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.

Monitoring bioaccumulation and toxic effects of hexachlorobenzene using the polyurethane foam unit method in the microbial communities of the Fuhe River, Wuhan

Hexachlorobenzene (HCB) is a chlorinated aromatic hydrocarbon that was widely used for seed dressing in prevention of fungal growth on crops, and also as a component of fireworks, ammunition, and synthetic rubbers. Because of its resistance to degradation and mobility, HCB is widely distributed throughout the environment and is accumulated through food chains in different ecosystems. In this study, a preliminary investigation was carried out on the bioaccumulation and the toxic effects of HCB in the microbial (protozoan in particular) communities in the Fuhe River, Wuhan, a water body receiving industrial wastewaters containing HCB and other pollutants, using the standardized polyurethane foam units (PFU) method. Field samples were taken from eight stations established along the Fuhe River in January and August 2006. The concentration ratios of HCB in microbial communities and in water were 9.66-18.64, and the microbial communities accumulated 13.29-56.88 mu g/L of HCB in January and 0.82-10.25 mu g/L HCB in August. Correlation analysis showed a negative correlation between the HCB contents in the microbial assemblage, and the number of species and the diversity index of the protozoan communities. This study demonstrated the applicability of the PFU method in monitoring the effects of HCB on the level of microbial communities.

Molecular phylogeny of Stentor (Ciliophora : Heterotrichea) based on small subunit ribosomal RNA sequences

To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally.

Reevaluation of the phylogenetic relationship between Mobilid and Sessilid peritrichs (Ciliophora, Oligohymenophorea) based on small subunit rRNA genes sequences

Based on morphological characters, peritrich ciliates (Class Olygohymenophorea, Subclass Peritrichia) have been subdivided into the Orders Sessilida and Mobilida. Molecular phylogenetic studies on peritrichs have been restricted to members of the Order Sessilida. In order to shed more light into the evolutionary relationships within peritrichs, the complete small subunit rRNA (SSU rRNA) sequences of four mobilid species, Trichodina nobilis, Trichodina heterodentata, Trichodina reticulata, and Trichodinella myakkae were used to construct phylogenetic trees using maximum parsimony, neighbor joining, and Bayesian analyses. Whatever phylogenetic method used, the peritrichs did not constitute a monophyletic group: mobilid and sessilid species did not cluster together. Similarity in morphology but difference in molecular data led us to suggest that the oral structures of peritrichs are the result of evolutionary convergence. In addition, Trichodina reticulata, a Trichodina species with granules in the center of the adhesive disc, branched separately from its congeners, Trichodina nobilis and Trichodina heterodentata, trichodinids without such granules. This indicates that granules in the adhesive disc might be a phylogenetic character of high importance within the Family Trichodinidae.

Cloning and sequencing of the growth hormone gene of large yellow croaker and its phylogenetic significance

Using conserved primers and the PCR reaction, the growth hormone (GH) gene and the 3'-UTR of the large yellow croaker (Pseudosciaena crocea) were amplified and sequenced. The gene structure was analyzed and compared to the GH genes of 5 other percoid fish downloaded from Genbank. Also the GH gene of the large yellow croaker and the genes from 14 Percoidei and 2 Labroidei species were aligned using Clustal X. A matrix of 564 bp was used to construct the phylogenetic tree using maximum parsimony and neighbor-joining methods. Phylogenetic trees by the two methods are identical in most of the clades with high bootstrap support. The results are also identical to those from morphological data. In general, this analysis does not support the monophyly of the families Centropomidae and Carangidae. But our GH gene tree indicates that the representative species of the families Sparidae and Sciaenidae are a monophyletic group.

Wafer-Level Testable High-Speed Silicon Microring Modulator Integrated with Grating Couplers

A wafer-level testable silicon-on-insulator-based microring modulator is demonstrated with high modulation speed, to which the grating couplers are integrated as the fiber-to-chip interfaces. Cost-efficient fabrications are realized with the help of optical structure and etching depth designs. Grating couplers and waveguides are patterned and etched together with the same slab thickness. Finally we obtain a 3-dB coupling bandwidth of about 60nm and 10 Gb/s nonreturn-to-zero modulation by wafer-level optical and electrical measurements.

