Studies on Pattern of Producing Transgenic Fish II. Several Elements Related to the Establishment of Transgenic Fish

A recombinant plasmid (pcMTsGH) containing the common carp methlothionein promoter (cMT) and salmon growth hormone gene (sGH) was microinjected into common carp fertilized eggs and embryos to optimize the conditions for producing transgenic fish. Several key elements were found to have significant effects on the integration of exogenous gene. These elements include forms and doses of pcMTsGH gene microinjected, stages of embryo at which microinjection conducted and pretreatment of fertilized eggs for microinjection. The results show that, in producing transgenic fish, (1) the best time for microinjection is late onecell stage, (2) the most appropriate dose of pcMTsGH gene for microinjection is around 106～107 copies; (3) the hatching rate rises up when the gene is microinjected into the eggs whose chorions are removed, but the integration rate has no significant changes before and after the removal of egg chorions.

Integration mechanisms of transgenes and population fitness of GH transgenic fish

It has been more than 20 years since the first batch of transgenic fish was produced. Five stable germ-line transmitted growth hormone (GH) transgenic fish lines have been generated. This paper reviews the mechanisms of integration and gene targeting of the transgene, as well as the viability, reproduction and transgenic approaches for the reproductive containment of GH-transgenic fish. Further, we propose that it should be necessary to do the following studies, in particularly, of the breeding of transgenic fish: to assess the fitness of transgenic fish in an aqueous environment with a large space and a complex structure; and to develop a controllable on-off strategy of reproduction in transgenic fish.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century

Preliminarily study on the maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on gastropods

The present study preliminarily examined the differences in maximum handling size, prey size and species selectivity of growth hormone transgenic and non-transgenic common carp Cyprinus carpio when foraging on four gastropods species( Bellamya aeruginosa, Radix auricularia, Parafossarulus sinensis and Alocinma longicornis) under laboratory conditions. In the maximum handling size trial, five fish from each age group(1-year-old and 2-year-old) and each genotype(transgenic and non-transgenic) of common carp were individually allowed to feed on B. aeruginosa with wide shell height range. The results showed that maximum handling size increased linearly with fish length, and there was no significant difference in maximum handling size between the two genotypes. In the size selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on three size groups of B. aeruginosa. The results show that the two genotypes of C. carpio favored the small-sized group over the large-sized group. In the species selection trial, three pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on thin-shelled B. aeruginosa and thick-shelled R. auricularia, and five pairs of 2-year-old transgenic and non-transgenic carp were individually allowed to feed on two gastropods species( P. sinensis and A. longicornis) with similar size and shell strength. The results showed that both genotypes preferred thin-shelled Radix auricularia rather than thick-shelled B. aeruginosa, but there were no significant difference in selectivity between the two genotypes when fed on P. sinensis and A. longicornis. The present study indicates that transgenic and non-transgenic C. carpio show similar selectivity of predation on the size-and species-limited gastropods. While this information may be useful for assessing the environmental risk of transgenic carp, it does not necessarily demonstrate that transgenic common carp might have lesser environmental impacts than non-transgenic carp.

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.

Fish genome manipulation and directional breeding

Aquaculture is one of the fastest developing agricultural industries worldwide.One of the most important factors for sustainable aquaculture is the development of high performing culture strains.Genome manipulation offers a powerful method to achieve rapid and directional breeding in fish.We review the history of fish breeding methods based on classical genome manipulation,including polyploidy breeding and nuclear transfer.Then,we discuss the advances and applications of fish directional breeding based on transgenic technology and recently developed genome editing technologies.These methods offer increased efficiency,precision and predictability in genetic improvement over traditional methods.

Application of immunohistochemistry technique in hydrobiological studies

There is growing demand for biotechniques in oceanobiological and hydrobiological studies.Immunohistochemistry(IHC)combines anatomical,immunological and biochemical techniques to identify specific antigen-antibody interactions.Detecting the target antigen with labeled antibodies is a multi-step process that requires optimization at every level to maximize the signal detection.Here we use zebrafish samples and optimize each IHC step,particularly antigen retrieval,transgenic reporter double staining and background counterstaining.The resulting images with high signal intensity lead us to recommend the resulting protocols for experiments with fish.