No long-term feeding toxicities on the health status in rats fed with cloned Korean native beef cattle (Hanwoo) meat.

This study was designed to undertake a risk assessment to identify the health status of rats fed with somatic cell nuclear transfer (SCNT)-cloned Korean native beef cattle (Hanwoo) meat for 26 weeks. The rats were randomly divided into 5 groups, each consisting of 12 male (142.6 ± 5.23 g) and 12 female (113.7 ± 6.31 g) rats each. The animals were fed commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) of normal cattle meat, and diets containing 5% (C-5) and 10% (C-10) of cloned cattle meat. The mortality; clinical signs; body weight; food consumption; urinary, hematology, blood biochemistry, and histopathological analyses; and absolute and relative organ weights were analyzed and compared. During the 26-week test period, health status-related factors of the rats fed on cloned Hanwoo meat were found to have no test substance-related toxicities. The only difference was the increased uterus weight in female C-10 rats as compared to their counterparts counterparts (p < .05). On the basis of these health status results, it can be postulated that no food consumption risks might arise from the long-term feeding of cloned cattle meat in rats.

Overexpression of Jazf1 induces cardiac malformation through the upregulation of pro-apoptotic genes in mice.

The transcription factor Juxtaposed with another zinc finger gene 1 (JAZF1) is a zinc finger protein that binds to the nuclear orphan receptor TR4. Recent evidence indicates that TR4 receptor functions as both a positive and negative regulator of transcription, but the role of JAZF1 in transcriptional mechanisms has not been elucidated. Recently, the incidence rate of congenital heart malformations was reported to be significantly elevated in patients who had neurofibromatosis 1 (NF1) with chromosomal microdeletion syndrome. Furthermore, Joined to JAZF1 (SUZ12) is expressed at high levels in the hearts of adult patients with NF1 microdeletion syndrome. Therefore, we hypothesized that ectopic expression of JAZF1 may lead to cardiac malformations that deleteriously affect the survival of neonates and adults. We sought to elucidate the role of JAZF1 in cardiac development using a Jazf1-overexpressing (Jazf1-Tg) mouse model. In Jazf1-Tg mice, Jazf1 mRNA expression was significantly elevated in the heart. Jazf1-Tg mice also showed cardiac defects, such as high blood pressure, electrocardiogram abnormalities, apoptosis of cardiomyocytes, ventricular non-compaction, and mitochondrial defects. In addition, we found that the expression levels of pro-apoptotic genes were elevated in the hearts of Jazf1-Tg mice. These findings suggest that Jazf1 overexpression may induce heart failure symptoms through the upregulation of pro-apoptotic genes in cardiomyocytes.

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer.

This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

Attenuation of cell cycle progression by 2,3,7,8-tetrachlorodibenzo-p-dioxin eliciting ovulatory blockade in gonadotropin-primed immature rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reduces ovulation rate in rats. The present study was to investigate whether TCDD alters the progression of cell cycle, and thus resulting in the blockade of ovulation in gonadotropin-primed, immature rats. The ovulation rate and ovarian weight were reduced in intact rats given TCDD (32 µg/kg BW in corn oil) by gavage one day before pregnant mare's serum gonadotropin (PMSG; 5 IU/rat) injection. Flow cytometry demonstrated that the percentage of granulosa cells in S-phase was increased at 24 h following PMSG treatment, but declined at 8 h following hCG treatment in corn oil-treated rats. Interestingly, the number of S-phase cells in TCDD-treated rats was reduced 24 and 48 h following PMSG treatment. TCDD, however, increased the percentage of cells in G2/M-phase at 24 h following PMSG treatment. TCDD inhibited the mRNA levels of Cdk2 at 0 h and 24 h, and cyclin D2 at 24 h and 48 h following PMSG treatment. Protein levels of aryl hydrocarbon receptor in granulosa cells were elevated in TCDD-treated rats at 12 h and 24 h following PMSG treatment. The present study indicates that TCDD reduces S-phase cells and inhibits levels of Cdk2 and cyclin D2 at 24 h following PMSG treatment, implying the ovulation-inhibiting action of TCDD may be exerted through the attenuation of cell cycle progression via AhR-mediated cascade.

