Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Vertical distribution and migration of planktonic rotifers in Xiangxi Bay of the three Gorges Reservoir, China

We investigated diel vertical migrations (DVM) and distributions of rotifers in summer, 2004 and spring, 2005, in Xiangxi Bay of the Three Gorges Reservoir, China. Water temperature, pH, conductivity, and phytoplankton were closely related to rotifer vertical distribution, while dissolved oxygen had no relationship with the vertical distribution of rotifers. The species composition and population density of rotifers changed significantly between seasons. However, rotifer vertical distributions in both seasons were similar. They aggregated at specific depths in the water column. All the rotifer species inhabited the surface layers (0.5-5 m). Generally, the rotifers did not display DVM except for Polyarthra vulgaris (in summer), which performed reverse migration. The reason that rotifers did not perform DVM may be explained by the low abundance of competitors and predators and the high density of food resources at the surface strata.

Digital data transmission using electro-optically modulated vertical-cavity surface-emitting laser with saturable absorber

An electro-optically (EO) modulated oxide-confined vertical-cavity surface-emitting laser (VCSEL) containing a saturable absorber in the VCSEL cavity is studied. The device contains an EO modulator section that is resonant with the VCSEL cavity. A type-II EO superlattice medium is employed in the modulator section and shown to result in a strong negative EO effect in weak electric fields. Applying the reverse bias voltages to the EO section allows triggering of short pulses in the device. Digital data transmission (return-to-zero pseudo-random bit sequence, 27-1) at 10Gb/s at bit-error-rates well below 10-9 is demonstrated. © 2014 AIP Publishing LLC.

Performance and mechanism of phosphorus removal in an integrated vertical flow constructed wetland treating eutrophic lake water

Phosphorus removal performance and a possible mechanism for the phosphorus removal from an eutrophic lake water were investigated using a medium-scale integrated vertical constructed wetland (combined vertical and reverse-vertical systems) from April, 11, 2001 to September, 28, 2004. Environmental factors affecting phosphorus removal and release profiles were monitored simultaneously under hydraulic loads from 400 to 2000 mm per day. The phosphorus removal rate varied with the environmental conditions. The removal rate for acidic influent water was superior to that for alkaline influent water. The substrate in the wetland chamber acted as a buffer to regulate the pH value of the water sample. As regards the water temperature, no significant differences were observed for the removal rate of total phosphorus (TP) and soluble reactive phosphorus (SRP) between low (lower than 15 degrees C) medium (16-25 degrees C) and high temperature (higher than 26 degrees C) conditions. Under a hydraulic load of 400 mm per day, the removal rate reached over 70%, the highest value achieved in this work. In addition, the highest hydraulic load of 2000 mm/d did not result in the lowest removal rate, as had been expected. After a two-year high hydraulic load test, the removal rate decreased significantly. Phosphorous release from the substrate was examined using a spatial sampling method. Depth profiles of total phosphorus and different states of phosphorus present in the substrate were recorded. This further study demonstrated that binding of phosphorus by iron and calcium might be another major factor in the removal and release of TP and SRP in this wetland system. The distribution of the speciated phosphorus showed that the amount of phosphorus captured in the substrate of the down-flow chamber was significantly higher than that captured in the up-flow chamber, suggesting that the up-flow chamber was the main source of phosphorus release in this constructed wetland.

Vertical mixing within the epilimnion modulates UVR-induced photoinhibition in tropical freshwater phytoplankton from southern China

1. The importance of vertical mixing in modulating the impact of UVR on phytoplankton photosynthesis was assessed in a tropical, shallow lake in southern China from late winter to mid-spring of 2005. 2. Daily cycles of fluorescence measurements (i.e. photosynthetic quantum yield, Y) were performed on both 'static' and in situ samples. Static samples were of surface water incubated at the surface of the lake under three radiation treatments - PAB (PAR + UVR, 280-700 nm), PA (PAR + UV-A, 320-700 nm) and P (PAR, 400-700 nm). In situ samples were collected every hour at three different depths - 0, 0.5 and 1 m. 3. The general daily pattern was of a significant decrease in Y from early morning towards noon, with partial recovery in the afternoon. Samples incubated under static conditions always had lower Y than those under in situ conditions at the same time of the day. 4. Under stratified conditions, no overall impact of UVR impact could be detected in situ when compared with the static samples. Further rapid vertical mixing not only counteracted the impact of UVR but also stimulated photosynthetic efficiency. 5. Based on these measurements of fluorescence, the mixing speed of cells moving within the epilimnion was estimated to range between 0.53 and 6.5 cm min(-1). 6. These data show that mixing is very important in modulating the photosynthetic response of phytoplankton exposed to natural radiation and, hence, strongly conditions the overall impact of UVR on aquatic ecosystems.

Framework for integration of informal waste management sector with the formal sector in Pakistan

Historically, waste pickers around the globe have utilised urban solid waste as a principal source of livelihood. Formal waste management sectors usually perceive the informal waste collection/recycling networks as backward, unhygienic and generally incompatible with modern waste management systems. It is proposed here that through careful planning and administration, these seemingly troublesome informal networks can be integrated into formal waste management systems in developing countries, providing mutual benefits. A theoretical framework for integration based on a case study in Lahore, Pakistan, is presented. The proposed solution suggests that the municipal authority should draw up and agree on a formal work contract with the group of waste pickers already operating in the area. The proposed system is assessed using the integration radar framework to classify and analyse possible intervention points between the sectors. The integration of the informal waste workers with the formal waste management sector is not a one dimensional or single step process. An ideal solution might aim for a balanced focus on all four categories of intervention, although this may be influenced by local conditions. Not all the positive benefits will be immediately apparent, but it is expected that as the acceptance of such projects increases over time, the informal recycling economy will financially supplement the formal system in many ways. © The Author(s) 2013.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Vertically-coupled microring laser array for dual-wavelength generation

We report the first demonstration of continuous-wave operation of a tunable, compact microring laser array based on a vertical-coupling architecture, well suited to larger-scale integration. Wavelength separation tunability from 4.9 to 6.3nm is observed. © 2006 Optical Society of America.

