Effective Condition for Whole Testis Cryopreservation of Endangered Miho Spine Loach (Cobitis choii) Through the Optimization of Mud Loach (Misgurnus mizolepis) Whole Testis Cryopreservation Condition.

　　BACKGROUND: Miho spine loach (Cobitis choii) is an endangered Korean endemic fish. Whole testis cryopreservation is a good way for species preservation, but needs to the sacrifice of a large number of fish to optimize the freezing condition. Considering this limitation, a surrogate fish species was used for the protocol development. OBJECTIVE: This study was to establish the effective condition for Miho spine loach whole testis cryopreservation by optimizing the conditions for whole testis cryopreservation in an allied species, mud loach (Misgurnus mizolepis). MATERIALS AND METHODS: The condition for whole testis cryopreservation was optimized in mud loach first, and then the optimal condition was applied to Miho spine loach testes. RESULTS: The optimal condition for mud loach testis cryopreservation consists of the freezing medium containing 1.3 M dimethyl sulfoxide, 6% fetal bovine serum and 0.3 M trehalose, -1 C/min cooling rate and 26 degree C thawing temperature, which also permits effective cryopreservation of Miho spine loach testes. CONCLUSION: An effective cryopreservation condition for whole testis of the endangered Miho spine loach has been established by using mud loach as a surrogate fish.

Derivation of the clonal-cell lines from Siberian sturgeon (Acipenser baerii) head-kidney cell lines and its applicability to foreign gene expression and virus culture.

This study was conducted to establish and characterize the clonal-cell lines from Siberian sturgeon Acipenser baerii head-kidney tissues and to evaluate its applicability as a research tool. From the culture of A. baerii head-kidney derived cells, 10 cell lines were established first and then eight clonal-cell lines were derived from clonal growth and colony expansion of two cell lines that showed significant high colony-forming ability. All eight clonal-cell lines were morphologically similar and grew stably under monolayer culture but their growth rates were significantly different. They possessed diploid DNA contents, expressed epithelial cell-related genes and showed strong anchorage dependency to substrates. When a clonal-cell line was transfected separately with three plasmid vectors including fluorescent reporter genes driven by cytomegalovirus, marine medaka Oryzias dancena ß-actin or A. baerii ß-actin promoter, the cell lines expressed fluorescent signals regardless of promoter types. The cells harbouring foreign genes could be expanded to stable cell lines under drug selection and then they additionally could form the extensively proliferating colonies at low-density culture. Finally, the clonal-cell lines showed the susceptibility to viral haemorrhagic septicaemia virus (VHSV). Collectively, the clonal-cell lines from A. baerii head kidney were established and these cell lines will be able to provide an excellent in vitro system for various biological studies in this fish species.

Shallow Population Genetic Structures of Thread-sail Filefish (Stephanolepis cirrhifer) Populations from Korean Coastal Waters.

Genetic diversities, population genetic structures and demographic histories of the thread-sail filefish Stephanolepis cirrhifer were investigated by nucleotide sequencing of 336 base pairs of the mitochondrial DNA (mtDNA) control region in 111 individuals collected from six populations in Korean coastal waters. A total of 70 haplotypes were defined by 58 variable nucleotide sites. The neighbor-joining tree of the 70 haplotypes was shallow and did not provide evidence of geographical associations. Expansion of S. cirrhifer populations began approximate 51,000 to 102,000 years before present, correlating with the period of sea level rise since the late Pleistocene glacial maximum. High levels of haplotype diversities (0.974±0.029 to 1.000±0.076) and nucleotide diversities (0.014 to 0.019), and low levels of genetic differentiation among populations inferred from pairwise population F ST values (-0.007 to 0.107), support an expansion of the S. cirrhifer population. Hierarchical analysis of molecular variance (AMOVA) revealed weak but significant genetic structures among three groups (F CT = 0.028, p<0.05), and no genetic variation within groups (0.53%; F SC = 0.005, p = 0.23). These results may help establish appropriate fishery management strategies for stocks of S. cirrhifer and related species.

Ubiquitin C-terminal hydrolase-L1 is a key regulator of tumor cell invasion and metastasis.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) catalyses the hydrolysis of ubiquitin ester and amide mainly in neuronal cells. Recently it was proposed as a marker with a potential role in carcinogenesis. However, the molecular mechanism underlying the biological function of UCH-L1 in tumor cells is poorly understood. We found that UCH-L1 is highly expressed in non-small lung cancer cell line H157, having high invasive potential, and that the expression of UCH-L1 in tumor cells enhances their invasive potential in vitro and in vivo. UCH-L1 changes cell morphology by regulating cell adhesion through Akt-mediated pathway. Suppressing UCH-L1 expression by RNAi significantly suppressed the invasion in vitro and in vivo, and the activation of Akt and downstream mitogen activated protein kinases c-Jun N-terminal kinases and p38, but not ERK. In Akt-negative mutants, overexpression of UCH-L1 does not affect the invasion and migration capability of H157 cells. These results suggest that UCH-L1 is a key molecule to regulate tumor-cell invasion by upstream activation of Akt.

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 beta-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 413 g and 41.5 cm) were larger and longer than even those of 12-year-old normal broodstock (maximum size reached to 89 g and 28 cm). 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 (10 g) 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.

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.

Genomic organization and sequence of the mud loach (Misgurnus mizolepis) growth hormone gene: a comparative analysis of teleost growth hormone genes.

The mud loach (Misgurnus mizolepis) growth hormone (GH) gene was cloned and a comparative analysis on its genomic organization was performed. Based on Southern analysis using various kinds of restriction endonucleases, the GH gene proved to exist as a single-copy gene in the mud loach. The complete nucleotide sequences of a 5.1 kb SacI/EcoRI genomic fragment containing the mud loach GH gene and its 5' flanking sequences as well as a mud loach GH cDNA obtained by rapid amplification of a reverse transcriptase-PCR have been determined. The GH gene spans 2.0 kb from the start codon to the polyadenylation signal, and contains five exons and four introns similar to those of carps and mammals. The evolutionary relation of the mud loach GH gene, inferred by comparative analyses of gene structures and sequences in each exon and intron of representative teleost GH genes, reflects the major phylogenetic groupings of teleost.