization of gene expression using stably expressed reference genes is essential in qRT-PCR. An evaluation of the stability of expression of reference genes has not yet been reported in blueberry. The objectives of this study were to develop an effective procedure for extracting RNA from different organs and to evaluate potential reference genes for qRT-PCR analyses in blueberry. RNA of high quality and yield was extracted from eight and six organs of rabbiteye and southern highbush blueberry, respectively, using a modified cetyltrimethyl ammonium bromide-based method. The expression stability of 12 reference genes was evaluated. UBIQUITIN-CONJUGATING ENZYME (UBC28), RNA HELICASE-LIKE (RH8), CLATHRIN ADAPTER COMPLEXES MEDIUM SUBUNIT FAMILY PROTEIN (CACSa), and POLYUBIQUITIN (UBQ3b) were the most stably expressed genes across multiple organs in both blueberry species. Further, the expression stability of the reference genes in the branch abscission zone following treatment with fruit abscission-inducing compounds was analyzed. CACSa, RH8, and UBC28 were the most stably expressed genes in the abscission zone under abscission-inducing conditions. We suggest a preliminary evaluation of UBC28, CACSa, RH8, and UBQ3b to identify the most suitable reference genes for the experimental conditions under consideration in blueberry.

  5. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    PubMed

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  6. Plant genetic transformation efficiency of selected Malaysian rice based on selectable marker gene (hptII).

    PubMed

    Htwe, Nwe Nwe; Ling, Ho Chai; Zaman, Faridah Qamaruz; Maziah, Mahmood

    2014-04-01

    Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.

  7. Efficient moment-based inference of admixture parameters and sources of gene flow.

    PubMed

    Lipson, Mark; Loh, Po-Ru; Levin, Alex; Reich, David; Patterson, Nick; Berger, Bonnie

    2013-08-01

    The recent explosion in available genetic data has led to significant advances in understanding the demographic histories of and relationships among human populations. It is still a challenge, however, to infer reliable parameter values for complicated models involving many populations. Here, we present MixMapper, an efficient, interactive method for constructing phylogenetic trees including admixture events using single nucleotide polymorphism (SNP) genotype data. MixMapper implements a novel two-phase approach to admixture inference using moment statistics, first building an unadmixed scaffold tree and then adding admixed populations by solving systems of equations that express allele frequency divergences in terms of mixture parameters. Importantly, all features of the model, including topology, sources of gene flow, branch lengths, and mixture proportions, are optimized automatically from the data and include estimates of statistical uncertainty. MixMapper also uses a new method to express branch lengths in easily interpretable drift units. We apply MixMapper to recently published data for Human Genome Diversity Cell Line Panel individuals genotyped on a SNP array designed especially for use in population genetics studies, obtaining confident results for 30 populations, 20 of them admixed. Notably, we confirm a signal of ancient admixture in European populations-including previously undetected admixture in Sardinians and Basques-involving a proportion of 20-40% ancient northern Eurasian ancestry. PMID:23709261

  8. Efficient transformation and artificial miRNA gene silencing in Lemna minor.

    PubMed

    Cantó-Pastor, A; Mollá-Morales, A; Ernst, E; Dahl, W; Zhai, J; Yan, Y; Meyers, B C; Shanklin, J; Martienssen, R

    2015-01-01

    Despite rapid doubling time, simple architecture and ease of metabolic labelling, a lack of genetic tools in the Lemnaceae (duckweed) has impeded the full implementation of this organism as a model for biological research. Here, we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via Agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a magnesium chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic L. minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae.

  9. Development of a rapid and efficient microinjection technique for gene insertion into fertilized salmonid eggs

    SciTech Connect

    Chandler, D.P.; Welt, M.; Leung, F.C.

    1990-10-01

    An efficient one-step injection technique for gene insertion into fertilized rainbow trout (Oncorhynchus mykiss) eggs is described, and basic parameters affecting egg survival are reported. Freshly fertilized rainbow trout eggs were injected in the perivitelline space with a recombinant mouse metallothionein-genomic bovine growth hormone (bGH) DNA construct using a 30-gauge hypodermic needle and a standard microinjection system. Relative to control, site of injection and DNA concentration did not affect the egg survival, but injections later than 3--4 hours post fertilization were detrimental. The injection technique permitted treatment of 100 eggs/hr with survivals up to 100%, resulting in a 4% DNA uptake rate as indicated by DNA dot blot analysis. Positive dot blot results also indicated that the injected DNA is able to cross the vitelline membrane and persist for 50--60 days post hatching, obviating the need for direct injection into the germinal disk. Results are consistent with previous transgenic fish work, underscoring the usefulness of the technique for generating transgenic trout and salmonids. 24 refs., 6 figs., 3 tabs.

  10. Efficient transformation and artificial miRNA gene silencing in Lemna minor.

    PubMed

    Cantó-Pastor, A; Mollá-Morales, A; Ernst, E; Dahl, W; Zhai, J; Yan, Y; Meyers, B C; Shanklin, J; Martienssen, R

    2015-01-01

    Despite rapid doubling time, simple architecture and ease of metabolic labelling, a lack of genetic tools in the Lemnaceae (duckweed) has impeded the full implementation of this organism as a model for biological research. Here, we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via Agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a magnesium chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic L. minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. PMID:24989135

  11. Efficient moment-based inference of admixture parameters and sources of gene flow.

    PubMed

    Lipson, Mark; Loh, Po-Ru; Levin, Alex; Reich, David; Patterson, Nick; Berger, Bonnie

    2013-08-01

    The recent explosion in available genetic data has led to significant advances in understanding the demographic histories of and relationships among human populations. It is still a challenge, however, to infer reliable parameter values for complicated models involving many populations. Here, we present MixMapper, an efficient, interactive method for constructing phylogenetic trees including admixture events using single nucleotide polymorphism (SNP) genotype data. MixMapper implements a novel two-phase approach to admixture inference using moment statistics, first building an unadmixed scaffold tree and then adding admixed populations by solving systems of equations that express allele frequency divergences in terms of mixture parameters. Importantly, all features of the model, including topology, sources of gene flow, branch lengths, and mixture proportions, are optimized automatically from the data and include estimates of statistical uncertainty. MixMapper also uses a new method to express branch lengths in easily interpretable drift units. We apply MixMapper to recently published data for Human Genome Diversity Cell Line Panel individuals genotyped on a SNP array designed especially for use in population genetics studies, obtaining confident results for 30 populations, 20 of them admixed. Notably, we confirm a signal of ancient admixture in European populations-including previously undetected admixture in Sardinians and Basques-involving a proportion of 20-40% ancient northern Eurasian ancestry.

  12. Thalidomide is more efficient than sodium butyrate in enhancing GATA-1 and EKLF gene expression in erythroid progenitors derived from HSCs with β-globin gene mutation

    PubMed Central

    Jalali Far, Mohammad Ali; Dehghani Fard, Ali; Hajizamani, Saiedeh; Mossahebi-Mohammadi, Majid; Yaghooti, Hamid; Saki, Najmaldin

    2016-01-01

    Background: Efficient induction of fetal hemoglobin (HbF) is considered as an effective therapeutic approach in beta thalassemia. HbF inducer agents can induce the expression of γ-globin gene and produce high levels of HbF via different epigenetic and molecular mechanisms. Thalidomide and sodium butyrate are known as HbF inducer drugs. Material and methods: CD133+ stem cells were isolated from umbilical cord blood of a newborn with minor β-thalassemia in order to evaluate the effects of these two drugs on the in vitro expression of GATA-1 and EKLF genes as erythroid transcription factors. CD133+ stem cells were expanded and differentiated into erythroid lineage and then treated with thalidomide and sodium butyrate and finally analyzed by quantitative real-time PCR. Statistical analysis was performed using student’s t-test by SPSS software. Results: Thalidomide and sodium butyrate increased GATA-1 and EKLF gene expression, compared to the non-treated control (P<0.05). Conclusion: Thalidomide was more efficient than sodium butyrate in augmenting expression of GATA-1 and EKLF genes. It seems that GATA-1 and EKLF have crucial roles in the efficient induction of HbF by thalidomide. PMID:27047649

  13. In Vivo Bio-distribution and Efficient Tumor Targeting of Gelatin/Silica Nanoparticles for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Zhao, Xueqin; Wang, Jun; Tao, SiJie; Ye, Ting; Kong, Xiangdong; Ren, Lei

    2016-04-01

    The non-viral gene delivery system is an attractive alternative to cancer therapy. The clinical success of non-viral gene delivery is hampered by transfection efficiency and tumor targeting, which can be individually overcome by addition of functional modules such as cell penetration or targeting. Here, we first engineered the multifunctional gelatin/silica (GS) nanovectors with separately controllable modules, including tumor-targeting aptamer AGRO100, membrane-destabilizing peptide HA2, and polyethylene glycol (PEG), and then studied their bio-distribution and in vivo transfection efficiencies by contrast resonance imaging (CRI). The results suggest that the sizes and zeta potentials of multifunctional gelatin/silica nanovectors were 203-217 nm and 2-8 mV, respectively. Functional GS-PEG nanoparticles mainly accumulated in the liver and tumor, with the lowest uptake by the heart and brain. Moreover, the synergistic effects of tumor-targeting aptamer AGRO100 and fusogenic peptide HA2 promoted the efficient cellular internalization in the tumor site. More importantly, the combined use of AGRO100 and PEG enhanced tumor gene expression specificity and effectively reduced toxicity in reticuloendothelial system (RES) organs after intravenous injection. Additionally, low accumulation of GS-PEG was observed in the heart tissues with high gene expression levels, which could provide opportunities for non-invasive gene therapy.

  14. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing.

    PubMed

    Park, Arnold; Hong, Patrick; Won, Sohui T; Thibault, Patricia A; Vigant, Frederic; Oguntuyo, Kasopefoluwa Y; Taft, Justin D; Lee, Benhur

    2016-01-01

    The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75-98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing. PMID:27606350

  15. CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.

    PubMed

    Bhattacharya, Dipankan; Marfo, Chris A; Li, Davis; Lane, Maura; Khokha, Mustafa K

    2015-12-15

    Congenital malformations are the major cause of infant mortality in the US and Europe. Due to rapid advances in human genomics, we can now efficiently identify sequence variants that may cause disease in these patients. However, establishing disease causality remains a challenge. Additionally, in the case of congenital heart disease, many of the identified candidate genes are either novel to embryonic development or have no known function. Therefore, there is a pressing need to develop inexpensive and efficient technologies to screen these candidate genes for disease phenocopy in model systems and to perform functional studies to uncover their role in development. For this purpose, we sought to test F0 CRISPR based gene editing as a loss of function strategy for disease phenocopy in the frog model organism, Xenopus tropicalis. We demonstrate that the CRISPR/Cas9 system can efficiently modify both alleles in the F0 generation within a few hours post fertilization, recapitulating even early disease phenotypes that are highly similar to knockdowns from morpholino oligos (MOs) in nearly all cases tested. We find that injecting Cas9 protein is dramatically more efficacious and less toxic than cas9 mRNA. We conclude that CRISPR based F0 gene modification in X. tropicalis is efficient and cost effective and readily recapitulates disease and MO phenotypes.

  16. Polyurethane dispersion containing quaternized ammonium groups: An efficient nanosize gene delivery carrier for A549 cancer cell line transfection.

    PubMed

    Yousefpour Marzbali, Mahsa; Yari Khosroushahi, Ahmad; Movassaghpour, AliAkbar; Yeganeh, Hamid

    2016-01-25

    A novel polyurethane containing cationic ammonium groups (QPU) was synthesized and used as vector for gene therapy and cancer gene targeting. The synthesized QPU was characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy methods. An agarose gel retardation electrophoresis assay was conducted to verify the complete complex formation between QPU and pDNA. The particles size and zeta potential of neat polymers, plasmid DNA, polymers/DNA polyplexes were determined by the dynamic light scattering technique. The polyplexes cytotoxicity was determined using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and its transfection efficiency was examined qualitatively by fluorescent microscopy and quantitatively by flow cytometery methods. The gel retardation assay, particle size and zeta potential measurements were confirmed that the synthesized cationic polymer could condense DNA efficiently in the physiologic condition. QPU polyplexes showed a significantly lower cytotoxicity compared to Polyfect polyplexes in the examined human cancerous (A549) or normal cells (KDR). Based on our findings, the transfection efficiency by QPU was 2.2 fold higher than Polyfect in the A549 cells whereas in the KDR cells, the cell transfection by Polyfect was 18.1 fold higher than QPU. Due to low cytotoxicity for normal cells and high transfection efficiency in cancer cells, the potential applicability of designed QPU as a non-viral gene carrier for targeting of cancer gene therapy was confirmed.

  17. Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction

    PubMed Central

    Li, Chao; Ding, Lei; Sun, Chiao-Wang; Wu, Li-Chen; Zhou, Dewang; Pawlik, Kevin M.; Khodadadi-Jamayran, Alireza; Westin, Erik; Goldman, Frederick D.; Townes, Tim M.

    2016-01-01

    CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (βA/[βS+βA]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications. PMID:27460639

  18. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing

    PubMed Central

    Park, Arnold; Hong, Patrick; Won, Sohui T; Thibault, Patricia A; Vigant, Frederic; Oguntuyo, Kasopefoluwa Y; Taft, Justin D; Lee, Benhur

    2016-01-01

    The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75–98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing. PMID:27606350

  19. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing

    PubMed Central

    Park, Arnold; Hong, Patrick; Won, Sohui T; Thibault, Patricia A; Vigant, Frederic; Oguntuyo, Kasopefoluwa Y; Taft, Justin D; Lee, Benhur

    2016-01-01

    The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75–98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing.

  20. Terminal modification on mPEG-dendritic poly-(l)-lysine cationic diblock copolymer for efficient gene delivery.

    PubMed

    Sheng, Ruilong; Xia, Kejia; Chen, Jian; Xu, Yuhong; Cao, Amin

    2013-01-01

    The development of new non-viral gene vectors with the advantages of low cytotoxicity and high gene transfection efficiency is a recent trend in gene therapy. In this work, we developed a series of termini-modified mPEG-dendritic poly-(l)-lysine cationic diblock copolymers (mPEG5k-DPL4-CG) by coupling various cationic groups to the dendritic skeleton. Their molecular structures were characterized by (1)H NMR, and the buffering capacities were measured by acid titration. The plasmid DNA (pDNA) binding affinities of the mPEG5k-DPL4-CG copolymers were investigated by EB displacement and agarose gel retardation assay, and the average particle size and surface charge of the polyplexes were analyzed by dynamic light scattering. Cytotoxicity and in vitro gene transfection were evaluated in several cell lines in the presence and absence of serum by the luciferase expression assay. The results indicated that the low molecular weight polyethylenimine (PEI800) termini-modified copolymer, mPEG5k-DPL4-PEI800, possessed high pDNA binding affinity, low cytotoxicity, and high gene transfection capability which were maintained in the presence of serum (10% FBS). It is worth noting that the gene delivery efficiency of the dendritic poly-(l)-lysine gene vector was enhanced by termini modification of suitable cationic blocks. The low cytotoxicity and serum-resistance properties of mPEG5k-DPL4-PEI800 make it a potential long-circulating gene vector in gene therapy applications.

  1. Successful PGD for late infantile neuronal ceroid lipofuscinosis achieved by combined chromosome and TPP1 gene analysis.