Vertical variation in dissolved alkaline phosphatase activity in a shallow eutrophic lake determined in reverse micelles

We evaluated the feasibility of microencapsulating dissolved alkaline phosphatase of a water body into reverse micelle systems prepared by hexadecyltrimethylammonium bromide as a surfactant in cyclohexane and 1-butanol as co-surfactant. The dissolved alkaline phosphatase activity within the micelle was described, including its kinetic parameters and the effects of pH and temperature on catalytic activity in surface, overlying and interstitial water of Lake Donghu. We found the similarities on the behavior of dissolved alkaline phosphatase of surface and interstitial water in reverse micelles, which was distinctly different from its behavior in the overlying water. This difference likely reflected the different origins of the dissolved alkaline phosphatase in the vertical profile of the lake. This system provides a novel tool with which to study the diversity and ecological significance of extracellular enzymes in aquatic environments.

Technology management tools: Generalization, integration and configuration

Managers in technology-intensive businesses need to make decisions in complex and dynamic environments. Many tools, frameworks and processes have been developed to support managers in these situations, leading to a proliferation of such approaches, with little consistency in terminology or theoretical foundation, and a lack of understanding of how such tools can be linked together to tackle management challenges in an integrated way. As a step towards addressing these issues, this paper proposes the concept of an integrated 'toolkit', incorporating generalized forms of three core technology management tools that support strategic planning (roadmapping, portfolio analysis and linked analysis grids). © 2006 World Scientific Publishing Company.

Minimalistic models of an energy-efficient vertical-hopping robot

The use of free vibration in elastic structure can lead to energy-efficient robot locomotion, since it significantly reduces the energy expenditure if properly designed and controlled. However, it is not well understood how to harness the dynamics of free vibration for the robot locomotion, because of the complex dynamics originated in discrete events and energy dissipation during locomotion. From this perspective, the goals of this paper are to propose a design strategy of hopping robot based on elastic curved beams and actuated rotating masses and to identify the minimalistic model that can characterize the basic principle of robot locomotion. Since the robot mainly exhibits vertical hopping, three 1-D models are examined that contain different configurations of simple spring-damper-mass components. The real-world and simulation experiments show that one of the models best characterizes the robot hopping, through analyzing the basic kinematics and negative works in actuation. Based on this model, the self-stability of hopping motion under disturbances is investigated, and design and control parameters are analyzed for the energy-efficient hopping. In addition, further analyses show that this robot can achieve the energy-efficient hopping with the variation in payload, and the source of energy dissipation of the robot hopping is investigated. © 1982-2012 IEEE.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp beta-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Minimalistic models of an energy efficient vertical hopping robot

The use of free vibration in elastic structure can lead to energy efficient robot locomotion, since it significantly reduces the energy expenditure if properly designed and controlled. However, it is not well understood how to harness the dynamics of free vibration for the robot locomotion, because of the complex dynamics originated in discrete events and energy dissipation during locomotion. From this perspective, this paper explores three minimalistic models of free vibration that can characterize the basic principle of robot locomotion. Since the robot mainly exhibits vertical hopping, three one-dimensional models are examined that contain different configurations of simple spring-damper-mass components. The self-stability of these models are also investigated in simulation. The real-world and simulation experiments show that one of the models best characterizes the robot hopping, through analyzing the basic kinematics and negative works in actuation. Based on this model, the control parameters are analyzed for the energy efficient hopping. © 2013 IEEE.

Large-payload climbing in complex vertical environments using thermoplastic adhesive bonds

Despite many approaches proposed in the past, robotic climbing in a complex vertical environment is still a big challenge. We present here an alternative climbing technology that is based on thermoplastic adhesive (TPA) bonds. The approach has a great advantage because of its large payload capacity and viability to a wide range of flat surfaces and complex vertical terrains. The large payload capacity comes from a physical process of thermal bonding, while the wide applicability benefits from rheological properties of TPAs at higher temperatures and intermolecular forces between TPAs and adherends when being cooled down. A particular type of TPA has been used in combination with two robotic platforms, featuring different foot designs, including heating/cooling methods and construction of footpads. Various experiments have been conducted to quantitatively assess different aspects of the approach. Results show that an exceptionally high ratio of 500% between dynamic payloads and body mass can be achieved for stable and repeatable vertical climbing on flat surfaces at a low speed. Assessments on four types of typical complex vertical terrains with a measure, i.e., terrain shape index ranging from -0.114 to 0.167, return a universal success rate of 80%-100%. © 2004-2012 IEEE.

Climbing vertical terrains with a self-contained robot

Vertical climbing on a variety of flat surfaces with a single robot has been previously demonstrated using vacuum suction, electrostatic adhesion, and biologically inspired approaches, etc. These methods generally have a low attachment strength, and it is not clear whether they can provide satisfactory attachment on vertical terrains with richer 3D features. Recent development of a climbing technology based on hot melt adhesives (HMAs) has shown its advantage with a high attachment strength through thermal bonding and viability to any solid surfaces. However, its feasibility for vertical climbing has only been proven on flat surfaces and with external energy supplies. This paper provides quantitative measurements for vertical climbing performance on five types of surfaces and terrains with a self-contained robot exploiting HMAs. We show that robust vertical climbing on multiple terrains can be achieved with reliable high-strength attachment. © 2012 IEEE.