Enhanced resistance to Aeromonas hydrophila infection and enhanced phagocytic activities in human lactoferrin-transgenic grass carp (Ctenopharyngodon idellus)

Human lactoferrin (hLF) is an iron-binding protein with antimicrobial and immunomodulatory activities. hLF cDNA was transferred into grass carp via electroporated sperm. The production of transgenic fish was as high as 55% tinder the best parameters. 2(11) pulses and 20-min incubation. The expression of the transgene was demonstrated by the detection of hLF mRNA by RT-PCR. We also investigated the response of G(0) transgenic grass carp to Aeromonas hydrophila infection. Serum lysozyme activities (P>0.05) and phagocytic activities of kidney cells (P<0.05) were measured in transgenic individuals. The transgenic fish not only cleared A. hydrophila significantly faster than the control carp (P<0.05), but also showed enhanced phagocytic activities. The result shows that hLF has immunomodulatory activities in hLF-transgenic grass carp. The transgenic grass carp exhibited enhanced immunity to A. hydrophila infection. These results reveal that the mechanisms of disease resistance are different between hLF-transgenic plants and hLF-transgenic grass carp. (C) 2004 Elsevier B.V. All rights reserved.

Transgenes in F-4 pMThGH-transgenic common carp (Cyprinus carpio L.) are highly polymorphic

To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F-4 generation of pMThGH transgenic common carp (Cyprinus carpio L,) and 33 recovered genes were analyzed. The restriction maps of these recovered genes were constructed by digestion with five kinds of enzymes. These transgenes can be classified into 4 types according to their restriction maps. Only one type of transgenes maintains its original molecular form, whereas the other three types are very different from the original one and vary each other on both molecular weight and restriction maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. The results of PCR amplification and Southern blot hybridization indicate that MThGH in Type I transgene keeps intact but most of its sequence has been deleted in other three types. All these results suggest that transgenes in F-4 generation of transgenic carp are highly polymorphic. Two DNA fragments concerning integration site of transgenes were cloned from recovered transgenes, and found to be homologous to the 5'UTR of beta -actin gene of common carp and mouse mRNA for receptor tyrosine kinase (RTK), respectively.

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.

Whole-body amino acid pattern of F-4 human growth hormone gene-transgenic red common carp (Cyprinus carpio) fed diets with different protein levels

F-4 generation of human growth hormone (hGH) gene-transgenic red common carp, and the non-transgenic controls were fed for 8 weeks on purified diets with 20%, 30% or 40% protein. Analysis of whole-body amino acids showed that the proportions of lysine, leucine, phenylalanine, valine and alanine, as percentages of body protein, increased significantly, while those of arginine, glutamic acid and tyrosine decreased, with increases in dietary protein level in at least one strain of fish. Proportions of the other amino acids were unaffected by the diets. The proportions of lysine and arginine were significantly higher, while those of leucine and alanine were lower in the transgenics than in the controls in at least one diet group. Proportions of the other amino acids were unaffected by strain. The results suggest that the whole-body amino acid profile of transgenic carp, when expressed as proportions of body protein, was in general, similar to that of the non-transgenic controls. (C) 2000 Elsevier Science B.V. All rights reserved.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

Growth and feed utilization by F-4 human growth hormone transgenic carp fed diets with different protein levels

The F-4 generation of human growth hormone (hGH) transgenic red common carp Cyprinus carpio had significantly higher growth rates than the non-transgenic controls. Protein and energy intakes were significantly higher in the transgenic carp than in the controls fed the 20% protein diet, but were not different between the two strains fed diets with 30 and 40% protein. Faecal protein loss, as a proportion of protein intake, was significantly lower in the transgenics than in the controls fed diets with 20 and 30% protein, but was not different between the two strains Fed diet with 40% protein. Faecal energy loss, as a proportion of energy intake, was significantly lower in the transgenics than in the controls fed diet with 20% protein, but was not different between the two strains fed diets with 30 and 40% protein. Recovered protein, as a proportion of protein intake, was significantly higher in the transgenics than in the controls fed all diets, whereas recovered energy was significantly higher in the transgenic fish fed the 40% protein diet. For fish fed each diet, the transgenics had significantly higher body contents of dry matter and protein, but lower contents of lipid than the controls. It was concluded that transgenics were more efficient in utilizing dietary protein than the controls. it a lower dietary protein level; transgenics achieved higher growth rates mainly by increasing feed intake; at higher levels of dietary protein, transgenics achieved higher growth rates mainly through a higher energy conversion efficiency. (C) 1998 The Fisheries Society of the British Isles.

Comparison of photobioreactors and optimal light regime for rapid vegetative propagation of transgenic Undaria pinnatifida gametophytes

BACKGROUND: Previously, tachyplesin gene (tac) has been successfully transferred into Undaria pinnatifida gametophytes using the method of microprojectile bombardment transformation. The objectives of this study were to compare and evaluate the performance of bubble-column and airlift bioreactors to determine a preferred configuration of bioreactor for vegetative propagation of transgenic U. pinnatifida gametophytes, and to then investigate the influence of light on vegetative propagation of these gametophytes, including incident light intensity, photoperiod and light quality to resolve the problems of rapid vegetative propagation within the selected bioreactor. RESULTS: Experimental results showed that final dry cell density in the airlift bioreactor was 12.7% higher than that in the bubble-column bioreactor under the optimal aeration rate of 1.2 L air min(-1) L-1 culture. And a maximum final dry cell density of 2830 mg L-1 was obtained within the airlift bioreactor using blue light at 40 mu mol m(-2) s(-1) with a light/dark cycle of 14/10 (h). Polymerase chain reaction (PCR) analysis indicated that genes (bar and tac) were not lost during rapid vegetative propagation within the airlift bioreactor. CONCLUSION: The airlift bioreactor was shown to be much more suitable for rapid vegetative propagation of transgenic U. pinnatifida gametophytes than the bubble-column bioreactor in the laboratory. The use of blue light allows improvement of vegetative propagation of transgenic U. pinnatifida gametophytes. (C) 2009 Society of Chemical Industry