WorldWideScience

Sample records for mammalian somatic cells

  1. Aneuploidy in mammalian somatic cells in vivo.

    Science.gov (United States)

    Cimino, M C; Tice, R R; Liang, J C

    1986-01-01

    Aneuploidy is an important potential source of human disease and of reproductive failure. Nevertheless, the ability of chemical agents to induce aneuploidy has been investigated only sporadically in intact (whole-animal) mammalian systems. A search of the available literature from the EMCT Aneuploidy File (for years 1970-1983) provided 112 papers that dealt with aneuploidy in mammalian somatic cells in vivo. 59 of these papers did not meet minimal criteria for analysis and were rejected from subsequent review. Of the remaining 53 papers that dealt with aneuploidy induction by chemical agents in mammalian somatic cells in vivo, only 3 (6%) contained data that were considered to be supported conclusively by adequate study designs, execution, and reporting. These 3 papers dealt with 2 chemicals, one of which, mercury, was negative for aneuploidy induction in humans, and the other, pyrimethamine, was positive in an experimental rodent study. The majority of papers (94%) were considered inconclusive for a variety of reasons. The most common reasons for calling a study inconclusive were (a) combining data on hyperploidy with those on hypoploidy and/or polyploidy, (b) an inadequate or unspecified number of animals and/or cells per animal scored per treatment group, and (c) poor data presentation such that animal-to-animal variability could not be assessed. Suggestions for protocol development are made, and the future directions of research into aneuploidy induction are discussed.

  2. Factors affecting the spontaneous mutational spectra in somatic mammalian cells

    Directory of Open Access Journals (Sweden)

    О.А. Ковальова

    2006-04-01

    Full Text Available  In our survey of references we are discussed the influence of factors biological origin on the spontaneous mutation specters in mammalian. Seasonal and age components influence on the frequence of cytogenetic anomalies. The immune and endocrinous systems are take part in control of the alteration of the spontaneous mutation specters. Genetical difference of sensibility in animal and human at the alteration of factors enviroment as and  genetical differences of repair systems activity are may influence on individual variation of spontaneous destabilization characters of chromosomal apparatus.

  3. Clastogenic activity of 2-chlorodeoxyadenosine in mammalian somatic cells

    Directory of Open Access Journals (Sweden)

    Gilmara Ausech Antonucci

    2005-01-01

    Full Text Available The base analogue 2-chlorodeoxyadenosine (2-CdA used for therapy in chronic resistant and advanced lymphoproliferative disorders, is cytotoxic for both dividing and non-dividing lymphocytes. The present work evaluated the clastogenic potential of this drug in vitro in human lymphocytes in culture and in vivo in BALB/c mice bone marrow cells. In human lymphocytes, the clastogenic effect of 2-CdA was studied in G1, S and G2 phases of the cell cycle, using three different concentrations (10, 20 and 40 mug/mL. The endpoints analyzed included mitotic index (MI, proliferation index (PI, sister chromatid exchange (SCE, and chromosomal aberration (CA. Statistical analysis by a variance (ANOVA test showed a significant increase (p < 0.05 in CA frequencies for cells treated during the S phase, but the MI did not vary. The concentrations tested did not produce a significant increase in the mean frequency of SCEs, nor did they change the cell PI in the G1 and S phases. The concentrations in vivo tested were 0.25, 0.375 and 0.5 mg/kg body weight. In this assay, alterations in CA frequencies and MI were not observed at the dose levels tested. Therefore, the results indicate a clastogenic effect of 2-CdA in human lymphocyte cultures.

  4. Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics--recent achievements.

    Science.gov (United States)

    Samiec, M; Skrzyszowska, M

    2011-01-01

    Somatic cell cloning technology in mammals promotes the multiplication of productively-valuable genetically engineered individuals, and consequently allows also for standardization of transgenic farm animal-derived products, which, in the context of market requirements, will have growing significance. Gene farming is one of the most promising areas in modern biotechnology. The use of live bioreactors for the expression of human genes in the lactating mammary gland of transgenic animals seems to be the most cost-effective method for the production/processing of valuable recombinant therapeutic proteins. Among the transgenic farm livestock species used so far, cattle, goats, sheep, pigs and rabbits are useful candidates for the expression of tens to hundreds of grams of genetically-engineered proteins or xenogeneic biopreparations in the milk. At the beginning of the new millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on recombinant human proteins. The ever-growing demand for such pharmaceutical or nutriceutical proteins is an important driving force for the development of safe and large-scale production platforms. The aim of this paper is to present an overall survey of the state of the art in investigations which provide the current knowledge for deciphering the possibilities of practical application of the transgenic mammalian species generated by somatic cell cloning in biomedicine, the biopharmaceutical industry, human nutrition/dietetics and agriculture.

  5. Cellular and molecular regulation of the activation of mammalian primordial follicles: somatic cells initiate follicle activation in adulthood.

    Science.gov (United States)

    Zhang, Hua; Liu, Kui

    2015-01-01

    The first small follicles to appear in the mammalian ovaries are primordial follicles. The initial pool of primordial follicles serves as the source of developing follicles and oocytes for the entire reproductive lifespan of the animal. Although the selective activation of primordial follicles is critical for female fertility, its underlying mechanisms have remained poorly understood. A search of PubMed was conducted to identify peer-reviewed literature pertinent to the study of mammalian primordial follicle activation, especially recent reports of the role of primordial follicle granulosa cells (pfGCs) in regulating this process. In recent years, molecular mechanisms that regulate the activation of primordial follicles have been elucidated, mostly through the use of genetically modified mouse models. Several molecules and pathways operating in both the somatic pfGCs and oocytes, such as the phosphatidylinositol 3 kinase (PI3K) and the mechanistic target of rapamycin complex 1 (mTORC1) pathways, have been shown to be important for primordial follicle activation. More importantly, recent studies have provided an updated view of how exactly signaling pathways in pfGCs and in oocytes, such as the KIT ligand (KL) and KIT, coordinate in adult ovaries so that the activation of primordial follicles is achieved. In this review, we have provided an updated picture of how mammalian primordial follicles are activated. The functional roles of pfGCs in governing the activation of primordial follicles in adulthood are highlighted. The in-depth understanding of the cellular and molecular mechanisms of primordial follicle activation will hopefully lead to more treatments of female infertility, and the current progress indicates that the use of existing primordial follicles as a source for obtaining fertilizable oocytes as a new treatment for female infertility is just around the corner. © The Author 2015. Published by Oxford University Press on behalf of the European Society of

  6. Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes

    Science.gov (United States)

    Wigglesworth, Karen; Lee, Kyung-Bon; O’Brien, Marilyn J.; Peng, Jia; Matzuk, Martin M.; Eppig, John J.

    2013-01-01

    Coordinated regulation of oocyte and ovarian follicular development is essential for fertility. In particular, the progression of meiosis, a germ cell-specific cell division that reduces the number of chromosomes from diploid to haploid, must be arrested until just before ovulation. Follicular somatic cells are well-known to impose this arrest, which is essential for oocyte–follicle developmental synchrony. Follicular somatic cells sustain meiotic arrest via the natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system, and possibly also via high levels of the purine hypoxanthine in the follicular fluid. Upon activation by the ligand NPPC, NPR2, the predominant guanylyl cyclase in follicular somatic cells, produces cyclic guanosine monophosphate (cGMP), which maintains meiotic arrest after transfer to the oocyte via gap junctions. Here we report that both the NPPC/NPR2 system and hypoxanthine require the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme required for the production of guanylyl metabolites and cGMP. Furthermore, oocyte-derived paracrine factors, particularly the growth differentiation factor 9–bone morphogenetic protein 15 heterodimer, promote expression of Impdh and Npr2 and elevate cGMP levels in cumulus cells. Thus, although the somatic compartment of ovarian follicles plays an essential role in the maintenance of oocyte meiotic arrest, as has been known for many years, this function of the somatic cells is surprisingly regulated by signals from the oocyte itself. PMID:23980176

  7. The possibilities of practical application of transgenic mammalian species generated by somatic cell cloning in pharmacology, veterinary medicine and xenotransplantology.

    Science.gov (United States)

    Samiec, M; Skrzyszowska, M

    2011-01-01

    Cloning of genetically-modified mammals to produce: 1) novel animal bioreactors expressing human genes in rens, urinary bladder and the male accessory sex glands, as well as 2) porcine organs suitable in pig-to-human xenotransplantology, could offer new advantages for biomedical purposes. So too does the generation and/or multiplication of genetically-engineered cloned animals in order to produce: 3) physiologically-relevant animal models of serious monogenic human diseases and 4) prion disease-resistant small as well as large animals (i.e., rodents, ruminants). The basic purpose of this paper is to overview current knowledge deciphering the possibilities of using transgenic specimens created by somatic cell nuclear transfer in medical pharmacology, veterinary medicine, agriculture, transplantational medicine and immunology.

  8. Bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Ross, Pablo J; Cibelli, Jose B

    2010-01-01

    Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

  9. Genetic damage in mammalian somatic cells exposed to radiofrequency radiation: a meta-analysis of data from 63 publications (1990-2005).

    Science.gov (United States)

    VIjayalaxmi; Prihoda, Thomas J

    2008-05-01

    During the last several decades, numerous researchers have examined the potential of in vitro and /or in vivo exposure of radiofrequency( RF) radiation to damage the genetic material in mammalian somatic cells. A meta-analysis of reported data was conducted to obtain a quantitative estimate ( with 95% confidence intervals) of genotoxicity in RF-radiation-exposed cells compared with sham-exposed/unexposed control cells. The extent of genotoxicity was assessed for various end points, including single- and double-strand breaks in the DNA, incidence of chromosomal aberrations, micronuclei and sister chromatid exchanges. Among the several variables in the experimental protocols used in individual investigations, the influence of three specific variables related to RF-radiation exposure characteristics was examined in the meta-analysis: frequency, specific absorption rate, and exposure as continuous-wave, pulsed-wave and occupationally exposed/cell phone users. The overall data indicated that (1) the difference between RF-radiation exposure was small with few exceptions; (2) at certain RF radiation exposure conditions, there were statistically significant increases in genotoxicity for some end points; and (3) the mean indices for chromosomal aberrations and micronuclei in RF-radiation -exposed and sham-/unexposed controls were within the spontaneous levels reported in the historical database. Considerable evidence for publication bias was found in the meta-analysis.

  10. Nonchromosomal cytogenetic analysis of mammal somatic cells

    Directory of Open Access Journals (Sweden)

    Kovalova O. А.

    2013-01-01

    Full Text Available The mutational events that take place in mammalian somatic cells influenced with different endogenous and exogenous factors are presented in this review. The nonchromosomal method of research allows taking into account the complex cell characteristics without time-consuming analysis of the chromosomes as such. As a result, the information can be obtained about the mitotic (phases of mitosis, the number of nuclei per cell, micronuclei, pathology of mitosis and vital (mitotic index, apoptosis cell statuses, as well as about the state of chromosomal integrity (the presence of nucleoplasmic bridges, nucleus protrusions, chromosome fragmentation, micronuclei. Depending on the material studied (erythrocytes and lymphocytes of peripheral blood, buccal cells, permanent cell lines etc., a complex of cytogenetic characteristics can be selected for each case which is the most informative for determination of the mutational spectra in mammalian somatic cells.

  11. Ophiobolin A from Bipolaris oryzae Perturbs Motility and Membrane Integrities of Porcine Sperm and Induces Cell Death on Mammalian Somatic Cell Lines

    Directory of Open Access Journals (Sweden)

    Ottó Bencsik

    2014-09-01

    Full Text Available Bipolaris oryzae is a phytopathogenic fungus causing a brown spot disease in rice, and produces substance that strongly perturbs motility and membrane integrities of boar spermatozoa. The substance was isolated from the liquid culture of the fungal strain using extraction and a multi-step semi-preparative HPLC procedures. Based on the results of mass spectrometric and 2D NMR techniques, the bioactive molecule was identified as ophiobolin A, a previously described sesterterpene-type compound. The purified ophiobolin A exhibited strong motility inhibition and viability reduction on boar spermatozoa. Furthermore, it damaged the sperm mitochondria significantly at sublethal concentration by the dissipation of transmembrane potential in the mitochondrial inner membrane, while the plasma membrane permeability barrier remained intact. The study demonstrated that the cytotoxicity of ophiobolin A toward somatic cell lines is higher by 1–2 orders of magnitude compared to other mitochondriotoxic mycotoxins, and towards sperm cells unique by replacing the progressive motility by shivering tail beating at low exposure concentration.

  12. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  13. Monitoring Milk Somatic Cell Counts

    Directory of Open Access Journals (Sweden)

    Gheorghe Şteţca

    2014-11-01

    Full Text Available The presence of somatic cells in milk is a widely disputed issue in milk production sector. The somatic cell counts in raw milk are a marker for the specific cow diseases such as mastitis or swollen udder. The high level of somatic cells causes physical and chemical changes to milk composition and nutritional value, and as well to milk products. Also, the mastitic milk is not proper for human consumption due to its contribution to spreading of certain diseases and food poisoning. According to these effects, EU Regulations established the maximum threshold of admitted somatic cells in raw milk to 400000 cells / mL starting with 2014. The purpose of this study was carried out in order to examine the raw milk samples provided from small farms, industrial type farms and milk processing units. There are several ways to count somatic cells in milk but the reference accepted method is the microscopic method described by the SR EN ISO 13366-1/2008. Generally samples registered values in accordance with the admissible limit. By periodical monitoring of the somatic cell count, certain technological process issues are being avoided and consumer’s health ensured.

  14. Somatic Cell Nuclear Transfer in the Mouse

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  15. Hemoglobins, programmed cell death and somatic embryogenesis.

    Science.gov (United States)

    Hill, Robert D; Huang, Shuanglong; Stasolla, Claudio

    2013-10-01

    Programmed cell death (PCD) is a universal process in all multicellular organisms. It is a critical component in a diverse number of processes ranging from growth and differentiation to response to stress. Somatic embryogenesis is one such process where PCD is significantly involved. Nitric oxide is increasingly being recognized as playing a significant role in regulating PCD in both mammalian and plant systems. Plant hemoglobins scavenge NO, and evidence is accumulating that events that modify NO levels in plants also affect hemoglobin expression. Here, we review the process of PCD, describing the involvement of NO and plant hemoglobins in the process. NO is an effector of cell death in both plants and vertebrates, triggering the cascade of events leading to targeted cell death that is a part of an organism's response to stress or to tissue differentiation and development. Expression of specific hemoglobins can alter this response in plants by scavenging the NO, thus, interrupting the death process. Somatic embryogenesis is used as a model system to demonstrate how cell-specific expression of different classes of hemoglobins can alter the embryogenic process, affecting hormone synthesis, cell metabolite levels and genes associated with PCD and embryogenic competence. We propose that plant hemoglobins influence somatic embryogenesis and PCD through cell-specific expression of a distinct plant hemoglobin. It is based on the premise that both embryogenic competence and PCD are strongly influenced by cellular NO levels. Increases in cellular NO levels result in elevated Zn(2+) and reactive-oxygen species associated with PCD, but they also result in decreased expression of MYC2, a transcription factor that is a negative effector of indoleacetic acid synthesis, a hormone that positively influences embryogenic competence. Cell-specific hemoglobin expression reduces NO levels as a result of NO scavenging, resulting in cell survival.

  16. Autophagy in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Kadija; Abounit; Tiziano; M; Scarabelli; Roy; B; McCauley

    2012-01-01

    Autophagy is a regulated process for the degradation of cellular components that has been well conserved in eukaryotic cells. The discovery of autophagy-regulating proteins in yeast has been important in understanding this process. Although many parallels exist between fungi and mammals in the regulation and execution of autophagy, there are some important differences. The preautophagosomal structure found in yeast has not been identified in mammals, and it seems that there may be multiple origins for autophagosomes, including endoplasmic reticulum, plasma membrane and mitochondrial outer membrane. The maturation of the phagophore is largely dependent on 5’-AMP activated protein kinase and other factors that lead to the dephosphorylation of mammalian target of rapamycin. Once the process is initiated, the mammalian phagophore elongates and matures into an autophagosome by processes that are similar to those in yeast. Cargo selection is dependent on the ubiquitin conjugation of protein aggregates and organelles and recognition of these conjugates by autophagosomal receptors. Lysosomal degradation of cargo produces metabolites that can be recycled during stress. Autophagy is an impor-tant cellular safeguard during starvation in all eukaryotes; however, it may have more complicated, tissue specific roles in mammals. With certain exceptions, autophagy seems to be cytoprotective, and defects in the process have been associated with human disease.

