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SummaryUndifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.

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.

In this paper, we wrote about the basic experimentation of Optical CT`s temperature characteristic to measure high-current in a super-high-voltage electric power equipment which is using Faraday effect. We used the 1310[nm] Laser Diode as the light source and PIN Photodiode as receiver. For the transmission line of light, we used 30[m] single mode fiber which could maintain the state of polarization in the optical fiber. For the experiment, the temperature transformation device make by aluminium. the The range of current was from 400[A] and 1300[A] and the range of temperature was from . In a same experimental condition, magnitude increased input current increase follow by increasing proportion of input current.

Ultraviolet ray A (UVA) is an electromagnetic light with a long wavelength from the sun. The penetration of UVA deep into the human dermis causes changes in cells, such as DNA fragmentation, apoptosis, and senescence, eventually leading a decline of proliferation and wound-healing ability. These changes induced by UVA exposure are similar to those seen in the process of stem cell differentiation. We postulated that the condition that reverses cellular differentiation may alleviate the UVA-induced damage in skin cells. Human dermal fibroblasts (HDF) could be reprogrammed to induced pluripotent stem cells (iPSC). Conditioned medium (CM) was prepared during the process of iPSC reprogramming (referred to as Repro-CM). The UVA-irradiated HDF were cultured in Repro-CM for 24 h. In comparison with CM prepared from the culture of normal HDF and iPSC (referred to as HDF-CM and iPSC-CM, respectively), effects of Repro-CM on UVA-irradiated cells were investigated. Viability, wound-healing ability, apoptosis, and senescence of HDF were analysed by WST-1 assay, scratch assay, Annexin V assay, and senescence-associated β-galactosidase assay, respectively. Upon recovering from the UVA-induced damage, viability and wound-healing ability of HDF were significantly different (P

Process of somatic cell reprogramming into induced pluripotent stem (iPS) cells can broadly be divided into an early stochastic phase and a later, hierarchical and more deterministic phase of gene activation. Between the stochastic onset and the deterministic progression while approaching pluripotency, cells upon reprogramming undergo a transient activation or repression of developmental regulators. In these intermediate cells yet acquiring pluripotency, many somatic gene expressions are lost, while various lineage-specific factors are activated. In the present study, we produced pancreatic β cells by exposing human dermal fibroblasts (HDF) at the intermediate phase of reprogramming to pancreas-specific culture conditions. To obtain the intermediate cells, reprogramming factor Oct4, Sox2, Klf4, and c-Myc were transduced into HDF using retroviruses. By Day 9 after transduction, the expression of fibroblast-associated Thy1 was lost, while the early reprogramming marker CD49d was activated, suggesting that the cells are at the early stage of intermediate phase. These cells were capable of differentiating into 3 germ layers when cultured in tri-differentiation medium (including 100 ng mL–1 Activin A, 0.1 ng mL–1 Wnt3 for endodermal differentiation; 20 ng mL–1 BMP4, 20 ng mL–1 Activin A for mesodermal differentiation; 10 μM SB431542, 600 ng mL–1 Dorsomorphin, and 35 ng mL–1 Noggin for ectodermal differentiation) and expressing lineage-specific markers Sox17 (endoderm), Brachyury (mesoderm), and Otx2 (ectoderm). Further, the endodermal cells derived from HDF at intermediate phase of reprogramming were cultured in pancreas-induction medium (KO DMEM with 1% insulin-transferrin-selenium (ITS), 10 ng mL–1 BMP4, 10 mM nicotinamide, 10 μM forskolin, 10 μM dexamethasone, and 50 ng mL–1 exendin-4). After 5 days in culture, cells expressed pancreatic progenitor marker Pdx1, as examined by immunostaining and fluorescence-activated cell sorting (FACS) analysis. After culture for additional 10 days, the cells formed spherical clusters and expressed pancreatic islet-specific genes including insulin, somatostatin, glucagon, Nkx6.1, and MafA, as determined by RT-PCR. Moreover, the production of insulin and C-peptide from the derived β cells was confirmed by immunocytochemistry and FACS analysis. In response to glucose stimulation, these cells secreted insulin and C-peptide, as measured by ELISA. The present study demonstrates that fully differentiated functional cells could be converted from somatic cells at intermediate phase of reprogramming by exposing cells to specific culture conditions. The results shown in this study may provide a faster and potentially safer way to produce functional pancreatic cells without isolation and differentiation of iPS cells.