Implication of human OCT4 transactivation domains for self-regulatory transcription.

OCT4 plays a crucial role in pluripotency and self-renewal of embryonic stem cells. OCT4 is also expressed in testicular germ cell tumors (GCTs), suggesting the important function of OCT4 as an oncogenic factor in GCTs. To understand the molecular mechanism of human OCT4 (hOCT4) in tumorigenesis as well as stemness, we identified hOCT4 transactivation domains in human embryonic carcinoma cells. Context analyses of heterologous GAL4 and natural hOCT4 revealed that each N-terminal domain or C-terminal domain independently stimulated transcriptional activity, and that both domains are required for synergistic transactivation by deletion mapping analysis. Dose-dependent overexpression of exogenous hOCT4 significantly decreased the transcriptional activity of the hOCT4 promoter. This inhibition was reversed by the removal of one or both domains. These results suggest that the inhibitory effect of hOCT4 is mediated by transactivation domains, and that the self-regulation of hOCT4 may be mediated via a negative feedback loop in pluripotent cells.

Cloning and characterization of 5'-untranslated region of porcine beta casein gene (CSN2).

beta-Casein (CSN2) is a major milk protein in most mammals. The CSN2 gene is generally induced by lactogenic hormones bound to its promoter. The expression of this gene can be enhanced by signal transducers and activators of transcription (STAT) and glucocorticoid receptor (GR). Here, we analyzed the promoter and intron 1 regions of the porcine CSN2 gene. The porcine CSN2 promoter and intron 1 regions (-3098bp to +2446bp) were cloned into the pGL3-Basic vector containing the luciferase reporter gene (pCSN2-PEI). Lactogenic signals induced the transcription of porcine CSN2. By using AG490, a Janus kinase (JAK) inhibitor, we demonstrated that STAT5 positively regulates the transcription of porcine CSN2. Further, seven STAT mutants were generated by site-directed mutagenesis. By performing electrophoretic mobility shift assays (EMSAs), we located a critical element for pCSN2-PEI transcription bound to STAT5 in the -102bp to -84bp region. The construct containing only the promoter region (pCSN2-P), however, did not exert any promotive effects on transcription in two cell types-a mouse mammary epithelial cell line (HC11) and porcine mammary gland epithelial cells (PMECs). Thus, the construct containing intron 1 of porcine CSN2 exerts an elevating effect on transcription. We suggest that the transcription of porcine CSN2 is regulated by lactogenic signals via the STAT5 site (-102bp to -84bp) and intron 1.

Effects of mannosylated glycopolymers on specific interaction to bone marrow hematopoietic and progenitor cells derived from murine species.

Poly[N-pvinylbenzyl-O-D-galactopyranosyl-(1-4)-D-glucoamide], poly[N-pvinylbenzyl-O-D-glucopyranosyl-(1-4)-D-glucoamide], and poly[N-p-vinylbenzyl-O-mannopyranosyl-(1-4)-D-gluconamide] (referred to as PVLA, PVMA, and PV-Man) are polystyrene derivatives that contain galactose, glucose, and mannose moieties, which interact with hematopoietic cells (HCs). To clarify the specific interaction between the glucopolymers and hematopoietic cells, glycopolymers labeled with fluorescent isothiocyanate (FITC) were used to follow the specific interaction, which was visualized by confocal laser microscopy. We found that PV-Man binds strongly to HCs, probably because of a specific interaction mediated by specific receptors present on the cell membrane, while some cytotoxicity when was observed when PV-Man interacted with the cell membrane. The fluorescence intensity between PV-Man and HCs was up to four-fold (0.14 +/- 0.04) that of PVMA and PVLA with hematopoietic HCs (0.033 +/- 0.01). Moreover, cellular fluorescence increased significantly with increasing incubation time and increasing polymer concentration. Using hematopoietic lineage-specific antibodies, cells were stained and analyzed by flow cytometry to confirm which HCs showed specific binding with glycopolymers, especially hematopoietic stem cells and progenitor cells (c-kit+), B-lymphocyte progenitor cells (B220+), monocyte cells (CD11b+), and erythrocytes (Ter119+).

