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.

Correcting the systematic error of the density functional theory calculation: the alternate combination approach of genetic algorithm and neural network

The alternate combinational approach of genetic algorithm and neural network (AGANN) has been presented to correct the systematic error of the density functional theory (DFT) calculation. It treats the DFT as a black box and models the error through external statistical information. As a demonstration, the AGANN method has been applied in the correction of the lattice energies from the DFT calculation for 72 metal halides and hydrides. Through the AGANN correction, the mean absolute value of the relative errors of the calculated lattice energies to the experimental values decreases from 4.93% to 1.20% in the testing set. For comparison, the neural network approach reduces the mean value to 2.56%. And for the common combinational approach of genetic algorithm and neural network, the value drops to 2.15%. The multiple linear regression method almost has no correction effect here.

Comparison and combination of several stress relief methods for cubic boron nitride films deposited by ion beam assisted deposition

Cubic boron nitride (c-BN) films were prepared by ion beam assisted deposition (IBAD) technique, and the stresses were primary estimated by measuring the frequency shifts in the infrared-absorption peaks of c-BN samples. To test the possible effects of other factors, dependencies of the c-BN transversal optical mode position on film thickness and c-BN content were investigated. Several methods for reducing the stress of c-BN films including annealing, high temperature deposition, two-stage process, and the addition of a small amount of Si were studied, in which the c-BN films with similar thickness and cubic phase content were used to evaluate the effects of the various stress relief methods. It was shown that all the methods can reduce the stress in c-BN films to various extents. Especially, the incorporation of a small amount of Si (2.3 at.%) can result in a remarkable stress relief from 8.4 to similar to 3.6 GPa whereas the c-BN content is nearly unaffected, although a slight degradation of the c-BN crystallinity is observed. The stress can be further reduced down below I GPa by combination of the addition of Si with the two-stage deposition process. (c) 2008 Elsevier B.V. All rights reserved.

Symmetry and optical transition rule for low-dimensional semiconductor system with spin-orbit interaction and magnetic field

Starting from effective mass Hamiltonian, we systematically investigate the symmetry of low-dimensional structures with spin-orbit interaction and transverse magnetic field. The position-dependent potentials are assumed to be space symmetric, which is ever-present in theory and experiment research. By group theory, we analyze degeneracy in different cases. Spin-orbit interaction makes the transition between Zeeman sub-levels possible, which is originally forbidden within dipole approximation. However, a transition rule given in this paper for the first time shows that the transition between some levels is forbidden for space symmetric potentials. (C) 2009 Elsevier Ltd. All rights reserved.