Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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 <it>dHuEPO </it>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.</p> <p>Conclusions</p> <p>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.</p

Expression of aldo-keto reductase family 1 member C1 (AKR1C1) gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy

<p>Abstract</p> <p>Background</p> <p>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 20alpha-hydroxysteroid dehydrogenase (20alpha-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.</p> <p>Methods</p> <p>Rapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine <it>20alpha-HSD </it>cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary.</p> <p>Results</p> <p>The porcine 20alpha-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 <it>AKR1C1 </it>gene (337 amino acids) reported recently, and only differed in the C-terminal region (the <it>AKR1C1 </it>gene has a longer C-terminal region than our sequence). The <it>20alpha-HSD </it>gene (from now on referred to as <it>AKR1C1</it>) cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of <it>AKR1C1 </it>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 <it>AKR1C1 </it>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.</p> <p>Conclusions</p> <p>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.</p

Molecular characterization of bovine placental and ovarian 20α-hydroxysteroid dehydrogenase

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

Yearly Difference in Normalized Seed Weight of Cultivated Iris dichotoma Pall. in Mongolia

Maximum of mean seed weight was observed at 1997 while normalized seed weight was signifi cantly higher at 1982 and 1995, compared with other years. Seed weight variation is positively correlated with mean seed weight for earlier rainy season years, excluding later rainy season years (1982 and 1995). Low and high seed weight variation associated for years with colder and wetter in May and with hotter and drier in May, suggesting drought in May might be eff ective on seed weight variation of I. dichotoma. Moderate normalized seed weight associated for years with hotter in June, while high and low normalized seed weight for years with cooler and cool in June, suggesting I. dichotoma might use both photosynthetic productions and under-ground-storage for seed maturity. Mean seed weight and seed weight variation were linked to precipitation amount, whereas normalized seed weight was linked to precipitation periodicity. Seed weight variation detected delay of regrowth, caused by early drought, while normalized seed weight suggested seed weight response to complete seed maturation after droughty season, using photosynthetic productions and under-ground-storage. The results suggest that normalized seed weight might be useful to recognize seed weight response of I. dichotoma for climatic factors, better than mean seed weight and seed weight variation

Influence of underground storage on seed weight of perennial plants in Mongolia.

There are a number of studies on relevance of photosynthetic contribution for seed weight. Here we demonstrate a contribution of plant underground storage on seed weight. We collected 348 seed samples of 230 species, in the forest-steppe, steppe, desert-steppe and desert zones in Mongolia, between 1981 and 2007. All species were categorized by growth form, flowering phenology and underground storage. Plant height was not different among categories of flowering phenology and underground storage. Seed weight was higher in large storage perennials than in small storage species. Correlation between seed weight and plant height was significantly positive in small storage perennials but it was insignificant in large storage perennials. For small storage perennials, correlation between seed weight and plant height was found in early and continuous flowering perennials while was absent in late flowering perennials. For large storage perennials, seed weight and plant height positively correlated in early flowering perennials, but those were not correlated and negatively correlated in continuous and late flowering perennials, respectively. The results suggest that early and continuous flowering perennials might use photosynthetic production for seed development, more intensive than late flowering perennials. Also, late flowering perennials more strongly use underground storage for seed development

The transcription factor Ap-1 regulates monkey 20α-hydroxysteroid dehydrogenase promoter activity in CHO cells

BACKGROUND: Monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) is a catabolic enzyme responsible for converting progesterone into biologically inactive 20α-hydroxyprogesterone, thereby playing a key role in the estrous cycle or pregnancy and allowing ovulation and parturition to occur in most mammalian animals. Monkey 20α-HSD was highly abundant in ovarian and placental tissues during the pre-ovulation and pre-parturition phase and was primarily localized in the syncytiotrophoblast of the placenta. In this study, we focused on the molecular characterization of the monkey 20α-HSD promoter region by conducting reporter assays in Chinese hamster ovary (CHO) K1 cells. RESULTS: A reporter assay using constructs of various lengths of the 5′-flanking region (-890-Luc, -513-Luc, -306-Luc, -273-Luc, and -70-Luc) revealed that a region corresponding to the activator protein 1 (Ap-1) located between -281 and -274 bp was essential for the transcriptional activity. Absence of the Ap-1 site in -273-Luc dramatically decreased the transcription levels to the control levels. When the reporter constructs were co-transfected with Ap-1 (Jun) and specificity protein (Sp-1) genes, the transcription activities of the constructs increased with the exception of -273 and -70, while that of the double construct was reduced compared to that of Ap-1 alone. Furthermore, mutational analysis demonstrated that a putative Ap-1 site played an important role in the expression of the reporter gene. These findings were confirmed by EMSA examining the interactions of the protein Ap-1 in a nuclear extract from CHO-K1 cells and the expression levels of the Ap-1 transcription factor in pre-parturition placenta and CHO-K1 cells. Although mut-1 and mut-2 of Ap-1 bound with nuclear extracts from CHO-K1 cells, the transcriptional activity of mut-3 was almost completely suppressed. CONCLUSIONS: Our results indicate that the Ap-1 site (-281 → -274) (5′-TGTCTCAT-3′) plays a crucial role in the activation of the monkey 20α-HSD gene. Thus, we demonstrated that monkey 20α-HSD promoter activity is regulated by the transcription factor Ap-1 in CHO-K1 cells

Mongolian academic botanic garden as an introduction center and ecological resource for plant biodiversity conservation

The main results and achievements of the introduction work carried out at the Botanic Garden of the Mongolia Academy of Sciences (BG MAS) for almost 50 years of complicated development with ups and downs are given. It shows modern positioning and value of the BG MAS as the main country’s introduction center, ecological resource and public educational organization, treasurer of the scientific knowledge for the conservation and rational use of plant genetic resources, which contributes to the growing process of new botanic gardens and nurseries formation in Mongolia

Rare species of psammophyte flora in transboundary areas of Southern Siberia and Mongolia

Data on distribution of 3 rare species which occur in sandy arrays of cross-border territories of Southern Siberia and Mongolia are provided: Iris psammocola, Iris loczyi (Iridaceae) and Vicia tsydenii (Fabaceae), a brief characteristic of their cenopopulation is given