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Non-metastatic cells 1 (NME1) or nm23 is the first metastasis suppressor gene discovered. It functions through various enzymatic activities and interacts with many intracellular proteins. The NME1 gene encodes two splicing variants, NME1 and NME1L. Most studies have focused on NME1 because of its abundance in cells. We previously reported NME1L-mediated suppression of NF-κB signaling by interacting with and inhibiting IKKβ. In this study, we demonstrated that NME1L, but not NME1, mediated inhibition of cell proliferation, although both NME1 and NME1L were involved in suppressing metastasis. A reporter gene assay was performed to determine the growth signaling pathway regulated by NME1L but none of the growth factors tested could induce an NF-κB-dependent luciferase expression except TNFα. Interestingly, SRE-reporter gene activation by IGF1 was significantly downregulated, along with reduction of ERK phosphorylation in NME1L expressing cells, compared to vector or NME1 expressing cells. NME1L directly interacted with KSR1, which is a scaffold for Raf-1, MEK, and ERK, that regulates ERK activation. Hence, NME1L plays a crucial role in regulation of cell proliferation by inhibiting IGF1-stimulated ERK phosphorylation through N-terminal 25 amino acid-mediated interaction with KSR1.

The purpose of this study was to analyze the reproductive ability of transgenic female dogs born bysomatic cell nuclear transfer and to determine inheritance of the red fluorescent protein (RFP) transgene. The four founder transgenic bitches (F0) reached puberty at 340.8 ± 39.6 days after birth and were bred with wild-type male dogs by natural mating or by artificial insemination. The bitches all became pregnant and successfully delivered 13 puppies (F1), of which two females were bred with wild-type dogs to deliver 7 offspring (F2), including 1 stillbirth. Among the 19 live offspring, 10 puppies showed emission of RFP under UV light and the presence of the RFP transgene was confirmed by genomic PCR and Southern blot analyses. In conclusion, transgenic RFP female dogs exhibited normal reproductive ability and expression of the transgene was demonstrated in F1 and F2 generations.

Genetically manipulated transparent animals were already explored in many species for in vivo study of gene expression, transplantation analysis and cancer biology. However, there are no reports about transparent animals as in vitro genetic resources. In the present study, fin-derived cells from glass catfish (Krytopterus bicirrhis), naturally transparent fish with a visible skeleton and internal organs, were isolated after culturing fin explants and characterized using cryopreservation and cell cycle analysis. The cells grew well in DMEM (Dulbecco's modified Eagle's medium) containing 1% (v/v) P/S (penicillin-streptomycin) and 10% (v/v) fetal bovine serum at 26°C and showed increased cryopreservation efficiency with the slow-freezing method in the presence of 15% dimethyl sulfoxide. In addition, cell cycle analysis was evaluated based on flow cytometric analysis, and culturing to confluence (85%) was more effective for synchronizing cells at the G(0)/G(1) stages than roscovitine treatment (75%). This is the first report about cell isolation from transparent animals. The results from testing the cell's viability following cryopreservation and subjecting the cells to cycle analysis can be useful tools for genetic resource management.

Synchronization of the donor cell cycle is an important factor for successful animal cloning by nuclear transfer. To improve the efficiency of porcine cloning, in the present report, we evaluated effects of contact inhibition, serum starvation and roscovitine treatment of donor cells on in vitro and in vivo developmental potency of cloned porcine embryos. Fibroblasts derived from a porcine foetus at day 30 of gestation were isolated and cultured to 70% confluency. Then, cells were either cultured to 100% confluency for contact inhibition, or cultured in 0.5% serum for 72 h for serum starvation or with 15 μM roscovitine for 24 h. Cells were most effectively synchronized at G0/G1 in the serum starvation group (87.5%) compared with the contact inhibition and roscovitine treatment groups (76.3% and 79.9% respectively p < 0.05). However, after somatic cell nuclear transfer followed by in vitro culture, the serum starvation group showed a significantly lower blastocyst formation rate (5.6%) compared with the contact inhibition and roscovitine treatment groups (11.6% and 20.0% respectively). Differential expression of apoptosis-related genes and the level of apoptosis in each treatment group explain the variation in developmental competence among the groups. Significantly higher level of apoptosis was observed in the serum starvation group. On the other hand, the roscovitine treatment group shows the lowest level of apoptosis and the best in vitro development among the groups. Cloned embryos derived from roscovitine-treated donor cells were transferred to surrogate pigs. Three healthy live piglets were produced. In conclusion, we suggest that roscovitine treatment of donor cells improves development of cloned porcine embryos and can raise the efficiency of cloned piglet production.

