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.

Hydrogen behavior in GaN epilayers grown by NH3-MBE

Hydrogen behavior in unintentionally doped GaN epilayers on sapphire substrates grown by NH3-MBE is investigated. Firstly, we find by using nuclear reaction analysis (NRA) that with increasing hydrogen concentration the background electron concentration increases, which suggests that there exists a hydrogen-related donor in undoped GaN, Secondly, Fourier transform infrared (FTIR) absorption and X-ray photoelectron spectroscopy (XPS) reveal Further that hydrogen atom is bound to nitrogen atom in GaN with a local vibrational mode at about 3211 cm(-1) Hence, it is presumed that the hydrogen-related complex Ga. . .H-N is a hydrogen-related donor candidate partly responsible for high n-type background commonly observed in GaN films. Finally, Raman spectroscopy results of the epilayers show that ill addition to the expected compressive biaxial strain, in some cases GaN films suffer from serious tensile biaxial strain. This anomalous behavior has been well interpreted in terms of interstitial hydrogen lattice dilation. (C) 2001 Elsevier Science B.V. All rights reserved.

Hydrogen-dependent lattice dilation in GaN

Using Raman spectroscopy we have analysed the strain status of GaN films grown on sapphire substrates by NH3 source molecular beam epitaxy (MBE). In addition to the expected compressive biaxial strain, in some cases GaN films grown on c-face sapphire substrates suffer from serious tensile biaxial strain. This anomalous behaviour has been well interpreted in terms of interstitial hydrogen-dependent lattice dilation. The hydrogen concentration in the films is measured by nuclear reaction analysis (NRA). With increasing hydrogen incorporation, the residual compressive biaxial strain is first further relaxed, and then turns into tensile strain when the hydrogen contaminant exceeds a critical concentration. The hydrogen incorporation during the growth process is found to be growth-rate dependent, and is supposed to be strain driven. We believe that the strain-induced interstitial incorporation is another way for strain relaxation during heteroepitaxy, besides the two currently well known mechanisms: formation of dislocations and growth front roughening.

High-concentration hydrogen in unintentionally doped GaN

We use nuclear reaction analysis to study hydrogen in unintentionally doped GaN, and high-concentration hydrogen, nearly 10(21) cm(-3), is detected. Accordingly, a broad but intense infrared absorption zone with a peak at 2962 cm(-1) is reported, which is tentatively assigned to the stretch mode of NH: Ga complex. The complex is assumed to be one candidate answering for background electrons in unintentionally doped GaN. (C) 1998 Elsevier Science B.V. All rights reserved.

Influence of rapid thermal annealing on the optical properties of gallium nitride grown by gas-source molecular-beam epitaxy

Raman scattering, photoluminescence (PL), and nuclear reaction analysis (MA) have been employed to investigate the effects of rapid thermal annealing (RTA) on GaN films grown on sapphire (0001) substrates by gas-source molecular-beam epitaxy, The Raman spectra showed the presence of the E-2 (high) mode of GaN and shift of this mode from 572 to 568 cm(-1) caused by annealing. The results showed that RTA has a significant effect on the strain relaxation caused by the lattice and thermal expansion misfit between the GaN epilayer and the substrate. The PL peak exhibited a blueshift in its energy position and a decrease in the full width at half maximum after annealing, indicating an improvement in the optical quality of the film. Furthermore, a green luminescence appeared after annealing and increased in intensity with increasing annealing time. This effect was attributed to H concentration variation in the GaN film, which was measured by NRA. A high H concentration exists in as-grown GaN, which can neutralize the deep level, and the H-bonded complex dissociates during RTA, This leads to the appearance of a luminescent peak in the PL spectrum. (C) 1998 American Institute of Physics.

Hydrogen behavior in GaN epilayers grown by NH3-MBE

Hydrogen behavior in unintentionally doped GaN epilayers on sapphire substrates grown by NH3-MBE is investigated. Firstly, we find by using nuclear reaction analysis (NRA) that with increasing hydrogen concentration the background electron concentration increases, which suggests that there exists a hydrogen-related donor in undoped GaN, Secondly, Fourier transform infrared (FTIR) absorption and X-ray photoelectron spectroscopy (XPS) reveal Further that hydrogen atom is bound to nitrogen atom in GaN with a local vibrational mode at about 3211 cm(-1) Hence, it is presumed that the hydrogen-related complex Ga. . .H-N is a hydrogen-related donor candidate partly responsible for high n-type background commonly observed in GaN films. Finally, Raman spectroscopy results of the epilayers show that ill addition to the expected compressive biaxial strain, in some cases GaN films suffer from serious tensile biaxial strain. This anomalous behavior has been well interpreted in terms of interstitial hydrogen lattice dilation. (C) 2001 Elsevier Science B.V. All rights reserved.

