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.

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.

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.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

Monolithic integration of sampled grating DBR with electroabsorption modulator by combining selective-area-growth MOCVD and quantum-well intermixing

We present the monolithic integration of a sampled-grating distributed Bragg reflector (SC-DBR) laser with a quantum-well electroabsorption modulator (QW-EAM) by combining ultra-low-pressure (55 mbar) selective-area-growth (SAG) metal-organic chemical vapour deposition (MOCVD) and quantum-well intermixing (QWI) for the first time. The QW-EAM and the gain section can be grown simultaneously by using SAG MOCVD technology. Meanwhile, the QWI technology offers an abrupt band-gap change between two functional sections, which reduces internal absorption loss. The experimental results show that the threshold current I-th = 62 mA, and output power reaches 3.6 mW. The wavelength tuning range covers 30 nm, and all the corresponding side mode suppression ratios are over 30 dB. The extinction ratios at available wavelength channels can reach more than 14 dB with bias of -5 V.

Design of novel three port optical gates scheme for the integration of large optical cavity electroabsorption modulators and evanescently-coupled photodiodes

This paper presents a novel scheme to monolithically integrate an evanescently-coupled uni-travelling carrier photodiode with a planar short multimode waveguide structure and a large optical cavity electroabsorption modulator based on a multimode waveguide structure. By simulation, both electroabsorption modulator and photodiode show excellent optical performances. The device can be fabricated with conventional photolithography, reactive ion etching, and chemical wet etching.

Optical response of high-level bandgap in one-dimensional photonic crystal applying in-plane integration

A new broadband filter, based on the high level bandgap in 1-D photonic crystals (PCs) of the form Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si is designed by the plane wave expansion method (PWEM) and the transfer matrix method (TMM) and fabricated by lithography. The optical response of this filter to normal-incident and oblique-incident light proves that utilizing the high-level bandgaps of PCs is an efficient method to lower the difficulties of fabricating PCs, increase the etching depth of semiconductor materials, and reduce the coupling loss at the interface between optical fibers and the PC device. (c) 2007 Society of Photo-Optical Instrumentation Engineers.

Optical response of high-order band gap photonic crystal applying in-plane in one-dimensional integration

A new broadband filter, based on the high-order band gap in one-dimensional photonic crystal (PCs) of the form Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si, has been designed by the plane wave expansion method (PWEM) and transfer matrix method (TMM) and fabricated by lithography. The optical response of this filter to normal-incident and oblique-incident light proves that utilizing the high-order band gaps of PCs is an efficient method to lower the difficulties of fabricating PCs, increase the etching depth of semiconductor materials, and reduce the coupling loss at the interface between optical fibers and PC device. (c) 2007 Elsevier B.V. All rights reserved.

Integration of a photonic crystal polarization beam splitter and waveguide bend

In this work, we present the design of an integrated photonic-crystal polarization beam splitter (PC-PBS) and a low-loss photonic-crystal 60 waveguide bend. Firstly, the modal properties of the PC-PBS and the mechanism of the low-loss waveguide bend are investigated by the two-dimensional finite-difference time-domain (FDTD) method, and then the integration of the two devices is studied. It shows that, although the individual devices perform well separately, the performance of the integrated circuit is poor due to the multi-mode property of the PC-PBS. By introducing deformed airhole structures, a single-mode PC-PBS is proposed, which significantly enhance the performance of the circuit with the extinction ratios remaining above 20dB for both transverse-electric (TE) and transverse-magnetic (TM) polarizations. Both the specific result and the general idea of integration design are promising in the photonic crystal integrated circuits in the future. (C) 2009 Optical Society of America

Monolithic integration of an InGaAsP-InP strained DFB laser and an electroabsorption modulator by ultra-low-pressure selective-area-growth MOCVD

The design and basic characteristics of a strained InGaAsP-InP multiple-quantum-well (MQW) DFB laser monolithically integrated with an electroabsorption modulator (EAM) by ultra-low-pressure (22 mbar) selective-area-growth (SAG) MOCVD are presented. A fundamental study of the controllability and the applicability of band-gap energy by using the SAG, method is performed. A large band-gap photoluminescence wavelength shift of 88 mn. was obtained with a small mask width variation (0-30 mu m). The technique is then applied to fabricate a high performance strained MQW EAM integrated with a DFB laser. The threshold current of 26 mA at CW operation of the device with DFB laser length of 300 mu m and EAM length of 150 mu m has been realized at a modulator bias of 0 V. The devices also exhibit 15 dB on/off ratio at an applied bias voltage of 5 V.