Recombinant human erythropoietin produced in milk of transgenic pigs.

We have developed a line of transgenic swine harboring recombinant human erythropoietin through microinjection into fertilized one cell pig zygotes. Milk from generations F1 and F2 transgenic females was analyzed, and hEPO was detected in milk from all lactating females at concentrations of approximately 877.9+/-92.8 IU/1 ml. The amino acid sequence of rhEPO protein in the transgenic pig milk matched that of commercial rhEPO produced from cultured animal cells. In addition, an F-36 cell line, which proliferates in the presence of hEPO or commercial EPO, was induced to synthesize erythroid by extracts from tg sow milk. This study provides evidence that production of purified rhEPO from transgenic pig milk is a potentially valuable technology, and can be used as a cost-effective alternative in clinical applications as well as providing other clinical advantages.

A paucity of structural integrity in cloned porcine blastocysts produced in vitro.

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P < 0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P < 0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P < 0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I: <20%; II: 20-40%; III: >40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.

Antiapoptotic effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant in H9c2 rat cardiomyocytes.

Granulocyte colony-stimulating factor (G-CSF) is used for heart failure therapy and promotes myocardial regeneration by inducing mobilization of bone marrow stem cells to the injured heart after myocardial infarction; however, this treatment has one weakness in that its biological effect is transient. In our previous report, we generated 5 mutants harboring N-linked glycosylation to improve its antiapoptotic activities. Among them, one mutant (Phe140Asn) had higher cell viability than wild-type hG-CSF in rat cardiomyocytes, even after treatment with an apoptotic agent (H2O2). Cells treated with this mutant significantly upregulated the antiapoptotic proteins, and experienced reductions in caspase 3 activity and PARP cleavage. Moreover, the total number of apoptotic cells was dramatically lower in cultures treated with mutant hG-CSF. Taken together, these results suggest that the addition of an N-linked glycosylation was successful in improving the antiapoptotic activity of hG-CSF, and that this mutated product will be a feasible therapy for patients who have experienced heart failure.

Proteins associated with reproductive disorders in testes of human erythropoietin gene-harboring transgenic boars.

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (ß-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal ß-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.

The effects of various antioxidants on the development of parthenogenetic porcine embryos.

The major objective of this study was to improve the development rate of parthenogenetic porcine embryos. In this study, the anti-oxidative and anti-apoptotic effects of three antioxidants, ß-mercaptoethanol (ß-ME), α-tocopherol, and extracellular superoxide dismutase (EC-SOD), were examined on the development of parthenogenetic porcine embryos. The development rate of parthenogenetic porcine embryos to the blastocyst stage was 8.1% for control; 19.1%, 14.6%, and 5.0% for 1, 3, and 5 µM ß-ME; 17.2% and 17.5% for 50 and 100 µM α-tocopherol and 12.0% and 4.0% for EC-SOD transgenic mouse embryonic fibroblast (Tg-MEF) and EC-SOD non-transgenic mouse embryonic fibroblast (NTg-MEF) conditioned medium at day 3, respectively. Here, ß-ME, α-tocopherol, and EC-SOD Tg-MEF conditioned medium increased the development rate of parthenogenetic porcine embryos to the blastocyst stage (P < 0.05). The average number of total cells and apoptotic cells at the blastocyst was analyzed at the optimal conditions of the three antioxidants. The three antioxidants increased the average number of total cells at the blastocyst, and they decreased apoptotic cells at the blastocyst as compared to control without supplementation (P < 0.05). When the reactive oxygen species levels in two-cell embryos after 1 µM ß-ME and 100 µM α-tocopherol treatment were examined, those were lower than control group (P < 0.05). In conclusion, it was found that the three antioxidants, ß-mercaptoethanol, α-tocopherol, and EC-SOD Tg-MEF, conditioned medium can play a role as a strong stimulator in the development of parthenogenetic porcine embryos.

