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.

Off-axis sonar beam pattern of free-ranging finless porpoises measured by a stereo pulse event data logger

The off-axis sonar beam patterns of eight free-ranging finless porpoises were measured using attached data logger systems. The transmitted sound pressure level at each beam angle was calculated from the animal's body angle, the water surface echo level, and the swimming depth. The beam pattern of the off-axis signals between 45 and 115 (where 0 corresponds to the on-axis direction) was nearly constant. The sound pressure level of the off-axis signals reached 162 dB re 1 mPa peak-to-peak. The surface echo level received at the animal was over 140 dB, much higher than the auditory threshold level of small odontocetes. Finless porpoises are estimated to be able to receive the surface echoes of off-axis signals even at 50-m depth. Shallow water systems (less than 50-m depth) are the dominant habitat of both oceanic and freshwater populations of this species. Surface echoes may provide porpoises not only with diving depth information but also with information about surface direction and location of obstacles (including prey items) outside the on-axis sector of the sonar beam. 2005 Acoustical Society of America.

Dynamics of photo-enhanced magneto-crystalline anisotropy in diluted ferromagnetic GaMnAs

By chopping a pump beam in conventional time-resolved Kerr rotation (TRKR) experiments and measuring the time evolution of M-shaped "major" hysteresis loops of magnetic linear dichroism (Delta MLD = MLDpump-on MLDpump-off), the differential MLD signal in the presence and the absence of the pump beam, we studied the dynamics of photo-enhanced magneto-crystalline anisotropy, and found that its very long recovering time (much longer than 13 ns) might reflect the nature of the coherent coupling between photo-excited holes and localized spins in the d shell of manganese.