Human protein C (hPC) is a regulator of homeostasis, suggesting its potential use as a therapy for many disease states. Protein C is a zymogen of a serine protease that is activated by thrombin. Protein C, also known as autoprothrombin ⅡA and blood coagulation factor ⅪⅤ, is a zymogenic (inactive) protein, the activated form of which plays an important role in regulating blood clotting, inflammation, cell death and maintaining the permeability of blood vessel walls in humans and other animals. hPC is a 62 KD disulfide- linked heterodimer consisting of a 21 KD light chain and a 41 KD heavy chain which circulates as an inactive zymogen in plasma. In this study, we focus on generation of hPC transgenic mice. hPC transgenic mice were produced by using micro-injection method. The hPC cDNA was cloned into pBC1 vector under goat β-casein promoter. One-cell stage embryos microinjected were transferred to 24 recipient mice on day 1 of the estrus cycle. We screened 61 mice by the PCR. Four line transgenic mice were identified and confirmed expression of protein C gene in mammary gland and several organ. We also analyzed the expression of mRNA and protein through the northern blot and western blot in mammary gland of hPC transgenic mice. hPC was localized in the alveolar epithelial cell by immunohistochemistry. Now, we are collecting the milk from the 2 found lines. After then, we are checking the activity of hPC produced from mice milk.

20ɑ-hydroxysteroid dehydrogenase (20ɑ-HSD) enzyme converts progesterone into biological inactive steroid, thus playing a key role in the termination of pregnancy or estrus cycle and allowing parturition and ovulation to occur in most mammalian animals. However, function and regulation of this enzyme has not known well in primate reproductive physiology. We previously demonstrated the expression level and localization of the 20α-HSD in the reproductive tissues of macaque monkeys of pre-ovulation and pre-parturition period. Also, we amplified about 2005 bp 5'-flanking region from placenta genomic DNA and examined methylation pattern and promoter activity. In present study, we focus on the analysis of molecular characterization of the promoter region by using reporter assay systems. We constructed of deleted mutants (— 890 bp; HSF-2), (— 513 bp; XFD), (— 276 bp; Ap-1) and (— 72 bp; Sp-1) and each mutants were cloned into pGL3-basic vector. These deletion mutants were transfected into CHO cells and co-transfected with Sp-1 or Ap-1 transcription factor plasmids. Compared to — 890 bp and 513 bp promoter fragments alone, transcription activity increased when these constructs were co-transfected with Sp-1 and Ap-1 factor. However, for the absence Ap-1 factor binding site in 276 bp fragment activity dramatically decreased in both transfections. Next, we constructed of 306 bp fragment which is including of Ap-1 binding site and nucleotides converted mutants of the Ap-1 factor binding site. In this result, 306 bp fragment's transcription activity was high as wild type. However, the mutant activity which converted Ap-1 site’s all nucleotide was significantly decreased. These findings are confirmed by gel-shift assay examining Ap-1 binding site on the 20 α-HSD gene upstream region and expression of Ap-1 factor was determined by RT-PCR and Western blot in pre-parturition period placenta and CHO-K1 cell line. Our results indicate that Ap-1 site (— 281 → — 274) (5'-TGTCTCAT-3') plays a crucial role for monkey 20 α- HSD gene transcription.

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. In this study, we analyzed by immunohistochemical methods adaptive mechanisms to excessive erythrocytosis in transgenic (tg) mice expressing dimeric human erythropoietin (dHuEPO) gene. Splenomegaly was observed over 11 21 times in the tg mice. The 2,672 candidate spleen‐gderived genes were identified through the microarray analysis method, and decreased genes were higher than increased genes in the spleen. The specific proteins in the increased and decreased genes were analyzed by immunohistochemical methods. Our results demonstrate that problems of abnormal splenomegaly would solve in tg mice overexpressing dHuEPO gene.