Dong Soo Kim

Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis.

Transgenic mud loaches (Misgurnus mizolepis), in which the entire transgene originated from the same species, have been generated by microinjecting the mud loach growth hormone (mlGH) gene fused to the mud loach β-actin promoter. Out of 4,100 eggs injected, 7.5% fish derived from the injected eggs showed dramatically accelerated growth, with a maximum of 35-fold faster growth than their non-transgenic siblings. Many fast-growing transgenic individuals showed extraordinary gigantism: their body weight and total length (largest fish attained to 413u2009g and 41.5u2009cm) were larger and longer than even those of 12-year-old normal broodstock (maximum size reached to 89u2009g and 28u2009cm). Of 46 transgenic founders tested, 30 individuals transmitted the transgene to next generation with a wide range of germ-line transmission frequencies ranging from 2% to 33%. The growth performance of the subsequent generation (F1) was also dramatically accelerated up to 35-fold, although the levels of enhanced growth were variable among transgenic lines. Three transgenic germ-lines up to F4 were established, showing the expected Mendelian inheritance of the transgene. Expression of GH mRNA in many tissues was detected by RT-PCR analyses. The time required to attain marketable size (10u2009g) in these transgenic lines was only 30–50 days after fertilization, while at least 6 months in non-transgenic fish. Besides growth enhancement, significantly improved feed-conversion efficiency up to 1.9-fold was also observed.

Induction of triploidy in mud loach (Misgurnus mizolepis) and its effect on gonad development and growth

Abstract Triploidy was induced in mud loach ( Misgurnus mizolepis ) by cold shocking fertilized eggs 5 min post-fertilization at 2°C for 15 to 60 min. Best results were obtained when eggs were shocked for 60 min; 98% of fish examined in that treatment were triploids. Triploidy was confirmed by erythrocyte measurements and chromosome counts. Diploids had 48 chromosomes, while triploids had 72. Histological analysis of 9-month-old triploid ovaries showed an appreciable number of oocytes at the chromatin nucleolus stage with considerable interstitial tissue. However, diploids had well developed oocytes. Diploid testes from diploid males exhibited normal spermatids and spermatozoa, while a few were seen in triploid males. Growth rate was evaluated over a 9-month growth trial. Although male and female triploids were slightly heavier than their diploid counterparts from the third to the ninth month, their growth rates were not significantly different compared to their diploid controls.

Genomic Cloning of Mud Loach Misgurnus mizolepis (Cypriniformes, Cobitidae) β-Actin Gene and Usefulness of Its Promoter Region for Fish Transgenesis

As a part of our effort to obtain a strong regulatory element for the construction of an autogenic mud loach transgenic vector, we isolated a genomic clone that contains an open reading frame encoding the β-actin gene, then examined the transcriptional activity of the upstream sequences, including the first intron, in transiently transfected cell lines. It showed that the upstream region has substantially strong transcriptional activity, and that both the proximal promoter and distal region of intron 1 play a crucial role in the activity. A similar result, based on fish growth, was obtained with the expression vectors containing the growth hormone gene of mud loach. These were driven by the regulatory region of the mud loach β-actin gene with various mutations, and were directly transferred into the trunk muscle of fish using an electrostimulation-mediated method. Fish weights were monitored over the next 4 weeks. These data suggest that the proximal promoter and the first intron enhancer of the mud loach β-actin gene are useful for autogenic mud loach transgenesis.

Transmission and expression of an integrated reporter construct in three generations of transgenic mud loach (Misgurnus mizolepis)

Abstract We produced transgenic mud loach lines by microinjecting a CAT reporter gene driven by the carp β-actin promoter into fertilized eggs and demonstrated successful integration, transmission to F 3 , with variable patterns of expression in different tissues. Microinjection yielded transgenic F 0 individuals with a frequency that ranged from 9.3 to 27.8% among microinjected groups. Of 63 transgenic founders identified, seven males and one female were found to transmit the transgene to F 1 offsprings. All founders were mosaic in their germ cells; consequently the transmission frequency ranged from 3.7 to 33.4%. Most, but not all, F 1 and F 2 transgenics transmitted the transgenic construct to F 2 and F 3 progeny, respectively, with expected Mendelian ratios. Immunohistochemical in situ analysis showed that the transgene pFV4bCAT was effectively expressed in transgenic germlines. Reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) revealed that the presence of CAT mRNA and the expression patterns in different tissues of adult individuals varied among each transgenic line; the highest level of CAT expression was observed in muscle from a transgenic F 1 line, corresponding to more than 89-fold compared to the basal control level.