High-concentration hydrogen in unintentionally doped GaN

We use nuclear reaction analysis to study hydrogen in unintentionally doped GaN, and high-concentration hydrogen, nearly 10(21) cm(-3), is detected. Accordingly, a broad but intense infrared absorption zone with a peak at 2962 cm(-1) is reported, which is tentatively assigned to the stretch mode of NH: Ga complex. The complex is assumed to be one candidate answering for background electrons in unintentionally doped GaN. (C) 1998 Elsevier Science B.V. All rights reserved.

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

Heat shock protein 90 (HSP90) works as a multi-functional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25A degrees C to 35A degrees C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.

Expression of the lacZ reporter gene in sporophytes of the seaweed Laminaria japonica (Phaeophyceae) by gametophyte-targeted transformation

The seaweed Laminaria japonica (Phaeophyceae) has a two-generation life cycle consisting of haploid gametophytes and diploid sporophytes. Female and/or male gametophytes were transformed using particle bombardment and the histological LacZ assay was performed on sporophytes generated by either parthenogenesis or inbreeding. Female gametophyte-targeted transformation resulted in similar lower efficiencies in both parthenogenetic and zygotic sporophytes, and only a chimeric expression pattern was observed. Male gametophyte-targeted transformation led to a higher efficiency, with 3.5% of the zygotic sporophytes stained completely blue (all-blue), implying the integration of lacZ at the one-cell stage. Polymerase chain reaction analysis using primers specific for a lacZ-vector juncture fragment and subsequent blotting indicated the presence of the introduced gene in the sporophytes. The method reported here has a potential for seaweed transformation using spore-based bombardment followed by the developmental process.

Characterization of 12 single nucleotide polymorphisms (SNPs) in Pacific abalone, Haliotis discus hannai

We report here for the first time 12 polymorphic single nucleotide polymorphisms (SNPs) in a commercially important gastropod, Pacific abalone (Haliotis discus hannai) that were identified by searching expressed sequence tag database. These SNP loci (seven nuclear and five mitochondrial SNPs) were polymorphic among 37 wild abalone individuals, based on a four-primer allele-specific polymerase chain reaction analysis. All loci had two alleles and the minor allele frequency ranged from 0.027 to 0.473. For the seven nuclear SNPs, the expected and observed heterozygosities ranged from 0.053 to 0.499 and from 0.054 to 0.811, respectively.

Thrombopoietin, flt3-ligand and c-kit-ligand modulate HOX gene expression in expanding cord blood CD133(+) cells

Haemopoietic stem/progenitor cell (HSPC) development is regulated by extrinsic and intrinsic stimuli. Extrinsic modulators include growth factors and cell adhesion molecules, whereas intrinsic regulation is achieved with many transcription factor families, of which the HOX gene products are known to be important in haemopoiesis. Umbilical cord blood CD133(+) HSPC proliferation potential was tested in liquid culture with 'TPOFLK' (thrombopoietin, flt-3 ligand and c-kit ligand, promoting HSPC survival and self-renewal), in comparison to 'K36EG' (c-kit-ligand, interleukins-3 and -6, erythropoietin and granulocyte colony-stimulating factor, inducing haemopoietic differentiation). TPOFLK induced a higher CD133(+) HSPC proliferation (up to 60-fold more, at week 8) and maintained a higher frequency of the primitive colony-forming cells than K36EG. Quantitative polymerase chain reaction analysis revealed opposite expression patterns for specific HOX genes in expanding cord blood CD133(+) HSPC. After 8 weeks in liquid culture, TPOFLK increased the expression of HOX B3, B4 and A9 (associated with uncommitted HSPC) and reduced the expression of HOX B8 and A10 (expressed in committed myeloid cells) when compared to K36EG. These results suggest that TPOFLK induces CD133(+) HSPC proliferation, self-renewal and maintenance, up-regulation of HOX B3, B4 and A9 and down-regulation of HOX B8 and A10 gene expression.

Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments (similar to 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.

Detection of transgenic DNA and protein, in rumen fluid, duodenal digesta, milk, blood and faeces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood, and feces of lactating dairy cows

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.