Publications (7)20.79 Total impact
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Dataset: repro paper (Jagaa et al)
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Article: Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin.
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ABSTRACT: Dimeric human erythropoietin (dHuEPO) peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg) mice expressing dHuEPO and to investigate the characteristics of these mice. A dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile), was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice. A high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47) of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11). We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females) were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleen-derived candidate genes; more genes were downregulated than upregulated (849/764). Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used for validating the results of the microarray analysis of mRNA expression. In conclusion, dHuEPO tg mice caused excessive erythrocytosis that led to abnormal blood composition, short lifespan, and abnormal splenomegaly. Further, we identified 2,672 genes associated with splenomegaly by microarray analysis. These results could be useful in the development of dHuEPO-producing tg animals.Reproductive Biology and Endocrinology 01/2012; 10:6. · 2.05 Impact Factor -
Article: Parthenogenetic embryonic stem cells with H19 siRNA-mediated knockdown as a potential resource for cell therapy.
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ABSTRACT: Embryonic stem (ES) cells are used in cell therapy and tissue engineering due to their ability to produce different cells types. However, studies of ES cells that are derived from fertilized embryos have raised concerns about the limitations imposed by ethical and political considerations. Therefore, many studies of stem cells use the stem cells that are derived from unfertilized oocytes and adult tissue. Although parthenogenetic embryonic stem (ESP) cells also avoid ethical and political dilemmas and can be used in cell-based therapy, the ESP cells exhibit growth retardation problems. Therefore, to investigate the potential for muscle growth from genetically modified ESP cells, we established four ES cell types, including normal embryonic stem (ESN) cells, ESP cells, ESP cells that overexpress the insulin-like growth factor 2 (Igf2) gene (ESI) and ESP cells with down-regulated H19 gene expression (ESH). Using these cells, we examined the expression profiles of genes that were related to imprinting and muscle using microarrays. The gene expression patterns of ESI and ESH cells were similar and were more closely related to the ESN pattern than that of the ESP cells. Differentiated ESH cells exhibited increased expression of bone morphologic protein 4 (BMP4), which is a mesoderm marker, compared with the differentiated ESI cells. We showed that Igf2 expression was induced by H19 silencing in the ESP cells via hypermethylation of the H19 imprinting control region 1 (ICR1). Moreover, the proportion of ESH-derived chimera was slightly higher than those produced from the ESP cells. In addition, we detected increased cell proliferation in the MEF cells following H19 knock-down. These results indicate that the ESH cells may be a source of cell-based therapy for conditions such as muscular atrophy.International Journal of Molecular Medicine 11/2011; 29(2):257-62. · 1.98 Impact Factor -
Article: Molecular characterization of bovine placental and ovarian 20α-hydroxysteroid dehydrogenase.
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ABSTRACT: The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme plays a critical role in the regulation of luteal function in female mammals. In this study, we conducted the characterization and functional analyses of bovine 20α-HSD from placental and ovarian tissues. The nucleotide sequence of bovine 20α-HSD showed significant homology to that of goats (96%), humans (84%), rabbits (83%), and mice (81%). The mRNA levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. Northern blot analysis revealed a 1.2 kb mRNA in the bovine placental and ovarian tissues. An antibody specific to bovine 20α-HSD was generated in a rabbit immunized with the purified, recombinant protein. Recombinant 20α-HSD protein produced in mammalian cells had a molecular weight of ∼37 kDa. Bacterially expressed bovine 20α-HSD protein showed enzymatic activity. The expression pattern of the 20α-HSD protein in the pre-parturition placenta and the CL1 stage of the estrous cycle was similar to the level of 20α-HSD mRNA expression. Immunohistochemical analysis also revealed that bovine 20α-HSD protein was intensively localized in the large luteal cells during the late estrous cycle.Reproduction 09/2011; 142(5):723-31. · 2.58 Impact Factor -
Article: Expression and localization of the 20α-hydroxysteroid dehydrogenase (HSD) enzyme in the reproductive tissues of the cynomolgus monkey Macaca fascicularis.