  17. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  18. Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

    OpenAIRE

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

    2014-01-01

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed n...

  19. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Science.gov (United States)

    2010-07-01

    ... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture... selected by resistance to ouabain. (2) Description. Cells in suspension or monolayer culture are exposed to...

  20. Mammalian Cell Culture Simplified.

    Science.gov (United States)

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  1. Technology of mammalian cell encapsulation

    NARCIS (Netherlands)

    Uludag, H; De Vos, P; Tresco, PA

    2000-01-01

    Entrapment of mammalian cells in physical membranes has been practiced since the early 1950s when it was originally introduced as a basic research tool. The method has since been developed based on the promise of its therapeutic usefulness in tissue transplantation. Encapsulation physically isolates

  2. Generation of Peroxisome-Deficient Somatic Animal Cell Mutants.

    Science.gov (United States)

    Okumoto, Kanji; Fujiki, Yukio

    2017-01-01

    Cell mutants with a genetic defect affecting various cellular phenotypes are widely utilized as a powerful tool in genetic, biochemical, and cell biological research. More than a dozen complementation groups of animal somatic mutant cells defective in peroxisome biogenesis have been successfully isolated in Chinese hamster ovary (CHO) cells and used as a model system reflecting fatal human severe genetic disorders named peroxisome biogenesis disorders (PBD). Isolation and characterization of peroxisome-deficient CHO cell mutants has allowed the identification of PEX genes and the gene products peroxins, which directly leads to the accomplishment of isolation of pathogenic genes responsible for human PBDs, as well as elucidation of their functional roles in peroxisome biogenesis. Here, we describe the procedure to isolate peroxisome-deficient mammalian cell mutants from CHO cells, by making use of an effective, photo-sensitized selection method.

  3. Embryonic stem cell-somatic cell fusion and postfusion enucleation.

    Science.gov (United States)

    Sumer, Huseyin; Verma, Paul J

    2015-01-01

    Embryonic stem (ES) cells are able to reprogram somatic cells following cell fusion. The resulting cell hybrids have been shown to have similar properties to pluripotent cells. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However it is unclear whether these changes can be sustained following removal of the dominant ES nucleus. In this chapter, methods are described for the cell fusion of mouse tetraploid ES cells with somatic cells and enrichment of the resulting heterokaryons. We next describe the conditions for the differential removal of the ES cell nucleus, allowing for the recovery of somatic cells.

  4. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek;

    2016-01-01

    One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...

  5. Human somatic cell nuclear transfer and cloning.

    Science.gov (United States)

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. China Succeeded in Somatic Cell Cloning

    Institute of Scientific and Technical Information of China (English)

    Song Jianlan

    2002-01-01

    @@ Chinese scientists have succeeded in cloning a colony of cattle from fully differentiated somatic cells. The news was announced jointly by the Chinese Academy of Sciences (CAS), National Natural Science Foundation of China (NSFC) and the government of Shandong Province at a press conference held on March 7, 2002.

  7. [Reprogramming of somatic cells. Problems and solutions].

    Science.gov (United States)

    Schneider, T A; Fishman, V S; Liskovykh, M A; Ponamartsev, S V; Serov, O L; Tomilin, A N; Alenina, N

    2014-01-01

    An adult mammal is composed of more than 200 different types of specialized somatic cells whose differentiated state remains stable over the life of the organism. For a long time it was believed that the differentiation process is irreversible, and the transition between the two types of specialized cells is impossible. The possibility of direct conversion of one differentiated cell type to another was first shown in the 80s of the last century in experiments on the conversion of fibroblasts into myoblasts by ectopic expression of the transcription factor MyoD. Surprisingly, this technology has remained unclaimed in cell biology for a long time. Interest in it revived after 200 thanks to the research of Novel Prize winner Shinya Yamanaka who has shown that a small set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is capable of restoring pluripotency in somatic cells which they lost in the process of differentiation. In 2010, using a similar strategy and the tissue-specific transcription factors Vierbuchen and coauthors showed the possibility of direct conversion of fibroblasts into neurons, i. e. the possibility of transdifferentiation of one type of somatic cells in the other. The works of these authoras were a breakthrough in the field of cell biology and gave a powerful impulse to the development of cell technologies for the needs of regenerative medicine. The present review discusses the main historical discoveries that preceded this work, evaluates the status of the problem and the progress in the development of methods for reprogramming at the moment, describes the main approaches to solving the problems of reprogramming of somatic cells into neuronal, and briefly discusses the prospect of application of reprogramming and transdifferentiation of cells for such important application areas as regenerative medicine, cell replacement therapy and drug screening.

  8. Dogs cloned from adult somatic cells.

    Science.gov (United States)

    Lee, Byeong Chun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hossein, M Shamim; Shamim, M Hossein; Kim, Jung Ju; Kang, Sung Keun; Schatten, Gerald; Hwang, Woo Suk

    2005-08-04

    Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

  9. Genome regulation in mammalian cells.

    Science.gov (United States)

    Puck, T T; Krystosek, A; Chan, D C

    1990-05-01

    A theory is presented proposing that genetic regulation in mammalian cells is at least a two-tiered effect; that one level of regulation involves the transition between gene exposure and sequestration; that normal differentiation requires a different spectrum of genes to be exposed in each separate state of differentiation; that the fiber systems of the cell cytoskeleton and the nuclear matrix together control the degree of gene exposure; that specific phosphorylation of these elements causes them to assume a different organizational network and to impose a different pattern of sequestration and exposure on the elements of the genome; that the varied gene phosphorylation mechanisms in the cell are integrated in this function; that attachment of this network system to specific parts of the chromosomes brings about sequestration or exposure of the genes in their neighborhood in a fashion similar to that observed when microtubule elements attach through the kinetochore to the centromeric DNA; that one function of repetitive sequences is to serve as elements for the final attachment of this fibrous network to the specific chromosomal loci; and that at least an important part of the calcium manifestation as a metabolic trigger of different differentiation states involves its acting as a binding agent to centers of electronegativity, in particular proteins and especially phosphorylated groups, so as to change the conformation of the fiber network that ultimately controls gene exposure in the mammalian cell. It would appear essential to determine what abnormal gene exposures and sequestrations are characteristic of each type of cancer; which agonists, if any, will bring about reverse transformation; and whether these considerations can be used in therapy.

  10. Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

    Directory of Open Access Journals (Sweden)

    Mora Xavier

    2007-05-01

    Full Text Available Abstract Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes in physiological salt (0.14 M NaCl at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl. The large differences in relative affinity of the H1 subtypes for

  11. Human embryonic stem cells derived by somatic cell nuclear transfer.

    Science.gov (United States)

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L; Wolf, Don; Mitalipov, Shoukhrat

    2013-06-06

    Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

  12. Defining viability in mammalian cell cultures

    OpenAIRE

    Browne, Susan M.; Al-Rubeai, Mohamed

    2011-01-01

    Abstract A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure me...

  13. Epigenetic reprogramming by somatic cell nuclear transfer in primates.

    Science.gov (United States)

    Sparman, Michelle; Dighe, Vikas; Sritanaudomchai, Hathaitip; Ma, Hong; Ramsey, Cathy; Pedersen, Darlene; Clepper, Lisa; Nighot, Prashant; Wolf, Don; Hennebold, Jon; Mitalipov, Shoukhrat

    2009-06-01

    We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X-inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte-specific factors.

  14. Baculoviruses as Vectors in Mammalian Cells

    Institute of Scientific and Technical Information of China (English)

    Chang-yong LIANG; Xin-wen CHEN

    2007-01-01

    The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy.

  15. Hacking the genetic code of mammalian cells.

    Science.gov (United States)

    Schwarzer, Dirk

    2009-07-06

    A genetic shuttle: The highlighted article, which was recently published by Schultz, Geierstanger and co-workers, describes a straightforward scheme for enlarging the genetic code of mammalian cells. An orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for a new amino acid can be evolved in E. coli and subsequently transferred into mammalian cells. The feasibility of this approach was demonstrated by adding a photocaged lysine derivative to the genetic repertoire of a human cell line.

  16. Human somatic cell nuclear transfer is alive and well.

    Science.gov (United States)

    Cibelli, Jose B

    2014-06-05

    In this issue, Chung et al. (2014) generate human embryonic stem cells by fusing an adult somatic cell to a previously enucleated human oocyte, in agreement with recent reports by the Mitalipov and Egli groups. We can now safely say that human somatic cell nuclear transfer is alive and well.

  17. Endangered wolves cloned from adult somatic cells.

    Science.gov (United States)

    Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hwang, Woo Suk; Hossein, Mohammad Shamim; Kim, Joung Joo; Shin, Nam Shik; Kang, Sung Keun; Lee, Byeong Chun

    2007-01-01

    Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.

  18. Wnt signalling pathway parameters for mammalian cells.

    Directory of Open Access Journals (Sweden)

    Chin Wee Tan

    Full Text Available Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated

  19. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.

    Science.gov (United States)

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A; Shen, Li; Inoue, Azusa; Zhang, Yi

    2014-11-06

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells and its removal by ectopically expressed H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.

  20. Mammalian Cell-Based Sensor System

    Science.gov (United States)

    Banerjee, Pratik; Franz, Briana; Bhunia, Arun K.

    Use of living cells or cellular components in biosensors is receiving increased attention and opens a whole new area of functional diagnostics. The term "mammalian cell-based biosensor" is designated to biosensors utilizing mammalian cells as the biorecognition element. Cell-based assays, such as high-throughput screening (HTS) or cytotoxicity testing, have already emerged as dependable and promising approaches to measure the functionality or toxicity of a compound (in case of HTS); or to probe the presence of pathogenic or toxigenic entities in clinical, environmental, or food samples. External stimuli or changes in cellular microenvironment sometimes perturb the "normal" physiological activities of mammalian cells, thus allowing CBBs to screen, monitor, and measure the analyte-induced changes. The advantage of CBBs is that they can report the presence or absence of active components, such as live pathogens or active toxins. In some cases, mammalian cells or plasma membranes are used as electrical capacitors and cell-cell and cell-substrate contact is measured via conductivity or electrical impedance. In addition, cytopathogenicity or cytotoxicity induced by pathogens or toxins resulting in apoptosis or necrosis could be measured via optical devices using fluorescence or luminescence. This chapter focuses mainly on the type and applications of different mammalian cell-based sensor systems.

  1. Specificities and genomic distribution of somatic mammalian histone H1 subtypes.

    Science.gov (United States)

    Millán-Ariño, Lluís; Izquierdo-Bouldstridge, Andrea; Jordan, Albert

    2016-03-01

    Histone H1 is a structural component of chromatin that may have a role in the regulation of chromatin dynamics. Unlike core histones, the linker histone H1 family is evolutionarily diverse and many organisms have multiple H1 variants or subtypes, distinguishable between germ-line and somatic cells. In mammals, the H1 family includes seven somatic H1 variants with a prevalence that varies between cell types and over the course of differentiation, H1.1 to H1.5 being expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. Until recently, it has not been known whether the different variants had specific roles in the regulation of nuclear processes or were differentially distributed across the genome. To address this, an increasing effort has been made to investigate divergent features among H1 variants, regarding their structure, expression patterns, chromatin dynamics, post-translational modifications and genome-wide distribution. Although H1 subtypes seem to have redundant functions, several reports point to the idea that they are also differently involved in specific cellular processes. Initial studies investigating the genomic distribution of H1 variants have started to suggest that despite a wide overlap, different variants may be enriched or preferentially located at different chromatin types, but this may depend on the cell type, the relative abundance of the variants, the differentiation state of the cell, or whether cells are derived from a neoplastic process. Understanding the heterogeneity of the histone H1 family is crucial to elucidate their role in chromatin organization, gene expression regulation and other cellular processes.

  2. Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

    OpenAIRE

    Engelhardt John F; Li Ziyi

    2003-01-01

    Abstract Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not...

  3. Recent advancements in cloning by somatic cell nuclear transfer.

    Science.gov (United States)

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-05

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

  4. Mammalian Non-CpG Methylation: Stem Cells and Beyond

    Directory of Open Access Journals (Sweden)

    Sara E. Pinney

    2014-11-01

    Full Text Available Although CpG dinucleotides remain the primary site for DNA methylation in mammals, there is emerging evidence that DNA methylation at non-CpG sites (CpA, CpT and CpC is not only present in mammalian cells, but may play a unique role in the regulation of gene expression. For some time it has been known that non-CpG methylation is abundant in plants and present in mammalian embryonic stem cells, but non-CpG methylation was thought to be lost upon cell differentiation. However, recent publications have described a role for non-CpG methylation in adult mammalian somatic cells including the adult mammalian brain, skeletal muscle, and hematopoietic cells and new interest in this field has been stimulated by the availability of high throughput sequencing techniques that can accurately measure this epigenetic modification. Genome wide assays indicate that non-CpG methylation is negligible in human fetal brain, but abundant in human adult brain tissue. Genome wide measurement of non-CpG methylation coupled with RNA-Sequencing indicates that in the human adult brain non-CpG methylation levels are inversely proportional to the abundance of mRNA transcript at the associated gene. Additionally specific examples where alterations in non-CpG methylation lead to changes in gene expression have been described; in PGC1α in human skeletal muscle, IFN-γ in human T-cells and SYT11 in human brain, all of which contribute to the development of human disease.

  5. Autofluorescence of viable cultured mammalian cells.

    Science.gov (United States)

    Aubin, J E

    1979-01-01

    The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.

  6. Genotype by environment interaction for somatic cell score across bulk milk somatic cell count and days in milk

    NARCIS (Netherlands)

    Calus, M.P.L.; Janss, L.L.G.; Veerkamp, R.F.

    2006-01-01

    The objective of this paper was to investigate the importance of a genotype x environment interaction (G x E) for somatic cell score (SCS) across levels of bulk milk somatic cell count (BMSCC), number of days in milk (DIM), and their interaction. Variance components were estimated with a model inclu

  7. Producing primate embryonic stem cells by somatic cell nuclear transfer.

    Science.gov (United States)

    Byrne, J A; Pedersen, D A; Clepper, L L; Nelson, M; Sanger, W G; Gokhale, S; Wolf, D P; Mitalipov, S M

    2007-11-22

    Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

  8. Translational research on reprogramming of somatic cells

    Institute of Scientific and Technical Information of China (English)

    Yanhua Li; Jiahui Yin; Bingbing Zhang; Ping Zhou; Bin Feng; Fangyi Zhang; Yongzhong Lin; Zhanhua Liang; Jianling Du; Minghui Lü; Tiezheng Zheng; Jie Lin; Siyu Liu; Hao Hong; Xing Meng; Dandan Xia; Yang Sun; Pan Wei; Nan Cai; Hongye Li; Shuang Wu; Hui Zhao; Changkai Sun; Yuyuan Li; Changyu Gao; Wei Li; Ye Dai; Junde Wang; Hui Zhao; Xiaoxin Tan; Lili Men; Hui Ma; Jun Xu; Xiaohan Yang; Zengchun Hu; Ling Wang; Hong Wang; Pin Sun; Huifang Guo; Guirong Song; Hui Liu1; Baoshuai Shan; Lu Han; Linlang Liang; Min Wang; Xiaochen Wang; Dan Wang; Guihua Chen; Jianting Chen; Xiangyou Sun; Jun Xue; Zhiqi Wang; Jing Wang; Yongqing Zhang; Dongfeng Cai; Mozhen Liu; Guiping Zhang; Guoming Luan; Jianli Wang; Ming Fan; Xuetao Cao; Chao Wan; Qigui Liu; Anchun Yin

    2014-01-01

    Cerebrovascular diseases,dementia,diabetes,malignant tumors and degenerative bone diseases remain high prevalence,incidence,disability and mortality rates.One important reason might be the slow or stagnated progress in translating and applying cytoprotection and cellular repair researches into clinical practice.Based on collaboration among biomedical re-searchers,database experts,computer programmers,statisticians and management engineers,this is the first study to apply quanti-tative comparison on the overall characteristics and partial correlation analysis on the large-scale complex information and data regarding the topic“mature cells can be reprogrammed to become pluripotent”proposed by Sir John B.Gurdon and Shinya Yamanaka who were jointly awarded with 2012 Nobel Prize in Physiology or Medicine,as well as articles that cited publications of the two Nobel Laureates to discuss the prospects of translating somatic cell reprogramming researches into clinical practice and cor-responding implementation strategies.The study found that there was statistically significant difference between the two Nobel Laureates with regard to the number,publication date,subject categories and scientific and technological focuses of their origi-nal researches.The study revealed the importance,objectives,approaches and research trends of translational medicine,especially translational neuroscience.The study also identified the challenges that China should overcome to improve its medical research management scheme.

