Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). (C) 2002 Wiley-Liss, Inc.

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the unclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation of gene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.

Nuclear transfer in leach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the leach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 degreesC in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days, It was found that gastrula cells incubated at 4 degreesC had the same totipotence as blastula cells, The optimal UV dosage for inactivation of the oocyte chromatin was 180-240 mJ cm(-2). Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.

The onset of foreign gene transcription in nuclear-transferred embryos of fish

The transcriptional onset of hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F-4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F-4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F-4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blastula stage and even 16-cell stage in the first, second and third cases, respectively. it looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

Nuclear transplantation with early-embryonic cells of transgenic fish

Like other transgenic animals, transgenic fishes produced by microinjection are transgenic mosaics. In order to produce homogenous transgenic fish, the transgenic blastula or gastrula cells were dissociated from Carassius auratus, Pengze var, and Cyprinus carpio, Huanghe var., and the nuclei were transferred into the mature eggs of the same species via microinjection or electro-fusion. Five nuclear-transferred Carassius auratus, Pengze var. and one Cyprinus carpio, Huanghe var. were obtained and the existence of the transgene was detected. The possibility of generating homogenous strain of transgenic fish by nuclear transplantation with transgenic early-embryonic cells is discussed.

Transport phenomena in radial flow MOCVD reactor with three concentric vertical inlets

Transport phenomena in radial flow metalorganic chemical vapor deposition (MOCVD) reactor with three concentric vertical inlets are studied by two-dimensional numerical modeling. By varying the parameters such as gas pressure, flow rates combination of multi-inlets, geometric shapes and sizes of reactor and flow distributor, temperatures of susceptor and ceiling, and susceptor rotation, the corresponding velocity, temperature, and concentration fields inside the reactor are obtained; the onset and change of flow recirculation cells under influences of those parameters are determined. It is found that recirculation cells, originated from flow separation near the bend of reactor inlets, are affected mainly by the reactor height and shape, the operating pressure, the flow rates combination of multi-inlets, and the mean temperature between susceptor and ceiling. By increasing the flow rate of mid-inlet and the mean temperature, decreasing the pressure, maintaining the reactor height below certain criteria, and trimming the bends of reactor wall and flow distributor to streamlined shape, the recirculation cells can be minimized so that smooth and rectilinear flow prevails in the susceptor region, which corresponds to smooth and rectilinear isotherms and larger reactant concentration near the susceptor. For the optimized reactor shape, the reactor size can be enlarged to diameter D = 40 cm and height H = 2 cm without flow recirculation. The susceptor rotation over a few hundred rpm around the reactor central axis will induce the recirculation cell near the exit and deflect the streamlines near the susceptor, which is not the case for vertical reactors. (c) 2006 Elsevier B.V. All rights reserved.

Influences of reactor pressure of GaN buffer layers on morphological evolution of GaN grown by MOCVD

The morphological evolution of GaN thin films grown on sapphire by metalorganic chemical vapor deposition was demonstrated to depend strongly on the growth pressure of GaN nucleation layer (NL). For the commonly used two-step growth process, a change in deposition pressure of NL greatly influences the growth mode and morphological evolution of the following GaN epitaxy. By means of atomic force microscopy and scanning electron microscope, it is shown that the initial density and the spacing of nucleation sites on the NL and subsequently the growth mode of FIT GaN epilayer may be directly controlled by tailoring the initial low temperature NL growth pressure. A mode is proposed to explain the TD reduction for NL grown at relatively high reactor pressure. (C) 2003 Elsevier B.V. All rights reserved.

Effects of reactor pressure on GaN nucleation layers and subsequent GaN epilayers grown on sapphire substrate

The influence of reactor pressure on GaN nucleation layer (NL) and the quality of subsequent GaN on sapphire is studied. The layers were grown by low-pressure metalorganic chemical vapor deposition (MOCVD) on c-plane sapphire substrates and investigated by in situ laser reflectometry, atomic force microscope, scanning electron microscope, X-ray diffraction and photoluminescence. With the increase of reactor pressure prior to high-temperature GaN growth, the size of GaN nuclei formed after annealing decreases, the spacing between nucleation sites increases and the coalescence of GaN nuclei is deferred. The optical and crystalline qualities of GaN epilayer were improved when NLs were deposited at high pressure. The elongated lateral overgrowth of GaN islands is responsible for the quality improvement. (C) 2003 Elsevier Science B.V. All rights reserved.

Operational Optimization of GaN Thin Film Growth Employing Numerical Simulation in a Showerhead MOCVD Reactor

A detailed reaction-tran sport model was studied in a showerhead reactor for metal organic chemical vapor deposition of GaN film by using computational fluid dynamics simulation. It was found that flat flow lines without swirl are crucial to improve the uniformity of the film growth, and thin temperature gradient above the suscptor can increase the film deposition rate. By above-mentioned research, we can employ higher h (the distance from the susceptor to the inlet), P (operational pressure) and the rate of susceptor rotation to improve the film growth.

Synthesis of biodiesel from waste cooking oil using immobilized lipase in fixed bed reactor

Waste cooking oil (WCO) is the residue from the kitchen, restaurants, food factories and even human and animal waste which not only harm people's health but also causes environmental pollution. The production of biodiesel from waste cooking oil to partially substitute petroleum diesel is one of the measures for solving the twin problems of environment pollution and energy shortage. In this project, synthesis of biodiesel was catalyzed by immobilized Candida lipase in a three-step fixed bed reactor. The reaction solution was a mixture of WCO, water, methanol and solvent (hexane). The main product was biodiesel consisted of fatty acid methyl ester (FAME), of which methyl oleate was the main component. Effects of lipase, solvent, water, and temperature and flow of the reaction mixture on the synthesis of biodiesel were analyzed. The results indicate that a 91.08% of FAME can be achieved in the end product under optimal conditions. Most of the chemical and physical characters of the biodiesel were superior to the standards for 0(#)diesel (GB/T 19147) and biodiesel (DIN V51606 and ASTM D-6751).

Lipase-catalyzed synthesis of biodiesel from acid oil in fixed bed reactor

Acid oil, which is a by-product in vegetable oil refining, mainly contains free fatty acids (FFAs) and acylglycerols and is a feedstock for production of biodiesel fuel now. The transesterification of acid oil and methanol to biodiesel was catalyzed by immobilized Candida lipase in fixed bed reactors. The reactant solution was a mixture of acid oil, water, methanol and solvent (hexane) and the main product was biodiesel composed of fatty acid methyl ester (FAME) of which the main component was methyl oleate. The effects of lipase content, solvent content, water content temperature and flow velocity of the reactant on the reaction were analyzed. The experimental results indicate that a maximum FAME content of 90.18% can be obtained in the end product under optimum conditions. Most of the chemical and physical properties of the biodiesel were superior to the standards for 0(#) diesel (GB/T 19147) and biodiesel (DIN V51606 and ASTM D6751).