We have established a new 4 stage epiblast isolation method from whole bovine cloned blastocysts without using immunosurgery. The new "peeling" method consists of dissolution of the zona pellucida (first stage), elimination of mural trophoblast (second stage), isolation of primitive endoderm and epiblast from polar trophoblast (third stage), and isolation of epiblast from primitive endoderm (fourth stage). The bovine cloned blastocyst consists of 4 different types of cells showing abundant alkaline phosphatase activity. The epiblast origin of isolated cells was confirmed by in vitro differentiation of isolated cells to tubulin beta3-positive neurons and by embryoid body formation. The bovine cloned blastocyst origin of isolated epiblasts was confirmed by microsatellite analysis and mitochondrial DNA sequencing analysis. This new method might accelerate establishment of somatic cell nuclear transfer derived embryonic stem cell lines from bovine and other mammals.

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos.

Several studies have previously been conducted regarding cell cycle synchronization in mammalian somatic cells. However, limited work has been performed on the control of cell cycle stages in the somatic cells of fish. The aim of this study was to determine the cell cycle arresting effects of several dimethyl sulfoxide (DMSO) concentrations for different times on different cell cycle stages of goldfish caudal fin-derived fibroblasts. Results demonstrated that the cycling cells or control group (68.29%) yields significantly higher (p < 0.05) arrest in G0/G1 phase compared with the group treated for 24 h with different concentrations (0.5%, 1.0% or 1.5%) of DMSO (64.88%, 65.70%, 64.22% respectively). The cell cycle synchronization in the treatment of cells with 1.0% DMSO at 48 h (81.14%) was significantly higher than that in the groups treated for 24 h (76.82%) and the control group (77.90%). Observations showed that treatment of DMSO resulted in an increase in the proportion of cells at G0/G1 phase for 48 h of culture. However, high levels of apoptotic cells can be detected after 48 h of culture treated with 1% concentration of DMSO.

To improve pig cloning efficiency, the present study evaluated the effect of ovulation status, seasonality and embryo transfer (ET) method on in vivo development of cloned porcine embryos. Cloned embryos were transferred to surrogate mothers on the same day of somatic cell nuclear transfer. In pre-ovulation stage (PO), pregnancy rate (PR) and delivery rate (DR) were 36.3% and 9.4%, respectively. In post-ovulation stage, 22.7% PR and 2.1% DR were recorded (both PR and DR are significantly higher in PO). When ET was performed during winter (December-February), spring (March-May), summer (June-August) and autumn (September-November), the PRs were 13.4%, 37.3%, 24.6% and 51.0%, while DRs were 0%, 12.7%, 4.3% and 7.8%, respectively. The highest PRs were recorded in autumn groups. However, DRs were significantly lower in autumn (7.8%) group compared with spring (12.7%) group. The PR was the lowest and no piglets were born in winter group, which might be because of the effect of low temperature during ET. To overcome the low PR in winter group, 0.25 ml straws were used for ET to minimize exposure time of embryos to ambient temperature. The straw ET group showed significantly higher PR in the winter group (23. 9%) compared with the conventional catheter-loading group (7.7%). We suggest that using PO recipient and ET in spring is the best condition for pig cloning. In addition, alternative method to reduce cold shock during ET in winter is necessary.