    PubMed

    Shen, Jiandong; Cram, David Stephen; Wu, Wei; Cai, Lingbo; Yang, Xiaoyu; Sun, Xueping; Cui, Yugui; Liu, Jiayin

    2013-08-01

    Late infantile neuronal ceroid lipofuscinosis (NCL-2) is a severe debilitating autosomal recessive disease caused by mutations in TPP1. There are no effective treatments, resulting in early childhood death. A couple with two affected children presented for reproductive genetic counselling and chose to undertake IVF and preimplantation genetic diagnosis (PGD) to avoid the possibility of another affected child. However, DNA testing revealed only one mutation in the proband inherited from mother. Linkage analysis identified five informative linked short tandem repeat markers to aid the genetic diagnosis. Following IVF, five cleavage-stage embryos were biopsied and blastomeres were first subjected to whole-genome amplification, then a series of down-stream molecular genetic analyses to diagnose TPP1 genotype and finally array comparative genomic hybridization (CGH) to assess the chromosomal ploidy of each embryo. Two unaffected euploid embryos were identified for transfer. One was transferred on day 5 resulting in an ongoing pregnancy. Confirmatory prenatal diagnosis by amniocentesis showed concordance of the embryo and fetal diagnosis. As far as is known, this is the first successful report of PGD for NCL-2 using double-factor PGD with simultaneous single-gene testing and array CGH to identify an unaffected and chromosomally normal embryo for transfer.

  2. Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9.

    PubMed

    Fang, Yufeng; Tyler, Brett M

    2016-01-01

    Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9-induced DNA double-strand breaks (DSBs) in P. sojae was mediated by non-homologous end-joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA was present, homology-directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR-mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.

  3. How Many Letters Should Preschoolers in Public Programs Know? The Diagnostic Efficiency of Various Preschool Letter-Naming Benchmarks for Predicting First-Grade Literacy Achievement

    PubMed Central

    Piasta, Shayne B.; Petscher, Yaacov; Justice, Laura M.

    2015-01-01

    Review of current federal and state standards indicates little consensus or empirical justification regarding appropriate goals, often referred to as benchmarks, for preschool letter-name learning. The present study investigated the diagnostic efficiency of various letter-naming benchmarks using a longitudinal database of 371 children who attended publicly funded preschools. Children’s uppercase and lowercase letter-naming abilities were assessed at the end of preschool, and their literacy achievement on 3 standardized measures was assessed at the end of 1st grade. Diagnostic indices (sensitivity, specificity, and negative and positive predictive power) were generated to examine the extent to which attainment of various preschool letter-naming benchmarks was associated with later risk for literacy difficulties. Results indicated generally high negative predictive power for benchmarks requiring children to know 10 or more letter names by the end of preschool. Balancing across all diagnostic indices, optimal benchmarks of 18 uppercase and 15 lowercase letter names were identified. These findings are discussed in terms of educational implications, limitations, and future directions. PMID:26346643

  4. Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments

    PubMed Central

    Munkácsy, Gyöngyi; Sztupinszki, Zsófia; Herman, Péter; Bán, Bence; Pénzváltó, Zsófia; Szarvas, Nóra; Győrffy, Balázs

    2016-01-01

    No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal–Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E−06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E−04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency. PMID:27673562

  5. Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments.

    PubMed

    Munkácsy, Gyöngyi; Sztupinszki, Zsófia; Herman, Péter; Bán, Bence; Pénzváltó, Zsófia; Szarvas, Nóra; Győrffy, Balázs

    2016-01-01

    No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal-Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E-06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E-04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency. PMID:27673562

  6. Codon optimization of genes for efficient protein expression in mammalian cells by selection of only preferred human codons.

    PubMed

    Inouye, Satoshi; Sahara-Miura, Yuiko; Sato, Jun-ichi; Suzuki, Takahiro

    2015-05-01

    A simple design method for codon optimization of genes to express a heterologous protein in mammalian cells is described. Codon optimization was performed by choosing only codons preferentially used in humans and with over 60% GC content, and the method was named the "preferred human codon-optimized method." To test our simple rule for codon optimization, the preferred human codon-optimized genes for six proteins containing photoproteins (aequorin and clytin II) and luciferases (Gaussia luciferase, Renilla luciferase, and firefly luciferases from Photinus pyralis and Luciola cruciata) were chemically synthesized and transiently expressed in Chinese hamster ovary-K1 cells. All preferred human codon-optimized genes showed higher luminescence activity than the corresponding wild-type genes. Our simple design method could be used to improve protein expression in mammalian cells efficiently.

  7. A versatile and efficient markerless gene disruption system for Acidithiobacillus thiooxidans: application for characterizing a copper tolerance related multicopper oxidase gene.

    PubMed

    Wen, Qing; Liu, Xiangmei; Wang, Huiyan; Lin, Jianqun

    2014-11-01

    The acidophilic bioleaching bacteria can usually survive in high concentrations of copper ions because of their special living environment. However, little is known about the copper homeostatic mechanisms of Acidithiobacillus thiooxidans, an important member of bioleaching bacteria. Here, a putative multicopper oxidase gene (cueO) was detected from the draft genome of A. thiooxidans ATCC 19377. The transcriptional level of cueO in response to 10 mM CuSO₄was upregulated 25.01 ± 2.59 folds. The response of P(cueO) to copper was also detected and might be stimulated by a putative CueR protein. Then, by using the counter-selectable marker lacZ and enhancing the expression of endonuclease I-SceI with tac promoter, a modified markerless gene disruption system was developed and the cueO gene disruption mutant (ΔcueO) of A. thiooxidans was successfully constructed with a markedly improved second homologous recombination frequency of 0.28 ± 0.048. The ΔcueO mutant was more sensitive to external copper and nearly completely lost the phenoloxidase activity; however, the activity could be restored after complementing the cueO gene. All results suggest the close relation of cueO gene to copper tolerance in A. thiooxidans. In addition, the developed efficient markerless gene knockout method can also be introduced into other Acidithiobacillus strains.

  8. An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus.

    PubMed

    Tamakoshi, M; Yaoi, T; Oshima, T; Yamagishi, A

    1999-04-15

    A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a delta pyrE strain by a two step method. First, the leuB gene was replaced with the pyrE gene. Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid. A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host. When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme. These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes. PMID:10227171

  9. Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

    PubMed

    Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

    2009-09-01

    Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

  10. Assessing the tobacco-rattle-virus-based vectors system as an efficient gene silencing technique in Datura stramonium (Solanaceae).

    PubMed

    Eftekhariyan Ghamsari, Mohammad Reza; Karimi, Farah; Mousavi Gargari, Seyed Latif; Hosseini Tafreshi, Seyed Ali; Salami, Seyed Alireza

    2014-12-01

    Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium. PMID:25070062

  11. Bioreducible PEI-functionalized glycol chitosan: A novel gene vector with reduced cytotoxicity and improved transfection efficiency.

    PubMed

    Taranejoo, Shahrouz; Chandrasekaran, Ramya; Cheng, Wenlong; Hourigan, Kerry

    2016-11-20

    Non-viral gene delivery has been well recognised as a potential way to address the main safety limitations of viral gene carriers. A new redox-responsive PEI derivative was designed, synthesized and evaluated for non-viral delivery applications of GFP DNA. Glycol chitosan was covalently attached to highly branched LMW PEI via bio-cleavable disulfide bonds to synthesize a new redox-responsive gene carrier (GCS-ss-PEI). Results showed the enhanced buffering capacity of GCS-ss-PEI, 43.1%, compared to the buffering capacities of both LMW PEI and HMW PEI, 23.2% and 31.5%, respectively, indicating more likely endosomal escape of the entrapped gene for GCS-ss-PEI. Moreover, electrophoretic gel retardation assay, performed to investigate the binding strength of GCS-ss-PEI to GFP DNA, showed stronger complexation with GFP DNA in GCS-ss-PEI at non-GSH condition. Employing GCS and incorporation of disulfide bonds in the structure of the PEI-based gene carrier resulted in improved redox-responsivity, reduced toxicity, enhanced endosomal escape and GFP DNA transfection. The facilitated intracellular gene release along with excellent redox-responsive characteristics and dropped cytotoxicity suggests the potential of GCS-ss-PEI as a candidate for developing highly efficient and safe gene vectors. PMID:27561483

  12. Inclusion of high molecular weight dextran in calcium phosphate-mediated transfection significantly improves gene transfer efficiency.

    PubMed

    Wu, C; Lu, Y

    2007-05-15

    Calcium phosphate-based mammalian cell transfection is a widely used gene transfer technology. To facilitate the efficiency of this gene transfer method, several polysaccharide compounds were tested and evaluated for their effectiveness in enhancing DNA transfection. Using a HIV-1-derived lentivirus vector plasmid as a gene transfer indicator, we demonstrated that the addition of high molecular weight dextran-500 at 0.6-1.2% in the 2x Hepes buffered saline (HBS) increased transfection efficiency by over 50% (as reflected by the number of GFP-positive cells) and increased the titer of resulting lentivirus vector particles even more (up to 4-fold). This enhancement of transfection efficiency was further increased when higher molecular weight dextran formulations were used in place of dextran-500, and also when dextran was used in combination with polybrene, another polycationic chemical compound. Examination of transfected cells showed that dextran had no apparent adverse effect on cell viability and growth. Our data represent the first report showing that dextran can be used to enhance calcium phosphate-mediated gene transfer; this may be useful in applications for the generation of high-titer virus vector stocks using transient transfection technology.

  13. I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

    PubMed

    Ouedraogo, Jean Paul; Arentshorst, Mark; Nikolaev, Igor; Barends, Sharief; Ram, Arthur F J

    2015-12-01

    Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.

  14. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis.

    PubMed

    Nakajima, Keisuke; Yaoita, Yoshio

    2015-01-16

    Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  15. Pyramiding, alternating or mixing: comparative performances of deployment strategies of nematode resistance genes to promote plant resistance efficiency and durability

    PubMed Central

    2014-01-01

    Background Resistant cultivars are key elements for pathogen control and pesticide reduction, but their repeated use may lead to the emergence of virulent pathogen populations, able to overcome the resistance. Increased research efforts, mainly based on theoretical studies, explore spatio-temporal deployment strategies of resistance genes in order to maximize their durability. We evaluated experimentally three of these strategies to control root-knot nematodes: cultivar mixtures, alternating and pyramiding resistance genes, under controlled and field conditions over a 3-years period, assessing the efficiency and the durability of resistance in a protected crop rotation system with pepper as summer crop and lettuce as winter crop. Results The choice of the resistance gene and the genetic background in which it is introgressed, affected the frequency of resistance breakdown. The pyramiding of two different resistance genes in one genotype suppressed the emergence of virulent isolates. Alternating different resistance genes in rotation was also efficient to decrease virulent populations in fields due to the specificity of the virulence and the trapping effect of resistant plants. Mixing resistant cultivars together appeared as a less efficient strategy to control nematodes. Conclusions This work provides experimental evidence that, in a cropping system with seasonal sequences of vegetable species, pyramiding or alternating resistance genes benefit yields in the long-term by increasing the durability of resistant cultivars and improving the long-term control of a soil-borne pest. To our knowledge, this result is the first one obtained for a plant-nematode interaction, which helps demonstrate the general applicability of such strategies for breeding and sustainable management of resistant cultivars against pathogens. PMID:24559060

  16. A novel dendrimer based on poly (L-glutamic acid) derivatives as an efficient and biocompatible gene delivery vector

    NASA Astrophysics Data System (ADS)

    Zeng, Xin; Pan, Shirong; Li, Jie; Wang, Chi; Wen, Yuting; Wu, Hongmei; Wang, Cuifeng; Wu, Chuanbin; Feng, Min

    2011-09-01

    Non-viral gene delivery systems based on cationic polymers have faced limitations related to their relative low gene transfer efficiency, cytotoxicity and system instability in vivo. In this paper, a flexible and pompon-like dendrimer composed of poly (amidoamine) (PAMAM) G4.0 as the inner core and poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) as the surrounding multiple arms was synthesized (MGI dendrimer). The novel MGI dendrimer was designed to combine the merits of size-controlled PAMAM G4.0 and the low toxicity and flexible chains of PLGE. In phosphate-buffered saline dispersions the well-defined DNA/MGI complex above a N/P ratio of 30 showed good stability with particle sizes of approximately 200 nm and a comparatively low polydispersity index. However, the particle size of the DNA/25 kDa polyethylenimine (DNA/PEI 25K) complex was larger than 700 nm under the same salt conditions. The shielding of the compact amino groups at the periphery of flexible PAMAM and biocompatible PLGE chains in MGI resulted in a dramatic decrease of the cytotoxicity compared to native PAMAM G4.0 dendrimer. The in vitro transfection efficiency of DNA/MGI dendrimer complex was higher than that of PAMAM G4.0 dendrimer. Importantly, in serum-containing medium, DNA/MGI complexes at their optimal N/P ratio maintained the same high levels of transfection efficiency as in serum-free medium, while the transfection efficiency of native PAMAM G4.0, PEI 25K and Lipofectamine 2000 were sharply decreased. In vivo gene delivery of pVEGF165/MGI complex into balloon-injured rabbit carotid arteries resulted in significant inhibition of restenosis by increasing VEGF165 expression in local vessels. Therefore, the pompon-like MGI dendrimer may be a promising vector candidate for efficient gene delivery in vivo.

  17. Efficient retrovirus-mediated transfer of cell-cycle control genes to transformed cells.

    PubMed

    Strauss, B E; Costanzi-Strauss, E

    1999-07-01

    The use of gene therapy continues to be a promising, yet elusive, alternative for the treatment of cancer. The origins of cancer must be well understood so that the therapeutic gene can be chosen with the highest chance of successful tumor regression. The gene delivery system must be tailored for optimum transfer of the therapeutic gene to the target tissue. In order to accomplish this, we study models of G1 cell-cycle control in both normal and transformed cells in order to understand the reasons for uncontrolled cellular proliferation. We then use this information to choose the gene to be delivered to the cells. We have chosen to study p16, p21, p53 and pRb gene transfer using the pCL-retrovirus. Described here are some general concepts and specific results of our work that indicate continued hope for the development of genetically based cancer treatments.

  18. Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.

    PubMed

    Plasencia, Anna; Soler, Marçal; Dupas, Annabelle; Ladouce, Nathalie; Silva-Martins, Guilherme; Martinez, Yves; Lapierre, Catherine; Franche, Claudine; Truchet, Isabelle; Grima-Pettenati, Jacqueline

    2016-06-01

    Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes. PMID:26579999

  19. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

    PubMed

    Gantz, Valentino M; Jasinskiene, Nijole; Tatarenkova, Olga; Fazekas, Aniko; Macias, Vanessa M; Bier, Ethan; James, Anthony A

    2015-12-01

    Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda. PMID:26598698

  20. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

    PubMed

    Gantz, Valentino M; Jasinskiene, Nijole; Tatarenkova, Olga; Fazekas, Aniko; Macias, Vanessa M; Bier, Ethan; James, Anthony A

    2015-12-01

    Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

  1. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi

    PubMed Central

    Gantz, Valentino M.; Tatarenkova, Olga; Fazekas, Aniko; Macias, Vanessa M.; Bier, Ethan; James, Anthony A.

    2015-01-01

    Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda. PMID:26598698

  2. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    PubMed

    Liu, Ning; Chen, Guo-Qing; Ning, Guo-Ao; Shi, Huan-Bin; Zhang, Chu-Long; Lu, Jian-Ping; Mao, Li-Juan; Feng, Xiao-Xiao; Liu, Xiao-Hong; Su, Zhen-Zhu; Lin, Fu-Cheng

    2016-01-01

    The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae. PMID:26686612

  3. Transcriptome-wide characterization of candidate genes for improving the water use efficiency of energy crops grown on semiarid land.

    PubMed

    Fan, Yangyang; Wang, Qian; Kang, Lifang; Liu, Wei; Xu, Qin; Xing, Shilai; Tao, Chengcheng; Song, Zhihong; Zhu, Caiyun; Lin, Cong; Yan, Juan; Li, Jianqiang; Sang, Tao

    2015-10-01

    Understanding the genetic basis of water use efficiency (WUE) and its roles in plant adaptation to a drought environment is essential for the production of second-generation energy crops in water-deficit marginal land. In this study, RNA-Seq and WUE measurements were performed for 78 individuals of Miscanthus lutarioriparius grown in two common gardens, one located in warm and wet Central China near the native habitats of the species and the other located in the semiarid Loess Plateau, the domestication site of the energy crop. The field measurements showed that WUE of M. lutarioriparius in the semiarid location was significantly higher than that in the wet location. A matrix correlation analysis was conducted between gene expression levels and WUE to identify candidate genes involved in the improvement of WUE from the native to the domestication site. A total of 48 candidate genes were identified and assigned to functional categories, including photosynthesis, stomatal regulation, protein metabolism, and abiotic stress responses. Of these genes, nearly 73% were up-regulated in the semiarid site. It was also found that the relatively high expression variation of the WUE-related genes was affected to a larger extent by environment than by genetic variation. The study demonstrates that transcriptome-wide correlation between physiological phenotypes and expression levels offers an effective means for identifying candidate genes involved in the adaptation to environmental changes. PMID:26175351

  4. Construction of a modular dihydrofolate reductase cDNA gene: Analysis of signals utilized for efficient expression

    SciTech Connect

    Kaufman, J.; Sharp, P.A.

