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.

Identification of a WSSV neutralizing scFv antibody by phage display technology and in vitro screening

White spot syndrome virus (WSSV) is one of the most significant viral pathogens causing high mortality and economic damage in shrimp aquaculture. Although intensive efforts were undertaken to detect and characterize WSSV infection in shrimp during the last decade, we still lack methods either to prevent or cure white spot disease. Most of the studies on neutralizing antibodies from sera have been performed using in vivo assays. For the first time, we report use of an in vitro screening method to obtain a neutralizing scFv antibody against WSSV from a previously constructed anti-WSSV single chain fragment variable region (scFv) antibody phage display library. From clones that were positive for WSSV by ELISA, 1 neutralizing scFv antibody was identified using an in vitro screening method based on shrimp primary lymphoid cell cultures. The availability of a neutralizing antibody against the virus should accelerate identification of infection-related genes and the host cell receptor, and may also enable new approaches to the prevention and cure of white spot disease.

On the meaning of integration in coastal zone management

This paper discusses the definition and use of the term ‘integrated management’ in the context of coastal and ocean resources. It identifies several components which appear to be needed to establish an integrated management system for a large area subject to multiple use and jurisdiction. It suggests that the basis of integrated management should be a clear articulation of common purpose which addresses long term needs and vision. Once developed, this common purpose should be securely established to provide the setting against which sectoral and agencies managers and the community conduct and co-ordinate their activities.

Integration' in the US coastal zone management program

The purpose of this paper is to examine the extent to which the existing US Coastal Zone Management (CZM) program represents Integrated Coastal Management (ICM). The actions taken at Rio de Janeiro in June 1992 as part of the United Nations Conference on Environment and Development (UNCED) could eventually impact the policies of the US in such a way as to encourage better integration of US coastal and ocean management efforts.

Experimental demonstration of distributed feedback semiconductor lasers based on reconstruction-equivalent-chirp technology

In this paper we report, to the best of our knowledge, the first experimental realization of distributed feedback (DFB) semiconductor lasers based on reconstruction-equivalent-chirp (REC) technology. Lasers with different lasing wavelengths are achieved simultaneously on one chip, which shows a potential for the REC technology in combination with the photonic integrated circuits (PIC) technology to be a possible method for monolithic integration, in that its fabrication is as powerful as electron beam technology and the cost and time-consuming are almost the same as standard holographic technology. (C) 2009 Optical Society of America