Isolation and characterization of Argonaute 2: A key gene of the RNA interference pathway in the rare minnow, Gobiocypris rarus

Argonaute 2 gene plays a pivotal role in RNAi in many species. Herein is the first report of the cloning and characterization of Argonaute 2 gene in fish. The full-length cDNA of Gobiocypris rarus Argonaute 2 (GrAgo2) consisted of 3073 nucleotides encoding 869 amino acid residues with a calculated molecular weight of 98.499 kDa and an estimated isoelectric point of 9.18. Analysis of the deduced amino acid sequence showed the presence of two signature domains, PAZ and Piwi. RT-PCR analysis indicated that GrAgo2 mRNA expression could be detected in widespread tissues. After infection with grass carp reovirus, GrAgo2 expression was up-regulated from 12 h post-injection (p < 0.05) and returned to control levels at 48 h post-injection (p > 0.05). These data imply that GrAgo2 is involved in antiviral defense in rare minnow. (C) 2008 Published by Elsevier Ltd.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Interference in transport through double barriers in interacting quantum wires

We investigate interference effects of the backscattering current through a double-barrier structure in an interacting quantum wire attached to noninteracting leads. Depending on the interaction strength and the location of the barriers, the backscattering current exhibits different oscillation and scaling characteristics with the applied voltage in the strong and weak interaction cases. However, in both cases, the oscillation behaviors of the backscattering current are mainly determined by the quantum mechanical interference due to the existence of the double barriers.

Paired interference 3-dB coupler based on SOI rib waveguides with anisotropic chemical wet etching

A 3-dB paired interference (PI) optical coupler in silicon-on-insulator (SOI) based on rib waveguides with trapezoidal cross section was designed with simulation by a modified finite-difference beam propagation method (FD-BPM) and fabricated by potassium hydroxide (KOH) anisotropic chemical wet etching. Theoretically, tolerances of width, length, and port distance are more than 1, 100, and 1 mu m, respectively. Smooth interface was obtained with the propagation loss of 1.1 dB/cm at the wavelength of 1.55 mu m. The coupler has a good uniformity of 0.2 dB and low excess loss of less than 2 dB.

Coulomb blockade double-dot Aharonov-Bohm interferometer: Harmonic decomposition of the interference pattern

For the solid-state double-dot interferometer, the phase shifted interference pattern induced by the interplay of inter-dot Coulomb correlation and multiple reflections is analyzed by harmonic decomposition. Unexpected result is uncovered, and is discussed in connection with the which-path detection and electron loss. (C) 2009 Elsevier B.V. All rights reserved.

New type of multimode-interference-type thermo-optic variable optical attenuator

A thermo-optic variable optical attenuator (VOA) based on a Mach-Zehnder interferometer and multimode-interference coupler is fabricated. Not a single-mode but a multimode waveguide is used as the input and output structures of the optical field, which greatly reduces the coupling loss of the VOA with a normal single-mode fiber. The insertion loss of the fabricated VOA is 2.52 to 2.82 dB at the wavelength of 1520 to 1570 nm. The polarization dependent loss is 0.28 to 0.45 dB at the same wavelength range. Its maximum attenuation range is up to 26.3 dB when its power consumption is 369 mW. The response frequency of the fabricated VOA is about 10 kHz. (C) 2004 Society of Photo-Optical Instrumentation Engineers.

Study on the internal reflection of the multimode-interference-type device

The internal reflection of the multimode-interference (MMI)-type device is calculated with the bidirectional beam propagation method. The calculated results indicate that the difference of the effective refractive indices between the core region and the surrounding region has a determining effect on the internal reflection of the MMI-type device. The output taper for the MMI-type combiner and splitter has a more evident effect on the internal reflection than the input taper. The internal reflection decreases with increasing the end width of the taper. For the MMI-type device with appropriate tapers, the internal reflection does not show evident degradation with the deviation of the length of the MMI region from its optimal value. (C) 2004 Society of Photo-Optical Instrumentation Engineers.

1x8 cascaded multimode interference splitter in silicon-on-insulator

A cascaded multimode interference 1 x 8 power splitter is proposed and fabricated in silicon-on-insulator material. The device consists of seven 1 x 2 power splitters arranged in a tree configuration. The cascaded splitter and its 1 x 2 splitter element have the power uniformity of approximately 1.5 dB and 0.3 dB, respectively.

Multimode interference 3-dB coupler in silicon-on-insulator based on silicon rib waveguides with trapezoidal cross section

A 3-dB multimode interference optical coupler based on rib waveguides with trapezoidal cross section was designed and fabricated on silicon-on-insulator wafer. Potassium hydroxide (KOH) anisotropic chemical etching of silicon was used to fabricate the waveguides to obtain smooth interface. A modified finite-difference beam propagation method was used to simulate the multimode rib waveguide with slope interfaces. The rms roughness of etching interface is as small as 1.49 nm. The propagation loss of the waveguide is 1.3 dB/cm at wavelength of 1.55 mum. The fabricated 3-dB coupler has a good uniformity of 0.2 dB.

Interference and non-interference of sidebands in a quantum dot with oscillating levels

When a quantum dot is suffering an AC gate voltage, the sidebands turn up beside the static levels of the dot. We formularized the conductance and current when the effective coupling between levels in the quantum dot induced by the hybrid terms is included using a bi-unitary transform method, and we investigated the interference of the photon sidebands of deferent levels. The interference occurs if the same sidebands of deferent levels overlap, which is possible only when the static levels lie close to and overlap with each other. The overlap of different photon sidebands leads to a simple non-coherent superposition. (C) 2005 Elsevier Ltd. All rights reserved.

Size tolerance analysis of a 4X4 tapered multimode interference coupler in a silicon-on-insulator structure

Size tolerance of a 4X4 general interference tapered multimode interference (MMI) coupler in a silicon-on-insulator (SOI) structure is investigated by means of a 2-D finite difference beam propagation method (2D-FDBPM), together with an effective refractive index method (EIM). The results show that the tapered multimode interference coupler exhibits relatively larger size tolerance when light is launched from the edgeport than from midport, though it has much better output power uniformity when light is launched from midport. Besides that, it can reduce the device length greatly. The 4X4 general interference tapered MMI coupler has a slightly larger size tolerance compared with a conventional straight multimode interference coupler. (C) 2003 Society of Photo-Optical Instrumentation Engineers.

Multimode-interference-type thermo-optic variable optical attenuator with a response frequency of 10 kHz

A thermo-optic variable optical attenuator based on a multimode interference coupler principle is fabricated. The propagation loss of the fabricated device is 1.6 to 3.8 dB at the wavelength range 1510 to 1610 nm, which is very near the calculated value (1.2 dB) by the finite difference beam propagation method. The maximum power consumption is 363 mW and the dynamic attenuation range is 0 to 26 dB. The response frequency of the fabricated attenuator is about 10 kHz. (C) 2003 Society of Photo-Optical Instrumentation Engineers.