    1982-11-01

    Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome, DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR/sup -/ cells to the DHFR/sup +/ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFr mRNA produced in amplified lines indicated the following: (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA generated a recombinant capable of transforming cells to the DHFR/sup +/ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer 72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.

  5. Chimeric Adenoviral Vectors Incorporating a Fiber of Human Adenovirus 3 Efficiently Mediate Gene Transfer into Prostate Cancer Cells

    PubMed Central

    Murakami, Miho; Ugai, Hideyo; Belousova, Natalya; Pereboev, Alexander; Dent, Paul; Fisher, Paul B.; Everts, Maaike; Curiel, David T.

    2010-01-01

    BACKGROUND We have developed a range of adenoviral (Ad) vectors based on human adenovirus serotype 5 (HAdV-5) displaying the fiber shaft and knob domains of species B viruses (HAdV-3, HAdV-11, or HAdV-35). These species B Ads utilize different cellular receptors than HAdV-5 for infection. We evaluated whether Ad vectors displaying species B fiber shaft and knob domains (Ad5F3Luc1, Ad5F11Luc1, and Ad5F35Luc1) would efficiently infect cancer cells of distinct origins, including prostate cancer. METHODS The fiber chimeric Ad vectors were genetically generated and compared with the original Ad vector (Ad5Luc1) for transductional efficiency in a variety of cancer cell lines, including prostate cancer cells and primary prostate epithelial cells (PrEC), using luciferase as a reporter gene. RESULTS Prostate cancer cell lines infected with Ad5F3Luc1 expressed higher levels of luciferase than Ad5Luc1, as well as the other chimeric Ad vectors. We also analyzed the transductional efficiency via monitoring of luciferase activity in prostate cancer cells when expressed as a fraction of the gene transfer in PrEC cells. In the PC-3 and DU145 cell lines, the gene transfer ratio of cancer cells versus PrEC was once again highest for Ad5F3Luc1. CONCLUSION Of the investigated chimeric HAdV-5/species B vectors, Ad5F3Luc1 was judged to be the most suitable for targeting prostate cancer cells as it showed the highest transductional efficiency in these cells. It is foreseeable that an Ad vector incorporating the HAdV-3 fiber could potentially be used for prostate cancer gene therapy. PMID:19902467

  6. Cationic star copolymers based on β-cyclodextrins for efficient gene delivery to mouse embryonic stem cell colonies.

    PubMed

    Loh, Xian Jun; Wu, Yun-Long

    2015-07-11

    A cationic star copolymer with a β-cyclodextrin core was developed for nonviral gene transfer to mouse embryonic stem cells (mESCs). The copolymer comprises poly(2-dimethyl aminoethyl methacrylate) as the cationic component and poly(2-hydroxyethyl methacrylate) as the non-toxic stealth component. These materials have very low toxicity and show highly efficient transfection to mESC colonies. PMID:26040469

  7. Re-Designed Recombinant Hepatitis B Virus Vectors Enable Efficient Delivery of Versatile Cargo Genes to Hepatocytes with Improved Safety

    PubMed Central

    Bai, Weiya; Cui, Xiaoxian; Chen, Ruidong; Tao, Shuai; Hong, Ran; Zhang, Jiming; Zhang, Junqi; Wang, Yongxiang; Xie, Youhua; Liu, Jing

    2016-01-01

    Hepatitis B virus (HBV) takes humans as its sole natural host, and productive infection in vivo is restricted exclusively to hepatocytes in the liver. Consequently, HBV-derived viral vectors are attractive candidates for liver-targeting gene therapies. Previously, we developed a novel recombinant HBV vector, designated 5c3c, from a highly replicative clinical isolate. 5c3c was demonstrated to be capable of efficiently delivering protein or RNA expression into infected primary tupaia hepatocytes (PTH), but the design of 5c3c imposes stringent restrictions on inserted sequences, which have limited its wider adoption. In this work, we addressed issues with 5c3c by re-designing the insertion strategy. The resultant vector, designated 5dCG, was more replicative than parental 5c3c, imposed no specific restrictions on inserted sequences, and allowed insertion of a variety of cargo genes without significant loss of replication efficiency. 5dCG-based recombinant HBV effectively delivered protein and RNA expression into infected PTH. Furthermore, due to the loss of functional core ORF, 5dCG vectors depend on co-infecting wild type HBV for replication and efficient expression of cargo genes. Development of the improved 5dCG vector makes wider applications of recombinant HBV possible, while dependence on co-infecting wild type HBV results in improved safety for certain in vivo applications. PMID:27171107

  8. Synthesis and evaluation of diethylethylamine-chitosan for gene delivery: composition effects on the in vitro transfection efficiency

    NASA Astrophysics Data System (ADS)

    Pansani Oliveira, Franciele de Paula; Pfeifer Dalla Picola, Isadora; Shi, Qin; Franciane Gonçalves Barbosa, Hellen; Aparecida de Oliveira Tiera, Vera; Fernandes, Júlio Cesar; José Tiera, Marcio

    2013-02-01

    Chitosan has been indicated as a safe and promising polycation vector for gene delivery. However its low transfection efficiency has been a challenging obstacle for its application. To address this limitation, we synthesized chitosan derivatives which had increasing amounts of diethylethylamine groups (DEAE) attached to the chitosan main chain. The plasmid DNA VR1412 (pDNA), encoding the ß-galactosidase (ß-gal) reporter gene was used to prepare nanoparticles with the chitosan derivatives, and the transfection studies were performed with HeLa cells. By means of dynamic light scattering and zeta potential measurements, it was shown that diethylethylamine-chitosan derivatives (DEAEx-CH) were able to condense DNA into small particles having a surface charge depending on the polymer/DNA ratio (N/P ratio). Nanoparticles prepared with derivatives containing 15 and 25% of DEAE groups (DEAE15-CH and DEAE25-CH) exhibited transfection efficiencies ten times higher than that observed with deacetylated chitosan (CH). For derivatives with higher degrees of substitution (DS), transfection efficiency decreased. The most effective carriers showed low cytotoxicity and good transfection activities at low charge ratios (N/P). Vectors with low DS were easily degraded in the presence of lysozyme at physiological conditions in vitro and the nontoxicity displayed by these vectors opens up new opportunities in the design of DEAE-chitosan-based nanoparticles for gene delivery.

  9. Bacteriophages of Staphylococcus aureus efficiently package various bacterial genes and mobile genetic elements including SCCmec with different frequencies.

    PubMed

    Mašlaňová, Ivana; Doškař, Jiří; Varga, Marian; Kuntová, Lucie; Mužík, Jan; Malúšková, Denisa; Růžičková, Vladislava; Pantůček, Roman

    2013-02-01

    Staphylococcus aureus is a serious human and veterinary pathogen in which new strains with increasing virulence and antimicrobial resistance occur due to acquiring new genes by horizontal transfer. It is generally accepted that temperate bacteriophages play a major role in gene transfer. In this study, we proved the presence of various bacterial genes of the S. aureus COL strain directly within the phage particles via qPCR and quantified their packaging frequency. Non-parametric statistical analysis showed that transducing bacteriophages φ11, φ80 and φ80α of serogroup B, in contrast to serogroup A bacteriophage φ81, efficiently package selected chromosomal genes localized in 4 various loci of the chromosome and 8 genes carried on variable elements, such as staphylococcal cassette chromosome SCCmec, staphylococcal pathogenicity island SaPI1, genomic islands vSaα and vSaβ, and plasmids with various frequency. Bacterial gene copy number per ng of DNA isolated from phage particles ranged between 1.05 × 10(2) for the tetK plasmid gene and 3.86 × 10(5) for the SaPI1 integrase gene. The new and crucial finding that serogroup B bacteriophages can package concurrently ccrA1 (1.16 × 10(4)) and mecA (1.26 × 10(4)) located at SCCmec type I into their capsids indicates that generalized transduction plays an important role in the evolution and emergence of new methicillin-resistant clones.

  10. Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain.

    PubMed

    Foureau, Emilien; Courdavault, Vincent; Rojas, Luisa Fernanda; Dutilleul, Christelle; Simkin, Andrew J; Crèche, Joël; Atehortùa, Lucia; Giglioli-Guivarc'h, Nathalie; Clastre, Marc; Papon, Nicolas

    2013-07-01

    The yeast, Candida guilliermondii, has been widely studied due to its biotechnological interest as well as its biological control potential. It integrates foreign DNA predominantly via ectopic events, likely through the well-known non-homologous end-joining (NHEJ) pathway involving the Ku70p/Ku80p heterodimer, Lig4p, Nej1p and Lif1p. This phenomenon remains highly deleterious for targeted gene knock-out strategies that require the homologous recombination process. Here, we have constructed a ku70 mutant strain derived from the ATCC 6260 reference strain of C. guilliermondii. Following a series of disruption attempts of various genes (FCY1, ADE2 and TRP5), using several previously described dominant selectable markers (URA5, SAT-1 and HPH#), we demonstrated that the efficiencies of homologous gene targeting in such a NHEJ-deficient strain was very high compared to the wild type strain. The C. guilliermondii ku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast. PMID:23463324

  11. Efficient conditional gene expression following transplantation of retrovirally transduced bone marrow stem cells.

    PubMed

    Chung, Jie-Yu; Mackay, Fabienne; Alderuccio, Frank

    2015-01-01

    Retroviral gene therapy combined with bone marrow stem cell transplantation can be used to generate mice with ectopic gene expression in the bone marrow compartment in a quick and cost effective manner when compared to generating and maintaining transgenic mouse lines. However a limitation of this procedure is the lack of cell specificity in gene expression that is associated with the use of endogenous retroviral promoters. Restricting gene expression to specific cell subsets utilising tissue-specific promoter driven retroviral vectors is a challenge. Here we describe the generation of conditional expression of retrovirally encoded genes in specific bone marrow derived cell lineages utilising a Cre-dependent retroviral vector. By utilising Lck and CD19 restricted Cre transgenic bone marrow stem cells, we generate chimeric animals with T or B lymphocyte restricted gene expression respectively. The design of the Cre-dependent retroviral vector enables expression of encoded MOG and GFP genes only in association with Cre mediated DNA inversion. Importantly this strategy does not significantly increase the size of the retroviral vector; as such we are able to generate bone marrow chimeric animals with significantly higher chimerism levels than previous studies utilising Cre-dependent retroviral vectors and Cre transgenic bone marrow stem cells. This demonstrates that the use of Cre-dependent retroviral vectors is able to yield high chimerism levels for experimental use and represent a viable alternative to generating transgenic animals.

  12. An Efficient Data Assimilation Schema for Restoration and Extension of Gene Regulatory Networks Using Time-Course Observation Data

    PubMed Central

    Mori, Tomoya; Yamaguchi, Rui; Imoto, Seiya; Miyano, Satoru; Akutsu, Tatsuya

    2014-01-01

    Abstract Gene regulatory networks (GRNs) play a central role in sustaining complex biological systems in cells. Although we can construct GRNs by integrating biological interactions that have been recorded in literature, they can include suspicious data and a lack of information. Therefore, there has been an urgent need for an approach by which the validity of constructed networks can be evaluated; simulation-based methods have been applied in which biological observational data are assimilated. However, these methods apply nonlinear models that require high computational power to evaluate even one network consisting of only several genes. Therefore, to explore candidate networks whose simulation models can better predict the data by modifying and extending literature-based GRNs, an efficient and versatile method is urgently required. We applied a combinatorial transcription model, which can represent combinatorial regulatory effects of genes, as a biological simulation model, to reproduce the dynamic behavior of gene expressions within a state space model. Under the model, we applied the unscented Kalman filter to obtain the approximate posterior probability distribution of the hidden state to efficiently estimate parameter values maximizing prediction ability for observational data by the EM-algorithm. Utilizing the method, we propose a novel algorithm to modify GRNs reported in the literature so that their simulation models become consistent with observed data. The effectiveness of our approach was validated through comparison analysis to the previous methods using synthetic networks. Finally, as an application example, a Kyoto Encyclopedia of Genes and Genomes (KEGG)-based yeast cell cycle network was extended with additional candidate genes to better predict the real mRNA expressions data using the proposed method. PMID:25244077

  13. Solid Lipid Nanoparticles as Efficient Drug and Gene Delivery Systems: Recent Breakthroughs

    PubMed Central

    Ezzati Nazhad Dolatabadi, Jafar; Valizadeh, Hadi; Hamishehkar, Hamed

    2015-01-01

    In recent years, nanomaterials have been widely applied as advanced drug and gene delivery nanosystems. Among them, solid lipid nanoparticles (SLNs) have attracted great attention as colloidal drug delivery systems for incorporating hydrophilic or lipophilic drugs and various macromolecules as well as proteins and nucleic acids. Therefore, SLNs offer great promise for controlled and site specific drug and gene delivery. This article includes general information about SLN structures and properties, production procedures, characterization. In addition, recent progress on development of drug and gene delivery systems using SLNs was reviewed. PMID:26236652

  14. SAFETY AND EFFICIENCY OF MODULATING PARACELLULAR PERMEABILITY TO ENHANCE AIRWAY EPITHELIAL GENE TRANSFER IN VIVO

    EPA Science Inventory


    ABSTRACT

    We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability. Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after luminal application of vehicle, ...

  15. Involvement of the AATn polymorphism of the CNR1 gene in the efficiency of procedural learning in humans.

    PubMed

    Ruiz-Contreras, Alejandra E; Delgado-Herrera, Maribel; García-Vaca, Paola A; Almeida-Rosas, Georgina A; Soria-Rodríguez, Gerardo; Soriano-Bautista, Alejandro; Cadena-Valencia, Jaime; Bazán-Frías, Jorge R; Gómez-López, Nardhy; Espejel-Núñez, Aurora; Vadillo-Ortega, Felipe; Carrillo-Sánchez, Karol; Verdín-Reyes, Juan C; March-Mifsut, Santiago; Méndez-Díaz, Mónica; Prospéro-García, Oscar

    2011-05-01

    Procedural learning refers to the acquisition of motor skills and the practice that refines their performance. The striatum participates in this learning through a function regulated by endocannabinoid signaling and other systems. This study relates the efficiency in learning a procedural task with the AATn polymorphism of the CNR1 gene, which encodes for the CB1 receptor. The mirror-drawing star task was solved by 99 healthy young subjects in three trials. The sample was divided into high- and low-performance groups based on performance efficiency. AAT12/14 carriers were more frequent in the former group, while there were more AAT12/13 carriers in the latter, which also made more errors/min. Therefore, we characterized two efficiency phenotypes: high- vs. low-performers associated with the two AATn genotypes, AAT12/14 vs. AAT12/13. The findings suggest that AATn polymorphism modifies CNR1 translation, indicating a different modulation of CB1.