Gonadotropin regulation of genes differentially expressed in response to PKCzeta inhibitor during ovulation in the rat.

AIMS: The aim of the present study was to characterize genes regulated by protein kinase C PKCzeta inhibitor in the preovulatory granulosa cells following LH stimulation in the rat ovary. MAIN METHODS: Annealing control primer (ACP)-based polymerase chain reaction (PCR) method was used to identify differentially expressed genes in granulosa cells of preovulatory follicles cultured in the presence of luteinizing hormone (LH) and myristoylated PKCzeta pseudosubstrate peptide or a similarly sized control peptide. KEY FINDINGS: Among the 16 genes identified, five (testin, glypican-4, retrovirus SC1, aminolevulinic acid synthase 1 and serum-inducible kinase) experienced rapid and transient stimulation of gene expression upon exposure to human chorionic gonadotropin (hCG) in the ovary of immature rats primed with pregnant mare's serum gonadotropin (PMSG). In situ hybridization analysis revealed that hCG administration induced expression of these five genes in granulosa cells of preovulatory follicles. The Western analysis showed that the protein levels of testin and serum-inducible kinase were also increased by hCG. Expression of the eleven remaining genes in the ovary remained high at 24-72 h following hCG treatment. SIGNIFICANCE: The present data demonstrate the gonadotropin stimulation of genes differentially expressed by PKCzeta inhibitor, implicating that PKCzeta pathway possibly plays a role in controlling the ovulation process.

Expression of ectodermal neural cortex 1 and its association with actin during the ovulatory process in the rat.

Ectodermal neural cortex (ENC) 1, a member of the kelch family of genes, is an actin-binding protein and plays a pivotal role in neuronal and adipocyte differentiation. The present study was designed to examine the gonadotropin regulation and action of ENC1 during the ovulatory process in immature rats. The levels of ENC1 mRNA and protein were stimulated by LH/human chorionic gonadotropin (hCG) within 3 h both in vivo and in vitro. In situ hybridization analysis revealed that ENC1 mRNA was localized not only in theca/interstitial cells but also in granulosa cells of preovulatory follicles but not of growing follicles in pregnant mare's serum gonadotropin/hCG-treated ovaries. LH-induced ENC1 expression was suppressed by a high dose of protein kinase C inhibitor RO 31-8220 (10 microM) but not by low doses of RO 31-8220 (0.1-1.0 microM), suggesting the involvement of atypical protein kinase C. ENC1 was detected in both nucleus and cytoplasm that was increased by LH/hCG treatment. Both biochemical and morphological analysis revealed that LH/hCG treatment increased actin polymerization within 3 h in granulosa cells. Interestingly, ENC1 physically associated with actin and treatment with cytochalasin D, an actin-depolymerizing agent, abolished this association. Confocal microscopy further demonstrated the colocalization of ENC1 with filamentous actin (F-actin). The present study demonstrates that LH/hCG stimulates ENC1 expression and increases F-actin formation in granulosa cells. The present study further shows the physical association of ENC1 and F-actin, implicating the role of ENC1 in cytoskeletal reorganization during the differentiation of granulosa cells.

Cytolytic assessment of hyperacute rejection and production of nuclear transfer embryos using hCD46-transgenic porcine embryonic germ cells.