Isogenic transgenic homozygous fish induced by artificial parthenogenesis

As a model system for vertebrate transgenesis, fish have many attractive advantages, especially with respect to the characteristics of eggs, allowing us to produce isogenic, transgenic, homozygous vertebrates by combining with chromosome-set manipulation. Here, we describe the large-scale production of isogenic transgenic homozygous animals using our experimental organism, the mud loach Misgurnus mizolepis, by the simple process of artificial parthenogenesis in a single generation. These isogenic fish have retained transgenic homozygous status in a stable manner during the subsequent 5 years, and exhibited increased levels of transgene expression. Furthermore, their isogenic nature was confirmed by cloned transgenic homozygous offspring produced via another step of parthenogenic reproduction of the isogenic homozygous transgenic fish. These results demonstrate that a combination of transgenesis and artificial parthenogenesis will make the rapid utilization of genetically pure homozygous transgenic system in vertebrate transgenesis possible.

Survival and growth of induced tetraploid mud loach

Tetraploid mud loach (Misgurnusmizolepis) were produced by heat shock(40.5°C for 3 min, 28 minpost-fertilization) and their survival andgrowth up to 9 months of age were examined. Theincidence of tetraploidy in 1-month-oldsurvivors of the heat shock was 22.6%.Tetraploids had a mean erythrocyte nuclearvolume of 38.8 µm3, modal chromosomenumber of 4n = 96, and average DNA content of5.6 pg/cell, all of which were double those ofdiploid control values. The survival rate oftetraploids was similar to that of diploidcontrols throughout the experiment (from 1 to 9months of age; P > 0.05), but theheat-shocked group had significantly highermortality during early developmental stages(P < 0.05). Growth rates of tetraploidswere significantly depressed until 7 months ofage (P < 0.05), but were not differentfrom diploid controls at ages 8 and 9 months(P > 0.05).

The complete mitochondrial genome of the Korean soft-shelled turtle Pelodiscus sinensis (Testudines, Trionychidae)

We isolated Korean soft-shelled turtle, Pelodiscus sinensis, mitochondrial DNA by long-polymerase chain reaction (long-PCR) with conserved primers and sequenced this mitochondrial genome (mitogenome) with primer walking using flanking sequences. The P. sinensis mitochondrial DNA has 17,042 bp and its structural organization is conserved compared to those of other reptiles and mammals. To unveil the phylogenetic relationship of the turtles, we used the NJ, MP, and ML analysis methods after inferring those sequences from the mitochondrial 16S rRNA gene. We also compared two P. sinensis variants from Korea and China using the mitochondrial genome. In this study, we report the basic characteristics of the P. sinensis mitochondrial genome, including structural organization and base composition of the rRNAs, tRNAs and protein-coding genes, as well as characteristics of tRNAs. These features are applicable for the study of phylogenetic relationships in turtles.

Lidocaine hydrochloride–sodium bicarbonate as an anesthetic for soft‐shelled turtle Pelodiscus sinensis

Attempts were made to understand how the different sizes (mean body weight of 4.1±0.8 g for small and 182.6±23.7 g for large) of the soft-shelled turtle Pelodiscus sinensis are affected by different temperature (25°C or 30°C), and different concentrations (700, 1000 and 1300 p.p.m.) of anesthetic lidocaine hydrochloride-sodium bicarbonate. The soft-shelled turtles were fed to satiation once daily. Three-way anova design (size × temperature × concentration of lidocaine hydrochloride) with duplicate was used. Twenty each of large and small soft-shelled turtles were distributed into six 30-L tanks. Exposure time of the soft-shelled turtle was affected by all factors (temperature, concentration and size). Exposure time of the soft-shelled turtle for anesthetizing decreased with increase in temperature and in concentration of lidocaine hydrochloride, and decrease in size. Recovery time for the soft-shelled turtle was also affected by all factors. Recovery time of the soft-shelled turtle increased with increase in temperature, concentration of lidocaine hydrochloride, and size. According to these results, lidocaine hydrochloride (1000 p.p.m.)-sodium bicarbonate seemed an effective anesthetic for sedating and handling the soft-shelled turtle. However, when an anesthetic is recommended for wide use, especially on commercially important edible organisms, the time organism takes to metabolize and excrete anesthetic chemicals from its body to a safe level must be given to allow fitness for human consumption.