  16. An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila.

    PubMed

    Larsen, Camilla; Franch-Marro, Xavier; Hartenstein, Volker; Alexandre, Cyrille; Vincent, Jean-Paul

    2006-11-21

    Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic males. Many animals expressing GFP in distinct patterns can be recovered with relatively little effort. As expected, many insertions cause loss of function. After insertion at a genomic location, specific parts of the transposon can be excised by FLP recombinase, thus allowing it to induce conditional misexpression of the tagged gene. Therefore, both gain- and loss-of-function studies can be carried out with a single insertion in a gene identified by virtue of its expression pattern. Using this promoter trap approach, we have identified a group of cells that innervate the calyx of the mushroom body and could thus define a previously unrecognized memory circuit. PMID:17093046

  17. Fast and Efficient Screening for Wheat Loss-of-Gene Mutants Using Multiplexed Melt Curve Analyses.

    PubMed

    Mieog, Jos C; Ral, Jean-Philippe F

    2016-01-01

    This study describes a new approach in the screening for loss-of-gene mutants in Heavy Ion Bombardment (HIB) mutant populations of genetically complex organisms such as hexaploid bread wheat using multiplexed single-color (SYBR Green) melt curve analyses. The assay was set up for three target genes to test its validity and applicability. For each gene, three genome-specific primer pairs (one for each genome) with distinct melt curves were developed and multiplexed. This allowed screening for "single null mutants" (plants with the target gene deleted in one of the three genomes) for all three genomes in a single reaction. The first two genes (α-Amylase 3 and Epsilon Cyclase) were used to test the approach as HIB null lines for all three genomes were already available for these. The third assay was successfully applied to identify new single null lines of the target gene α-Amylase 2 in an in-house HIB wheat collection. The use of SYBR Green greatly reduced the time and/or cost investment compared to other techniques and the approach proved highly suitable for high-throughput applications. PMID:27459606

  18. Fast and Efficient Screening for Wheat Loss-of-Gene Mutants Using Multiplexed Melt Curve Analyses

    PubMed Central

    Mieog, Jos C.; Ral, Jean-Philippe F.

    2016-01-01

    This study describes a new approach in the screening for loss-of-gene mutants in Heavy Ion Bombardment (HIB) mutant populations of genetically complex organisms such as hexaploid bread wheat using multiplexed single-color (SYBR Green) melt curve analyses. The assay was set up for three target genes to test its validity and applicability. For each gene, three genome-specific primer pairs (one for each genome) with distinct melt curves were developed and multiplexed. This allowed screening for “single null mutants” (plants with the target gene deleted in one of the three genomes) for all three genomes in a single reaction. The first two genes (α-Amylase 3 and Epsilon Cyclase) were used to test the approach as HIB null lines for all three genomes were already available for these. The third assay was successfully applied to identify new single null lines of the target gene α-Amylase 2 in an in-house HIB wheat collection. The use of SYBR Green greatly reduced the time and/or cost investment compared to other techniques and the approach proved highly suitable for high-throughput applications. PMID:27459606

  19. Guanidinylated block copolymers for gene transfer: A comparison with amine-based materials for in vitro and in vivo gene transfer efficiency

    PubMed Central

    Choi, Jennifer L.; Tan, James-Kevin Y.; Sellers, Drew L.; Wei, Hua; Horner, Philip J.; Pun, Suzie H.

    2015-01-01

    There is currently no cure for neuron loss in the brain, which can occur due to traumatic injury or neurodegenerative disease. One method proposed to enhance neurogenesis in the brain is gene transfer to neural progenitor cells. In this work, a guanidine-based copolymer was synthesized and compared to an amine-based copolymer analog previously shown to effectively deliver genes in the murine brain. The guanidine-based copolymer was more efficient at gene transfer to immortalized, cultured cell lines; however, the amine-based copolymer was more effective at gene transfer in the brain. DNA condensation studies revealed that the nucleic acid complexes formed with the guanidine-based copolymer were more susceptible to unpackaging in the presence of heparin sulfate proteoglycans compared to complexes formed with the amine-based copolymer. Therefore, polyplexes formed from the amine-based copolymer may be more resistant to destabilization by the heparan sulfate proteoglycans present in the stem cell niches of the brain. PMID:25907042

  20. Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast.

    PubMed

    Lee, Nicholas C O; Larionov, Vladimir; Kouprina, Natalay

    2015-04-30

    Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5-2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.

  1. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

    PubMed Central

    Zebec, Ziga; Zink, Isabelle Anna; Kerou, Melina; Schleper, Christa

    2016-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR) locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs) leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms. PMID:27507792

  2. Effects of the Surface Charge of Stem Cell Membranes and DNA/Polyethyleneimine Nanocomplexes on Gene Transfection Efficiency.

    PubMed

    Kim, Da Yeon; Kwon, Jin Seon; Lee, Jae Hyeok; Jin, Ling Mei; Kim, Jae Ho; Kim, Moon Suk

    2015-03-01

    In this work, we examined the effects of the surface charge of stem cell membranes and DNA/polyethyleneimine (PEI) nanocomplexes on gene transfection efficiency, because PEI was one of the most reliable and efficient carriers, and rat bone marrow mesenchymal stem cells (rBMSCs) and rat muscle-derived stem cells (rMDSCs) were one of the readily accessible and plentiful sources of stem cells. Thus, we compared the efficiency of DNA transfection in rBMSCs and rMDSCs using the PEI as a gene carrier. Transfection efficiency was evaluated on the basis of electrostatic interaction between negatively charged stem cell membranes and positively charged DNA/PEI nanocomplexes. DNA was fully complexed with PEI at negative-to-positive (NIP) charge ratios greater than 2, as confirmed by gel electrophoresis and fluorescence measurements. DNA and PEI formed spherical nanocomplexes ranging in diameter from 150 nm to 500 nm. The positive surface charge of DNA/PEI nanocomplexes increased with an increasing N/P charge ratio, as measured using dynamic light scattering and a single-walled carbon nanotube-based field-effect transistor device. rBMSCs and rMDSCs both carried a negative surface charge, with rBMSCs being more negatively charged. The transfection efficiency of rMDSCs measured using DNA/PEI nanocomplexes was very low (1%-5%) at most of the N/P charge ratios tested, whereas better efficiencies were observed with rBMSCs (1%-17%). Nanocomplexes with high NIP charge ratios were cytotoxic to both rBMSCs and rMDSCs. Collectively, the results indicate that rBMSCs were more effectively transfected with DNA/PEI nanocomplexes than were rMDSCs, reflecting the higher negative charge of rBMSC membranes that facilitate the interaction with positively charged DNA/PEI nanocomplexes. PMID:26307834

  3. Use of combined steam-water and organic rankine cycles for achieving better efficiency of gas turbine units and internal combustion engines

    NASA Astrophysics Data System (ADS)

    Gotovskiy, M. A.; Grinman, M. I.; Fomin, V. I.; Aref'ev, V. K.; Grigor'ev, A. A.

    2012-03-01

    Innovative concepts of recovering waste heat using low-boiling working fluids, due to which the the efficiency can be increased to 28-30%, are presented. If distributed generation of electricity or combined production of heat and electricity is implemented, the electrical efficiency can reach 58-60% and the fuel heat utilization factor, 90%.

  4. Estimating Gene Expression and Codon-Specific Translational Efficiencies, Mutation Biases, and Selection Coefficients from Genomic Data Alone.

    PubMed

    Gilchrist, Michael A; Chen, Wei-Chen; Shah, Premal; Landerer, Cedric L; Zaretzki, Russell

    2015-05-14

    Extracting biologically meaningful information from the continuing flood of genomic data is a major challenge in the life sciences. Codon usage bias (CUB) is a general feature of most genomes and is thought to reflect the effects of both natural selection for efficient translation and mutation bias. Here we present a mechanistically interpretable, Bayesian model (ribosome overhead costs Stochastic Evolutionary Model of Protein Production Rate [ROC SEMPPR]) to extract meaningful information from patterns of CUB within a genome. ROC SEMPPR is grounded in population genetics and allows us to separate the contributions of mutational biases and natural selection against translational inefficiency on a gene-by-gene and codon-by-codon basis. Until now, the primary disadvantage of similar approaches was the need for genome scale measurements of gene expression. Here, we demonstrate that it is possible to both extract accurate estimates of codon-specific mutation biases and translational efficiencies while simultaneously generating accurate estimates of gene expression, rather than requiring such information. We demonstrate the utility of ROC SEMPPR using the Saccharomyces cerevisiae S288c genome. When we compare our model fits with previous approaches we observe an exceptionally high agreement between estimates of both codon-specific parameters and gene expression levels ([Formula: see text] in all cases). We also observe strong agreement between our parameter estimates and those derived from alternative data sets. For example, our estimates of mutation bias and those from mutational accumulation experiments are highly correlated ([Formula: see text]). Our estimates of codon-specific translational inefficiencies and tRNA copy number-based estimates of ribosome pausing time ([Formula: see text]), and mRNA and ribosome profiling footprint-based estimates of gene expression ([Formula: see text]) are also highly correlated, thus supporting the hypothesis that selection against

  5. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

    PubMed Central

    Richardson, C. D.; Ray, G. J.; Bray, N. L.; Corn, J. E.

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  6. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes.

    PubMed

    Richardson, C D; Ray, G J; Bray, N L; Corn, J E

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  7. Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing

    PubMed Central

    Fischer, Konrad; Kraner-Scheiber, Simone; Petersen, Björn; Rieblinger, Beate; Buermann, Anna; Flisikowska, Tatiana; Flisikowski, Krzysztof; Christan, Susanne; Edlinger, Marlene; Baars, Wiebke; Kurome, Mayuko; Zakhartchenko, Valeri; Kessler, Barbara; Plotzki, Elena; Szczerbal, Izabela; Switonski, Marek; Denner, Joachim; Wolf, Eckhard; Schwinzer, Reinhard; Niemann, Heiner; Kind, Alexander; Schnieke, Angelika

    2016-01-01

    Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age. PMID:27353424

  8. Recycling Gene Carrier with High Efficiency and Low Toxicity Mediated by L-Cystine-Bridged Bis(β-cyclodextrin)s

    PubMed Central

    Zhang, Yu-Hui; Chen, Yong; Zhang, Ying-Ming; Yang, Yang; Chen, Jia-Tong; Liu, Yu

    2014-01-01

    Constructing safe and effective gene delivery carriers is becoming highly desirable for gene therapy. Herein, a series of supramolecular crosslinking system were prepared through host-guest binding of adamantyl-modified low molecular weight of polyethyleneimine with L-cystine-bridged bis(β-cyclodextrin)s and characterized by 1H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, flow cytometry and confocal fluorescence microscopy. The results showed that these nanometersized supramolecular crosslinking systems exhibited higher DNA transfection efficiencies and lower cytotoxicity than the commercial DNA carrier gold standard (25 kDa bPEI) for both normal cells and cancer cells, giving a very high DNA transfection efficiency up to 54% for 293T cells. Significantly, this type of supramolecular crosslinking system possesses a number of enzyme-responsive disulfide bonds, which can be cleaved by reductive enzyme to promote the DNA release but recovered by oxidative enzyme to make the carrier renewable. These results demonstrate that these supramolecular crosslinking systems can be used as promising gene carriers. PMID:25503268

  9. Recycling Gene Carrier with High Efficiency and Low Toxicity Mediated by L-Cystine-Bridged Bis(β-cyclodextrin)s

    NASA Astrophysics Data System (ADS)

    Zhang, Yu-Hui; Chen, Yong; Zhang, Ying-Ming; Yang, Yang; Chen, Jia-Tong; Liu, Yu

    2014-12-01

    Constructing safe and effective gene delivery carriers is becoming highly desirable for gene therapy. Herein, a series of supramolecular crosslinking system were prepared through host-guest binding of adamantyl-modified low molecular weight of polyethyleneimine with L-cystine-bridged bis(β-cyclodextrin)s and characterized by 1H NMR titration, electron microscopy, zeta potential, dynamic light-scattering, gel electrophoresis, flow cytometry and confocal fluorescence microscopy. The results showed that these nanometersized supramolecular crosslinking systems exhibited higher DNA transfection efficiencies and lower cytotoxicity than the commercial DNA carrier gold standard (25 kDa bPEI) for both normal cells and cancer cells, giving a very high DNA transfection efficiency up to 54% for 293T cells. Significantly, this type of supramolecular crosslinking system possesses a number of enzyme-responsive disulfide bonds, which can be cleaved by reductive enzyme to promote the DNA release but recovered by oxidative enzyme to make the carrier renewable. These results demonstrate that these supramolecular crosslinking systems can be used as promising gene carriers.

  10. A P450 gene associated with robust resistance to DDT in ciliated protozoan, Tetrahymena thermophila by efficient degradation.

    PubMed

    Feng, Lifang; Fu, Chengjie; Yuan, Dongxia; Miao, Wei

    2014-04-01

    Analysis of metabolic mechanisms of dichlorodiphenyltrichloroethane (DDT) accumulation and degradation in microorganisms, which could be used to reduce its hazard to higher organisms at the higher in the food chain, have not been investigated. Robust resistance to DDT (grows well in 256 mg/L DDT) and a surprising ability to degrade DDT (more than 70% DDT within 4h) were found in the ciliated protozoan Tetrahymena thermophila. A P450 gene (CYP5013C2) was found to respond specifically to DDT treatment. In the presence of 256 mg/L DDT, cells with overexpressing CYP5013C2 (p450-OE) grew faster and degraded DDT more efficiently than wild-type (WT) cells, while cells with CYP5013C2 partially knocked down (p450-KD) grew slower and exhibited reduced ability to degrade DDT compared to WT cells. Both dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD) were detected in cells after exposure to DDT, and the concentration of DDD in the p450-OE strain gradually decreased from 0.5 to 4h. Thus, we argue that this P450 gene (CYP5013C2), by efficiently degrading DDT to DDD, is associated with robust resistance to DDT in Tetrahymena, and that a strain overexpressing this gene has the potential to serve as bioreactor that degrades environmental DDT.

  11. Quaternary ammonium salt containing soybean oil: an efficient nanosize gene delivery carrier for halophile green microalgal transformation.

    PubMed

    Akbari, Fariba; Yari Khosroushahi, Ahmad; Yeganeh, Hamid

    2015-01-01

    Dunaliella salina, a halophile green microalga, is considered a robust photobioreactor and a remarkable cost beneficial system for the production of therapeutic recombinant proteins. In this study, with low overall cost, a proper cationic lipid was synthesized from renewable soybean oil as an efficient gene delivery carrier for D. salina cells to create appropriate protein-producing transformed cell lines. To obtain an effective carrier, quaternary ammonium salt containing soybean oil (QASSO) was synthesized through the ring opening reaction of the epoxy groups of epoxidized soybean oil with diethylamine. QASSO was characterized using nuclear magnetic resonance and Fourier-transform infrared instruments. QASSO was used to prepare nanolipoplex construct using plasmid DNA molecules containing green fluorescent protein (GFP) as reporter gene. These nanolipoplexes (QASSO-pGFP, N/P=3) and QASSO had diameter of 63.62 and 110.63 nm, and zeta potential of -68.89 and 48.25 mV at pH 7.0, respectively. Results indicated the GFP gene expression and cytoplasmic accumulation of GFP protein in the transformants after incubation under desirable conditions for 48 h and 1 week. The transformation efficiency was quantitatively assayed by flow cytometry, which yielded transformations of 58.87% and 48.34% for QASSO and 38.32% and a negligible percentage for Polyfect® after 48 h and 1 week incubation, respectively.

  12. Efficient and rapid uptake of magnetic carbon nanotubes into human monocytic cells: implications for cell-based cancer gene therapy.

    PubMed

    Gul-Uludag, Hilal; Lu, Weibing; Xu, Peng; Xing, James; Chen, Jie

    2012-05-01

    Monocyte-based gene therapies in cancer have been hampered by either the resistance of these cells to non-viral molecular delivery methods or their poor trafficking to the tumor site after their ex vivo manipulations. Magnetic nanoparticles (MNP)-loaded genetically engineered monocytes can efficiently delivered to tumor site by external magnetic field, but they are not ideal delivery tools due to their spherical shape. Hence, we have investigated the cellular uptake efficiency and cytotoxicity of fluorescein isothiocyanate (FITC)-labelled magnetic carbon nanotubes (FITC-mCNT) in human monocytic leukemia cell line THP-1 for application in cell-based gene therapy against cancer. Uptake of FITC-mCNT into THP-1 cells reached 100% only 1 h after the delivery. Confocal imaging confirmed that FITC-mCNT entered the cell cytoplasm and even into the nucleus. FITC-mCNT uptake did not compromise cell viability. This delivery system might therefore enhance cell-based cancer gene therapies.