  9. Cholesterol, the central lipid of mammalian cells

    NARCIS (Netherlands)

    Maxfield, F. R.; van Meer, G.

    2010-01-01

    Despite its importance for mammalian cell biology and human health, there are many basic aspects of cholesterol homeostasis that are not well understood. Even for the well-characterized delivery of cholesterol to cells via lipoproteins, a novel regulatory mechanism has been discovered recently, invo

  10. Regulation of mammalian cell differentiation by long non-coding RNAs.

    Science.gov (United States)

    Hu, Wenqian; Alvarez-Dominguez, Juan R; Lodish, Harvey F

    2012-11-06

    Differentiation of specialized cell types from stem and progenitor cells is tightly regulated at several levels, both during development and during somatic tissue homeostasis. Many long non-coding RNAs have been recognized as an additional layer of regulation in the specification of cellular identities; these non-coding species can modulate gene-expression programmes in various biological contexts through diverse mechanisms at the transcriptional, translational or messenger RNA stability levels. Here, we summarize findings that implicate long non-coding RNAs in the control of mammalian cell differentiation. We focus on several representative differentiation systems and discuss how specific long non-coding RNAs contribute to the regulation of mammalian development.

  11. Somatic cells initiate primordial follicle activation and govern the development of dormant oocytes in mice.

    Science.gov (United States)

    Zhang, Hua; Risal, Sanjiv; Gorre, Nagaraju; Busayavalasa, Kiran; Li, Xin; Shen, Yan; Bosbach, Benedikt; Brännström, Mats; Liu, Kui

    2014-11-03

    The majority of oocytes in the mammalian ovary are dormant oocytes that are enclosed in primordial follicles by several somatic cells, which we refer to as primordial follicle granulosa cells (pfGCs). Very little is known, however, about how the pfGCs control the activation of primordial follicles and the developmental fates of dormant oocytes. By targeting molecules in pfGCs with several mutant mouse models, we demonstrate that the somatic pfGCs initiate the activation of primordial follicles and govern the quiescence or awakening of dormant oocytes. Inhibition of mTORC1 signaling in pfGCs prevents the differentiation of pfGCs into granulosa cells, and this arrests the dormant oocytes in their quiescent states, leading to oocyte death. Overactivation of mTORC1 signaling in pfGCs accelerates the differentiation of pfGCs into granulosa cells and causes premature activation of all dormant oocytes and primordial follicles. We further show that pfGCs trigger the awakening of dormant oocytes through KIT ligand (KITL), and we present an essential communication network between the somatic cells and germ cells that is based on signaling between the mTORC1-KITL cascade in pfGCs and KIT-PI3K signaling in oocytes. Our findings provide a relatively complete picture of how mammalian primordial follicles are activated. The microenvironment surrounding primordial follicles can activate mTORC1-KITL signaling in pfGCs, and these cells trigger the awakening of dormant oocytes and complete the process of follicular activation. Such communication between the microenvironment, somatic cells, and germ cells is essential to maintaining the proper reproductive lifespan in mammals. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Ricin Trafficking in Plant and Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Robert A. Spooner

    2011-06-01

    Full Text Available Ricin is a heterodimeric plant protein that is potently toxic to mammalian and many other eukaryotic cells. It is synthesized and stored in the endosperm cells of maturing Ricinus communis seeds (castor beans. The ricin family has two major members, both, lectins, collectively known as Ricinus communis agglutinin ll (ricin and Ricinus communis agglutinin l (RCA. These proteins are stored in vacuoles within the endosperm cells of mature Ricinus seeds and they are rapidly broken down by hydrolysis during the early stages of post-germinative growth. Both ricin and RCA traffic within the plant cell from their site of synthesis to the storage vacuoles, and when they intoxicate mammalian cells they traffic from outside the cell to their site of action. In this review we will consider both of these trafficking routes.

  13. Cell-surface remodelling during mammalian erythropoiesis.

    Science.gov (United States)

    Wraith, D C; Chesterton, C J

    1982-10-15

    Current evidence suggests that the major cell-surface modification occurring during mammalian erythropoiesis could be generated by two separate mechanisms: either selective loss of membrane proteins during enucleation or endocytosis at the subsequent reticulocyte and erythrocyte stages. The former idea was tested by collecting developing rabbit erythroid cells before and after the enucleation step and comparing their cell-surface protein composition via radiolabelling and electrophoresis. Few changes were observed. Our data thus lend support to the endocytosis mechanism.

  14. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.

    Science.gov (United States)

    Dupuy, Adam J; Akagi, Keiko; Largaespada, David A; Copeland, Neal G; Jenkins, Nancy A

    2005-07-14

    Transposons have provided important genetic tools for functional genomic screens in lower eukaryotes but have proven less useful in higher eukaryotes because of their low transposition frequency. Here we show that Sleeping Beauty (SB), a member of the Tc1/mariner class of transposons, can be mobilized in mouse somatic cells at frequencies high enough to induce embryonic death and cancer in wild-type mice. Tumours are aggressive, with some animals developing two or even three different types of cancer within a few months of birth. The tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of genes and pathways that induce disease. SB transposition can easily be controlled to mutagenize any target tissue and can therefore, in principle, be used to induce many of the cancers affecting humans, including those for which little is known about the aetiology. The uses of SB are also not restricted to the mouse and could potentially be used for forward genetic screens in any higher eukaryote in which transgenesis is possible.

  15. Acoustophoretic Synchronization of Mammalian Cells in Microchannels

    DEFF Research Database (Denmark)

    Thévoz, P.; Adams, J.D.; Shea, H.

    2010-01-01

    We report the first use of ultrasonic standing waves to achieve cell cycle phase synchronization in mammalian cells in a high-throughput and reagent-free manner. The acoustophoretic cell synchronization (ACS) device utilizes volume-dependent acoustic radiation force within a microchannel...... to selectively purify target cells of desired phase from an asynchronous mixture based on cell cycle-dependent fluctuations in size. We show that ultrasonic separation allows for gentle, scalable, and label-free synchronization with high G1 phase synchrony (84%) and throughput (3 × 106 cells/h per microchannel)....

  16. Acoustophoretic Synchronization of Mammalian Cells in Microchannels

    DEFF Research Database (Denmark)

    Thévoz, P.; Adams, J.D.; Shea, H.

    2010-01-01

    We report the first use of ultrasonic standing waves to achieve cell cycle phase synchronization in mammalian cells in a high-throughput and reagent-free manner. The acoustophoretic cell synchronization (ACS) device utilizes volume-dependent acoustic radiation force within a microchannel...... to selectively purify target cells of desired phase from an asynchronous mixture based on cell cycle-dependent fluctuations in size. We show that ultrasonic separation allows for gentle, scalable, and label-free synchronization with high G1 phase synchrony (84%) and throughput (3 × 106 cells/h per microchannel)....

  17. E-nose identification of milk somatic cell count

    OpenAIRE

    İNALPULAT, Melis; Kızıl, Ünal; Bilgücü, Ertuğrul; GENÇ, Levent

    2016-01-01

    Mastitis is a common disease among dairy animals which causes serious economic losses. It can be diagnosed via diverse clinical findings, while milk somatic cell count (SCC) is accepted as a key indicator. However, determination of SCC with traditional methods is time consuming and laborious. This paper focuses on the ability of electronic nose (e-nose) system containing 12 different metal oxide sensors (MOS) to discriminate milks with somatic cell counts (SCC) above a th...

  18. The fungicide mancozeb induces toxic effects on mammalian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Paro, Rita [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy); Tiboni, Gian Mario [Department of Medicine and Aging, Section of Reproductive Sciences, University “G. D' Annunzio”, Chieti-Pescara (Italy); Buccione, Roberto [Tumor Cell Invasion Laboratory, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti (Italy); Rossi, Gianna; Cellini, Valerio [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy); Canipari, Rita [Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Section of Histology and Embryology, School of Pharmacy and Medicine, “Sapienza” University of Rome, Rome (Italy); Cecconi, Sandra, E-mail: sandra.cecconi@cc.univaq.it [Department of Health Sciences, University of L' Aquila, Via Vetoio, L' Aquila (Italy)

    2012-04-15

    The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant. Highlights: ► The fungicide mancozeb affects oocyte spindle morphology and fertilization rate. ► We investigated the toxic effects of mancozeb on mouse and human granulosa cells. ► Granulosa cells modify their morphology and expression level of p53. ► Mancozeb induces a premalignant-like status in exposed cells.

  19. Cell-penetrating peptides: From mammalian to plant cells

    OpenAIRE

    Eudes, François; Chugh, Archana

    2008-01-01

    Internalization of cell-penetrating peptides, well described in mammalian cell system, has recently been reported in a range of plant cells by three independent groups. Despite fundamental differences between animal cell and plant cell composition, the CPP uptake pattern between the mammalian system and the plant system is very similar. Tat, Tat-2 pVEC and transportan internalisation is concentration dependent and non saturable, enhanced at low temperature (4°C), and receptor independent. The...

  20. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    Institute of Scientific and Technical Information of China (English)

    Amjad Riaz; Xiaoyang Zhao; Xiangpeng Dai; Wei Li; Lei Liu; Haifeng Wan; Yang Yu; Liu Wang; Qi Zhou

    2011-01-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem(ES)cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved.Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  1. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    Science.gov (United States)

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  2. Epigenetic regulation of the mammalian cell.

    Directory of Open Access Journals (Sweden)

    Keith Baverstock

    Full Text Available BACKGROUND: Understanding how mammalian cells are regulated epigenetically to express phenotype is a priority. The cellular phenotypic transition, induced by ionising radiation, from a normal cell to the genomic instability phenotype, where the ability to replicate the genotype accurately is compromised, illustrates important features of epigenetic regulation. Based on this phenomenon and earlier work we propose a model to describe the mammalian cell as a self assembled open system operating in an environment that includes its genotype, neighbouring cells and beyond. Phenotype is represented by high dimensional attractors, evolutionarily conditioned for stability and robustness and contingent on rules of engagement between gene products encoded in the genetic network. METHODOLOGY/FINDINGS: We describe how this system functions and note the indeterminacy and fluidity of its internal workings which place it in the logical reasoning framework of predicative logic. We find that the hypothesis is supported by evidence from cell and molecular biology. CONCLUSIONS: Epigenetic regulation and memory are fundamentally physical, as opposed to chemical, processes and the transition to genomic instability is an important feature of mammalian cells with probable fundamental relevance to speciation and carcinogenesis. A source of evolutionarily selectable variation, in terms of the rules of engagement between gene products, is seen as more likely to have greater prominence than genetic variation in an evolutionary context. As this epigenetic variation is based on attractor states phenotypic changes are not gradual; a phenotypic transition can involve the changed contribution of several gene products in a single step.

  3. Metabolic-flux analysis of mammalian-cell culture.

    NARCIS (Netherlands)

    Bonarius, H.P.J.

    1998-01-01

    In the biopharmaceutical industry mammalian cells are cultivated for the production of recombinant glycoproteins, vaccines, and monoclonal antibodies. In contrast to other expression systems, such as prokaryotes or yeasts, mammalian cells are able to glycosylate and fold therapeutic proteins correct

  4. Metabolic-flux analysis of mammalian-cell culture

    NARCIS (Netherlands)

    Bonarius, H.P.J.

    1998-01-01

    In the biopharmaceutical industry mammalian cells are cultivated for the production of recombinant glycoproteins, vaccines, and monoclonal antibodies. In contrast to other expression systems, such as prokaryotes or yeasts, mammalian cells are able to glycosylate and fold therapeutic

  5. [DNA homologous recombination repair in mammalian cells].

    Science.gov (United States)

    Popławski, Tomasz; Błasiak, Janusz

    2006-01-01

    DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.

  6. Cell lineage analysis of the mammalian female germline.

    Directory of Open Access Journals (Sweden)

    Yitzhak Reizel

    Full Text Available Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote. We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

  7. Cell lineage analysis of the mammalian female germline.

    Science.gov (United States)

    Reizel, Yitzhak; Itzkovitz, Shalev; Adar, Rivka; Elbaz, Judith; Jinich, Adrian; Chapal-Ilani, Noa; Maruvka, Yosef E; Nevo, Nava; Marx, Zipora; Horovitz, Inna; Wasserstrom, Adam; Mayo, Avi; Shur, Irena; Benayahu, Dafna; Skorecki, Karl; Segal, Eran; Dekel, Nava; Shapiro, Ehud

    2012-01-01

    Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

  8. The influence of interspecies somatic cell nuclear transfer on epigenetic enzymes transcription in early embryos

    Directory of Open Access Journals (Sweden)

    Martin Morovic

    2016-10-01

    Full Text Available One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a genes in early embryonic stages of interspecies (bovine, porcine nuclear transfer embryos (iSCNT by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly infl uenced by the ooplasmic environment.

  9. Chlorpromazine inhibits mitosis of mammalian cells.

    Science.gov (United States)

    Boder, G B; Paul, D C; Williams, D C

    1983-09-01

    Chlorpromazine (CPZ) at minimally effective concentrations accumulates mammalian cells in mitosis without lethal effects on the cells. Star-metaphase morphology similar to effects seen with classical antimitotic compounds probably results from the preferential action of CPZ on a specific class of microtubules--the pole-to-pole microtubules of the mitotic spindle. At CPZ concentrations of 8 X 10(-6) M, flow cytometry indicates no effect of CPZ on the progress of cells through phases of the cell cycle other than mitosis (M). These results suggest a possible mechanism for toxic side effects of CPZ in man such as granulocytopenia and light sensitization.

  10. Somatic Embryogenesis of Lilium from Microbulb Transverse Thin Cell Layers.

    Science.gov (United States)

    Marinangeli, Pablo

    2016-01-01

    A reliable somatic embryogenesis protocol is a prerequisite for application of other plant biotechniques. Several protocols were reported for genus Lilium, with variable success. Between them, transverse Thin Cell Layers (tTCL) were used efficiently to induce indirect somatic embryogenesis of Lilium. Somatic embryogenesis potential is dependent on the genotype, explant, and culture medium composition, especially as for plant growth regulators and environmental conditions. Usually, the process comprises three phases: embryogenic callus induction, embryogenic callus proliferation and somatic embryo germination. Somatic embryo germination can be achieved in light or dark. In the first case, complete plantlets are formed, with green leaves and pseudobulb in the base. In darkness, microbulbs are formed from single somatic embryos or clusters. A last phase of microbulb enlargement allows plantlets or microbulbs to increase their biomass. These enlarged microbulbs do not need special acclimatization conditions when transferred to soil and quickly produce sturdy plants. This chapter describes a protocol for somatic embryogenesis of Lilium using tTCL from microbulbs.

  11. Sex determination in mammalian germ cells

    Directory of Open Access Journals (Sweden)

    Cassy M Spiller

    2015-06-01

    Full Text Available Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatogenesis. Here, we discuss what we know so far about the regulation of sex-specific differentiation of germ cells, considering extrinsic molecular cues produced by somatic cells, as well as critical intrinsic changes within the germ cells. This review focuses almost exclusively on our understanding of these events in the mouse model.

  12. Isolation of genomic DNA from mammalian cells.

    Science.gov (United States)

    Koh, Cheryl M

    2013-01-01

    The isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique with numerous downstream applications. The isolated DNA can be used as a template for PCR, cloning, and genotyping and to generate genomic DNA libraries. It can also be used for sequencing to detect mutations and other alterations, and for DNA methylation analyses. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Generation of bovine transgenics using somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Stice Steven L

    2003-11-01

    Full Text Available Abstract The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This review summarizes the benefits of using somatic cell nuclear transfer to create bovine transgenics as well as the possible opportunities this method creates for the future.