Animals with a targeted disruption of genes can be produced by somatic cell nuclear transfer (SCNT). However, difficulties in clonal selection of somatic cells with a targeted mutation often result in heterogeneous nuclear donor cells, including gene-targeted and non-targeted cells, and impose a risk of producing undesired wildtype cloned animals after SCNT. In addition, the efficiency of cloning by SCNT has remained extremely low. Most cloned embryos die in utero, and the few that develop to term show a high incidence of postnatal death and abnormalities. In the present study, resurrection of an alpha-1,3-galactosyltransferase (αGT) gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts was attempted. Three cloned piglets were produced from the first round of SCNT, including one stillborn and two who died immediately after birth due to respiratory distress syndrome and cardiac dysfunction. Among the three piglets, two were confirmed to be αGT gene-targeted. Fibroblasts derived from postmortem ear skin biopsies were used as nuclear donor cells for the second round of SCNT, and a piglet was produced. As expected, PCR and Southern analyses confirmed that the piglet produced from recloning was αGT gene-targeted. Currently, the piglet is fourteen months of age, and no overt health problems have been observed. Results from the present study demonstrate that loss of an invaluable animal, such as a gene-targeted miniature pig, may be rescued by recloning, with assurance of the desired genetic modification.

SummaryHuman complement regulatory protein hCD46 may reduce the hyperacute rejection (HAR) in pig-to-human xenotransplantation. In this study, anhCD46gene 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.

Gene targeting is a precise manipulation of endogenous gene by introduction of exogenous DNA and has contributed greatly to the elucidation of gene functions. Conventional gene targeting has been achieved through a use of embryonic stem cells. However, such procedure is often long, tedious, and expensive. This study was carried out to develop a simple procedure of gene targeting using E. coli recombinase A (RecA) and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene in mouse embryos. The single-stranded oligonucleotide to target an exon 3 of HPRT was 74 bases in length including phosphorothioate linkages at each terminus to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except a single nucleotide that induces a mismatch between an introduced oligonucleotide and endogenous HPRT gene. Endogenous repairing of such mismatch would give rise to the conversion of TAT to TAG stop codon thereby losing the function of the target gene. Before an introduction into zygotes, single-stranded oligonucleotides were bound to RecA to enhance the homologous recombination. The RecA-oligonucleotide complex was microinjected into the pronucleus of zygote. Individual microinjected embryos developed to the blastocyst stage were analyzed for the expected nucleotide conversion using polymerase chain reaction (PCR) and subsequent sequencing. The conversion of TAT to TAG stop codon was detected in three embryos among 48 tested blastocysts (6.25% in frequency). The result suggests that the gene targeting was feasible by relatively easier and direct method.

The generation of neural stem cells (NSC) from somatic cells may provide unlimited source of neuronal cells for autologous transplantation to patients of neurological disorders. Recently, direct conversion of fibroblasts into NSC by epigenetic reprogramming has been reported (Han et al. 2012 Cell Stem Cell 10, 465–472; Thier et al. 2012 Cell Stem Cell 10, 473–479; Ring et al. 2012 Cell Stem Cell 11, 100–109). These reprogrammed cells are referred to as induced neural stem cells (iNSC) and they share the characteristics of NSC in their morphology, molecular marker expressions, and capacity to differentiate into neurons, astrocytes, and oligodendrocytes. One of the procedures to convert fibroblasts into iNSC is restriction of Oct4 activity to the initial phase of reprogramming, while Sox2, Klf4, and c-Myc are constitutively expressed. In the present study, we examined the effect of Oct4 in reprogramming of human fibroblasts into iNSC. Oct4 protein was modified by the addition of poly-arginine protein transduction domain to easily penetrate into the cell membrane. We transduced Oct4 protein, in contrast to the previous reports where the Oct4 gene was virally introduced. First, human fibroblasts were transfected by retroviral vectors carrying the genes encoding Sox2, Klf4, and c-Myc. Then, transfected cells were cultured in ReNcell NSC maintenance medium containing Oct4 protein. After 4 days, Oct4 protein was removed from the medium. With Oct4 protein transduction, 21 flat colonies were formed from 4 × 105 fibroblasts. These colonies were picked and passaged for subculture and later became iNSC. However, in the absence of Oct4 protein, no colonies were obtained from the same number of fibroblasts that were initially plated. Approximately 40 days after transduction of reprogramming factors, cluster of iNSC were obtained. These cells expressed molecular markers of human NSC, including Nestin, Sox2, Pax6, and Blbp. Moreover, these iNSC could differentiate into neurons, astrocytes, and oligodendrocytes in vitro. Results of the present study demonstrate that transduction of exogenous Oct4 protein may be essential to the direct conversion of human fibroblasts into iNSC using a combination of reprogramming factors Sox2, Klf4, and c-Myc.