  13. Influence of lipid components on gene delivery by polycation liposomes: Transfection efficiency, intracellular kinetics and in vivo tumor inhibition.

    PubMed

    Chen, Jinliang; Sun, Xiaoyi; Yu, Zhenwei; Gao, Jianqing; Liang, Wenquan

    2012-01-17

    Transfection efficiency of non-viral gene vectors is influenced by many factors, including chemical makeup, cellular uptake pathway and intracellular delivery. To investigate the effect of lipid saturation on transfection efficiency of polycation liposomes (PCLs), a soybean phospholipids (SPL), egg phospholipids (EPL) and hydrogenated soybean phosphatidylcholine (HSPC) series was used to prepare PCLs. Testing these PCLs in a luciferase assay indicated that with increasing saturation (SPLgene expression decreased. The effect of protamine combined with these PCLs was also studied in different cell lines. Improved transfection because of protamine incorporation was dependent on lipid saturation and on the cell line tested. The kinetics of cellular uptake and intracellular distribution was studied using flow cytometry and laser scanning confocal microscope, which showed that naked oligonucleotide (ODN) and PCLs/ODN complexes became equilibrium after 4h incubation. PCLs containing SPL (PCLs-S) and 1,2-dieleoyl-sn-glycero-3-phosphoethanolamine (PCLs-D) increased uptake rates by 2.20- and 5.45-fold, respectively. Furthermore, pCMV-IL-12 transfection mediated by PCLs-D showed excellent tumor inhibition efficiency compared with control and naked pCMV-IL-12 treatments in vivo. PMID:22119962

  14. Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle.

    PubMed

    Nalbantoglu, J; Larochelle, N; Wolf, E; Karpati, G; Lochmuller, H; Holland, P C

    2001-05-01

    Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 +/- 121 in transgenic mice versus 8 +/- 4 in nontransgenic littermates) as well as the 25-fold increase in overall beta-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer-chicken beta-actin gene promoter), differential transducibility was still evident (893 +/- 149 versus 153 +/- 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 +/- 130 versus 14 +/- 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.

  15. EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

    PubMed Central

    Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A.; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forné, Thierry; Feil, Robert; Weber, Michael

    2016-01-01

    The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

  16. Efficient inhibition of ovarian cancer by degradable nanoparticle-delivered survivin T34A gene

    PubMed Central

    Luo, Li; Du, Ting; Zhang, Jiumeng; Zhao, Wei; Cheng, Hao; Yang, Yuping; Wu, Yujiao; Wang, Chunmei; Men, Ke; Gou, Maling

    2016-01-01

    Gene therapy has promising applications in ovarian cancer therapy. Blocking the function of the survivin protein could lead to the growth inhibition of cancer cells. Herein, we used degradable heparin–polyethyleneimine (HPEI) nanoparticles to deliver a dominant-negative human survivin T34A (hs-T34A) gene to treat ovarian cancer. HPEI nanoparticles were characterized and were found to have a dynamic diameter of 66±4.5 nm and a zeta potential of 27.1±1.87 mV. The constructed hs-T34A gene expression plasmid could be effectively delivered into SKOV3 ovarian carcinoma cells by HPEI nanoparticles with low cytotoxicity. Intraperitoneal administration of HPEI/hs-T34A complexes could markedly inhibit tumor growth in a mouse xenograft model of SKOV3 human ovarian cancer. Moreover, according to our results, apparent apoptosis of cancer cells was observed both in vitro and in vivo. Taken together, the prepared HPEI/hs-T34A formulation showed potential applications in ovarian cancer gene therapy. PMID:26893558

  17. Inseminating fresh or cryopreserved semen for maximum efficiency: implications for gene banks and industry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Developing germplasm collections in gene banks for animal genetic resources requires establishing germplasm collection goals, that consider capturing the genetic diversity of the population in question and the amount of germplasm required for reconstitution of the population or for other purposes. C...

  18. Gateway Vectors for Efficient Artificial Gene Assembly In Vitro and Expression in Yeast Saccharomyces cerevisiae

    PubMed Central

    Giuraniuc, Claudiu V.; MacPherson, Murray; Saka, Yasushi

    2013-01-01

    Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene’s functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology. PMID:23675537

  19. Efficient inhibition of ovarian cancer by degradable nanoparticle-delivered survivin T34A gene.

    PubMed

    Luo, Li; Du, Ting; Zhang, Jiumeng; Zhao, Wei; Cheng, Hao; Yang, Yuping; Wu, Yujiao; Wang, Chunmei; Men, Ke; Gou, Maling

    2016-01-01

    Gene therapy has promising applications in ovarian cancer therapy. Blocking the function of the survivin protein could lead to the growth inhibition of cancer cells. Herein, we used degradable heparin-polyethyleneimine (HPEI) nanoparticles to deliver a dominant-negative human survivin T34A (hs-T34A) gene to treat ovarian cancer. HPEI nanoparticles were characterized and were found to have a dynamic diameter of 66±4.5 nm and a zeta potential of 27.1±1.87 mV. The constructed hs-T34A gene expression plasmid could be effectively delivered into SKOV3 ovarian carcinoma cells by HPEI nanoparticles with low cytotoxicity. Intraperitoneal administration of HPEI/hs-T34A complexes could markedly inhibit tumor growth in a mouse xenograft model of SKOV3 human ovarian cancer. Moreover, according to our results, apparent apoptosis of cancer cells was observed both in vitro and in vivo. Taken together, the prepared HPEI/hs-T34A formulation showed potential applications in ovarian cancer gene therapy. PMID:26893558

  20. TFIIS.h, a new target of p53, regulates transcription efficiency of pro-apoptotic bax gene.

    PubMed

    Liao, Jun-Ming; Cao, Bo; Deng, Jun; Zhou, Xiang; Strong, Michael; Zeng, Shelya; Xiong, Jianping; Flemington, Erik; Lu, Hua

    2016-01-01

    Tumor suppressor p53 transcriptionally regulates hundreds of genes involved in various cellular functions. However, the detailed mechanisms underlying the selection of p53 targets in response to different stresses are still elusive. Here, we identify TFIIS.h, a transcription elongation factor, as a new transcriptional target of p53, and also show that it can enhance the efficiency of transcription elongation of apoptosis-associated bax gene, but not cell cycle-associated p21 (CDKN1A) gene. TFIIS.h is revealed as a p53 target through microarray analysis of RNAs extracted from cells treated with or without inauhzin (INZ), a p53 activator, and further confirmed by RT-q-PCR, western blot, luciferase reporter, and ChIP assays. Interestingly, knocking down TFIIS.h impairs, but overexpressing TFIIS.h promotes, induction of bax, but not other p53 targets including p21, by p53 activation. In addition, overexpression of TFIIS.h induces cell death in a bax- dependent fashion. These findings reveal a mechanism by which p53 utilizes TFIIS.h to selectively promote the transcriptional elongation of the bax gene, upsurging cell death in response to severe DNA damage.

  1. TFIIS.h, a new target of p53, regulates transcription efficiency of pro-apoptotic bax gene

    PubMed Central

    Liao, Jun-Ming; Cao, Bo; Deng, Jun; Zhou, Xiang; Strong, Michael; Zeng, Shelya; Xiong, Jianping; Flemington, Erik; Lu, Hua

    2016-01-01

    Tumor suppressor p53 transcriptionally regulates hundreds of genes involved in various cellular functions. However, the detailed mechanisms underlying the selection of p53 targets in response to different stresses are still elusive. Here, we identify TFIIS.h, a transcription elongation factor, as a new transcriptional target of p53, and also show that it can enhance the efficiency of transcription elongation of apoptosis-associated bax gene, but not cell cycle-associated p21 (CDKN1A) gene. TFIIS.h is revealed as a p53 target through microarray analysis of RNAs extracted from cells treated with or without inauhzin (INZ), a p53 activator, and further confirmed by RT-q-PCR, western blot, luciferase reporter, and ChIP assays. Interestingly, knocking down TFIIS.h impairs, but overexpressing TFIIS.h promotes, induction of bax, but not other p53 targets including p21, by p53 activation. In addition, overexpression of TFIIS.h induces cell death in a bax- dependent fashion. These findings reveal a mechanism by which p53 utilizes TFIIS.h to selectively promote the transcriptional elongation of the bax gene, upsurging cell death in response to severe DNA damage. PMID:27005522

  2. Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes

    PubMed Central

    Chamorro, Cristina; Mencía, Angeles; Almarza, David; Duarte, Blanca; Büning, Hildegard; Sallach, Jessica; Hausser, Ingrid; Del Río, Marcela; Larcher, Fernando; Murillas, Rodolfo

    2016-01-01

    Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction. PMID:27045209

  3. Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting.

    PubMed

    Chen, Fengjiao; Wang, Ying; Yuan, Yilin; Zhang, Wei; Ren, Zijian; Jin, Yong; Liu, Xiaorui; Xiong, Qiang; Chen, Qin; Zhang, Manling; Li, Xiaokang; Zhao, Lihua; Li, Ze; Wu, Zhaoqiang; Zhang, Yanfei; Hu, Feifei; Huang, Juan; Li, Rongfeng; Dai, Yifan

    2015-08-20

    Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic IgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the IgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.

  4. Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

    NASA Astrophysics Data System (ADS)

    Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2014-08-01

    We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

  5. Improving the Efficiency of Homologous Gene Replacement by Disrupting the NHEJ Pathway for Gene KusA in the Oleaginous Fungus Mortierella alpina

    NASA Astrophysics Data System (ADS)

    Krueger, Kathleen; Dai, Ziyu; Uzuner, Uger

    2012-11-01

    Mortierella alpina, a oleaginous filamentous fungus, is one of industrial fungal strains known for the production of arachidonic acid. It is also of particular interest for hydrocarbon biofuel production since it is able to produce up to 50% of its mass in rich, long-chain polyunsaturated fatty acids [PUFA's]. In addition to high fatty acid production, M. alpina like many other oleaginous fungi, already have mechanisms for accumulating significant concentrations of hydrophobic compounds making it a naturally equipped candidate to handle potential toxic concentrations of hydrocarbons. The goal of this study was to develop an efficient transformation method for this strain, hence allowing researchers to further manipulate these fungi for further improvement of lipid production. Included was optimization of best culture medium for growth and maintenance, optimal conditions for protoplast generation, and replacement of the homologous KusA gene. A successful deletion of KusA gene within biotechnologically important M. alpina could enable homologous recombination of other genes of interest in a higher frequency. This capacity may also improve the advancing the production of microbial oils for bioenergy and arachidonic acid human health applications.

  6. Synthesis and Evaluation of Tetramethylguanidinium-Polyethylenimine Polymers as Efficient Gene Delivery Vectors

    PubMed Central

    Mahato, Manohar; Yadav, Santosh; Kumar, Pradeep; Sharma, Ashwani Kumar

    2014-01-01

    Previously, we demonstrated that 6-(N,N,N′,N′-tetramethylguanidinium chloride)-hexanoyl-polyethylenimine (THP) polymers exhibited significantly enhanced transfection efficiency and cell viability. Here, in the present study, we have synthesized a series of N,N,N′,N′-tetramethylguanidinium-polyethylenimine (TP1-TP5) polymers via a single-step reaction involving peripheral primary amines of bPEI and varying amounts of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). These polymers were found to interact efficiently with negatively charged pDNA and formed stable complexes in the size range of ~240–450 nm. Acid-base titration profiles revealed improved buffering capacity of TP polymers as compared to bPEI. Transfection and cytotoxicity assays performed with TP/pDNA complexes on HEK293, CHO, and HeLa cells showed significantly higher transfection efficiency and cell viability with one of the complexes, TP2/pDNA complex, exhibited the highest transfection efficiency (~1.4–2.3-fold) outcompeting native bPEI and the commercially available transfection reagent, Lipofectamine 2000. Compared to previously reported THP polymers, the transfection efficiency of TP/pDNA complexes was found to be lower, as examined by flow cytometry. These results highlight the importance of the hydrophobic C-6 linker in THP polymers in forming compact nanostructures with pDNA, which might lead to efficient uptake and internalization of the complexes; however, the projected TP polymers offer an advantage of their rapid and economical one-step synthesis. PMID:24864245

  7. The enhancement of gene silencing efficiency with chitosan-coated liposome formulations of siRNAs targeting HIF-1α and VEGF.

    PubMed

    Şalva, Emine; Turan, Suna Özbaş; Eren, Fatih; Akbuğa, Jülide

    2015-01-15

    RNA interference (RNAi) holds considerable promise as a novel therapeutic strategy in the silencing of disease-causing genes. The development of effective delivery systems is important for the use of small interfering RNA (siRNA) as therapy. In the present study, we investigated the effect on breast cancer cell lines and the co-delivery of liposomes containing siHIF1-α and siVEGF. In order to achieve the co-delivery of siHIF1-α and siVEGF and to obtain lower cytotoxicity, higher transfection and silencing efficiency, in this study, we used chitosan-coated liposomal formulation as the siRNA delivery system. The obtained particle size and zeta potential values show that the chitosan coating process is an effective parameter for particle size and the zeta potential of liposomes. The liposome formulations loaded with siHIF1-α and siVEGF showed good stability and protected siRNA from serum degradation after 24-h of incubation. The expression level of VEGF mRNA was markedly suppressed in MCF-7 and MDA-MB435 cells transfected with chitosan-coated liposomes containing HIF1-α and VEGF siRNA, respectively (95% and 94%). In vitro co-delivery of siVEGF and siHIF1-α using chitosan-coated liposome significantly inhibited VEGF (89%) and the HIF1-α (62%) protein expression when compared to other liposome formulations in the MDA-MB435 cell. The co-delivery of siVEGF and siHIF1-α was greatly enhanced in the vitro gene silencing efficiency. In addition, chitosan-coated liposomes showed 96% cell viability. Considering the role of VEGF and HIF1-α in breast cancer, siRNA-based therapies with chitosan coated liposomes may have some promises in cancer therapy.

  8. Examining the Impact of an Integrative Method of Using Technology on Students' Achievement and Efficiency of Computer Usage and on Pedagogical Procedure in Geometry

    ERIC Educational Resources Information Center

    Gurevich, Irina; Gurev, Dvora

    2012-01-01

    In the current study we follow the development of the pedagogical procedure for the course "Constructions in Geometry" that resulted from using dynamic geometry software (DGS), where the computer became an integral part of the educational process. Furthermore, we examine the influence of integrating DGS into the course on students' achievement and…

  9. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

    PubMed

    Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

    2015-02-01

    Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line.

  10. Efficient gene therapy-based method for the delivery of therapeutics to primate cortex.

    PubMed

    Kells, Adrian P; Hadaczek, Piotr; Yin, Dali; Bringas, John; Varenika, Vanja; Forsayeth, John; Bankiewicz, Krystof S

    2009-02-17

    Transduction of the primate cortex with adeno-associated virus (AAV)-based gene therapy vectors has been challenging, because of the large size of the cortex. We report that a single infusion of AAV2 vector into thalamus results in widespread expression of transgene in the cortex through transduction of widely dispersed thalamocortical projections. This finding has important implications for the treatment of certain genetic and neurodegenerative diseases.

  11. Identification of candidate genes underlying an iron efficiency QTL in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prevalent on calcareous soils in the United States and abroad, iron deficiency is among the most common and severe nutritional stresses in plants. In soybean commercial plantings, identification and use of iron efficient genotypes has proven to be the best form of managing this soil-related plant st...

  12. Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles.

    PubMed

    Yan, Jingjing; Du, Yong-Zhong; Chen, Feng-Ying; You, Jian; Yuan, Hong; Hu, Fu-Qiang

    2013-07-01

    A stearic acid-grafted chitosan oligosaccharide (CS-SA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection, and excellent cellular internalization, a CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, bovine serum albumin (BSA) with a different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporated into a CS-SA based gene delivery system. CS-SA/DNA binary complexes and CS-SA/BSA/DNA ternary complexes were then prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However, referring to the transfection activity, the BSA of different isoelectric point value (pI) had a distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA(4.7)/DNA complexes which showed the highest transfection efficiency. On the contrary, CS-SA/BSA(9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA(4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake. The aforementioned results indicated that the acid group in protein played an important role in enhancing the transfection efficiency of CS/BSA/DNA complexes, and the study provided guidelines in the design of an efficient vector for DNA transfection.