  14. Generation of Mouse Haploid Somatic Cells by Small Molecules for Genome-wide Genetic Screening

    Directory of Open Access Journals (Sweden)

    Zheng-Quan He

    2017-08-01

    Full Text Available The recent success of derivation of mammalian haploid embryonic stem cells (haESCs has provided a powerful tool for large-scale functional analysis of the mammalian genome. However, haESCs rapidly become diploidized after differentiation, posing challenges for genetic analysis. Here, we show that the spontaneous diploidization of haESCs happens in metaphase due to mitotic slippage. Diploidization can be suppressed by small-molecule-mediated inhibition of CDK1 and ROCK. Through ROCK inhibition, we can generate haploid somatic cells of all three germ layers from haESCs, including terminally differentiated neurons. Using piggyBac transposon-based insertional mutagenesis, we generated a haploid neural cell library harboring genome-wide mutations for genetic screening. As a proof of concept, we screened for Mn2+-mediated toxicity and identified the Park2 gene. Our findings expand the applications of mouse haploid cell technology to somatic cell types and may also shed light on the mechanisms of ploidy maintenance.

  15. Manipulating Somatic Cells to Remove Barriers in Induced Pluripotent Stem Cell Reprogramming

    OpenAIRE

    Chung, Julia

    2013-01-01

    Development leads unidirectionally towards a more restricted cell fate that is usually stable. However, it has been proven that developmental systems are reversible by the success of animal cloning of a differentiated somatic genome through somatic cell nuclear transfer (SCNT). Recently, reprogramming of somatic cells to a pluripotent embryonic stem cell (ESC)-like state by introducing defined transcripton factor has been achieved, resulting in the generation of induced pluripotent stem cells...

  16. Interpretation of reprogramming to predict the success of somatic cell cloning.

    Science.gov (United States)

    Eckardt, Sigrid; McLaughlin, K John

    2004-07-01

    In the context of mammalian somatic cell cloning, the term reprogramming refers to the processes that enable a somatic cell nucleus to adopt the role of a zygotic nucleus. Gene re-expression is one measure of reprogramming if correlated with subsequent developmental potential. This paper describes several experiments utilizing pre-implantation gene expression to evaluate reprogramming and clone viability. We have established a direct correlation between Oct4 expression in mouse clones at the blastocyst stage and their potential to maintain pluripotent embryonic cells essential for post-implantation development. Furthermore, the quality of gene expression in clones dramatically improves when genetically identical clones are combined in clone-clone aggregate chimeras. Clone--clone aggregates exhibit a higher developmental potential than single clones both in vitro and in vivo. This could be mediated by complementation between blastomeres from epigenetically different clones within the aggregate rather than by the increase in cell number resulting from aggregation. We also discuss the use of tetraploid embryos as a model to evaluate reprogramming using gene expression and demonstrate that somatic cell nuclei can be reprogrammed by blastomeres to re-express embryonic specific genes but not to contribute to post-implantation development.

  17. Somatic mutations of KIT in familial testicular germ cell tumours

    NARCIS (Netherlands)

    Rapley, EA; Hockley, S; Warren, W; Johnson, L; Huddart, R; Crockford, G; Forman, D; Leahy, MG; Oliver, DT; Tucker, K; Friedlander, M; Phillips, KA; Hogg, D; Jewett, MAS; Lohynska, R; Daugaard, G; Richard, S; Heidenreich, A; Geczi, L; Bodrogi, [No Value; Olah, E; Ormiston, WJ; Daly, PA; Looijenga, LHJ; Guilford, P; Aass, N; Fossa, SD; Heimdal, K; Tjulandin, SA; Liubchenko, L; Stoll, H; Weber, W; Einhorn, L; Weber, BL; McMaster, M; Greene, MH; Bishop, DT; Easton, D; Stratton, M

    2004-01-01

    Somatic mutations of the KIT gene have been reported in mast cell diseases and gastrointestinal stromal tumours. Recently, they have also been found in mediastinal and testicular germ cell tumours (TGCTs), particularly in cases with bilateral disease. We screened the KIT coding sequence ( except exo

  18. Reprogramming to pluripotency can conceal somatic cell chromosomal instability.

    NARCIS (Netherlands)

    Hamada, M.; Malureanu, L.A.; Wijshake, T.; Zhou, W.; Deursen, J.M.A. van

    2012-01-01

    The discovery that somatic cells are reprogrammable to pluripotency by ectopic expression of a small subset of transcription factors has created great potential for the development of broadly applicable stem-cell-based therapies. One of the concerns regarding the safe use of induced pluripotent stem

  19. Somatic cell reprogramming-free generation of genetically modified pigs

    Science.gov (United States)

    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-ichiro; Otoi, Takeshige

    2016-01-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs. PMID:27652340

  20. Buffalo milk: proteins electrophoretic profile and somatic cell count

    Directory of Open Access Journals (Sweden)

    S. Mattii

    2011-03-01

    Full Text Available Water buffalo milk differs from the cow’s milk for greater fat and protein content, very important features in cheese making. Proteins, casein and whey-proteins in particular, are the most important factors determining cheese yield. Several previous research discussed the rule of SCC in cow milk production (Varisco, 1999 and the close relationship existing between cow’s milk cheese yield and somatic cell count (Barbano, 2000. In particular the inverse correlation between cheese yields and somatic cells’content have been demonstrated. In Italy the regulation in force DPR 54/97 acknowledges what expressed in EEC 46/92 Directive (Tripodi, 1999 without fixing the limit threshold of somatic cells for buffalo’s milk....

  1. Somatic cell reprogramming-free generation of genetically modified pigs.

    Science.gov (United States)

    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-Ichiro; Otoi, Takeshige

    2016-09-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

  2. Global Splicing Pattern Reversion during Somatic Cell Reprogramming

    Directory of Open Access Journals (Sweden)

    Sho Ohta

    2013-10-01

    Full Text Available Alternative splicing generates multiple transcripts from a single gene, and cell-type-specific splicing profiles are important for the properties and functions of the cells. Recently, somatic cells have been shown to undergo dedifferentiation after the forced expression of transcription factors. However, it remains unclear whether somatic cell splicing is reorganized during reprogramming. Here, by combining deep sequencing with high-throughput absolute qRT-PCR, we show that somatic splicing profiles revert to pluripotent ones during reprogramming. Remarkably, the splicing pattern in pluripotent stem cells resembles that in testes, and the regulatory regions have specific characteristics in length and sequence. Furthermore, our siRNA screen has identified RNA-binding proteins that regulate splicing events in iPSCs. We have then demonstrated that two of the RNA-binding proteins, U2af1 and Srsf3, play a role in somatic cell reprogramming. Our results indicate that the drastic alteration in splicing represents part of the molecular network involved in the reprogramming process.

  3. Mammalian synthetic biology for studying the cell.

    Science.gov (United States)

    Mathur, Melina; Xiang, Joy S; Smolke, Christina D

    2017-01-02

    Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

  4. Stimulus-triggered Fate Conversion of Somatic Cells into Pluripotency in Chronic Wounds in Human Beings?

    Directory of Open Access Journals (Sweden)

    Basavraj S. Nagoba

    2015-10-01

    Full Text Available Bone-marrow derived stem cells are multi potential or totipotent and are able to differentiate into numerous cell types. Their application is indicated in various reconstructive and restorative surgeries for rapid healing. A technique for creating cells that have the embryonic ability to turn into almost any cell type in the mammalian body has been reported. Recently, an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposing to sublethal stimuli such as citrate based acidic medium has been reported. With the concept of creating acidic environment in chronic infected wounds to make a condition unsuitable for growth and multiplication of bacteria using 3% citric acid has been reported. It would be interesting to study whether the phenomenon of pluripotency takes place in chronic infected wounds in human beings following the application of 3% citric acid and plays an important role in formation of healthy granulation tissue.

  5. RNAa is conserved in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Vera Huang

    Full Text Available BACKGROUND: RNA activation (RNAa is a newly discovered mechanism of gene activation triggered by small double-stranded RNAs termed 'small activating RNAs' (saRNAs. Thus far, RNAa has only been demonstrated in human cells and is unclear whether it is conserved in other mammals. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we evaluated RNAa in cells derived from four mammalian species including nonhuman primates (African green monkey and chimpanzee, mouse, and rat. Previously, we identified saRNAs leading to the activation of E-cadherin, p21, and VEGF in human cells. As the targeted sequences are highly conserved in primates, transfection of each human saRNA into African green monkey (COS1 and chimpanzee (WES cells also resulted in induction of the intended gene. Additional saRNAs targeting clinically relevant genes including p53, PAR4, WT1, RB1, p27, NKX3-1, VDR, IL2, and pS2 were also designed and transfected into COS1 and WES cells. Of the nine genes, p53, PAR4, WT1, and NKX3-1 were induced by their corresponding saRNAs. We further extended our analysis of RNAa into rodent cell types. We identified two saRNAs that induced the expression of mouse Cyclin B1 in NIH/3T3 and TRAMP C1 cells, which led to increased phosphorylation of histone H3, a downstream marker for chromosome condensation and entry into mitosis. We also identified two saRNAs that activated the expression of CXCR4 in primary rat adipose-derived stem cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that RNAa exists in mammalian species other than human. Our findings also suggest that nonhuman primate disease models may have clinical applicability for validating RNAa-based drugs.

  6. Stn1 is critical for telomere maintenance and long-term viability of somatic human cells.

    Science.gov (United States)

    Boccardi, Virginia; Razdan, Neetu; Kaplunov, Jessica; Mundra, Jyoti J; Kimura, Masayuki; Aviv, Abraham; Herbig, Utz

    2015-06-01

    Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging-associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA-mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long-term viability of normal somatic mammalian cells.

  7. Optogenetics for gene expression in mammalian cells.

    Science.gov (United States)

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  8. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Tak, Hyosun, E-mail: chuberry@naver.com [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Rho, Mina, E-mail: minarho@hanyang.ac.kr [Department of Computer Science, Hanyang University, Seoul 133-791 (Korea, Republic of); Chang, Hae Ryung, E-mail: heyhae@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Yon Hui, E-mail: yhkim@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Kyung Tae, E-mail: bioktkim@ncc.re.kr [Molecular Epidemiology Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Balch, Curt, E-mail: curt.balch@gmail.com [Medical Sciences Program, Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Bloomington, IN 47405 (United States); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Nam, Seungyoon, E-mail: seungyoon.nam@ncc.re.kr [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of)

    2014-03-28

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.

  9. Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer.

    Science.gov (United States)

    Long, Charles R; Westhusin, Mark E; Golding, Michael C

    2014-02-01

    Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection.

  10. Engineered Trehalose Permeable to Mammalian Cells.

    Directory of Open Access Journals (Sweden)

    Alireza Abazari

    Full Text Available Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre and trehalose tetraacetate (4-O-Ac-Tre. Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

  11. Germ cells and the origins of mammalian pluripotent cells

    NARCIS (Netherlands)

    Kuijk, E.W.

    2009-01-01

    Mammalian embryonic stem (ES) cells originate from preimplantation embryos and can be propagated indefinitely without loss of pluripotency; i.e. the potential to develop into any embryonic cell type. ES cells have been described for mouse, rhesus monkey, and human. There is considerable interest in

  12. Development of porcine tetraploid somatic cell nuclear transfer embryos is influenced by oocyte nuclei.

    Science.gov (United States)

    Fu, Bo; Liu, Di; Ma, Hong; Guo, Zhen-Hua; Wang, Liang; Li, Zhong-Qiu; Peng, Fu-Gang; Bai, Jing

    2016-02-01

    Cloning efficiency in mammalian systems remains low because reprogramming of donor cells is frequently incomplete. Nuclear factors in the oocyte are removed by enucleation, and this removal may adversely affect reprogramming efficiency. Here, we investigated the role of porcine oocyte nuclear factors during reprogramming. We introduced somatic cell nuclei into intact MII oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. We then examined the influence of the oocyte nucleus on tetraploid SCNT embryo development by assessing characteristics including pronucleus formation, cleavage rate, and blastocyst formation. Overall, tetraploid SCNT embryos have a higher developmental competence than do standard diploid SCNT embryos. Therefore, we have established an embryonic model in which a fetal fibroblast nucleus and an oocyte metaphase II plate coexist. Tetraploid SCNT represents a new research platform that is potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.

  13. Functional evaluation of ES-somatic cell hybrids in vitro and in vivo.

    Science.gov (United States)

    Sumer, Huseyin; Kim, Kitai; Liu, Jun; Ng, Kitwa; Daley, George Q; Verma, Paul J

    2014-06-01

    Embryonic stem cells (ESCs) have previously been reported to reprogram somatic cells following fusion. The resulting ES-somatic cell hybrids have been shown to adopt the transcriptional profile of ESCs, suggesting that the pluripotent program is dominant. ES-somatic cell hybrids have most characteristics of pluripotent cells in vitro; however, it remains unclear whether the somatic genome is an active partner in the hybrid cells or simply retained predominately as silent cargo. Furthermore, the functional properties of ES-somatic cell hybrids in vivo have been limited to studies on their contribution to teratomas and developing embryos/chimeras. The extent of their pluripotency remains largely unclear. Here we determined that the somatic genome is actively transcribed by generating ES-somatic cell hybrids using Rag2-deficient ESCs fused to autologous wild-type somatic cells. Rag2 expression was detected during in vitro differentiation, suggesting that the somatic genome follows the correct temporal cues during differentiation. Furthermore, ES-somatic cell hybrids maintain their tetraploid state following 4 weeks of differentiation in vivo and are immune tolerated when transferred into matched individuals. The ES-somatic cell hybrids can efficiently differentiate into hematopoietic precursors in both myeloid and lymphoid lineages in vitro, suggesting that the somatic genome is actively transcribed following cell fusion based reprogramming. However, the ES-somatic cell hybrids showed an altered hematopoietic potential following in vitro differentiation and were unable to show hematopoietic engraftment in a mouse model.

  14. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer

    OpenAIRE

    Young-Hee Jeong; Hanlin Lu; Chi-Hun Park; Meiyan Li; Huijuan Luo; Joung Joo Kim; Siyang Liu; Kyeong Hee Ko; Shujia Huang; In Sung Hwang; Mi Na Kang; Desheng Gong; Kang Bae Park; Eun Ji Choi; Jung Hyun Park

    2016-01-01

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, e...

  15. Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Domenico Iuso

    Full Text Available The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT. Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

  16. The Somatic Genomic Landscape of Chromophobe Renal Cell Carcinoma

    NARCIS (Netherlands)

    Davis, Caleb F; Ricketts, Christopher J; Wang, Min; Yang, Lixing; Cherniack, Andrew D; Shen, Hui; Buhay, Christian; Kang, Hyojin; Kim, Sang Cheol; Fahey, Catherine C; Hacker, Kathryn E; Bhanot, Gyan; Gordenin, Dmitry A; Chu, Andy; Gunaratne, Preethi H; Biehl, Michael; Seth, Sahil; Kaipparettu, Benny A; Bristow, Christopher A; Donehower, Lawrence A; Wallen, Eric M; Smith, Angela B; Tickoo, Satish K; Tamboli, Pheroze; Reuter, Victor; Schmidt, Laura S; Hsieh, James J; Choueiri, Toni K; Hakimi, A Ari; Chin, Lynda; Meyerson, Matthew; Kucherlapati, Raju; Park, Woong-Yang; Robertson, A Gordon; Laird, Peter W; Henske, Elizabeth P; Kwiatkowski, David J; Park, Peter J; Morgan, Margaret; Shuch, Brian; Muzny, Donna; Wheeler, David A; Linehan, W Marston; Gibbs, Richard A; Rathmell, W Kimryn; Creighton, Chad J

    2014-01-01

    We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared

  17. Somatic cell count distributions during lactation predict clinical mastitis

    NARCIS (Netherlands)

    Green, M.J.; Green, L.E.; Schukken, Y.H.; Bradley, A.J.; Peeler, E.J.; Barkema, H.W.; Haas, de Y.; Collis, V.J.; Medley, G.F.

    2004-01-01

    This research investigated somatic cell count (SCC) records during lactation, with the purpose of identifying distribution characteristics (mean and measures of variation) that were most closely associated with clinical mastitis. Three separate data sets were used, one containing quarter SCC (n =

  18. Somatic cell count distributions during lactation predict clinical mastitis

    NARCIS (Netherlands)

    Green, M.J.; Green, L.E.; Schukken, Y.H.; Bradley, A.J.; Peeler, E.J.; Barkema, H.W.; Haas, de Y.; Collis, V.J.; Medley, G.F.