Fabrication and performance of an index-coupled DFB laser with sampled grating

An index-coupled DFB laser with a sampled grating has been designed and fabricated. The key concept of the approaches is to utilize the +1st-order reflection of the sampled grating for laser operation, and use a conventional holographic exposure combined with the usual photolithography to form the sampled grating. The typical threshold current of the sampled grating DFB laser is 25 mA, and the optical output is about 10 mW at the injected current of 100 mA. The lasing wavelength of the device is 1.5314 mu m, which is the +1st-order peak of the sampled grating.

InP Based PD/EAM Integrated Photonic Switch

A new compact three-port InP based PD/EAM (photo-detector/electro-absorption modulator) integrated photonic switch is reported. The device demonstrates bi-directional wavelength conversion over 20 nm at 2.5 Gbit/s with a low input optical power of about 20 mW.

Measurement of a reciprocal four-port transmission line structure using the 16-term error model

A new method to measure reciprocal four-port structures, using a 16-term error model, is presented. The measurement is based on 5 two-port calibration standards connected to two of the ports, while the network analyzer is connected to the two remaining ports. Least-squares-fit data reduction techniques are used to lower error sensitivity. The effect of connectors is deembedded using closed-form equations. (C) 2007 Wiley Periodicals, Inc.

Optical microwave generation using two parallel DFB lasers integrated with Y-branch waveguide coupler

A new device of two parallel distributed feedback (DFB) lasers integrated monolithically with Y-branch waveguide coupler was fabricated by means of quantum well intermixing. Optical microwave signal was generated in the Y-branch waveguide coupler through frequency beating of the two laser modes coming from two DFB laser in parallel, which had a small difference in frequency. Continuous rapid tuning of optical microwave signal from 13 to 42 GHz were realized by adjusting independently the driving currents injected into the two DFB lasers.

Improving the optical profile of electroabsorption modulator using single-sided large optical cavity structure

An electroabsorption modulator with large optical cavity was designed and fabricated successfully. Both the simulated and experimental results show that, the larger optical cavity structure introduced could obviously improve the optical profile of EA modulator, the traditional elliptical near-field spot becomes more rounded, so it will match better with the optical fiber and is beneficial for raising the coupling efficiency.

Design and Characterization of Evanescently Coupled Uni-Traveling Carrier Photodiodes with a Multimode Diluted Waveguide Structure

A new evanescently coupled uni-traveling carrier photodiode (EC-UTC-PD) is designed, fabricated and characterized, which incorporates a multimode diluted waveguide structure and UTC active waveguide structure together. A high responsivity of 0.68A/W at 1.55-mu m without an anti-reflection coating, a linear photocurrent responsivity of more than 21 mA, and a large-1 dB vertical alignment tolerance of 2.5 mu m are achieved.

Monolithic optical gates based on integration of evanescently-coupled uni-traveling-carrier photodiodes and electroabsorption modulators

We report on chip-scale optical gates based on the integration of evanescent waveguide unitraveling-carrier photodiodes (EC-UTC-PDs) and intra-step quantum well electroabsorption modulators (IQW-EAMs) on n-InP substrates. These devices exhibit simultaneously 2.1 GHz and -16.2 dB RF-gain at 21 GHz with a 450 Omega thin-film resistor and a bypass capacitor integrated on a chip.

Micro Fabry-Perot interferometers in silica fibers machined by femtosecond laser

Micro Fabry-Perot (F-P) interferometers (MFPIs) are machined in a single-mode fiber (SMF) and a photonic crystal fiber (PCF) by using a near-infrared femtosecond laser, respectively. The strain and temperature characteristics of the two MFPIs with an identical cavity length are investigated and the experimental results show that the strain sensitivity of the PCF-based MFPI is smaller than that of the SMF-based MFPI due to their different waveguide structures, while the two MFPIs have close temperature sensitivities which are much smaller than that of an in-line SMF etalon sensor reported previously. These MFPIs in silica fibers are compact, stable, inexpensive, capable for mass-production and easy fabrication, offering great potentials for wide sensing applications. (c) 2007 Optical Society of America.