Comparative analysis of immune related genes between domestic pig and germ-free minipig / 한국실험동물학회지

Recently, minipig has been considered as an animal model that is appropriate for human disease model to study toxicology, pharmacology, and xenotransplantation. Nevertheless, minipigs are bred in various environment according to their use. Here, we suggest that minipigs used for research should be bred in well-controlled facility, comparing immune status of pigs raised in different breeding environment. DNA microarray was performed with ear skin and placenta of Landrace domestic pigs (DPs) and Minnesota germ-free minipigs (GPs). Their immune transcriptome was analyzed by gene ontology (GO) annotation database, based on criteria of |log2 fold change| ≥1 with P ≤ 0.05. As a result, we found that immune related genes in the ear skin of DPs were highly activated, compared to GPs. On the other hand, no significant s were found in the placenta. Quantitative real-time PCR (qRT-PCR) was performed in five candidate immune genes. Their fold changes were consistent with the results from DNA microarray (P ≤ 0.05). In conclusion, we experimentally proved that porcine immune system was affected by different breeding environment, suggesting the importance of controlling microbes in animal room for the qualified research.

Comparative analysis of immune related genes between domestic pig and germ-free minipig / 한국실험동물학회지

Recently, minipig has been considered as an animal model that is appropriate for human disease model to study toxicology, pharmacology, and xenotransplantation. Nevertheless, minipigs are bred in various environment according to their use. Here, we suggest that minipigs used for research should be bred in well-controlled facility, comparing immune status of pigs raised in different breeding environment. DNA microarray was performed with ear skin and placenta of Landrace domestic pigs (DPs) and Minnesota germ-free minipigs (GPs). Their immune transcriptome was analyzed by gene ontology (GO) annotation database, based on criteria of |log2 fold change| ≥1 with P ≤ 0.05. As a result, we found that immune related genes in the ear skin of DPs were highly activated, compared to GPs. On the other hand, no significant s were found in the placenta. Quantitative real-time PCR (qRT-PCR) was performed in five candidate immune genes. Their fold changes were consistent with the results from DNA microarray (P ≤ 0.05). In conclusion, we experimentally proved that porcine immune system was affected by different breeding environment, suggesting the importance of controlling microbes in animal room for the qualified research.

Cryopreservation of Oocytes and Embryos by Vitrification / 대한불임학회지

Life can be kept in suspended animation either before fertilization at oocyte stage or after fertilization at different stages of embryonic development for a variety of reasons. It not only has potential applications in fertility preservation and management in human but also has important roles in the preservation and management of animal genetic resources, low-cost international movement of selected genetics, and rapid dissemination of germplasm through assisted reproductive technologies (ART) and genetic engineering. Currently, slow-freezing and vitrification are the two approaches by which oocytes and embryos can be cryopreserved for long-term storage. Both of these methods have their own advantages and disadvantages but allow the cryopreservation of oocytes and embryos with comparable efficiency. Vitrification of oocyte and embryos, although proven successful just 13 years after slow-freezing, is generally considered an emerging technology and appears to slow gain acceptance in both animal and human ART despite having controversial storage and contamination issues. In this manuscript, we discuss the basic techniques of oocyte/embryo cryopreservation and review the current status and recent developments in vitrification.

Molecular Characterization of Porcine Endogenous Retrovirus gag Genes from Pigs in Korea

Xenotransplantation, as a potential solution to the shortage of human organs, is associated with a number of concerns including immunologic rejection and xenogenic infection. While the pigs are considered the most suitable organ source for xenotransplantation, there is a potential public health risk due to zoonosis. Among the known porcine zoonotic microbes, Porcine Endogenous Retrovirus (PERV) is the most considerable virus. PERV belongs to the Gammaretrovirus and has been divided into three groups (A, B, and C). To characterize the gag of PERVs, we isolated the genomic DNAs from three pig breeds (Birkshire, Duroc, and Yorkshire) and two types of SPF miniature pigs. About 1.5 kb fragments covering full length of gag were amplified and cloned into T-vector. A total of 38 clones were obtained and sequenced. Nucleotide sequences were analyzed and phylogenetic trees were constructed from the nucleotide and deduced amino acids. PERV-A, -B and -C were present in the proportion of 47, 19 and 34%, respectively. Regardless of origin or subgroups, gag clones showed highly homology in nucleotide and deduced amino acid sequences. Deduced amino acids sequence alignments showed typical conserve sequences, Cys-His box and processing sites. Among analyzed clones, about 28% of isolates had the correct open reading frame. To test the functional expression of Gag protein, gag was subcloned into expression vector and confirmed its expression in HeLa cell. This research provides the fundamental information about molecular characteristics of gag gene and functional Gag protein related xenotropic PERVs.

Characterization of a brain tumor cell line established from transgenic mice expressing the vasopressin SV-40 T antigen

We previously reported that transgenic mice produced with a transgene consisting of the SV40 T antigen and vasopressin without the 3'-flanking region exhibit brain tumors and lymphoma. In this study, transgenic mice were produced with the fusion gene containing the SV40 T antigen and the whole vasopressin gene with the 3'-flanking region. Six transgenic mice were generated, five which died after 2-6 weeks. The remaining founder mouse was investigated for fusion gene expression and tumor progression at the age of 6 weeks. Brain tumor cells were characterized for phenotypes and transgene expression. During in vitro cell cultures, the phenotypic appearances at 10, 20, and 30 passages were as a uniform monolayer with similar growth rates. The site of SV40 T antigen integration was in the A2 region of chromosome 11, and SV40 T antigen was expressed at the same level in cells of both earlier and later passages. Thirty passages were probably insufficient to reach crisis and immortalization. These cells enriched brain tumor cell compositions with astrocytes and neuronal cells.