    2004-01-01

    This research investigated somatic cell count (SCC) records during lactation, with the purpose of identifying distribution characteristics (mean and measures of variation) that were most closely associated with clinical mastitis. Three separate data sets were used, one containing quarter SCC (n = 14

  19. Protein and genome evolution in Mammalian cells for biotechnology applications.

    Science.gov (United States)

    Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

    2009-06-01

    Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

  20. Lineage development of cell fusion hybrids upon somatic reprogramming

    OpenAIRE

    2011-01-01

    Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2011 Somatic cell reprogramming has been extensively studied over the last years and opened new perspectives in the use of pluripotent cells for regenerative biomedical purposes. Spontaneous cell fusion has been suggested to be involved in regenerative processes in vivo. Strong evidences support the hypothesis that the reprogrammed hybrids resulting from the fusion between a pluripote...

  1. File list: Unc.Emb.10.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.10.AllAg.Gonadal_somatic_cells mm9 Unclassified Embryo Gonadal somatic cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.10.AllAg.Gonadal_somatic_cells.bed ...

  2. File list: ALL.Emb.50.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.Gonadal_somatic_cells mm9 All antigens Embryo Gonadal somatic cell...s SRX685753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.Gonadal_somatic_cells.bed ...

  3. File list: Unc.Emb.20.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.20.AllAg.Gonadal_somatic_cells mm9 Unclassified Embryo Gonadal somatic cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.20.AllAg.Gonadal_somatic_cells.bed ...

  4. File list: ALL.Emb.10.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.10.AllAg.Gonadal_somatic_cells mm9 All antigens Embryo Gonadal somatic cell...s SRX685753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.10.AllAg.Gonadal_somatic_cells.bed ...

  5. File list: Unc.Emb.05.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.05.AllAg.Gonadal_somatic_cells mm9 Unclassified Embryo Gonadal somatic cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.05.AllAg.Gonadal_somatic_cells.bed ...

  6. File list: ALL.Emb.20.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.Gonadal_somatic_cells mm9 All antigens Embryo Gonadal somatic cell...s SRX685753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Gonadal_somatic_cells.bed ...

  7. File list: Unc.Emb.50.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.50.AllAg.Gonadal_somatic_cells mm9 Unclassified Embryo Gonadal somatic cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.50.AllAg.Gonadal_somatic_cells.bed ...

  8. File list: ALL.Emb.05.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.05.AllAg.Gonadal_somatic_cells mm9 All antigens Embryo Gonadal somatic cell...s SRX685753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.05.AllAg.Gonadal_somatic_cells.bed ...

  9. File list: His.Gon.05.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Gon.05.AllAg.Testicular_somatic_cells mm9 Histone Gonad Testicular somatic cell...s SRX591729,SRX591717 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Gon.05.AllAg.Testicular_somatic_cells.bed ...

  10. File list: His.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Gon.50.AllAg.Testicular_somatic_cells mm9 Histone Gonad Testicular somatic cell...s SRX591729,SRX591717 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  11. File list: His.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Gon.20.AllAg.Testicular_somatic_cells mm9 Histone Gonad Testicular somatic cell...s SRX591729,SRX591717 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  12. File list: His.Gon.10.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Gon.10.AllAg.Testicular_somatic_cells mm9 Histone Gonad Testicular somatic cell...s SRX591729,SRX591717 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Gon.10.AllAg.Testicular_somatic_cells.bed ...

  13. [Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

    Science.gov (United States)

    Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

    2006-12-01

    Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

  14. Cell fate regulation in early mammalian development

    Science.gov (United States)

    Oron, Efrat; Ivanova, Natalia

    2012-08-01

    Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

  15. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    NARCIS (Netherlands)

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated doma

  16. Derivation of induced pluripotent stem cells from pig somatic cells.

    Science.gov (United States)

    Ezashi, Toshihiko; Telugu, Bhanu Prakash V L; Alexenko, Andrei P; Sachdev, Shrikesh; Sinha, Sunilima; Roberts, R Michael

    2009-07-07

    For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after approximately 22 days, providing an overall reprogramming efficiency of approximately 0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of approximately 17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.

  17. File list: Pol.Emb.50.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.50.AllAg.Gonadal_somatic_cells mm9 RNA polymerase Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.50.AllAg.Gonadal_somatic_cells.bed ...

  18. File list: DNS.Emb.05.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.05.AllAg.Gonadal_somatic_cells mm9 DNase-seq Embryo Gonadal somatic cells h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.05.AllAg.Gonadal_somatic_cells.bed ...

  19. File list: His.Emb.05.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.05.AllAg.Gonadal_somatic_cells mm9 Histone Embryo Gonadal somatic cells SRX...685753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.05.AllAg.Gonadal_somatic_cells.bed ...

  20. File list: His.Emb.10.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.10.AllAg.Gonadal_somatic_cells mm9 Histone Embryo Gonadal somatic cells SRX...685753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.10.AllAg.Gonadal_somatic_cells.bed ...

  1. File list: His.Emb.20.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.20.AllAg.Gonadal_somatic_cells mm9 Histone Embryo Gonadal somatic cells SRX...685753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.20.AllAg.Gonadal_somatic_cells.bed ...

  2. File list: Oth.Emb.20.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.AllAg.Gonadal_somatic_cells mm9 TFs and others Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.20.AllAg.Gonadal_somatic_cells.bed ...

  3. File list: Oth.Emb.10.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.AllAg.Gonadal_somatic_cells mm9 TFs and others Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.10.AllAg.Gonadal_somatic_cells.bed ...

  4. File list: Pol.Emb.20.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.20.AllAg.Gonadal_somatic_cells mm9 RNA polymerase Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.20.AllAg.Gonadal_somatic_cells.bed ...

  5. File list: Oth.Emb.50.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.AllAg.Gonadal_somatic_cells mm9 TFs and others Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.50.AllAg.Gonadal_somatic_cells.bed ...

  6. File list: Pol.Emb.05.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.05.AllAg.Gonadal_somatic_cells mm9 RNA polymerase Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.05.AllAg.Gonadal_somatic_cells.bed ...

  7. File list: Pol.Emb.10.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.10.AllAg.Gonadal_somatic_cells mm9 RNA polymerase Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.10.AllAg.Gonadal_somatic_cells.bed ...

  8. File list: His.Emb.50.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.50.AllAg.Gonadal_somatic_cells mm9 Histone Embryo Gonadal somatic cells SRX...685753 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.50.AllAg.Gonadal_somatic_cells.bed ...

  9. File list: DNS.Emb.10.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.10.AllAg.Gonadal_somatic_cells mm9 DNase-seq Embryo Gonadal somatic cells h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.10.AllAg.Gonadal_somatic_cells.bed ...

  10. File list: Pol.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.50.AllAg.Testicular_somatic_cells mm9 RNA polymerase Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  11. File list: Oth.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.50.AllAg.Testicular_somatic_cells mm9 TFs and others Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  12. File list: DNS.Gon.10.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Gon.10.AllAg.Testicular_somatic_cells mm9 DNase-seq Gonad Testicular somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Gon.10.AllAg.Testicular_somatic_cells.bed ...

  13. File list: ALL.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Gon.20.AllAg.Testicular_somatic_cells mm9 All antigens Gonad Testicular somatic... cells SRX591728,SRX591729,SRX591717,SRX591716 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  14. File list: Oth.Gon.10.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.10.AllAg.Testicular_somatic_cells mm9 TFs and others Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.10.AllAg.Testicular_somatic_cells.bed ...

  15. File list: Unc.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Gon.50.AllAg.Testicular_somatic_cells mm9 Unclassified Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  16. File list: ALL.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Gon.50.AllAg.Testicular_somatic_cells mm9 All antigens Gonad Testicular somatic... cells SRX591728,SRX591729,SRX591717,SRX591716 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  17. File list: Unc.Gon.10.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Gon.10.AllAg.Testicular_somatic_cells mm9 Unclassified Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Gon.10.AllAg.Testicular_somatic_cells.bed ...

  18. File list: DNS.Gon.05.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Gon.05.AllAg.Testicular_somatic_cells mm9 DNase-seq Gonad Testicular somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Gon.05.AllAg.Testicular_somatic_cells.bed ...

  19. File list: Pol.Gon.10.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.10.AllAg.Testicular_somatic_cells mm9 RNA polymerase Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Gon.10.AllAg.Testicular_somatic_cells.bed ...

  20. File list: Pol.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.20.AllAg.Testicular_somatic_cells mm9 RNA polymerase Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  1. File list: Oth.Gon.05.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.05.AllAg.Testicular_somatic_cells mm9 TFs and others Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.05.AllAg.Testicular_somatic_cells.bed ...

  2. File list: Unc.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Gon.20.AllAg.Testicular_somatic_cells mm9 Unclassified Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  3. File list: ALL.Gon.05.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Gon.05.AllAg.Testicular_somatic_cells mm9 All antigens Gonad Testicular somatic... cells SRX591729,SRX591728,SRX591717,SRX591716 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Gon.05.AllAg.Testicular_somatic_cells.bed ...

  4. File list: Unc.Gon.05.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Gon.05.AllAg.Testicular_somatic_cells mm9 Unclassified Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Gon.05.AllAg.Testicular_somatic_cells.bed ...

  5. File list: DNS.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Gon.50.AllAg.Testicular_somatic_cells mm9 DNase-seq Gonad Testicular somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  6. File list: Pol.Gon.05.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.05.AllAg.Testicular_somatic_cells mm9 RNA polymerase Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Gon.05.AllAg.Testicular_somatic_cells.bed ...

  7. File list: Oth.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.20.AllAg.Testicular_somatic_cells mm9 TFs and others Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  8. File list: DNS.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Gon.20.AllAg.Testicular_somatic_cells mm9 DNase-seq Gonad Testicular somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  9. Somatic cell nuclear transfer cloning: practical applications and current legislation.

    Science.gov (United States)

    Niemann, H; Lucas-Hahn, A

    2012-08-01

    Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

  10. Mitochondrial toxicity of triclosan on mammalian cells

    Directory of Open Access Journals (Sweden)

    Charmaine Ajao

    2015-01-01

    Full Text Available Effects of triclosan (5-chloro-2′-(2,4-dichlorophenoxyphenol on mammalian cells were investigated using human peripheral blood mono nuclear cells (PBMC, keratinocytes (HaCaT, porcine spermatozoa and kidney tubular epithelial cells (PK-15, murine pancreatic islets (MIN-6 and neuroblastoma cells (MNA as targets. We show that triclosan (1–10 μg ml−1 depolarised the mitochondria, upshifted the rate of glucose consumption in PMBC, HaCaT, PK-15 and MNA, and subsequently induced metabolic acidosis. Triclosan induced a regression of insulin producing pancreatic islets into tiny pycnotic cells and necrotic death. Short exposure to low concentrations of triclosan (30 min, ≤1 μg/ml paralyzed the high amplitude tail beating and progressive motility of spermatozoa, within 30 min exposure, depolarized the spermatozoan mitochondria and hyperpolarised the acrosome region of the sperm head and the flagellar fibrous sheath (distal part of the flagellum. Experiments with isolated rat liver mitochondria showed that triclosan impaired oxidative phosphorylation, downshifted ATP synthesis, uncoupled respiration and provoked excessive oxygen uptake. These exposure concentrations are 100–1000 fold lower that those permitted in consumer goods. The mitochondriotoxic mechanism of triclosan differs from that of valinomycin, cereulide and the enniatins by not involving potassium ionophoric activity.

  11. Reprogramming to pluripotency can conceal somatic cell chromosomal instability.

    Directory of Open Access Journals (Sweden)

    Masakazu Hamada

    Full Text Available The discovery that somatic cells are reprogrammable to pluripotency by ectopic expression of a small subset of transcription factors has created great potential for the development of broadly applicable stem-cell-based therapies. One of the concerns regarding the safe use of induced pluripotent stem cells (iPSCs in therapeutic applications is loss of genomic integrity, a hallmark of various human conditions and diseases, including cancer. Structural chromosome defects such as short telomeres and double-strand breaks are known to limit reprogramming of somatic cells into iPSCs, but whether defects that cause whole-chromosome instability (W-CIN preclude reprogramming is unknown. Here we demonstrate, using aneuploidy-prone mouse embryonic fibroblasts (MEFs in which chromosome missegregation is driven by BubR1 or RanBP2 insufficiency, that W-CIN is not a barrier to reprogramming. Unexpectedly, the two W-CIN defects had contrasting effects on iPSC genomic integrity, with BubR1 hypomorphic MEFs almost exclusively yielding aneuploid iPSC clones and RanBP2 hypomorphic MEFs karyotypically normal iPSC clones. Moreover, BubR1-insufficient iPSC clones were karyotypically unstable, whereas RanBP2-insufficient iPSC clones were rather stable. These findings suggest that aneuploid cells can be selected for or against during reprogramming depending on the W-CIN gene defect and present the novel concept that somatic cell W-CIN can be concealed in the pluripotent state. Thus, karyotypic analysis of somatic cells of origin in addition to iPSC lines is necessary for safe application of reprogramming technology.

  12. TCPs, WUSs, and WINDs: families of transcription factors that regulate shoot meristem formation, stem cell maintenance, and somatic cell differentiation.

    Science.gov (United States)

    Ikeda, Miho; Ohme-Takagi, Masaru

    2014-01-01

    In contrast to somatic mammalian cells, which cannot alter their fate, plant cells can dedifferentiate to form totipotent callus cells and regenerate a whole plant, following treatment with specific phytohormones. However, the regulatory mechanisms and key factors that control differentiation-dedifferentiation and cell totipotency have not been completely clarified in plants. Recently, several plant transcription factors that regulate meristem formation and dedifferentiation have been identified and include members of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP), WUSCHEL (WUS), and WOUND INDUCED DEDIFFERENTIATION (WIND1) families. WUS and WIND positively control plant cell totipotency, while TCP negatively controls it. Interestingly, TCP is a transcriptional activator that acts as a negative regulator of shoot meristem formation, and WUS is a transcriptional repressor that positively maintains totipotency of the stem cells of the shoot meristem. We describe here the functions of TCP, WUS, and WIND transcription factors in the regulation of differentiation-dedifferentiation by positive and negative transcriptional regulators.

  13. The role of p53 in limiting somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Yan Liu; Ruben Hoya-Arias; Stephen D Nimer

    2009-01-01

    @@ The first successful generation of induced pluripotent stem(iPS)cells from somatic cells was accomplished by introducing four genes into the cell,0ct3/4,Sox2,Klf4,and c-myc [1].While a tour-de-force,this approach to iPS cell generation is inefficient,and unlikely to be directly translated into therapeutic use since it involves the use of retroviruses to introduce these genes into the cell.Subsequent studies have used non-integrating genetic elements,chemical compounds,or proteins rather than DNA to bypass concerns about retroviral insertional mutagenesis [2-5].

  14. DNA methylation supports intrinsic epigenetic memory in mammalian cells.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation.

  15. [Retrotransposons: selfish DNA or active epigenetic players in somatic cells?].

    Science.gov (United States)

    Guidez, Fabien

    2014-01-01

    Transposable elements (TE) represent around 40% of the human genome. They are endogenous mobile DNA sequences able to jump and duplicate in the host genome. TE have long been considered as "junk" DNA but are now believed to be important regulators of gene expression by participating to the establishment of the DNA methylation profile. Recent advances in genome sequencing reveals a higher transposition frequency and TE driven gene expression in somatic cells than previously thought. As TE propagation is deleterious and may be involved in oncogenic mechanisms, host cells have developed silencing mechanisms mainly described in germinal and embryonic cells. However, somatic cells are also proned to TE transposition and use specific mechanisms involving tumor suppressor proteins including p53, Rb and PLZF. These transcription factors specifically target genomic retrotransposon sequences, histone deacetylase and DNA methylase activities, inducing epigenetic modifications related to gene silencing. Thus, these transcription factors negatively regulate TE expression by the formation of DNA methylation profil in somatic cells possibly associated with oncogenic mechanisms.

  16. [Inducing brain regeneration from within: in vivo reprogramming of endogenous somatic cells into neurons].

