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Jung E.-M.,Chungbuk National University | Son H.Y.,SooAm Biotech Research Foundation | Jeung M.K.,Chungbuk National University | Jeung M.K.,SooAm Biotech Research Foundation | And 3 more authors.
International Journal of Molecular Medicine | Year: 2011

Some human embryonic stem cell lines have shown genomic instabilities over long-term culture. To study the controversial origin of the SCNT-hES-1 line, which was derived from autologous somatic cell nuclear transfer (SCNT), we compared the expression and methylation patterns of imprinted genes in the SCNT-hES-1 cells with the donor's somatic cells by semi-quantitative RT-PCR, real-time PCR and bisulfite sequencing. Examined imprinted genes were H19, GNAS, SLC22A18, UBE3A and ZNF264 for maternally expressed genes, and IGF2, SNRPN, PEG3, PEG10, MEST, MAGEL2 and ARHI for paternally expressed genes, respectively. We found that the expression of imprinted genes in the SCNT-hES-1 cell line is comparable to that in the donor's somatic cells, and that its methylation patterns are similar to those of other SCNT-products. Therefore, the present study indicates that the SCNT-hES-1 line was derived from SCNT.

Kim Y.-K.,SooAm Biotech Research Foundation | Lee G.-S.,Kangwon National University | Jung E.-M.,Chungbuk National University | Hyun S.-H.,Chungbuk National University | And 2 more authors.
Molecular Medicine Reports | Year: 2012

Type 2 diabetes mellitus (T2DM) is one of the most common complex metabolic disorders in humans, and is characterized by hyperglycemia and metabolic alterations. In T2DM, fasting hyperglycemia is attributed to excessive hepatic glucose production, and increased gluconeogenesis has been ascribed to increased transcriptional expression of phosphoenolpyruvate carboxykinase (PEPCK). In this study, we analyzed porcine PEPCK promoter activities to generate liver-specific expression vectors. We generated miniature pig fibroblasts overexpressing PEPCK via transgenes to provide an animal model of human T2DM. Various regions of the promoter showed high levels of activity in the presence of glucocorticoids, a PEPCK gene inducer, in liver cells compared to a positive control promoter. The selected promoter region for a liver-specific expression system was adopted based on the current targeting vector containing two selection markers, green fluorescence protein and a neomycin-resistance gene. The linearized vector was introduced into pig fibroblasts which facilitated liver-specific PEPCK overexpression and screening according to the two selection markers. In the present study, we used a liposome-mediated transfection protocol rather than a virus-mediated gene delivery system, since the virus may cause side effects. Following transfection, 46 colonies out of 33 transfection trials had positively integrated the overexpression vector, indicating that a relatively high transfection efficiency rate was obtained by the liposomal-mediated system. Thus, we recommend the optimal liver-specific expression system for safe and effective transfection of pig cells. We plan to use these cells for somatic cell nuclear transfer to produce piglets that overexpress liver-specific PEPCK as an animal model for human T2DM.

Jung E.-M.,Chungbuk National University | Kim Y.-K.,SooAm Biotech Research Foundation | Lee G.-S.,Kangwon National University | Hyun S.-H.,Chungbuk National University | And 2 more authors.
Molecular Medicine Reports | Year: 2012

Diabetes mellitus is a metabolic disease caused by impaired insulin secretion from the pancreatic β cells and increased insulin resistance in peripheral tissues. Recently, the overexpression of inducible cyclic AMP (cAMP) early repressor (ICER) Iγ in rodent pancreatic β cells was found to induce insulin deficiency and glucagon overproduction similar to that found in human diabetes mellitus. ICER Iγ with only a DNA binding domain interrupts the transcriptional regulation of the cAMP responsive element-binding protein (CREB) target genes. Based on this information, we hypothesized that the overexpression of ICER Iγ, the most powerful competitor to CREB, could be useful for generating a pig model of diabetes. First, we evaluated the promoter activities of the human insulin gene for the β cell-specific overexpression of ICER Iγ in the pig pancreas. The maximum promoter activity region [-1,431 nucleotides (nt) to +1 nt, +1 = the transcriptional start site] of the insulin gene presented an activity level 3-fold higher than a promoterless construct. Second, ICER Iγ overexpression controlled by this promoter region significantly blocked the glucose-mediated insulin transcription, such as that regulated by the viral promoter in the pancreatic β cell line, MIN6. This suggests that the human insulin promoter may facilitate the overexpression of ICER Iγ in porcine pancreatic β cells. In addition, the overexpression of ICER Iγ in porcine β cells may induce human-like type 1 diabetes mellitus in pigs. In the present study, we generated transgenic fibroblasts containing ICER Iγ cDNA controlled by the human insulin promoter, as well as two screening markers, the green fluorescence protein and the neomycin resistance gene. These fibroblasts may provide a source for somatic cell nuclear transfer to generate a pig model that mimics human diabetes mellitus.

Jung E.-M.,Chungbuk National University | An B.-S.,Pusan National University | Kim Y.-K.,SooAm Biotech Research Foundation | Jeong Y.-H.,SooAm Biotech Research Foundation | And 2 more authors.
Molecular Medicine Reports | Year: 2013

Metabolic syndrome arises from a combination of disorders that increase the risk of cardiovascular disease and diabetes. In previous studies, it was observed that overexpression of 11β -hydroxysteroid dehydrogenase type 1 (11β-HSD1) induced obesity and the insulin resistance that accompanies metabolic syndrome in rodent adipose tissue. Based on these observations, it was hypothesized that overexpression of 11β-HSD1 may be suitable for the generation of a porcine model of metabolic syndrome. It was evaluated that promoter activities of the porcine adipose fatty acid-binding protein (aP2) gene generates adipose tissue-specific 11β-HSD1 expression. In adipose tissue, the maximum promoter activity (-2,826 to +51 nt) of aP2 was 200-fold higher than that of a promoterless construct. In addition, 11β-HSD1 transcriptional levels were significantly increased following the introduction of the aP2 promoter into 3T3-L1 adipocytes. These observations indicate that the aP2 promoter may facilitate 11β-HSD1 overexpression in porcine adipose tissue. Transgenic fibroblasts were generated containing 11β-HSD1 cDNA controlled by the aP2 promoter with two screening markers, green fluorescence protein and a neomycin-resistance gene. It was hypothesized that transgenic fibroblasts may be useful for generating a porcine model of metabolic syndrome.

Park C.-H.,SooAm Biotech Research Foundation | Park C.-H.,South Korean National Institute of Animal Science | Park C.-H.,Seoul National University | Uh K.-J.,SooAm Biotech Research Foundation | And 7 more authors.
PLoS ONE | Year: 2011

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos. © 2011 Park et al.

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