Human complement regulatory protein hCD46 may reduce the hyperacute rejection (HAR) in pig-to-human xenotransplantation. In this study, an hCD46 gene was introduced into porcine embryonic germ (EG) cells. Treatment of human serum did not affect the survival of hCD46-transgenic EG cells, whereas the treatment significantly reduced the survival of non-transgenic EG cells (p < 0.01). The transgenic EG cells presumably capable of alleviating HAR were transferred into enucleated oocytes. Among 235 reconstituted oocytes, 35 (14.9%) developed to the blastocyst stage. Analysis of individual embryos indicated that 80.0% (28/35) of embryos contained the transgene hCD46. The result of the present study demonstrates resistance of hCD46-transgenic EG cells against HAR, and the usefulness of the transgenic approach may be predicted by this cytolytic assessment prior to actual production of transgenic pigs. Subsequently performed EG cell nuclear transfer gave rise to hCD46-transgenic embryos. Further study on the transfer of these embryos to recipients may produce hCD46-transgenic pigs.

Production of recombinant human von Willebrand factor in the milk of transgenic pigs.

Von Willebrand factor (vWF), a large multimeric glycoprotein present in blood plasma, is a blood protein of the coagulation system. It is defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura-hemolytic uremic syndrome and heyde's syndrome. We have developed a line of transgenic swine harboring recombinant human von Willebrand factor (rhvWF) cDNA through microinjection of fertilized one-cell pig zygotes. Expression of rhvWF in the mammary gland and secretion of rhvWF into the milk of the transgenic swine were confirmed by immunohistochemical and western blot analyses, respectively, and rhvWF proteins were detected in milk from all lactating founder females at concentrations that were 28- to 56-folds greater than that in circulating human plasma. The amino acid sequence of rhvWF protein in the transgenic pig milk matched that of vWF produced from human blood plasma. This study provides evidence that production of rhvWF from transgenic pig milk is a potentially valuable technology and can be used as a cost-effective alternative in clinical applications.

Interaction of glycopolymers with human hematopoietic cells from cord blood and peripheral blood.

Polystyrene derivatives, poly[N-pvinylbenzyl-O-D-glucopyranosyl-(1-4)-D-glucoamide] (PV Maltose) and poly[N-p-vinylbenzyl-O-mannopyranosyl-(1-4)-D-glucoamide] (PV Mannose), which contain glucose and mannose moieties, respectively, have the specific binding ability with murine hematopoietic cells. In this study, we confirm the ability of these glycopolymers to interact specifically with human hematopoietic stem cells (HSCs) and mature cells derived from human cord blood (CB) and peripheral blood (PB). Using fluorescence isothiocyanate (FITC)-labeled glycopolymers, we observed that 98% to 93% of hematopoietic cells interacted very strongly with PV Mannose, and 63% of CB and 29% PB interacted with PV Maltose. Both glycopolymers bound better to cells from CB than from PB. Cytotoxic studies revealed that a 0.1 mM dose of PV Mannose induced apoptosis in 20% CB cells, in contrast to 3-5% PB cells. Furthermore, we demonstrated that all of CD34(+) HSCs of both origins bound specifically to PV Mannose, whereas 33-47% bound to PV Maltose. In addition, the majority of B cells (CD19(+)), T cells (CD3(+)), monocytes (CD14(+)), and erythrocytes (CD235a(+)) bound to PV Mannose, but a lower percentage interacted with PV Maltose. In vivo study, bone marrow, spleen, and liver tissues in NOD-SCID mice injected with PV Mannose conjugated CB, were detected PV Mannose positive hematopoietic cells. These data suggest that the use of PV Mannose and PV Maltose might be used for gene and drug delivery for hematopoietic cells and thus, may be useful in therapeutic settings.

Transcriptional regulation of human Oct4 by steroidogenic factor-1.