    Science.gov (United States)

    Heinrich, Christophe; Rouaux, Caroline

    2015-01-01

    In order to overcome the quasi-total inability of the mammalian central nervous system to regenerate in response to injuries, and in parallel to the studies dedicated to prevent neuronal loss under these circumstances, alternative approaches based on the programming of pluripotent cells or the reprogramming of somatic cells into neurons have recently emerged. These uniquely combine growing knowledge of the mechanisms that underlie neurogenesis and neuronal specification during development to the most recent findings of the molecular and epigenetic mechanisms that govern the acquisition and maintenance of cellular identity. Here, we discuss the possibility to instruct the regeneration of the central nervous system from within for therapeutic purposes, in light of the recent works reporting on the generation of neurons by direct conversion of various cerebral cell types in vitro and in vivo. © 2015 médecine/sciences – Inserm.

  17. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi

    1997-06-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  18. The different shades of mammalian pluripotent stem cells

    NARCIS (Netherlands)

    Kuijk, E.W.; Lopes, S.M.; Geijsen, N.; Macklon, N.; Roelen, B.A.

    2011-01-01

    BACKGROUND: Pluripotent stem cells have been derived from a variety of sources such as from the inner cell mass of preimplantation embryos, from primordial germ cells, from teratocarcinomas and from male germ cells. The recent development of induced pluripotent stem cells demonstrates that somatic c

  19. Regulation of L-threonine dehydrogenase in somatic cell reprogramming.

    Science.gov (United States)

    Han, Chuanchun; Gu, Hao; Wang, Jiaxu; Lu, Weiguang; Mei, Yide; Wu, Mian

    2013-05-01

    Increasing evidence suggests that metabolic remodeling plays an important role in the regulation of somatic cell reprogramming. Threonine catabolism mediated by L-threonine dehydrogenase (TDH) has been recognized as a specific metabolic trait of mouse embryonic stem cells. However, it remains unknown whether TDH-mediated threonine catabolism could regulate reprogramming. Here, we report TDH as a novel regulator of somatic cell reprogramming. Knockdown of TDH inhibits, whereas induction of TDH enhances reprogramming efficiency. Moreover, microRNA-9 post-transcriptionally regulates the expression of TDH and thereby inhibits reprogramming efficiency. Furthermore, protein arginine methyltransferase (PRMT5) interacts with TDH and mediates its post-translational arginine methylation. PRMT5 appears to regulate TDH enzyme activity through both methyltransferase-dependent and -independent mechanisms. Functionally, TDH-facilitated reprogramming efficiency is further enhanced by PRMT5. These results suggest that TDH-mediated threonine catabolism controls somatic cell reprogramming and indicate the importance of post-transcriptional and post-translational regulation of TDH.

  20. Somatic cell bovine cloning: Effect of donor cell and recipients

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Adult somatic cell nuclear transfer was conducted by using cultured ear fibroblast cells obtained from a Holstein female cow (GN) and a Galoway herd bull (GLV). The percentages of reconstructed eggs developed into blastocysts were similar in GN (23.98%, 123 of 513) and in GLV groups (29.55%, 138 of 467). However, the rate of reconstructed female (GN) embryos developed into term was higher than that of male (GLV) (8.02% and 1.82%, respectively). Three kinds of cows, Luxi Yellow cows, Holstein heifers and Holstein cows with normal reproductive records were used as recipients. When the reconstructed embryos from GN were transferred, there was no difference in the pregnancy rate among three kinds of recipients, but the abortion rate of Luxi Yellow cows was significantly higher (85.71%) than in the other two groups (14.29% and 0%, respectively; P < 0.05). And the percentages of newborn calves in transferred embryos were significantly different between Luxi Yellow cows and Holstein breed (1.54%, 10.39% and 20.0%, respectively, P < 0.05). However, when reconstructed embryos from GLV were transferred, there was no difference among three kinds of recipients in the pregnancy rate, the abortion rate and the delivery rate.

  1. Mitochondrial inheritance is mediated by microtubules in mammalian cell division.

    Science.gov (United States)

    Lawrence, Elizabeth; Mandato, Craig

    2013-11-01

    The mitochondrial network fragments and becomes uniformly dispersed within the cytoplasm when mammalian cells enter mitosis. Such morphology and distribution of mitochondria was previously thought to facilitate the stochastic inheritance of mitochondria by daughter cells. In contrast, we recently reported that mitochondria in dividing mammalian cells are inherited by an ordered mechanism of inheritance mediated by microtubules. We showed that mitochondria are progressively enriched at the cell equator and depleted at the poles throughout division. Furthermore, the mitochondrial distribution during division is dependent on microtubules, indicating an ordered inheritance strategy. The microtubule-mediated positioning of mitochondria in dividing mammalian cells may have functional consequences for cell division and/or mitochondrial inheritance.

  2. Self-Renewal and Pluripotency Acquired through Somatic Reprogramming to Human Cancer Stem Cells.

    OpenAIRE

    2012-01-01

    Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re-plating followed by...

  3. The neurosteroid dehydroepiandrosterone could improve somatic cell reprogramming.

    Science.gov (United States)

    Shoae-Hassani, Alireza; Sharif, Shiva; Verdi, Javad

    2011-10-01

    Expression of four major reprogramming transgenes, including Oct4, Sox2, Klf4 and c-myc, in somatic cells enables them to have pluripotency. These cells are iPSC (induced pluripotent stem cell) that currently show the greatest potential for differentiation into cells of the three germ lineages. One of the issues facing the successful reprogramming and clinical translation of iPSC technology is the high rate of apoptosis after the reprogramming process. Reprogramming is a stressful process, and the p53 apoptotic pathway plays a negative role in cell growth and self-renewal. Apoptosis via the p53 pathway serves as a major barrier in nuclear somatic cell reprogramming during iPSC generation. DHEA (dehydroepiandrosterone) is an abundant steroid that is produced at high levels in the adrenal cells, and withdrawal of DHEA increases the levels of p53 in the epithelial and stromal cells, resulting in increased levels of apoptotic cells; meanwhile, DHEA decreases cellular apoptosis. DHEA could improve the efficacy of reprogramming yield due to a decrease in apoptosis via the p53 pathway and an increase in cell viability.

  4. Normal somatic cell count and subclinical mastitis in Murrah buffaloes.

    Science.gov (United States)

    Dhakal, I P

    2006-03-01

    This study was conducted to investigate the normal somatic cell count (SCC) and to define subclinical mastitis in Murrah buffaloes. Data were collected from 60 clinically normal buffaloes stationed at five farms of Chitwan Nepal and Buffalo Research Center, Hissar, India. Somatic cell count was measured using the Newman-Lampert staining technique. The upper limit of SCC was determined >or=200 000/ml of milk based on the mean +/- 2SD of a total SCC. Abnormal data of the SCC was repeatedly removed, which lie beyond the values of more than mean + 2SD until all the data come to lie within (mean + 2SD). Averages of SCC of right front and right hind quarters were significantly higher than left front and left hind quarters. Nearly 94% of California mastitis test (CMT) negative quarters were having somatic cells >or=200 000/ml. The mean SCC of CMT positive quarter was significantly higher (P CMT negative quarters. Subclinical mastitis was diagnosed on the basis of samples with SCCs >or=200 000/ml with positive bacterial cultures. Subclinical mastitis was found in 21.7% buffaloes and 8% of the quarter foremilk samples. Neutrophil counts were significantly higher in subclinical mastitis milk.

  5. Cell transformation and mutability of different genetic loci in mammalian cells by metabolically activated carcinogenic polycylic hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.

    1977-01-01

    Treatment of experimental animals with chemical carcinogens, including some polycyclic hydrocarbons, can result in the formation of malignant tumors. The process whereby some chemicals induce malignancy is as yet unknown. However, in a model system using mammalian cells in culture, it was possible to show that the chemical carcinogens induce malignant transformation rather than select for pre-existing tumor cells. In the process of the in vitro cell transformation, the normal cells, which have an oriented pattern of cell growth, a limited life-span in vitro, and are not tumorigenic, are converted into cells that have a hereditary random pattern of cell growth, the ability to grow continuously in culture, and the ability to form tumors in vivo. This stable heritable phenotype of the transformed cells is similar to that of cells derived from spontaneous or experimentally induced tumors. Such stable heritable phenotype changes may arise from alteration in gene expression due to a somatic mutation after interaction of the carcinogen with cellular DNA. In the present experiments we have shown that metabolically activated carcinogenic polycyclic hydrocarbons which have been shown to bind to cellular DNA induce somatic mutations at different genetic loci in mammalian cells and that there is a relationship between the degree of mutant induction and the degree of carcinogenicity of the different hydrocarbons tested.

  6. Somatic cell nuclear transfer in horses.

    Science.gov (United States)

    Galli, Cesare; Lagutina, Irina; Duchi, Roberto; Colleoni, Silvia; Lazzari, Giovanna

    2008-07-01

    The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo-matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.

  7. Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Gómez, Martha C; Pope, C Earle

    2015-01-01

    In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

  8. A versatile expression vector system for mammalian cell factories

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Hansen, Bjarne Gram

    The development of the field of mammalian cell factories requests fast and high-throughput methods which means high need for simpler and more efficient cloning techniques. This project applies the ligation-free USERTM (uracil-specific excision reagent) cloning technique to construct mammalian...

  9. Adult somatic progenitor cells and hematopoiesis in oysters.

    Science.gov (United States)

    Jemaà, Mohamed; Morin, Nathalie; Cavelier, Patricia; Cau, Julien; Strub, Jean Marc; Delsert, Claude

    2014-09-01

    Long-lived animals show a non-observable age-related decline in immune defense, which is provided by blood cells that derive from self-renewing stem cells. The oldest living animals are bivalves. Yet, the origin of hemocytes, the cells involved in innate immunity, is unknown in bivalves and current knowledge about mollusk adult somatic stem cells is scarce. Here we identify a population of adult somatic precursor cells and show their differentiation into hemocytes. Oyster gill contains an as yet unreported irregularly folded structure (IFS) with stem-like cells bathing into the hemolymph. BrdU labeling revealed that the stem-like cells in the gill epithelium and in the nearby hemolymph replicate DNA. Proliferation of this cell population was further evidenced by phosphorylated-histone H3 mitotic staining. Finally, these small cells, most abundant in the IFS epithelium, were found to be positive for the stemness marker Sox2. We provide evidence for hematopoiesis by showing that co-expression of Sox2 and Cu/Zn superoxide dismutase, a hemocyte-specific enzyme, does not occur in the gill epithelial cells but rather in the underlying tissues and vessels. We further confirm the hematopoietic features of these cells by the detection of Filamin, a protein specific for a sub-population of hemocytes, in large BrdU-labeled cells bathing into gill vessels. Altogether, our data show that progenitor cells differentiate into hemocytes in the gill, which suggests that hematopoiesis occurs in oyster gills. © 2014. Published by The Company of Biologists Ltd.

  10. Modelling of Mammalian cells and cell culture processes.

    Science.gov (United States)

    Sidoli, F R; Mantalaris, A; Asprey, S P

    2004-01-01

    Mammalian cell cultures represent the major source for a number of very high-value biopharmaceutical products, including monoclonal antibodies (MAbs), viral vaccines, and hormones. These products are produced in relatively small quantities due to the highly specialised culture conditions and their susceptibility to either reduced productivity or cell death as a result of slight deviations in the culture conditions. The use of mathematical relationships to characterise distinct parts of the physiological behaviour of mammalian cells and the systematic integration of this information into a coherent, predictive model, which can be used for simulation, optimisation, and control purposes would contribute to efforts to increase productivity and control product quality. Models can also aid in the understanding and elucidation of underlying mechanisms and highlight the lack of accuracy or descriptive ability in parts of the model where experimental and simulated data cannot be reconciled. This paper reviews developments in the modelling of mammalian cell cultures in the last decade and proposes a future direction - the incorporation of genomic, proteomic, and metabolomic data, taking advantage of recent developments in these disciplines and thus improving model fidelity. Furthermore, with mammalian cell technology dependent on experiments for information, model-based experiment design is formally introduced, which when applied can result in the acquisition of more informative data from fewer experiments. This represents only part of a broader framework for model building and validation, which consists of three distinct stages: theoretical model assessment, model discrimination, and model precision, which provides a systematic strategy from assessing the identifiability and distinguishability of a set of competing models to improving the parameter precision of a final validated model.

  11. A stochastic model of epigenetic dynamics in somatic cell reprogramming

    Directory of Open Access Journals (Sweden)

    Max eFloettmann

    2012-06-01

    Full Text Available Somatic cell reprogramming has dramatically changed stem cell research inrecent years. The high pace of new findings in the field and an ever increasingamount of data from new high throughput techniques make it challengingto isolate core principles of the process. In order to analyze suchmechanisms, we developed an abstract mechanistic model of a subset of theknown regulatory processes during cell differentiation and production of inducedpluripotent stem cells. This probabilistic Boolean network describesthe interplay between gene expression, chromatin modifications and DNAmethylation. The model incorporates recent findings in epigenetics and reproducesexperimentally observed reprogramming efficiencies and changes inmethylation and chromatin remodeling. It enables us to investigate in detail,how the temporal progression of the process is regulated. It also explicitlyincludes the transduction of factors using viral vectors and their silencing inreprogrammed cells, since this is still a standard procedure in somatic cellreprogramming. Based on the model we calculate an epigenetic landscape.Simulation results show good reproduction of experimental observations duringreprogramming, despite the simple stucture of the model. An extensiveanalysis and introduced variations hint towards possible optimizations of theprocess, that could push the technique closer to clinical applications. Fasterchanges in DNA methylation increase the speed of reprogramming at theexpense of efficiency, while accelerated chromatin modifications moderatelyimprove efficiency.

  12. Notch2 is required in somatic cells for breakdown of ovarian germ-cell nests and formation of primordial follicles

    Science.gov (United States)

    2013-01-01

    Background In the mouse ovary, oocytes initially develop in clusters termed germ-cell nests. Shortly after birth, these germ-cell nests break apart, and the oocytes individually become surrounded by somatic granulosa cells to form primordial follicles. Notch signaling plays essential roles during oogenesis in Drosophila, and recent studies have suggested that Notch signaling also plays an essential role during oogenesis and ovary development in mammals. However, no in vivo loss-of-function studies have been performed to establish whether Notch family receptors have an essential physiological role during normal ovarian development in mutant mice. Results Female mice with conditional deletion of the Notch2 gene in somatic granulosa cells of the ovary exhibited reduced fertility, accompanied by the formation of multi-oocyte follicles, which became hemorrhagic by 7 weeks of age. Formation of multi-oocyte follicles resulted from defects in breakdown of the primordial germ-cell nests. The ovaries of the Notch2 conditional mutant mice had increased numbers of oocytes, but decreased numbers of primordial follicles. Oocyte numbers in the Notch2 conditional mutants were increased not by excess or extended cellular proliferation, but as a result of decreased oocyte apoptosis. Conclusions Our work demonstrates that Notch2-mediated signaling in the somatic-cell lineage of the mouse ovary regulates oocyte apoptosis non-cell autonomously, and is essential for regulating breakdown of germ-cell nests and formation of primordial follicles. This model provides a new resource for studying the developmental and physiological roles of Notch signaling during mammalian reproductive biology. PMID:23406467

  13. Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Engelhardt John F

    2003-11-01

    Full Text Available Abstract Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret.

  14. Reproductive semi-cloning respecting biparental embryo origin: embryos from syngamy between a gamete and a haploidized somatic cell.

    Science.gov (United States)

    Tesarik, J

    2002-08-01

    Embryos formed by somatic cell nuclear transfer to enucleated oocytes (cloning) have given rise to viable offspring in several mammalian species. The possibility of future application of this technique to human assisted reproduction (reproductive cloning) has been widely debated. On this background there is current discussion of the potential for a cloning-derived technique, which aims at syngamy between a gamete nucleus from one parent and a somatic cell nucleus from the other. Critical analysis of the clinical indications, the current state of the art, biological concerns and ethical considerations relative to this technique, called here reproductive semi-cloning, are presented. Such a technique requires validation by further research before it can be considered as a treatment option. This debate explores issues raised by the technique.

  15. The different shades of mammalian pluripotent stem cells

    NARCIS (Netherlands)

    Kuijk, E.W.; Lopes, S.M.; Geijsen, N.; Macklon, N.S.; Roelen, B.A.J.