  13. Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles.

    PubMed

    Yan, Jingjing; Du, Yong-Zhong; Chen, Feng-Ying; You, Jian; Yuan, Hong; Hu, Fu-Qiang

    2013-07-01

    A stearic acid-grafted chitosan oligosaccharide (CS-SA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection, and excellent cellular internalization, a CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, bovine serum albumin (BSA) with a different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporated into a CS-SA based gene delivery system. CS-SA/DNA binary complexes and CS-SA/BSA/DNA ternary complexes were then prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However, referring to the transfection activity, the BSA of different isoelectric point value (pI) had a distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA(4.7)/DNA complexes which showed the highest transfection efficiency. On the contrary, CS-SA/BSA(9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA(4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake. The aforementioned results indicated that the acid group in protein played an important role in enhancing the transfection efficiency of CS/BSA/DNA complexes, and the study provided guidelines in the design of an efficient vector for DNA transfection. PMID:23679858

  14. Gene therapy of Hunter syndrome: evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS).

    PubMed

    Friso, A; Tomanin, R; Zanetti, A; Mennuni, C; Calvaruso, F; La Monica, N; Marin, O; Zacchello, F; Scarpa, M

    2008-10-01

    Mucopolysaccharidosis type II (MPSII) is an inherited disorder due to a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The disease is characterized by a considerable deposition of heparan- and dermatan-sulfate, causing a general impairment of physiological functions. Most of the therapeutic protocols proposed so far are mainly based upon enzyme replacement therapy which is very expensive. There is a requirement for an alternative approach, and to this aim, we evaluated the feasibility of muscle electro gene transfer (EGT) performed in the IDS-knockout (IDS-ko) mouse model. EGT is a highly efficient method of delivering exogenous molecules into different tissues. More recently, pre-treatment with bovine hyaluronidase has shown to further improve transfection efficiency of muscle EGT. We here show that, by applying such procedure, IDS was very efficiently produced inside the muscle. However, no induced IDS activity was measured in the IDS-ko mice plasma, in contrast to matched healthy controls. In the same samples, an anticipated and rapidly increasing immune response against the recombinant protein was observed in the IDS-ko vs control mice, although reaching the same levels at 5 weeks post-injection. Additional experiments performed on healthy mice showed a significant contribution of hyaluronidase pre-treatment in increasing the immune response.

  15. How Many Letters Should Preschoolers in Public Programs Know? The Diagnostic Efficiency of Various Preschool Letter-Naming Benchmarks for Predicting First-Grade Literacy Achievement

    ERIC Educational Resources Information Center

    Piasta, Shayne B.; Petscher, Yaacov; Justice, Laura M.

    2012-01-01

    Review of current federal and state standards indicates little consensus or empirical justification regarding appropriate goals, often referred to as benchmarks, for preschool letter-name learning. The present study investigated the diagnostic efficiency of various letter-naming benchmarks using a longitudinal database of 371 children who attended…

  16. Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

    PubMed

    Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-01

    The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

  17. Efficient in vivo gene transfer by intraperitoneal injection of plasmid DNA and calcium carbonate microflowers in mice.

    PubMed

    Fumoto, Shintaro; Nakajima, Sayuri; Mine, Toyoharu; Yoshikawa, Naoki; Kitahara, Takashi; Sasaki, Hitoshi; Miyamoto, Hirotaka; Nishida, Koyo

    2012-07-01

    Gene transfer to intraperitoneal organs is thought to be a promising approach to treat such conditions as peritoneal fibrosis and peritoneal dissemination of cancers. We previously discovered that simple instillation of naked plasmid DNA (pDNA) onto intraperitoneal organs such as the liver and stomach could effectively transfer foreign genes in mice. In this study, we developed a novel nonviral method to enhance transfection efficiency of naked pDNA to intraperitoneal organs using a calcium carbonate suspension containing pDNA. Using commercially available calcium carbonate, we successfully transfected pDNA to the stomach. Handling of commercially available calcium carbonate, however, was troublesome owing to rapid precipitation and caking. To obtain slowly settling particles of calcium carbonate, we tried to synthesize novel versions of such particles and succeeded in creating flower-shaped particles, named calcium carbonate microflowers. Sedimentation of calcium carbonate microflowers was sufficiently slow for in vivo experiments. Moreover, the transfection efficiency of the suspension of calcium carbonate microflowers to the stomach was more effective than that of commercially available calcium carbonate, especially at low concentrations. Intraperitoneal injection of the suspension of calcium carbonate microflowers containing pDNA greatly enhanced naked pDNA transfer to whole intraperitoneal organs in mice. Furthermore, lactate dehydrogenase activities in intraperitoneal fluid and plasma were not raised by the suspension of calcium carbonate microflowers.

  18. Efficient Clinical Scale Gene Modification via Zinc Finger Nuclease–Targeted Disruption of the HIV Co-receptor CCR5

    PubMed Central

    Maier, Dawn A.; Brennan, Andrea L.; Jiang, Shuguang; Binder-Scholl, Gwendolyn K.; Lee, Gary; Plesa, Gabriela; Zheng, Zhaohui; Cotte, Julio; Carpenito, Carmine; Wood, Travis; Spratt, S. Kaye; Ando, Dale; Gregory, Philip; Holmes, Michael C.; Perez, Elena. E.; Riley, James L.; Carroll, Richard G.; June, Carl H.

    2013-01-01

    Abstract Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4+ T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >1010 CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection. PMID:23360514

  19. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.

    PubMed

    Arazoe, Takayuki; Miyoshi, Kennosuke; Yamato, Tohru; Ogawa, Tetsuo; Ohsato, Shuichi; Arie, Tsutomu; Kuwata, Shigeru

    2015-12-01

    CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi. PMID:26039904

  20. Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5.

    PubMed

    Maier, Dawn A; Brennan, Andrea L; Jiang, Shuguang; Binder-Scholl, Gwendolyn K; Lee, Gary; Plesa, Gabriela; Zheng, Zhaohui; Cotte, Julio; Carpenito, Carmine; Wood, Travis; Spratt, S Kaye; Ando, Dale; Gregory, Philip; Holmes, Michael C; Perez, Elena E; Riley, James L; Carroll, Richard G; June, Carl H; Levine, Bruce L

    2013-03-01

    Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

  1. [Construction and selection of the most efficient siRNA interfering plasmid specific to mouse Qa-1 gene].

    PubMed

    Cheng, Zi-He; Fang, Jian-Pei; Xu, Hong-Gui; Pan, Qiu-Hui

    2008-10-01

    This study was purposed to construct three siRNA eukaryotic expression vector specific to mouse Qa-1 gene, to investigate its silencing effect on Qa-1 gene and to select the most efficient siRNA plasmid specific to mouse Qa-1 gene. Three siRNA peptides specific to mouse Qa-1 through siRNA Web design tools of Ambion company were chosed. Jingsai Company helped to complete the siRNA eukaryotic expression vector. The mouse NIH3T3 cells cultured in RPMI 1640 medium with 10% fetal bovine serum were divided into four groups: three groups of the cells were transfected with lipofectamine 2000 reagent and three different siRNA eukaryotic expression vectors, while one group cells were transfected with lipofectamine 2000 reagent alone as negative control. Cells were collected at 24, 48, 72 hours after transfection; the RNA level of Qa-1 was detected by RT-PCR, and the expression position was examined with flow cytometry analysis by using anti-Qa-1 monoclonal antibody. The results indicated that the constructed three siRNA eukaryotic expression vectors were found to be specific to mouse Qa-1 gene. The sequence analysis showed that the sequence was identical to what chosed from web tools. NIH3T3 cells in vitro were adhered in culture that cell shape appeared to change after transfection. RT-PCR and flow cytometry analysis by using anti-Qa-1 monoclonal antibody approved that both Qa-1 RNA and the expression of Qa-1 on cell surface decreased. The decreased levels in the three groups were different. At 24, 48 and 72 hours, the expression of Qa-1 on NIH3T3 cells decreased as in the following: H2-T231: 60.9%, 81.9%, 43.6%; H2-T232: 64.5%, 73.9%, 61.1%; H2-T233: 61.9%, 71.2%, 47.5%. H2-T232 was most efficient one in all three time points. It is concluded that all three siRNA eukaryotic expression vectors selected can successfully suppress the expression of the Qa-1, and from them H2-T232 is most efficient. PMID:18928620

  2. Dye-sensitized solar cells employing a single film of mesoporous TiO2 beads achieve power conversion efficiencies over 10%.

    PubMed

    Sauvage, Frédéric; Chen, Dehong; Comte, Pascal; Huang, Fuzhi; Heiniger, Leo-Philipp; Cheng, Yi-Bing; Caruso, Rachel A; Graetzel, Michael

    2010-08-24

    Dye-sensitized solar cells employing mesoporous TiO(2) beads have demonstrated longer electron diffusion lengths and extended electron lifetimes over Degussa P25 titania electrodes due to the well interconnected, densely packed nanocrystalline TiO(2) particles inside the beads. Careful selection of the dye to match the dye photon absorption characteristics with the light scattering properties of the beads have improved the light harvesting and conversion efficiency of the bead electrode in the dye-sensitized solar cell. This has resulted in a solar to electric power conversion efficiency (PCE) of greater than 10% (10.6% for Ru(II)-based dye C101 and 10.7% using C106) for the first time using a single screen-printed titania layer cell construction (that is, without an additional scattering layer).

  3. Identification of genes associated with nitrogen-use efficiency by genome-wide transcriptional analysis of two soybean genotypes

    PubMed Central

    2011-01-01

    Background Soybean is a valuable crop that provides protein and oil. Soybean requires a large amount of nitrogen (N) to accumulate high levels of N in the seed. The yield and protein content of soybean seeds are directly affected by the N-use efficiency (NUE) of the plant, and improvements in NUE will improve yields and quality of soybean products. Genetic engineering is one of the approaches to improve NUE, but at present, it is hampered by the lack of information on genes associated with NUE. Solexa sequencing is a new method for estimating gene expression in the transcription level. Here, the expression profiles were analyzed between two soybean varieties in N-limited conditions to identify genes related to NUE. Results Two soybean genotypes were grown under N-limited conditions; a low-N-tolerant variety (No.116) and a low-N-sensitive variety (No.84-70). The shoots and roots of soybeans were used for sequencing. Eight libraries were generated for analysis: 2 genotypes × 2 tissues (roots and shoots) × 2 time periods [short-term (0.5 to 12 h) and long-term (3 to 12 d) responses] and compared the transcriptomes by high-throughput tag-sequencing analysis. 5,739,999, 5,846,807, 5,731,901, 5,970,775, 5,476,878, 5,900,343, 5,930,716, and 5,862,642 clean tags were obtained for the eight libraries: L1, 116-shoot short-term; L2 84-70-shoot short-term; L3 116-shoot long-term; L4 84-70-shoot long-term; L5 116-root short-term; L6 84-70-root short-term; L7 116-root long-term;L8 84-70-root long-term; these corresponded to 224,154, 162,415, 191,994, 181,792, 204,639, 206,998, 233,839 and 257,077 distinct tags, respectively. The clean tags were mapped to the reference sequences for annotation of expressed genes. Many genes showed substantial differences in expression among the libraries. In total, 3,231genes involved in twenty-two metabolic and signal transduction pathways were up- or down-regulated. Twenty-four genes were randomly selected and confirmed their expression

  4. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  5. Highly Efficient Gene Suppression by Chemically Modified 27 Nucleotide Double-Stranded RNAs

    NASA Astrophysics Data System (ADS)

    Kubo, Takanori; Zhelev, Zhivko; Bakalova, Rumiana; Ohba, Hideki

    2008-02-01

    RNA interference (RNAi) technology, described by Fire and Mello in 1998, is a powerful tool for the suppression of gene expression in mammalian cells. RNAi technology has several advantages over other chemical and genetic drugs. However, several problems in RNAi technology, such as cellular delivery, nuclease stability, and side effects, should be solved before applying it in the clinic. In this study, we focused on the development of novel chemically modified 27 nucleotide (nt) double-stranded RNAs (dsRNAs) with improved biological properties. Our chemically modified 27 nt dsRNAs exhibited an enhanced RNAi activity and a markedly increased stability in cell culture medium (containing 10% serum) in comparison with widely used 21 nt siRNAs and recently reported nonmodified 27 nt dsRNAs. The chemically modified 27 nt dsRNAs also exhibited a strong high long-term gene silencing effect after the 7 d treatment of viable cells. The chemically modified 27 nt dsRNAs in specific positions could be processed to 21 nt siRNAs by a recombinant Dicer enzyme. We suggested that the chemically modified 27 nt dsRNAs could be used for therapeutic applications (as genetic drugs) and bioanalyses.

  6. An Efficient LCM-Based Method for Tissue Specific Expression Analysis of Genes and miRNAs

    PubMed Central

    Gautam, Vibhav; Singh, Archita; Singh, Sharmila; Sarkar, Ananda K.

    2016-01-01

    Laser Capture Microdissection (LCM) is a powerful tool to isolate and study gene expression pattern of desired and less accessible cells or tissues from a heterogeneous population. Existing LCM-based methods fail to obtain high quality RNA including small RNAs from small microdissected plant tissue and therefore, are not suitable for miRNA expression studies. Here, we describe an efficient and cost-effective method to obtain both high quality RNA and miRNAs from LCM-derived embryonic root apical meristematic tissue, which is difficult to access. We have significantly modified and improved the tissue fixation, processing, sectioning and RNA isolation steps and minimized the use of kits. Isolated RNA was checked for quality with bioanalyzer and used for gene expression studies. We have confirmed the presence of 19-24 nucleotide long mature miRNAs using modified stem-loop RT-PCR. This modified LCM-based method is suitable for tissue specific expression analysis of both genes and small RNAs (miRNAs). PMID:26861910

  7. Mitochondrial genome of the Komodo dragon: efficient sequencing method with reptile-oriented primers and novel gene rearrangements.

    PubMed

    Kumazawa, Yoshinori; Endo, Hideki

    2004-04-30

    The mitochondrial genome of the Komodo dragon (Varanus komodoensis) was nearly completely sequenced, except for two highly repetitive noncoding regions. An efficient sequencing method for squamate mitochondrial genomes was established by combining the long polymerase chain reaction (PCR) technology and a set of reptile-oriented primers designed for nested PCR amplifications. It was found that the mitochondrial genome had novel gene arrangements in which genes from NADH dehydrogenase subunit 6 to proline tRNA were extensively shuffled with duplicate control regions. These control regions had 99% sequence similarity over 700 bp. Although snake mitochondrial genomes are also known to possess duplicate control regions with nearly identical sequences, the location of the second control region suggested independent occurrence of the duplication on lineages leading to snakes and the Komodo dragon. Another feature of the mitochondrial genome of the Komodo dragon was the considerable number of tandem repeats, including sequences with a strong secondary structure, as a possible site for the slipped-strand mispairing in replication. These observations are consistent with hypotheses that tandem duplications via the slipped-strand mispairing may induce mitochondrial gene rearrangements and may serve to maintain similar copies of the control region.

  8. Mitochondrial genome of the Komodo dragon: efficient sequencing method with reptile-oriented primers and novel gene rearrangements.

    PubMed

    Kumazawa, Yoshinori; Endo, Hideki

    2004-04-30

    The mitochondrial genome of the Komodo dragon (Varanus komodoensis) was nearly completely sequenced, except for two highly repetitive noncoding regions. An efficient sequencing method for squamate mitochondrial genomes was established by combining the long polymerase chain reaction (PCR) technology and a set of reptile-oriented primers designed for nested PCR amplifications. It was found that the mitochondrial genome had novel gene arrangements in which genes from NADH dehydrogenase subunit 6 to proline tRNA were extensively shuffled with duplicate control regions. These control regions had 99% sequence similarity over 700 bp. Although snake mitochondrial genomes are also known to possess duplicate control regions with nearly identical sequences, the location of the second control region suggested independent occurrence of the duplication on lineages leading to snakes and the Komodo dragon. Another feature of the mitochondrial genome of the Komodo dragon was the considerable number of tandem repeats, including sequences with a strong secondary structure, as a possible site for the slipped-strand mispairing in replication. These observations are consistent with hypotheses that tandem duplications via the slipped-strand mispairing may induce mitochondrial gene rearrangements and may serve to maintain similar copies of the control region. PMID:15449544

  9. Jumping the nuclear envelop barrier: Improving polyplex-mediated gene transfection efficiency by a selective CDK1 inhibitor RO-3306.