목 적 : 포도알균 열상 피부 증후군(Staphylococcal Scalded Skin Syndrome)은 포도알균의 피부박탈성 독소에 의하여 전신성 수포와 낙설을 일으키는 포도알균의 피부 감염증의 한 형태이다. 환자들의 임상양상과 치료의 결과, 합병증 등을 알고 자 본 연구를 시행하였다. 방 법 : 2002년 2월부터 2005년 12월까지 특징적인...

Constitutive expression of A transgene often results in serious physiological disturbances in transgenic animals. For instance, systemic overexpression of human growth hormone in transgenic pigs has resulted in detrimental side effects in general health and reproductive performance. One of the solutions to such problem would be inducible expression of a transgene that may restrict production of foreign proteins from transgenic animals only when needed. In this study, a retrovirus vector was designed to express the green fluorescent protein (GFP) gene under the control of the tetracycline-inducible promoter. Transformation of porcine fetal fibroblast cells was achieved by infection of the cells with the vector and subsequent antibiotic selection. To induce transgene expression, transformed porcine fetal fibroblast cells were cultured in medium supplemented with doxycycline for 48 h. Induction of the GFP gene was verified by the emission of fluorescence from transformed cells. Nuclei of transformed cells with or without doxycycline treatment were transferred into enucleated oocytes, and the induction efficiency was analyzed by monitoring fluorescent emission during development of reconstituted embryos to the blastocyst stage. In addition, differences in the rates of blastocyst development between experimental groups were analyzed by Student's t-test. Blastocyst formation of nuclear transfer embryos using transformed cells with tetracycline-inducible retrovirus vector (12.0%, 128/1072) was not significantly different (P > 0.05) from that with non-inducible control vectors (13.7%, 41/300), suggesting that an introduction of tetracycline-inducible retrovirus vector was not particularly harmful to the development of nuclear transfer embryos. Also, the blastocyst development rate of nuclear transfer embryos after induction of transgene by doxycycline (12.1%, 99/815) was not significantly different (P > 0.05) from that of the non-induced counterparts (11.3%, 29/257), suggesting that the induction of transgene did not affect the development of transgenic clone embryos. In a majority of embryos, high expression of the GFP gene was observed in cloned embryos with transgene induction, whereas poor or no GFP expression was detected in non-induced controls. The results from this study suggest that tetracycline-inducible expression of transgenes in nuclear transfer embryos may be used for production of foreign proteins in transgenic animals in a more controlled manner than with conventional procedures. Further experiments on transfer of cloned embryos carrying such an inducible transgene to recipients may enable production of transgenic pigs with fewer side effects from unregulated expression of the transgene.

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including their morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of both in vitro and in vivo differentiation have been established (Shim H et al. 1997 Biol. Reprod. 57, 1089–1095). Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide an inexhaustible source of karyoplasts in nuclear transfer (NT). This would be particularly advantageous in maintaining nuclear donor cells carrying a transgene. In addition, genome-wide demethylation of DNA occurs in pre-implantation embryos as well as PGC. Nuclear transfer embryos using EG cells rather than somatic cells may be close to embryos from normal fertilization in their DNA methylation status. If combined with NT technique, EG cells may potentially be useful for genetic manipulation in pigs. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblast and EG cells were compared. Two different techniques were used to perform NT. When conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes was used, the rates of development to the blastocyst stage were 16.8% (59/351) and 14.1% (50/354) in EG and somatic cell NT, respectively. In piezo-driven micromanipulation (Honolulu method) involving direct injection of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 7.5% (12/160), respectively. Although the differences between EG and somatic cell NT were statistically insignificant, the rates of blastocyst development in EG cell NT were comparable to the somatic cell counterpart regardless of NT methods used in the present study. To investigate if EG cells can be used for transgenesis in pigs, GFP gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29/137, 21.2%), and all embryos that developed to the blastocyst stage expressed GFP, based on observation under fluorescence microscope. In this study, the possibility of using EG cells as karyoplast donors in NT procedure was tested. The results suggest that EG cell NT may be used as an alternative to somatic cell NT, and transgenic pig embryos may be produced using EG cells. This research was supported by a grant (SC14033) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.