    2011-01-01

    The different shades of mammalian pluripotent stem cells Abstract BACKGROUND Pluripotent stem cells have been derived from a variety of sources such as from the inner cell mass of preimplantation embryos, from primordial germ cells, from teratocarcinomas and from male germ cells. The recent developm

  16. Generation of cloned calves from different types of somatic cells

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; DAI Yunping; ZHU Huabing; WANG Haiping; WANG Lili; LI Rong; WAN Rong; LIU Ying; LINing

    2004-01-01

    Six types of bovine somatic cell lines,including a granulosa cell line of Chinese red-breed yellow cattle(YGR),a granulosa cell line of Holstein cow(HGR),two skin fibroblast cell lines of two adult Holstein cows respectively(AFB1 and AFB2),a skin fibroblast cell line(FFB)and an oviduct epithelial cell line(FOV)of a Holstein fetus,were established.Somatic cell nuclear transfer(SCNT)was carried out using these cells as nuclei donor,and a total of 12 healthy calves were cloned.The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated.(i)There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR(33.2% and 35.1%,respectively).Pregnancy rates of them were 33.3% and 30.2%,respectively; and birth rates were 16.7%and 11.6%,respectively.(ii)Development rates to the blastocyst stage for SCNT embryos from diffetent individuals(AFB1 and AFB2)differed significantly(27.9% and 39.4%,respectively,P <0.05).Pregnancy rates of them were 36.2% and 36.4%,respectively; and birth rates were 14.9% and 27.3%,respectively.(iii)There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus(37.9% and 41.5%,respectively,P < 0.05).Pregnancy rates of them were 45.7% and 24.1%,respectively; and birth rates were 22.9 % and 10.3%,respectively.Finally,developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows(HGR,AFB,FFB and FOV)were compared.For in vitro development stage,development rates to the blastocyst stage for SCNT embryos from HGR,AFB,FFB and FOV were 35.1%A,29.4%B,37.9%A and 41.5%C,respectively(pABC<0.05); for in vivo development stage,pregnancy rates of them were 30.2%,36.2%,45.7%and 24.1%,respectively; and birth rates of them were 11.6%,17.2%,22.9% and 10.3% respectively.

  17. Applications of somatic cell nuclear transfer in goats

    Directory of Open Access Journals (Sweden)

    Ton Yoisungnern

    2014-06-01

    Full Text Available A number of animals with genetically identical appearance can be produced by somatic cell nuclear transfer (SCNT. From current advancement of SCNT and molecular techniques, production of a transgenic animal becomes easier. Although cloning efficiency in goat is low, the ability to propagate genetically identical animals, with a gene or genes of interest, would be important for increasing productivity and ultimately the economic livelihood. In this paper, the potential applications and uses of SCNT technology like production of transgenic goat for production of quality milk and meat are discussed.

  18. Reconstruction of human embryos derived from somatic cells

    Institute of Scientific and Technical Information of China (English)

    LU Changfu; LIN Ge; XIE Changqing; GONG Fei; ZHOU Hong; TAN Yueqiu; LU Guangxiu

    2003-01-01

    Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into MⅡ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyte activation and 2PN formation, we removed the female PN. By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation, and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into theblastocyst stage in vitro.

  19. Recent Progress of Somatic Cell Nuclear Transfer in Pigs

    Institute of Scientific and Technical Information of China (English)

    Xu Xiaoming; Dou Zhongying

    2005-01-01

    Research in the field of somatic cell nuclear transfer (SCNT) and transgenic cloning in pigs has become a global hotspot, because porcine organs probably can be the first source of donor organs for human xenotransplantation. In recent years, though great progress has been made in porcine SCNT, the efficiency of nuclear transfer remains very low (<1% ). Thus, it is necessary to improve the procedure of nuclear transfer and to investigate some basic problems further. Recent progress and the related problems of SCNT in pigs are reviewed and analyzed so as to offer some beneficial illumination to researchers.

  20. Local Actions of Melatonin in Somatic Cells of the Testis

    Directory of Open Access Journals (Sweden)

    Mónica Beatriz Frungieri

    2017-05-01

    Full Text Available The pineal hormone melatonin regulates testicular function through the hypothalamic-adenohypophyseal axis. In addition, direct actions of melatonin in somatic cells of the testis have been described. Melatonin acts as a local modulator of the endocrine activity in Leydig cells. In Sertoli cells, melatonin influences cellular growth, proliferation, energy metabolism and the oxidation state, and consequently may regulate spermatogenesis. These data pinpoint melatonin as a key player in the regulation of testicular physiology (i.e., steroidogenesis, spermatogenesis mostly in seasonal breeders. In patients with idiopathic infertility, melatonin exerts anti-proliferative and anti-inflammatory effects on testicular macrophages, and provides protective effects against oxidative stress in testicular mast cells. Consequently, melatonin is also involved in the modulation of inflammatory and oxidant/anti-oxidant states in testicular pathology. Overall, the literature data indicate that melatonin has important effects on testicular function and male reproduction.

  1. Induced DNA damage by dental resin monomers in somatic cells.

    Science.gov (United States)

    Arossi, Guilherme Anziliero; Lehmann, Mauricio; Dihl, Rafael Rodrigues; Reguly, Maria Luiza; de Andrade, Heloisa Helena Rodrigues

    2010-02-01

    The present in vivo study investigated the genotoxicity of four dental resin monomers: triethyleneglycoldimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA), urethanedimethacrylate (UDMA) and bisphenol A-glycidylmethacrylate (BisGMA). The Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly's wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. The present findings provide evidence that the mechanistic basis underlying the genotoxicity of UDMA and TEGDMA is related to homologous recombination and gene/chromosomal mutation. A genotoxic pattern can correspondingly be discerned for both UDMA and TEGDMA: their genotoxicity is attributed respectively to 49% and 44% of mitotic recombination, as well as 51% and 56% of mutational events, including point and chromosomal alterations. The monomer UDMA is 1.6 times more active than TEGDMA to induce mutant clones per treatment unit. BisGMA and HEMA had no statistically significant effect on total spot frequencies - suggesting no genotoxic action in the SMART assay. The clinical significance of these observations has to be interpreted for data obtained in other bioassays.

  2. Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.

    Directory of Open Access Journals (Sweden)

    Foad J Rouhani

    2016-04-01

    Full Text Available The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs, their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.

  3. Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.

    Directory of Open Access Journals (Sweden)

    Foad J Rouhani

    2016-04-01

    Full Text Available The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs, their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.

  4. Cell Cycle Activity of Neurogenic and Oligodendrogenic Progenitors in the Diseased Mammalian Brain

    Directory of Open Access Journals (Sweden)

    Sara eBragado Alonso

    2014-02-01

    Full Text Available Basic research during embryonic development has led to the identification of general principles governing cell cycle progression, proliferation and differentiation of mammalian neural stem cells (NSC. These findings were recently translated to the adult brain in an attempt to identify the overall principles governing stemness in the two contexts and allowing us to manipulate the expansion of NSC for regenerative therapies. However, and despite a huge literature on embryonic neural precursors, very little is known about cell cycle parameters of adult neural, or any other somatic, stem cell. In this review, we briefly discuss the long journey of NSC research from embryonic development to adult homeostasis, aging and therapy with a specific focus on their quiescence and cell cycle length in physiological conditions and neurological disorders. Particular attention is given to a new important player in the field, oligodendrocyte progenitors, while discussing the limitation hampering further development in this challenging area.

  5. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

    OpenAIRE

    Klein, U.; Klein, G.; Ehlin-Henriksson, B.; Rajewsky, K.; Küppers, R.

    1995-01-01

    BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells a...

  6. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    Directory of Open Access Journals (Sweden)

    Paves Heiti

    2004-04-01

    Full Text Available Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

  7. Low density of membrane particles in auditory hair cells of lizards and birds suggests an absence of somatic motility.

    Science.gov (United States)

    Köppl, Christine; Forge, Andrew; Manley, Geoffrey A

    2004-11-08

    Hair cells are the mechanoreceptive cells of the vertebrate lateral line and inner ear. In addition to their sensory function, hair cells display motility and thus themselves generate mechanical energy, which is thought to enhance sensitivity. Two principal cellular mechanism are known that can mediate hair-cell motility in vitro. One of these is based on voltage-dependent changes of an intramembrane protein and has so far been demonstrated only in outer hair cells of the mammalian cochlea. Correlated with this, the cell membranes of outer hair cells carry an extreme density of embedded particles, as revealed by freeze fracturing. The present study explored the possibility of membrane-based motility in hair cells of nonmammals, by determining their density of intramembrane particles. Replicas of freeze-fractured membrane were prepared from auditory hair cells of a lizard, the Tokay gecko, and a bird, the barn owl. These species were chosen because of independent evidence for active cochlear mechanics, in the form of spontaneous otoacoustic emissions. For quantitative comparison, mammalian inner and outer hair cells, as well as vestibular hair, cells were reevaluated. Lizard and bird hair cells displayed median densities of 2,360 and 1,880 intramembrane particles/microm2, respectively. This was not significantly different from the densities in vestibular and mammalian inner hair cells; however, it was about half the density in of mammalian outer hair cells. This suggests that nonmammalian hair cells do not possess high densities of motor protein in their membranes and are thus unlikely to be capable of somatic motility. 2004 Wiley-Liss, Inc.

  8. Targeting cellular memory to reprogram the epigenome, restore potential, and improve somatic cell nuclear transfer.

    Science.gov (United States)

    Eilertsen, K J; Power, R A; Harkins, L L; Misica, P

    2007-03-01

    Successful cloning by somatic cell nuclear transfer (SCNT) is thought to require reprogramming of a somatic nucleus to a state of restored totipotentiality [Dean, W., Santos, F., Reik, W., 2003. Epigenetic programming in early mammalian development and following somatic cell nuclear transfer. Semin. Cell. Dev. Biol. 14, 93-100; Jouneau, A., Renard, J.P., 2003. Reprogramming in nuclear transfer. Curr. Opin. Genet. Dev. 13, 486-491; ]. Though SCNT-induced reprogramming is reminiscent of the reprogramming that occurs after fertilization, reprogramming a differentiated nucleus to an embryonic state is delayed and incomplete in comparison (for review, see ). This is likely due to the existence of an epigenetic-based cellular memory, or program, that serves to regulate global patterns of gene expression, and is the basis of a genome defense mechanism that silences viruses and transposons. The mechanisms of this memory include CpG methylation and modification of histones. Recent evidence by Feng et al. [Feng, Y.-Q., Desprat, R., Fu, H., Olivier, E., Lin, C.M., Lobell, A., Gowda, S.N., Aladjem, M.I., Bouhasira, E.E., 2006. DNA methylation supports intrinsic epigenetic memory in mammalian cells. PLOS Genet. 2, 0461-0470], using a transgenic experimental system, indicates that these marks may be acquired in more than one order and thus, silent heterochromatic structure can be initiated by either methylation of CpG dinucleotides or by histone modifications. In this system, however, CpG methylation appears to differ from histone modifications because it bestows a persistent epigenetic, or cellular, memory. In other words, CpG methylation can independently confer cellular memory, whereas histone modifications appear to be limited in this capacity. Therefore, in the context of genomic reprogramming induced by SCNT, efficient demethylation is likely a key (if not the only) rate-limiting step to improving the efficiency and outcomes of SCNT cloning. This review discusses the

  9. Cdc45 limits replicon usage from a low density of preRCs in mammalian cells.

    Science.gov (United States)

    Wong, Philip G; Winter, Sherry L; Zaika, Elena; Cao, Thinh V; Oguz, Umut; Koomen, John M; Hamlin, Joyce L; Alexandrow, Mark G

    2011-03-01

    Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4-5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of

  10. Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Yeoman, Richard R; Nusser, Kevin D; Wolf, Don P

    2002-05-01

    Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

  11. Injection molded pinched flow fractionation device for enrichment of somatic cells in cow milk

    DEFF Research Database (Denmark)

    Jensen, Marie Pødenphant; Marie, Rodolphe; Olesen, Tom;

    2014-01-01

    In this paper the continuous microfluidic separation technique pinched flow fractionation is applied to the enrichment of somatic cells from cow milk. Somatic cells were separated from the smallest fat particles and proteins thus better imaging and analysis of the cells can be achieved...

  12. Amino acids in the cultivation of mammalian cells.

    Science.gov (United States)

    Salazar, Andrew; Keusgen, Michael; von Hagen, Jörg

    2016-05-01

    Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure.

  13. Stable protein expression in mammalian cells using baculoviruses.

    Science.gov (United States)

    Lackner, Andreas; Kreidl, Emanuel; Peter-Vörösmarty, Barbara; Spiegl-Kreinecker, Sabine; Berger, Walter; Grusch, Michael

    2012-01-01

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been widely used in biotechnology for protein expression in insect cells. Baculoviruses use arthropods as their natural hosts and are unable to replicate in mammalian cells. However, AcMNPV is able to enter many mammalian cell types and can be used for transgene expression if engineered to contain suitable expression cassettes. In this chapter, we describe the construction and application of a recombinant baculovirus containing a bicistronic expression cassette that can be used for stable protein expression in mammalian cells. As an example, the generation of glioblastoma and hepatocellular carcinoma cell lines stably expressing green fluorescent protein after puromycin selection is shown.

  14. Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

    Science.gov (United States)

    Hwang, Jeong Ho; Kim, Sang Eun; Gupta, Mukesh Kumar; Lee, HoonTaek

    2016-08-01

    Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos.

  15. Production of transgenic calves by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; WAN Rong; HUANG Yinghua; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Lili; WANG Haiping; TANG Bo; LIU Ying; LI Rong

    2004-01-01

    Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.

  16. An internal ribosome entry site (IRES mutant library for tuning expression level of multiple genes in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Esther Y C Koh

    Full Text Available A set of mutated Encephalomyocarditis virus (EMCV internal ribosome entry site (IRES elements with varying strengths is generated by mutating the translation initiation codons of 10(th, 11(th, and 12(th AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells.

  17. Agronomic traits and RAPD analysis of two mutants derived from rice somatic cell culturing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Genetic variation, including agronomic trait variation, often occurs in somatic cell culturing. In this study, we compared the main agronomic traits of two rice mutants, M3 and M14, which were derived from Shenxiangjing 5 somatic cell culturing. Significant differences were found between the two mutants and the wild rice Shenxiangjing 5 (Table 1). Results were as follows:

  18. Expression and purification of splicing proteins from mammalian cells.

    Science.gov (United States)

    Allemand, Eric; Hastings, Michelle L

    2014-01-01

    Pre-mRNA splicing is a complex process that is carried out by a large ribonucleoprotein enzyme, termed the spliceosome, which comprises up to 200 proteins. Despite this complexity, the role of individual spliceosomal proteins in the splicing reaction has been successfully investigated using cell-free assays. In many cases, the splicing factor of interest must be expressed and purified in order to study its function in vitro. Posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination of splicing factors are important for activity. Thus, their purification from mammalian cells presents numerous advantages. Here, we describe a method for expression and purification of splicing proteins from mammalian cells.

  19. Somatic cell and factors which affect their count in milk

    Directory of Open Access Journals (Sweden)

    Zrinka Čačić

    2003-01-01

    Full Text Available Milk quality is determined by chemical composition, physical characteristics and hygienic parameters. The main indicators of hygienic quality of milk are total number of microorganisms and somatic cell count (SCC. Environmental factors have the greatest influence on increasing SCC. The most important environmental parameters are status of udder infection, age of cow, stage of lactation, number of lactation, breed, housing, geographicalarea and seasons, herd size, stress, heavy physical activity and, milking. A farmer (milk producer himself can control a great number of environmental factors using good management practise and permanent education. Since SCC participate in creating the price of milk, it is necessary to inform milk producers how to organise their production so that they would produce maximum quantity of good hygienic quality milk.