    PubMed

    Zhou, Xuefei; Liu, Xiangrui; Zhao, Bingxiang; Liu, Xin; Zhu, Dingcheng; Qiu, Nasha; Zhou, Quan; Piao, Ying; Zhou, Zhuxian; Tang, Jianbin; Shen, Youqing

    2016-07-28

    Successful transfection of plasmid DNA (pDNA) requires intranuclear internalization of pDNA effectively and the nuclear envelope appears to be one of the critical intracellular barriers for polymer mediated pDNA delivery. Polyethylenimine (PEI), as the classic cationic polymer, compact the negatively charged pDNA tightly and make up stable polyplexes. The polyplexes are too large to enter the nuclear through nuclear pores and it is believed that the nuclear envelope breakdown in mitosis could facilitate the nuclear entry of polyplexes. To jump the nuclear envelope barrier, we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle and increased the proportion of mitotic cells which have disappeared nuclear envelope during transfection. Herein, we show that RO-3306 remarkably increases the transfection efficiency of PEI polyplexes through enhanced nuclear localization of PEI and pDNA. However, RO-3306 is less effective to the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) which responses to cellular stimuli and releases free pDNA in cytoplasm. Our findings not only offer new opportunities for improving non-viral based gene delivery but also provide theoretical support for the rational design of novel functional polymers for gene delivery. We also report current data showing that RO-3306 synergizes TRAIL gene induced apoptosis in cancer cells.

  10. Targeted modifications in adeno-associated virus serotype 8 capsid improves its hepatic gene transfer efficiency in vivo.

    PubMed

    Sen, Dwaipayan; Gadkari, Rupali A; Sudha, Govindarajan; Gabriel, Nishanth; Kumar, Yesupatham Sathish; Selot, Ruchita; Samuel, Rekha; Rajalingam, Sumathi; Ramya, V; Nair, Sukesh C; Srinivasan, Narayanaswamy; Srivastava, Alok; Jayandharan, Giridhara R

    2013-04-01

    Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (~9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in

  11. Rhizobium nodM and nodN genes are common nod genes: nodM encodes functions for efficiency of nod signal production and bacteroid maturation.

    PubMed Central

    Baev, N; Schultze, M; Barlier, I; Ha, D C; Virelizier, H; Kondorosi, E; Kondorosi, A

    1992-01-01

    Earlier, we showed that Rhizobium meliloti nodM codes for glucosamine synthase and that nodM and nodN mutants produce strongly reduced root hair deformation activity and display delayed nodulation of Medicago sativa (Baev et al., Mol. Gen. Genet. 228:113-124, 1991). Here, we demonstrate that nodM and nodN genes from Rhizobium leguminosarum biovar viciae restore the root hair deformation activity of exudates of the corresponding R. meliloti mutant strains. Partial restoration of the nodulation phenotypes of these two strains was also observed. In nodulation assays, galactosamine and N-acetylglucosamine could substitute for glucosamine in the suppression of the R. meliloti nodM mutation, although N-acetylglucosamine was less efficient. We observed that in nodules induced by nodM mutants, the bacteroids did not show complete development or were deteriorated, resulting in decreased nitrogen fixation and, consequently, lower dry weights of the plants. This mutant phenotype could also be suppressed by exogenously supplied glucosamine, N-acetylglucosamine, and galactosamine and to a lesser extent by glucosamine-6-phosphate, indicating that the nodM mutant bacteroids are limited for glucosamine. In addition, by using derivatives of the wild type and a nodM mutant in which the nod genes are expressed at a high constitutive level, it was shown that the nodM mutant produces significantly fewer Nod factors than the wild-type strain but that their chemical structures are unchanged. However, the relative amounts of analogs of the cognate Nod signals were elevated, and this may explain the observed host range effects of the nodM mutation. Our data indicate that both the nodM and nodN genes of the two species have common functions and confirm that NodM is a glucosamine synthase with the biochemical role of providing sufficient amounts of the sugar moiety for the synthesis of the glucosamine oligosaccharide signal molecules. Images PMID:1447128

  12. The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants.

    PubMed Central

    Crespi, M; Vereecke, D; Temmerman, W; Van Montagu, M; Desomer, J

    1994-01-01

    Three virulence loci (fas, att, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed that fas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants. PMID:8169198

  13. Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid.

    PubMed

    Ito, Tomoko; Iida-Tanaka, Naoko; Koyama, Yoshiyuki

    2008-05-01

    Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells. PMID:18446606

  14. Simple, Efficient CRISPR-Cas9-Mediated Gene Editing in Mice: Strategies and Methods.

    PubMed

    Low, Benjamin E; Kutny, Peter M; Wiles, Michael V

    2016-01-01

    Genetic modification of almost any species is now possible using approaches based on targeted nucleases. These novel tools now bypass previous limited species windows, allowing precision nucleotide modification of the genome at high efficiency, rapidly and economically. Here we focus on the modification of the mouse genome; the mouse, with its short generation time and comparatively low maintenance/production costs is the perfect mammal with which to probe the genome to understand its functions and complexities. Further, using targeted nucleases combined with homologous recombination, it is now possible to precisely tailor the genome, creating models of human diseases and conditions directly and efficiently in zygotes derived from any mouse strain. Combined these approaches make it possible to sequentially and progressively refine mouse models to better reflect human disease, test and develop therapeutics. Here, we briefly review the strategies involved in designing targeted nucleases (sgRNAs) providing solutions and outlining in detail the practical processes involved in precision targeting and modification of the mouse genome and the establishing of new precision genetically modified mouse lines. PMID:27150082

  15. Efficient Gene Delivery by Sonoporation Is Associated with Microbubble Entry into Cells and the Clathrin-Dependent Endocytosis Pathway.

    PubMed

    Delalande, Anthony; Leduc, Chloé; Midoux, Patrick; Postema, Michiel; Pichon, Chantal

    2015-07-01

    Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble-cell interactions do not occur randomly during sonoporation; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

  16. Transcriptomics-assisted quantitative trait locus fine mapping for the rapid identification of a nodulin 26-like intrinsic protein gene regulating boron efficiency in allotetraploid rapeseed.

    PubMed

    Hua, Yingpeng; Zhang, Didi; Zhou, Ting; He, Mingliang; Ding, Guangda; Shi, Lei; Xu, Fangsen

    2016-07-01

    Allotetraploid rapeseed (Brassica napus L., An An Cn Cn , 2n = 4x = 38) is extraordinarily susceptible to boron (B) deficiency, a ubiquitous problem causing severe losses in seed yield. The breeding of B-efficient rapeseed germ plasm is a cost-effective and environmentally friendly strategy for the agricultural industry; however, genes regulating B efficiency in allotetraploid rapeseed have not yet been isolated. In this research, quantitative trait locus (QTL) fine mapping and digital gene expression (DGE) profiling were combined to identify the candidate genes underlying the major-effect QTL qBEC-A3a, which regulates B efficiency. Comparative phenotype analyses of the near-isogenic lines (NILs) indicated that qBEC-A3a plays a significant role in improving B efficiency under B deficiency. Exploiting QTL fine mapping and DGE analyses revealed a nodulin 26-like intrinsic protein (NIP) gene, which encodes a likely boric acid channel. The gene co-expression network for putative B transporters also highlighted its central role in the efficiency of B uptake. An integration of whole-genome re-sequencing (WGS) with bulked segregant analysis (BSA) authenticated the emerging availability of QTL-seq for the QTL analyses in allotetraploid rapeseed. Transcriptomics-assisted QTL mapping and comparative genomics provided novel insights into the rapid identification of quantitative trait genes (QTGs) in plant species with complex genomes. PMID:26934080

  17. A nuclear localization signal in the matrix of spleen necrosis virus (SNV) does not allow efficient gene transfer into quiescent cells with SNV-derived vectors

    SciTech Connect

    Caron, Marie-Christine; Caruso, Manuel . E-mail: manuel.caruso@crhdq.ulaval.ca

    2005-08-01

    A major limitation in gene therapy for vectors derived from Moloney murine leukemia virus (MLV) is that they only deliver genes into dividing cells. In this study, a careful comparison of spleen necrosis virus (SNV)-derived vectors with MLV and human immunodeficiency virus (HIV)-1 retroviral vectors indicated that SNV vectors can deliver genes 4-fold more efficiently than MLV vectors into aphidicolin-arrested cells, although at a 25-fold lower efficiency than HIV-1-derived vectors. Furthermore, the addition of a NLS in the SNV matrix (MA) that mimics the one located in HIV-1 MA did not increase the ability of SNV vectors to transfer genes into arrested cells. Also, we found that the RD114 envelope was able to pseudotype SNV viral particles in a very efficient manner.

  18. Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9.

    PubMed

    Lv, Qingyan; Yuan, Lin; Deng, Jichao; Chen, Mao; Wang, Yong; Zeng, Jian; Li, Zhanjun; Lai, Liangxue

    2016-04-26

    CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models. Myostatin (MSTN) is a negative regulator of muscle mass, related to muscle growth and differentiation. The knockout of MSTN with the desired phenotype of double muscle has been successfully generated in mice, goats, pigs and cattle, but not in rabbits. In this study, the MSTN knockout (KO) rabbits were generated by co-injection of Cas9 mRNA and sgRNA into zygotes. The typical phenotype of double muscle with hyperplasia or hypertrophy of muscle fiber was observed in MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation, suggesting that the mutation of MSTN could be stably inherited in the MSTN KO rabbits. In summary, we have successfully generated MSTN KO rabbits using CRISPR/Cas9 system with high efficiency, which is a reliable and effective animal model for the study of muscle development and related diseases.

  19. Codon-optimized human sodium iodide symporter (opt-hNIS) as a sensitive reporter and efficient therapeutic gene.

    PubMed

    Kim, Young-Hwa; Youn, Hyewon; Na, Juri; Hong, Kee-Jong; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

    2015-01-01

    To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. 125I uptake by opt-hNIS-expressing cells was 1.6~2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and 125I uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. 131I treatment reduced tumor sizes of hNIS- and opt-hNIS-expressing tumors to 0.57- and 0.27- fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene.

  20. Achieving extremely concentrated aqueous dispersions of graphene flakes and catalytically efficient graphene-metal nanoparticle hybrids with flavin mononucleotide as a high-performance stabilizer.

    PubMed

    Ayán-Varela, M; Paredes, J I; Guardia, L; Villar-Rodil, S; Munuera, J M; Díaz-González, M; Fernández-Sánchez, C; Martínez-Alonso, A; Tascón, J M D

    2015-05-20

    The stable dispersion of graphene flakes in an aqueous medium is highly desirable for the development of materials based on this two-dimensional carbon structure, but current production protocols that make use of a number of surfactants typically suffer from limitations regarding graphene concentration or the amount of surfactant required to colloidally stabilize the sheets. Here, we demonstrate that an innocuous and readily available derivative of vitamin B2, namely the sodium salt of flavin mononucleotide (FMNS), is a highly efficient dispersant in the preparation of aqueous dispersions of defect-free, few-layer graphene flakes. Most notably, graphene concentrations in water as high as ∼50 mg mL(-1) using low amounts of FMNS (FMNS/graphene mass ratios of about 0.04) could be attained, which facilitated the formation of free-standing graphene films displaying high electrical conductivity (∼52000 S m(-1)) without the need of carrying out thermal annealing or other types of post-treatment. The excellent performance of FMNS as a graphene dispersant could be attributed to the combined effect of strong adsorption on the sheets through the isoalloxazine moiety of the molecule and efficient colloidal stabilization provided by its negatively charged phosphate group. The FMNS-stabilized graphene sheets could be decorated with nanoparticles of several noble metals (Ag, Pd, and Pt), and the resulting hybrids exhibited a high catalytic activity in the reduction of nitroarenes and electroreduction of oxygen. Overall, the present results should expedite the processing and implementation of graphene in, e.g., conductive inks, composites, and hybrid materials with practical utility in a wide range of applications. PMID:25915172

  1. Achieving extremely concentrated aqueous dispersions of graphene flakes and catalytically efficient graphene-metal nanoparticle hybrids with flavin mononucleotide as a high-performance stabilizer.

    PubMed

    Ayán-Varela, M; Paredes, J I; Guardia, L; Villar-Rodil, S; Munuera, J M; Díaz-González, M; Fernández-Sánchez, C; Martínez-Alonso, A; Tascón, J M D

    2015-05-20

    The stable dispersion of graphene flakes in an aqueous medium is highly desirable for the development of materials based on this two-dimensional carbon structure, but current production protocols that make use of a number of surfactants typically suffer from limitations regarding graphene concentration or the amount of surfactant required to colloidally stabilize the sheets. Here, we demonstrate that an innocuous and readily available derivative of vitamin B2, namely the sodium salt of flavin mononucleotide (FMNS), is a highly efficient dispersant in the preparation of aqueous dispersions of defect-free, few-layer graphene flakes. Most notably, graphene concentrations in water as high as ∼50 mg mL(-1) using low amounts of FMNS (FMNS/graphene mass ratios of about 0.04) could be attained, which facilitated the formation of free-standing graphene films displaying high electrical conductivity (∼52000 S m(-1)) without the need of carrying out thermal annealing or other types of post-treatment. The excellent performance of FMNS as a graphene dispersant could be attributed to the combined effect of strong adsorption on the sheets through the isoalloxazine moiety of the molecule and efficient colloidal stabilization provided by its negatively charged phosphate group. The FMNS-stabilized graphene sheets could be decorated with nanoparticles of several noble metals (Ag, Pd, and Pt), and the resulting hybrids exhibited a high catalytic activity in the reduction of nitroarenes and electroreduction of oxygen. Overall, the present results should expedite the processing and implementation of graphene in, e.g., conductive inks, composites, and hybrid materials with practical utility in a wide range of applications.

  2. Achieving significantly enhanced visible-light photocatalytic efficiency using a polyelectrolyte: the composites of exfoliated titania nanosheets, graphene, and poly(diallyl-dimethyl-ammonium chloride)

    NASA Astrophysics Data System (ADS)

    Zhang, Qian; An, Qi; Luan, Xinglong; Huang, Hongwei; Li, Xiaowei; Meng, Zilin; Tong, Wangshu; Chen, Xiaodong; Chu, Paul K.; Zhang, Yihe

    2015-08-01

    A high-performance visible-light-active photocatalyst is prepared using the polyelectrolyte/exfoliated titania nanosheet/graphene oxide (GO) precursor by flocculation followed by calcination. The polyelectrolyte poly(diallyl-dimethyl-ammonium chloride) serves not only as an effective binder to precipitate GO and titania nanosheets, but also boosts the overall performance of the catalyst significantly. Unlike most titania nanosheet-based catalysts reported in the literature, the composite absorbs light in the UV-Vis-NIR range. Its decomposition rate of methylene blue is 98% under visible light. This novel strategy of using a polymer to enhance the catalytic performance of titania nanosheet-based catalysts affords immense potential in designing and fabricating next-generation photocatalysts with high efficiency.A high-performance visible-light-active photocatalyst is prepared using the polyelectrolyte/exfoliated titania nanosheet/graphene oxide (GO) precursor by flocculation followed by calcination. The polyelectrolyte poly(diallyl-dimethyl-ammonium chloride) serves not only as an effective binder to precipitate GO and titania nanosheets, but also boosts the overall performance of the catalyst significantly. Unlike most titania nanosheet-based catalysts reported in the literature, the composite absorbs light in the UV-Vis-NIR range. Its decomposition rate of methylene blue is 98% under visible light. This novel strategy of using a polymer to enhance the catalytic performance of titania nanosheet-based catalysts affords immense potential in designing and fabricating next-generation photocatalysts with high efficiency. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr03256c

  3. Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach.