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ABSTRACT: This study was conducted to characterize and functionally analyze the monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) in the ovary, placenta, and oviduct. We focused on 20α-HSD mRNA expression and protein localization in monkey reproductive tissues and the molecular characterization of the promoter region. Reverse transcription-polymerase chain reaction (RT-PCR) monkey 20α-HSD mRNA was more strongly detected in the ovary at pre-ovulation than in the placenta and oviduct at pre-parturition. The mRNA was approximately 1.2kb in size and the expression was high in the ovary, which was the same as the RT-PCR result. We also produced His tagged 20α-HSD proteins by using an Escherichia coli expression system. In a western blot for the 20α-HSD protein, only 1 band of approximately 37-kDa was detected in the ovary, oviduct tissue, and recombinant protein produced in the Chinese hamster ovary (CHO) cell line. However, in the placenta, additional 2 bands (35 and 39 kDa) were detected. Immunohistochemical analyses suggested that the monkey 20α-HSD protein was localized mainly in the syncytiotrophoblast of the placenta and the isthmus cells of the oviduct. According to promoter analyses with the enhanced green fluorescent protein (EGFP) gene, the monkey 20α-HSD promoter was efficiently expressed in the CHO-K1 cell line; however, the promoter was not expressed in bovine fetal fibroblast (bFF) cell. Taken together, our study showed that the 20α-HSD mRNA and protein are coordinately expressed in the ovary at pre-ovulation and in the placenta and oviduct at pre-parturition. Therefore, monkey 20α-HSD in the placenta, ovary and oviduct plays an important role in the estrous cycle, pregnancy, and parturition.The Journal of steroid biochemistry and molecular biology 07/2011; 127(3-5):337-44. · 2.66 Impact Factor -
Article: Expression of aldo-keto reductase family 1 member C1 (AKR1C1) gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy.
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ABSTRACT: The aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPH-dependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) is a member of AKR family. The aims of this study were to determine its expression in the ovary and uterus endometrium during the estrous cycle and pregnancy. Rapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine 20 alpha-HSD cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20 alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary. The porcine 20 alpha-HSD cDNA is 957 bp in length and encodes a protein of 319 amino acids. The cloned cDNA was virtually the same as the porcine AKR1C1 gene (337 amino acids) reported recently, and only differed in the C-terminal region (the AKR1C1 gene has a longer C-terminal region than our sequence). The 20 alpha-HSD gene (from now on referred to as AKR1C1) cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of AKR1C1 genes from other animals. Porcine AKR1C1 mRNA was expressed on day 5, 10, 12, 15 of the cycle and 0-60 of pregnancy in the ovary. The mRNA was also specifically detected in the uterine endometrium on day 30 of pregnancy. Western blot analysis indicated that the pattern of AKR1C1 protein in the ovary during the estrous cycle and uterus during early pregnancy was similar to that of AKR1C1 mRNA expression. The recombinant protein produced in CHO cells was detected at approximately 37 kDa. Immunohistochemical analysis also revealed that pig AKR1C1 protein was localized in the large luteal cells in the early stages of the estrous cycle and before parturition. Our study demonstrated that AKR1C1 mRNA and protein are coordinately expressed in the luteal cell of ovary throughout the estrous cycle and in the uterus on day 30 of pregnancy. Thus, the porcine AKR1C1 gene might control important mechanisms during the estrous cycle.Reproductive Biology and Endocrinology 01/2011; 9:139. · 2.05 Impact Factor -
Article: Activation of histone deacetylase 2 by inducible heat shock protein 70 in cardiac hypertrophy.
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ABSTRACT: Diverse cardiac diseases induce cardiac hypertrophy, which leads to dilatation and heart failure. We previously reported that hypertrophy can be blocked by class I histone deacetylase (HDAC) inhibitor, which prompted us to investigate the regulatory mechanism of class I HDACs. Cardiac hypertrophy was introduced by aortic banding, by infusion of isoproterenol or angiotensin II, or by swimming. Hypertrophic stimuli transiently elevated the activity of histone deacetylase-2 (Hdac2), a class I HDAC. In cardiomyocytes, forced expression of Hdac2 simulated hypertrophy in an Akt-dependent manner, whereas enzymatically inert Hdac2 H141A failed to do so. Hypertrophic stimuli induced the expression of heat shock protein (Hsp)70. The induced Hsp70 physically associated with and activated Hdac2. Hsp70 overexpression produced a hypertrophic phenotype, which was blocked either by siHdac2 or by a dominant negative Hsp70DeltaABD. In Hsp70.1(-/-) mice, cardiac hypertrophy and Hdac2 activation were significantly blunted. Heat shock either to cardiomyocytes or to mice activated Hdac2 and induced hypertrophy. However, heat shock-induced Hdac2 activation was blunted in the cardiomyocytes isolated from Hsp70.1(-/-) mice. These results suggest that the induction of Hsp70 in response to diverse hypertrophic stresses and the ensuing activation of HDAC2 trigger cardiac hypertrophy, emphasizing HSP70/HDAC2 as a novel mechanism regulating hypertrophy.Circulation Research 11/2008; 103(11):1259-69. · 9.49 Impact Factor
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2011–2012
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Hankyong National University
Anseong, Gyeonggi, South Korea
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