A bistable, self-latching inverter by the monolithic integration of resonant tunnelling diode and high electron mobility transistor

This paper reports that the structures of AlGaAs/InGaAs high electron mobility transistor (HEMT) and AlAs/GaAs resonant tunnelling diode (RTD) are epitaxially grown by molecular beam epitaxy ( MBE) in turn on a GaAs substrate. An Al0.24Ga0.76As chair barrier layer, which is grown adjacent to the top AlAs barrier, helps to reduce the valley current of RTD. The peak-to-valley current ratio of fabricated RTD is 4.8 and the transconductance for the 1-mu m gate HEMT is 125mS/mm. A static inverter which consists of two RTDs and a HEMT is designed and fabricated. Unlike a conventional CMOS inverter, the novel inverter exhibits self-latching property.

Monolithic integration of electroabsorption modulator and DFB laser for 10-Gb/s transmission

A strained InGaAsP-InP multiple-quantum-well DFB laser monolithically integrated with electroabsorption modulator by ultra-low-pressure (22 mbar) selective-area-growth is presented. The integrated chip exhibits superior characteristics, such as low threshold current of 19 mA, single-mode operation around 1550 nm range with side-mode suppression ratio over 40 dB, and larger than 16 dB extinction ratio when coupled into a single-mode fiber. More than 10 GHz modulation bandwidth is also achieved. After packaged in a compact module, the device successfully performs 10-Gb/s NRZ transmission experiments through 53.3 km of standard fiber with 8.7 dB dynamic extinction ratio. A receiver sensitivity of -18.9 dBm at bit-error-rate of 10(-1)0 is confirmed. (c) 2005 Elsevier B.V. All rights reserved.

Investigation progress on key photonic integration for application in optical communication network

Proceeding from the consideration of the demands from the functional architecture of high speed, high capacity optical communication network, this paper points out that photonic integrated devices, including high speed response laser source, narrow band response photodetector high speed wavelength converter, dense wavelength multi/demultiplexer, low loss high speed response photo-switch and multi-beam coupler are the key components in the system. The, investigation progress in the laboratory will be introduced.

Wafer bonding technique used for the integration of cubic GaN/GaAs (001) with Si substrate

We successfully used the metal mediated-wafer bonding technique in transferring the as-grown cubic GaN LED structure of Si substrate. The absorbing GaAs substrate was removed by using the chemical solutions of NH4OH : H2O2=1 : 10. SEM and PL results show that wafer bonding technique could transfer the cubic GaN epilayers uniformly to Si without affecting the physical and optical properties of epilayers. XRD result shows that there appeared new peaks related to AgGa2 and Ni4N diffraction, indicating that the metals used as adhesive and protective layers interacted with the p-GaN layer during the long annealing process. It is just the reaction that ensures the reliability of the integration of GaN with metal and minor contact resistance on the interface.

Photonic switch and NOT logic gate based on the hybrid integration of a GaAsVCSEL and a GaAs MISS

The hybrid integrated photonic switch and not logic gate based on the integration of a GaAs VCSEL (Vertical Cavity Surface Emitting Lasers) and a MISS (Metal-Insulator-Semiconductor Switches) device are reported. The GaAs VCSEL is fabricated by selective etching and selective oxidation. The Ultra-Thin semi-Insulating layer (UTI) of the GaAs MISS is formed by using oxidation of A1As that is grown by MBE. The accurate control of UTI and the processing compatibility between VCSEL and MISS are solved by this procedure. Ifa VCSEL is connected in series with a MISS, the integrated device can be used as a photonic switch, or a light amplifier. A low switching power (10 mu W) and a good on-off ratio (17 dB contrast) have been achieved. If they are connected in parallel, they perform a photonic NOT gate operation.