    PubMed

    Vergara, M Natalia; Gutierrez, Christian; Canto-Soler, M Valeria

    2015-01-01

    The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols(1). In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility. PMID:26556302

  4. A Highly Efficient Gene Expression Programming (GEP) Model for Auxiliary Diagnosis of Small Cell Lung Cancer

    PubMed Central

    Si, Hongzong; Liu, Shihai; Li, Xianchao; Gao, Caihong; Cui, Lianhua; Li, Chuan; Yang, Xue; Yao, Xiaojun

    2015-01-01

    Background Lung cancer is an important and common cancer that constitutes a major public health problem, but early detection of small cell lung cancer can significantly improve the survival rate of cancer patients. A number of serum biomarkers have been used in the diagnosis of lung cancers; however, they exhibit low sensitivity and specificity. Methods We used biochemical methods to measure blood levels of lactate dehydrogenase (LDH), C-reactive protein (CRP), Na+, Cl-, carcino-embryonic antigen (CEA), and neuron specific enolase (NSE) in 145 small cell lung cancer (SCLC) patients and 155 non-small cell lung cancer and 155 normal controls. A gene expression programming (GEP) model and Receiver Operating Characteristic (ROC) curves incorporating these biomarkers was developed for the auxiliary diagnosis of SCLC. Results After appropriate modification of the parameters, the GEP model was initially set up based on a training set of 115 SCLC patients and 125 normal controls for GEP model generation. Then the GEP was applied to the remaining 60 subjects (the test set) for model validation. GEP successfully discriminated 281 out of 300 cases, showing a correct classification rate for lung cancer patients of 93.75% (225/240) and 93.33% (56/60) for the training and test sets, respectively. Another GEP model incorporating four biomarkers, including CEA, NSE, LDH, and CRP, exhibited slightly lower detection sensitivity than the GEP model, including six biomarkers. We repeat the models on artificial neural network (ANN), and our results showed that the accuracy of GEP models were higher than that in ANN. GEP model incorporating six serum biomarkers performed by NSCLC patients and normal controls showed low accuracy than SCLC patients and was enough to prove that the GEP model is suitable for the SCLC patients. Conclusion We have developed a GEP model with high sensitivity and specificity for the auxiliary diagnosis of SCLC. This GEP model has the potential for the wide use

  5. Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9

    PubMed Central

    Lv, Qingyan; Yuan, Lin; Deng, Jichao; Chen, Mao; Wang, Yong; Zeng, Jian; Li, Zhanjun; Lai, Liangxue

    2016-01-01

    CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models. Myostatin (MSTN) is a negative regulator of muscle mass, related to muscle growth and differentiation. The knockout of MSTN with the desired phenotype of double muscle has been successfully generated in mice, goats, pigs and cattle, but not in rabbits. In this study, the MSTN knockout (KO) rabbits were generated by co-injection of Cas9 mRNA and sgRNA into zygotes. The typical phenotype of double muscle with hyperplasia or hypertrophy of muscle fiber was observed in MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation, suggesting that the mutation of MSTN could be stably inherited in the MSTN KO rabbits. In summary, we have successfully generated MSTN KO rabbits using CRISPR/Cas9 system with high efficiency, which is a reliable and effective animal model for the study of muscle development and related diseases. PMID:27113799

  6. Efficient removal of antibiotics in surface-flow constructed wetlands, with no observed impact on antibiotic resistance genes.

    PubMed

    Berglund, Björn; Khan, Ghazanfar Ali; Weisner, Stefan E B; Ehde, Per Magnus; Fick, Jerker; Lindgren, Per-Eric

    2014-04-01

    Recently, there have been growing concerns about pharmaceuticals including antibiotics as environmental contaminants. Antibiotics of concentrations commonly encountered in wastewater have been suggested to affect bacterial population dynamics and to promote dissemination of antibiotic resistance. Conventional wastewater treatment processes do not always adequately remove pharmaceuticals causing environmental dissemination of low levels of these compounds. Using constructed wetlands as an additional treatment step after sewage treatment plants have been proposed as a cheap alternative to increase reduction of wastewater contaminants, however this means that the natural microbial community of the wetlands becomes exposed to elevated levels of antibiotics. In this study, experimental surface-flow wetlands in Sweden were continuously exposed to antibiotics of concentrations commonly encountered in wastewater. The aim was to assess the antibiotic removal efficiency of constructed wetlands and to evaluate the impact of low levels of antibiotics on bacterial diversity, resistance development and expression in the wetland bacterial community. Antibiotic concentrations were measured using liquid chromatography-mass spectrometry and the effect on the bacterial diversity was assessed with 16S rRNA-based denaturing gradient gel electrophoresis. Real-time PCR was used to detect and quantify antibiotic resistance genes and integrons in the wetlands, during and after the exposure period. The results indicated that the antibiotic removal efficiency of constructed wetlands was comparable to conventional wastewater treatment schemes. Furthermore, short-term treatment of the constructed wetlands with environmentally relevant concentrations (i.e. 100-2000 ng×l(-1)) of antibiotics did not significantly affect resistance gene concentrations, suggesting that surface-flow constructed wetlands are well-suited for wastewater treatment purposes.

  7. Efficient delivery of C/EBP beta gene into human mesenchymal stem cells via polyethylenimine-coated gold nanoparticles enhances adipogenic differentiation

    PubMed Central

    Joydeep, Das; Choi, Yun-Jung; Yasuda, Hideyo; Han, Jae Woong; Park, Chankyu; Song, Hyuk; Bae, Hojae; Kim, Jin-Hoi

    2016-01-01

    The controlled differentiation of stem cells via the delivery of specific genes encoding appropriate differentiation factors may provide useful models for regenerative medicine and aid in developing therapies for human patients. However, the majority of non-viral vectors are not efficient enough to manipulate difficult-to-transfect adult human stem cells in vitro. Herein, we report the first use of 25 kDa branched polyethylenimine-entrapped gold nanoparticles (AuPEINPs) and covalently bound polyethylenimine-gold nanoparticles (AuMUAPEINPs) as carriers for efficient gene delivery into human mesenchymal stem cells (hMSCs). We determined a functional application of these nanoparticles by transfecting hMSCs with the C/EBP beta gene, fused to EGFP, to induce adipogenic differentiation. Transfection efficacy with AuPEINPs and AuMUAPEINPs was 52.3% and 40.7%, respectively, which was 2.48 and 1.93 times higher than that by using Lipofectamine 2000. Luciferase assay results also demonstrated improved gene transfection efficiency of AuPEINPs/AuMUAPEINPs over Lipofectamine 2000 and polyethylenimine. Overexpression of exogenous C/EBP beta significantly enhanced adipogenesis in hMSCs as indicated by both of Oil Red O staining and mRNA expression analyses. Nanoparticle/DNA complexes exhibited favorable cytocompatibility in hMSCs. Taken together, AuPEINPs and AuMUAPEINPs potentially represent safe and highly efficient vehicles for gene delivery to control hMSC differentiation and for therapeutic gene delivery applications. PMID:27677463

  8. Use of boron cluster-containing redox nanoparticles with ROS scavenging ability in boron neutron capture therapy to achieve high therapeutic efficiency and low adverse effects.

    PubMed

    Gao, Zhenyu; Horiguchi, Yukichi; Nakai, Kei; Matsumura, Akira; Suzuki, Minoru; Ono, Koji; Nagasaki, Yukio

    2016-10-01

    A boron delivery system with high therapeutic efficiency and low adverse effects is crucial for a successful boron neutron capture therapy (BNCT). In this study, we developed boron cluster-containing redox nanoparticles (BNPs) via polyion complex (PIC) formation, using a newly synthesized poly(ethylene glycol)-polyanion (PEG-polyanion, possessing a (10)B-enriched boron cluster as a side chain of one of its segments) and PEG-polycation (possessing a reactive oxygen species (ROS) scavenger as a side chain of one of its segments). The BNPs exhibited high colloidal stability, selective uptake in tumor cells, specific accumulation, and long retention in tumor tissue and ROS scavenging ability. After thermal neutron irradiation, significant suppression of tumor growth was observed in the BNP-treated group, with only 5-ppm (10)B in tumor tissues, whereas at least 20-ppm (10)B is generally required for low molecular weight (LMW) (10)B agents. In addition, increased leukocyte levels were observed in the LMW (10)B agent-treated group after thermal neutron irradiation, and not in BNP-treated group, which might be attributed to its ROS scavenging ability. No visual metastasis of tumor cells to other organs was observed 1 month after irradiation in the BNP-treated group. These results suggest that BNPs are promising for enhancing the BNCT performance. PMID:27467416

  9. Use of boron cluster-containing redox nanoparticles with ROS scavenging ability in boron neutron capture therapy to achieve high therapeutic efficiency and low adverse effects.

    PubMed

    Gao, Zhenyu; Horiguchi, Yukichi; Nakai, Kei; Matsumura, Akira; Suzuki, Minoru; Ono, Koji; Nagasaki, Yukio

    2016-10-01

    A boron delivery system with high therapeutic efficiency and low adverse effects is crucial for a successful boron neutron capture therapy (BNCT). In this study, we developed boron cluster-containing redox nanoparticles (BNPs) via polyion complex (PIC) formation, using a newly synthesized poly(ethylene glycol)-polyanion (PEG-polyanion, possessing a (10)B-enriched boron cluster as a side chain of one of its segments) and PEG-polycation (possessing a reactive oxygen species (ROS) scavenger as a side chain of one of its segments). The BNPs exhibited high colloidal stability, selective uptake in tumor cells, specific accumulation, and long retention in tumor tissue and ROS scavenging ability. After thermal neutron irradiation, significant suppression of tumor growth was observed in the BNP-treated group, with only 5-ppm (10)B in tumor tissues, whereas at least 20-ppm (10)B is generally required for low molecular weight (LMW) (10)B agents. In addition, increased leukocyte levels were observed in the LMW (10)B agent-treated group after thermal neutron irradiation, and not in BNP-treated group, which might be attributed to its ROS scavenging ability. No visual metastasis of tumor cells to other organs was observed 1 month after irradiation in the BNP-treated group. These results suggest that BNPs are promising for enhancing the BNCT performance.

  10. Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells.

    PubMed

    Böttcher, Romy; Hollmann, Manuel; Merk, Karin; Nitschko, Volker; Obermaier, Christina; Philippou-Massier, Julia; Wieland, Isabella; Gaul, Ulrike; Förstemann, Klaus

    2014-06-01

    The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5'-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated.

  11. Ferulic acid, an efficient inhibitor of type B trichothecene biosynthesis and Tri gene expression in Fusarium liquid cultures.

    PubMed

    Boutigny, Anne-Laure; Barreau, Christian; Atanasova-Penichon, Vessela; Verdal-Bonnin, Marie-Noëlle; Pinson-Gadais, Laëtitia; Richard-Forget, Florence

    2009-01-01

    The effect of ferulic acid, the most abundant phenolic acid in wheat bran, was studied in vitro on type B trichothecene biosynthesis by Fusarium. It was demonstrated that ferulic acid is an efficient inhibitor of mycotoxin production by all strains of Fusarium tested, including different chemotypes and species. To analyse the mechanism of toxin biosynthesis inhibition by ferulic acid, expression of representative Tri genes, involved in the trichothecene biosynthesis pathway, was monitored by real-time RT-PCR. A decrease in the level of Tri gene expression was measured, suggesting that inhibition of toxin synthesis by ferulic acid could be regulated at the transcriptional level. Moreover, toxin production was shown to be reduced proportionally to the initial amount of ferulic acid added in the culture medium. Addition of ferulic acid either at the spore germination step or to a mycelial culture resulted in the same final inhibitory effect on mycotoxin accumulation. A cumulative inhibitory effect on trichothecene biosynthesis was even observed with successive supplementation of ferulic acid. Ferulic acid, which content varies among wheat varieties, could then play an important role in modulating trichothecene biosynthesis by Fusarium in some wheat varieties.

  12. Safe, Efficient, and Reproducible Gene Therapy of the Brain in the Dog Models of Sanfilippo and Hurler Syndromes

    PubMed Central

    Ellinwood, N Matthew; Ausseil, Jérôme; Desmaris, Nathalie; Bigou, Stéphanie; Liu, Song; Jens, Jackie K; Snella, Elizabeth M; Mohammed, Eman EA; Thomson, Christopher B; Raoul, Sylvie; Joussemet, Béatrice; Roux, Françoise; Chérel, Yan; Lajat, Yaouen; Piraud, Monique; Benchaouir, Rachid; Hermening, Stephan; Petry, Harald; Froissart, Roseline; Tardieu, Marc; Ciron, Carine; Moullier, Philippe; Parkes, Jennifer; Kline, Karen L; Maire, Irène; Vanier, Marie-Thérèse; Heard, Jean-Michel; Colle, Marie-Anne

    2011-01-01

    Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome. PMID:21139569

  13. Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16.

    PubMed

    Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

    2014-09-30

    The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

  14. Reprint of "versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16".

    PubMed

    Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

    2014-12-20

    The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

  15. Interaction of ACTN3 gene polymorphism and muscle imbalance effects on kinematic efficiency in combat sports athletes

    PubMed Central

    Lee, Namju; Park, Sok

    2016-01-01

    [Purpose] The purpose of this study was to determine the interaction of ACTN3 gene polymorphism and muscle imbalance effects on kinematic efficiency changes in combat sports athletes. [Methods] Six types of combat sports athletes (Judo, Taekwondo, boxing, kendo, wrestling, and Korean Ssi-reum) participated in the study. ATCN3 gene polymorphism and muscle imbalance in lower extremity were evaluated followed by analysis of differences of moment in hip, knee, and ankle joint during V-cut jumping and stop. To examine the moment difference due to an interaction of ATCN3 polymorphism and muscle imbalance, all participants were divided into 4 groups (R+MB, R+MIB, X+MB, and X+MIB). [Results] There was no significant difference of hip, knee, and ankle joint moment in R allele and X allele during V-cut jumping and stop based on ACTN3 gene polymorphism. Otherwise, muscle imbalance of knee moment in X-axis and ground reaction force of knee in Z-axis showed a higher significance in muscle imbalance during V-cut jumping and stop compared to muscle balance (p<0.05). In addition, joint analysis showed that muscle imbalance in X allele group had significantly higher knee moment of V-cut ground reaction force in X-axis and higher ankle moment of jumping ground reaction force in X and Z-axis compared to muscle balance with R and/or X group (p <0.05). [Conclusion] This study confirmed that muscle imbalance in lower extremity of combat athletes might induce higher risk factors of sports injury incidence than genetic factor and training might reduce the ratio of sports injury risk incidence. PMID:27508148

  16. Efficient reinforcement learning of a reservoir network model of parametric working memory achieved with a cluster population winner-take-all readout mechanism.

    PubMed

    Cheng, Zhenbo; Deng, Zhidong; Hu, Xiaolin; Zhang, Bo; Yang, Tianming

    2015-12-01

    The brain often has to make decisions based on information stored in working memory, but the neural circuitry underlying working memory is not fully understood. Many theoretical efforts have been focused on modeling the persistent delay period activity in the prefrontal areas that is believed to represent working memory. Recent experiments reveal that the delay period activity in the prefrontal cortex is neither static nor homogeneous as previously assumed. Models based on reservoir networks have been proposed to model such a dynamical activity pattern. The connections between neurons within a reservoir are random and do not require explicit tuning. Information storage does not depend on the stable states of the network. However, it is not clear how the encoded information can be retrieved for decision making with a biologically realistic algorithm. We therefore built a reservoir-based neural network to model the neuronal responses of the prefrontal cortex in a somatosensory delayed discrimination task. We first illustrate that the neurons in the reservoir exhibit a heterogeneous and dynamical delay period activity observed in previous experiments. Then we show that a cluster population circuit decodes the information from the reservoir with a winner-