Effects of Neurotrophic Factors on the Generation of Functional Dopamine Secretory Neurons Derived from in vitro Differentiated Human Embryonic Stem Cells / 대한불임학회지

OBJECTIVE: This study was to examine the in vitro neural cell differentiation patterns of human embryonic stem (hES) cells following treatment of various neurotrophic factors [basic fibroblast growth factor (bFGF), retinoic acid (RA), brain derived neurotrophic factor (BDNF) and transforming growth factor (TGF)-alpha], particulary in dopaminergic neuron formation. METHODS: The hES cells were induced to differentiate by bFGF and RA. Group I) In bFGF induction method, embryoid bodies (EBs, for 4 days) derived from hES were plated onto gelatin dish, selected for 8 days in ITSFn medium and expanded at the presence of bFGF (10 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14 and 21 days. Group II) For RA induction, EBs were exposed of RA (10-6 M) for 4 days and allowed to differentiate in N2 medium for 7, 14 and 21 days. Group III) To examine the effects of additional neurotrophic factors, bFGF or RA induced cells were exposed to either BDNF (10 ng/ml) or TGF-alpha (10 ng/ml) during the 21 days of final differentiation. Neuron differentiation and dopamine secretion were examined by indirect immunocytochemistry and HPLC, respectively. RESULTS: The bFGF or RA treated hES cells were resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with BDNF or TGF-alpha during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression of a dopaminergic neuron marker, compared to control (p<0.05). In contrast, no effect was observed on the rate of mature neuron (NF-200) or glutamic acid decarboxylase-positive neurons. Immunocytochemistry and HPLC analyses revealed the higher levels of TH expression (20.3%) and dopamine secretion (265.5+/-62.8 pmol/mg) in bFGF and TGF-alpha sequentially treated hES cells than those in RA or BDNF treated hES cells. CONCLUSION: These results indicate that the generation of dopamine secretory neurons from in vitro differentiated hES cells can be improved by TGF-alpha addition in the bFGF induction protocol.

Functional Cardiomyocytes Formation Derived from Parthenogenetic Mouse Embryonic Stem Cells / 대한불임학회지

OBJECTIVE: This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. MATERIALS AND METHODS: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to defect cardiomyocytes (anti-sarcomeric alpha-actinin Ab, 1: 100; anti-cardiac troponin I Ab, 1: 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. RESULTS: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3% at 13 days and 69.8% at 15 days, respectively. Also the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and mES02,4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric alpha-actinin Ab and cardiac specific anti-cardiac troponin I Ab. CONCLUSION: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

Parthenogenetic Mouse Embryonic Stem Cells have Similar Characteristics to In Vitro Fertilization mES Cells / 대한불임학회지

OBJECTIVE: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. MATERIAL AND METHODS: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and 5microgram/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal perm of hybrid F1 male mice(1x106/ml). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, blastocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identity ES cells, the surface markers alkaline phosphatase, SSEA-1, 3, 4 and Oct4 staining were examined in replated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. RESULTS: Although the cleavage rate (> or =2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic blastocysts (9.6+/-3.1, 35.1+/-5.2) were significantly lower than those of IVF blastocysts (19.5+/-4.7, 63.2+/-13.0) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-1 and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac differentiation derived from mES or P-mES cells was confirmed. CONCLUSION: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

Telomerase Activity and Expression of MIB-1 and bcl-2 in Human Chorionic Villi from Early and Term Normal Pregnancy

Telomerase is an enzyme that maintains telomeres and prevents telomere shortening, and may be linked with cellular proliferation or the aging process. The purpose was to examine telomerase activity in human chorionic villi from early and term normal pregnancies, and to analyze the correlation of telomerase activity (TA) with MIB-1 & bcl-2. A total of 37 placentae were obtained from 16 early and 21 term pregnancies. TA was assayed by telomeric repeat amplification protocol, and immunohistochemical staining was performed for MIB-1 & bcl-2 expression. TA & MIB-1 expression were strong in early placenta, but bcl-2 was highly expressed in term placentae. Thirteen (81.25%) of 16 early placentae showed TA, but only 2 (9.52%) of 21 term placentae expressed TA (p<0.01). MIB-1 was observed in nuclei of cytotrophoblast, and the expression rate was 16.09% in early placentae and 2.87% in term placentae (p<0.01). bcl-2 was observed only in the cytoplasm of syncytiotrophoblast. Term placenta demonstrated stronger expression of bcl-2 compared to early placentae (p<0.05). These findings suggest that TA, MIB-1 & bcl-2 expression are critically regulated over the course of gestation: cytotrophoblast, main cells of early chorionic villi, may be a common source of telomerase and proliferative activity. The TA showed good correlation with cellular proliferative activity. Syncytiotrophoblast, may be a main source of bcl-2 expression which is stronger in the term placentae.

Effect of cell-free human amniotic fluid on the develpment of 2-cell stage mouse embryos in vitro / 대한산부인과학회잡지

No abstract available.

Effect of cell-free human amniotic fluid on the develpment of 2-cell stage mouse embryos in vitro / 대한산부인과학회잡지

No abstract available.