Oct4 encodes a transcription factor that is involved in the maintenance of self-renewal in stem cells. Recently, the molecular mechanisms that regulate Oct4 expression have come under investigation. In this study, we demonstrate that the orphan nuclear receptor steroidogenic factor-1 (SF-1) behaves as a transcriptional activator of human Oct4 (hOct4) through direct interaction with a SF-1 binding element in the hOct4 proximal promoter. We found that Oct4 and SF-1 were co-expressed in undifferentiated human embryonal carcinoma NCCIT cells and downregulated during retinoic acid-mediated differentiation. We examined the functional role played by SF-1 in regulation of hOct4 transcription using a luciferase reporter assay and Western blot analysis. Overexpression of SF-1 increased up to about threefold hOct4 promoter activity and endogenous hOct4 protein expression. Sequence analysis of the hOct4 promoter revealed that the transcriptional activity was closely linked to Conserved Regions 1 (CR1) and 2 (CR2), which contain three putative SF-1-binding sites (1st, 2nd, and 3rd SF-1). Binding assays and mutagenesis of binding sites indicated that the 1st and 2nd SF-1 elements (in CR1 and CR2, respectively) might be important cis-regulatory elements in hOct4 promoter activity. However, differences in response to SF-1 overexpression between wild-type and mutant hOct4 promoters revealed that the 1st SF-1 element is the key binding site for SF-1-mediated transcriptional activation. Thus, our data indicate that SF-1 plays a crucial role in the regulation of hOct4 transcription through direct binding to the 1st SF-1 in CR1 of the hOct4 proximal promoter.

Extracellular ATP is involved in the induction of apoptosis in murine hematopoietic cells.

Extracellular nucleotides have multiple biological actions in processes such as proliferation, differentiation, chemotaxis, and cytokine secretion through P2X receptors on the cell surface. To determine the biological activity of adenosine triphosphate (ATP) and the expression of P2 nucleotide receptors in murine bone marrow-derived hematopoietic cells and stem cells/progenitor cells, we investigated the effects of ATP in assays of cell proliferation and cell death in vitro. Our results demonstrated that several subtypes of P2X receptors were expressed on hematopoietic cells and that P2X7, in particular, was partially expressed in hematopoietic stem cells/progenitor cells. In addition, stimulation of hematopoietic cells with high concentrations of ATP caused severe inhibition of cell proliferation despite the presence of cytokine stimulation. We analyzed the apoptotic effects of stimulation with several different dosages of ATP and confirmed the enhanced apoptotic activity in hematopoietic cells and progenitor cells. Antagonists, against P2X receptors and ATP, suramin and oxidized ATP, inhibited the induction of cell death for murine hematopoietic cells. Our data suggest that extracellular nucleotides may provide a novel and powerful tool for regulating the cell fate of hematopoietic stem cells.

Optimization of 25 kDa linear polyethylenimine for efficient gene delivery.

A 25-kDa linear polyethylenimine (25 kDa L-PEI) has proven to be efficient and versatile agent for gene delivery. Therefore, we determined the optimal transfection conditions of 25 kDa L-PEI and examined whether it has comparable transfection efficiency with other commercially available reagents, ExGen 500, LipofectAMINE 2000, and Effectene by using EGFP expression vector in different cell lines. Transfection efficiency and cytotoxicity were measured by flow cytometry. First of all, we determined the optimal ratio of nitrogen to phosphorous (N/P) and DNA concentration. With the increase of N/P ratio and DNA amounts, transfection efficiency increased with a slight variation in cell types. The optimal amounts of 25 kDa L-PEI were determined at N/P ratio 40 and DNA concentration varied among the cell types. In addition, 25 kDa L-PEI worked efficiently and was less toxic than other reagents. However, the efficiency and toxicity of all these reagents varied according to cell types as well as the ratio of DNA to reagents and the amounts of DNA. Our finding illustrates the importance of optimal transfection conditions of 25 kDa L-PEI to obtain maximal transgene expression with less cytotoxicity. Importantly, the optimization of those conditions may make possible to perform transfection cost-effectively and efficiently.