  20. Somatic Stem Cells and Their Dysfunction in Endometriosis

    Science.gov (United States)

    Djokovic, Dusan; Calhaz-Jorge, Carlos

    2015-01-01

    Emerging evidence indicates that somatic stem cells (SSCs) of different types prominently contribute to endometrium-associated disorders such as endometriosis. We reviewed the pertinent studies available on PubMed, published in English language until December 2014 and focused on the involvement of SSCs in the pathogenesis of this common gynecological disease. A concise summary of the data obtained from in vitro experiments, animal models, and human tissue analyses provides insights into the SSC dysregulation in endometriotic lesions. In addition, a set of research results is presented supporting that SSC-targeting, in combination with hormonal therapy, may result in improved control of the disease, while a more in-depth characterization of endometriosis SSCs may contribute to the development of early-disease diagnostic tests with increased sensitivity and specificity. Key message: Seemingly essential for the establishment and progression of endometriotic lesions, dysregulated SSCs, and associated molecular alterations hold a promise as potential endometriosis markers and therapeutic targets. PMID:25593975

  1. A conditional Orco requirement in the somatic cyst cells for maintaining spermatids in a tight bundle in Drosophila testis

    Indian Academy of Sciences (India)

    Pankaj Dubey; Prakash Joti; Krishanu Ray

    2016-06-01

    Odorant receptors (OR) heterodimerizes with the OR co-receptor (Orco), forming specific odorant-gated cation channels, which are key to odor reception at the olfactory sensory neurons (OSN). Mammalian ORs are expressed in many other tissues, including testis. However, their biological implications are yet to be fully ascertained. In the mosquito, Orco is localized along the sperm tail and is indicated to maintain fidelity. Here, we show that orco expresses in Drosophila testis. The levels are higher in the somatic cyst cells. The orco-null mutants are perfectly fertile at 25°C. At 28°C, the coiled spermatid bundles are severely disrupted. The loss of Orco also disrupts the actin cap, which forms inside the head cyst cell at the rostral ends of the spermatid nuclei after coiling, and plays a key role in preventing the abnormal release of spermatids from the cyst enclosure. Both the defects are rescued by the somatic cyst cell-specific expression of the UAS-orco transgene. These results highlight a novel role of Orco in the somatic tissue during sperm release.

  2. Birth of Beagle dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Hossein, Mohammad Shamim; Jeong, Yeon Woo; Park, Sun Woo; Kim, Joung Joo; Lee, Eugine; Ko, Kyeong Hee; Hyuk, Park; Hoon, Song Seung; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hwang, Woo Suk

    2009-09-01

    The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 micros electrical stimulation, chemically activated after 1h of fusion using 10 microM calcium ionophore for 4 min and cultured 4h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4-10, 11-25 and 26-40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P>0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4-10 embryos (n=7) and 26-40 embryos (n=12) while pregnancy was detected in 23.53% recipients received a group of 11-25 embryos (n=17). Among them, five (1.76%) live puppies were born (P<0.05). These data show an increase in the overall efficiency of SCNT in canine species.

  3. Deterministic direct reprogramming of somatic cells to pluripotency.

    Science.gov (United States)

    Rais, Yoach; Zviran, Asaf; Geula, Shay; Gafni, Ohad; Chomsky, Elad; Viukov, Sergey; Mansour, Abed AlFatah; Caspi, Inbal; Krupalnik, Vladislav; Zerbib, Mirie; Maza, Itay; Mor, Nofar; Baran, Dror; Weinberger, Leehee; Jaitin, Diego A; Lara-Astiaso, David; Blecher-Gonen, Ronnie; Shipony, Zohar; Mukamel, Zohar; Hagai, Tzachi; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Tanay, Amos; Amit, Ido; Novershtern, Noa; Hanna, Jacob H

    2013-10-03

    Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

  4. CYTOLOGICAL QUALITY OF GOAT MILK ON THE BASIS OF THE SOMATIC CELL COUNT

    Directory of Open Access Journals (Sweden)

    Henryka BERNACKA

    2007-07-01

    Full Text Available The aim of the present paper was to evaluate the cytological quality of goat milk based on the somatic cell count in respective months of lactation. Besides there was defined the effect of somatic cell on the milk production and chemical composition of milk. The research covered goats of color improved breed in the 2nd and 3rd lactation. Daily milk yield, chemical composition of milk and its somatic cell count were defined based on monthly morning and evening control milkings from both teats, following the A4 method applied in District Animal Evaluation Stations. The research indicated that the greater the somatic cell count in milk, the lower the daily milk yield, however the greater the somatic cell count, the greater the percentage content of fat and dry matter and the lower the content of lactose.

  5. Stress-mediated p38 activation promotes somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Xinxiu Xu; Quan Wang; Yuan Long; Ru Zhang; Xiaoyuan Wei; Mingzhe Xing; Haifeng Gu

    2013-01-01

    Environmental stress-mediated adaptation plays essential roles in the evolution of life.Cellular adaptation mechanisms usually involve the regulation of chromatin structure,transcription,mRNA stability and translation,which eventually lead to efficient changes in gene expression.Global epigenetic change is also involved in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells by defined factors.Here we report that environmental stress such as hyperosmosis not only facilitates four factor-mediated reprogramming,but also enhances two or one factor-induced iPS cell generation.Hyperosmosis-induced p38 activation plays a critical role in this process.Constitutive active p38 mimics the positive effect of hyperosmosis,while dominant negative p38 and p38 inhibitor block the effect of hyperosmosis.Further study indicates stress-mediated p38 activation may promote reprogramming by reducing the global DNA methylation level and enhancing the expression of pluripotency genes.Our results demonstrate how simple environmental stress like hyperosmosis helps to alter the fate of cells via intracellular signaling and epigenetic modulation.

  6. Chromosome engineering: generation of mono- and dicentric isochromosomes in a somatic cell hybrid system.

    Science.gov (United States)

    Higgins, A W; Schueler, M G; Willard, H F

    1999-08-01

    The most common isochromosome found in humans involves the long arm of the X, i(Xq), and is associated with a subset of Turner syndrome cases. To study the formation and behavior of isochromosomes in a more tractable experimental system, we have developed a somatic cell hybrid model system that allows for the selection of mono- or dicentric isochromosomes involving the short arm of the X, i(Xp). Simultaneous positive and negative counterselection of a mouse/human somatic cell hybrid containing a human X chromosome, selecting for retention of the UBE1 locus in Xp but against the HPRT locus in Xq, results in a variety of abnormalities of the X chromosome involving deletions of Xq. We have generated 70 such "Pushmi-Pullyu" hybrids derived from seven independent X chromosomes. Cytogenetic analysis of these hybrids using fluorescence in situ hybridization showed i(Xp) chromosomes in approximately 19% of the hybrids. Southern blot and polymerase chain reaction analyses of the Pushmi-Pullyu hybrids revealed a distribution of breakpoints along Xq. The distance between the centromeres of the dicentric i(Xp)s generated ranged from approximately 2 Mb to approximately 20 Mb. To examine centromeric activity in these dicentric i(Xp)s, we used indirect immunofluorescence with antibodies to centromere protein E (CENP-E). CENP-E was detected at only one of the centromeres of a dicentric i(Xp) with approximately 2-3 Mb of Xq DNA. In contrast, CENP-E was detected at both centromeres of a dicentric i(Xp) with approximately 14 Mb of Xq DNA. Two other dicentric i(Xp) chromosomes were heterogeneous with respect to centromeric activity, suggesting that centromeric activity and chromosome stability of dicentric chromosomes may be more complicated than previously thought. The Pushmi-Pullyu model system presented in this study may provide a tool for examining the structure and function of mammalian centromeres.

  7. Somatic deletions implicated in functional diversity of brain cells of individuals with schizophrenia and unaffected controls.

    Science.gov (United States)

    Kim, Junho; Shin, Jong-Yeon; Kim, Jong-Il; Seo, Jeong-Sun; Webster, Maree J; Lee, Doheon; Kim, Sanghyeon

    2014-01-22

    While somatic DNA copy number variations (CNVs) have been identified in multiple tissues from normal people, they have not been well studied in brain tissues from individuals with psychiatric disorders. With ultrahigh depth sequencing data, we developed an integrated pipeline for calling somatic deletions using data from multiple tissues of the same individual or a single tissue type taken from multiple individuals. Using the pipelines, we identified 106 somatic deletions in DNA from prefrontal cortex (PFC) and/or cerebellum of two normal controls subjects and/or three individuals with schizophrenia. We then validated somatic deletions in 18 genic and in 1 intergenic region. Somatic deletions in BOD1 and CBX3 were reconfirmed using DNA isolated from non-pyramidal neurons and from cells in white matter using laser capture microdissection (LCM). Our results suggest that somatic deletions may affect metabolic processes and brain development in a region specific manner.

  8. Somatic Embryogenesis from Cell Suspension Cultures of Aspen Clone

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Suspension cultures initiated from callus derived from petiole explants of aspen hybrid (Populus tremuloides × P.tremula) produced somatic embryos. Callus was induced on a MS medium supplemented with 5 mg·L-1 2,4-D and 0.05 mg·L-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10 mg·L-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10 mg·L-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.

  9. The mechanism of gene targeting in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Yinan Kan

    2014-04-01

    Full Text Available Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB repair known as homologous recombination (HR. The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

  10. The mechanism of gene targeting in human somatic cells.

    Science.gov (United States)

    Kan, Yinan; Ruis, Brian; Lin, Sherry; Hendrickson, Eric A

    2014-04-01

    Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

  11. Fungal cell gigantism during mammalian infection.

    Directory of Open Access Journals (Sweden)

    Oscar Zaragoza

    2010-06-01

    Full Text Available The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

  12. Fungal cell gigantism during mammalian infection.

    Science.gov (United States)

    Zaragoza, Oscar; García-Rodas, Rocío; Nosanchuk, Joshua D; Cuenca-Estrella, Manuel; Rodríguez-Tudela, Juan Luis; Casadevall, Arturo

    2010-01-01

    The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

  13. Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes).

    Science.gov (United States)

    Bubenshchikova, Ekaterina; Kaftanovskaya, Elena; Motosugi, Nami; Fujimoto, Takafumi; Arai, Katsutoshi; Kinoshita, Masato; Hashimoto, Hisashi; Ozato, Kenjiro; Wakamatsu, Yuko

    2007-12-01

    Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.

  14. Somatic cell nuclear transfer and derivation of embryonic stem cells in the mouse.

    Science.gov (United States)

    Markoulaki, Styliani; Meissner, Alexander; Jaenisch, Rudolf

    2008-06-01

    Addressing the fundamental questions of nuclear equivalence in somatic cells has fascinated scientists for decades and has resulted in the development of somatic cell nuclear transfer (SCNT) or animal cloning. SCNT involves the transfer of the nucleus of a somatic cell into the cytoplasm of an egg whose own chromosomes have been removed. In the mouse, SCNT has not only been successfully used to address the issue of nuclear equivalence, but has been used as a model system to test the hypothesis that embryonic stem cells (ESCs) derived from NT blastocysts have the potential to correct--through genetic manipulations--degenerative diseases. This paper aims to provide a comprehensive description of SCNT in the mouse and the derivation of ESCs from blastocysts generated by this technique. SCNT is a very challenging and inefficient procedure because it is technically complex, it bypasses the normal events of gamete interactions and egg activation, and it depends on adequate reprogramming of the somatic cell nucleus in vivo. Improvements in any or all those aspects may enhance the efficiency and applicability of SCNT. ESC derivation from SCNT blastocysts, on the other hand, requires the survival of only a few successfully reprogrammed cells, which have the capacity to proliferate indefinitely in vitro, maintain correct genetic and epigenetic status, and differentiate into any cell type in the body--characteristics that are essential for transplantation therapy or any other in vivo application.

  15. TCPs, WUSs, and WINDs: Families of transcription factors that regulate shoot meristem formation, stem cell maintenance, and somatic cell differentiation

    Directory of Open Access Journals (Sweden)

    Miho eIkeda

    2014-09-01

    Full Text Available In contrast to somatic mammalian cells, which cannot alter their fate, plant cells can dedifferentiate to form totipotent callus cells and regenerate a whole plant, following treatment with specific phytohormones. However, the regulatory mechanisms and key factors that control differentiation-dedifferentiation and cell totipotency have not been completely clarified in plants. Recently, several plant transcription factors that regulate meristem formation and dedifferentiation have been identified and include members of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP, WUSCHEL (WUS, and WOUND INDUCED DEDIFFERENTIATION (WIND1 families. WUS and WIND positively control plant cell totipotency, while TCP negatively controls it. Interestingly, TCP is a transcriptional activator that acts as a negative regulator of shoot meristem formation, and WUS is a transcriptional repressor that positively maintains totipotency of the stem cells of the shoot meristem. We describe here the functions of TCP, WUS and WIND transcription factors in the regulation of differentiation-dedifferentiation by positive and negative transcriptional regulators.

  16. File list: InP.Emb.10.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.10.AllAg.Gonadal_somatic_cells mm9 Input control Embryo Gonadal somatic cel...ls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.10.AllAg.Gonadal_somatic_cells.bed ...

  17. File list: NoD.Emb.10.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.10.AllAg.Gonadal_somatic_cells mm9 No description Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.10.AllAg.Gonadal_somatic_cells.bed ...

  18. File list: NoD.Emb.05.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.05.AllAg.Gonadal_somatic_cells mm9 No description Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.05.AllAg.Gonadal_somatic_cells.bed ...

  19. File list: NoD.Emb.50.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.50.AllAg.Gonadal_somatic_cells mm9 No description Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.50.AllAg.Gonadal_somatic_cells.bed ...

  20. File list: InP.Emb.50.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.Gonadal_somatic_cells mm9 Input control Embryo Gonadal somatic cel...ls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.Gonadal_somatic_cells.bed ...

  1. File list: InP.Emb.05.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.05.AllAg.Gonadal_somatic_cells mm9 Input control Embryo Gonadal somatic cel...ls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.05.AllAg.Gonadal_somatic_cells.bed ...

  2. File list: NoD.Emb.20.AllAg.Gonadal_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.20.AllAg.Gonadal_somatic_cells mm9 No description Embryo Gonadal somatic ce...lls http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.20.AllAg.Gonadal_somatic_cells.bed ...

  3. File list: NoD.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Gon.20.AllAg.Testicular_somatic_cells mm9 No description Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  4. File list: NoD.Gon.05.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Gon.05.AllAg.Testicular_somatic_cells mm9 No description Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Gon.05.AllAg.Testicular_somatic_cells.bed ...

  5. File list: NoD.Gon.10.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Gon.10.AllAg.Testicular_somatic_cells mm9 No description Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Gon.10.AllAg.Testicular_somatic_cells.bed ...

  6. File list: InP.Gon.10.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Gon.10.AllAg.Testicular_somatic_cells mm9 Input control Gonad Testicular somatic... cells SRX591728,SRX591716 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Gon.10.AllAg.Testicular_somatic_cells.bed ...

  7. File list: NoD.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Gon.50.AllAg.Testicular_somatic_cells mm9 No description Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  8. File list: InP.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Gon.20.AllAg.Testicular_somatic_cells mm9 Input control Gonad Testicular somatic... cells SRX591728,SRX591716 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  9. File list: InP.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Gon.50.AllAg.Testicular_somatic_cells mm9 Input control Gonad Testicular somatic... cells SRX591728,SRX591716 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  10. Regulators of DNA methylation in mammalian cells

    OpenAIRE

    Termanis, Ausma

    2013-01-01

    Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly t...

  11. PepGMV Rep-Protein Expression in Mammalian Cells

    Science.gov (United States)

    Chapa-Oliver, Angela María; Mejía-Teniente, Laura; García-Gasca, Teresa; Guevara-Gonzalez, Ramon Gerardo; Torres-Pacheco, Irineo

    2012-01-01

    The Geminiviruses genome is a small, single strand DNA that replicates in the plant cell nucleus. Analogous to animal DNA viruses, Geminiviruses depend on the host replication machinery to amplify their genomes and only supply the factors required to initiate their replication. Consequently, Geminiviruses remove the cell-cycle arrest and induce the host replication machinery using an endocycle process. They encode proteins, such as the conserved replication-associated proteins (Rep) that interact with retinoblastoma-like proteins in plants and alter the cell division cycle in yeasts. Therefore, the aim of this work is to analyze the impact of Pepper Golden Mosaic Virus (PepGMV) Rep protein in mammalian cells. Results indicate that the pTracer-SV40:Rep construction obtained in this work can be used to analyze the Rep protein effect in mammalian cells in order to compare the cell cycle regulation mechanisms in plants and animals. PMID:23170183

  12. Wave characterization for mammalian cell culture: residence time distribution.

    Science.gov (United States)

    Rodrigues, Maria Elisa; Costa, Ana Rita; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2012-02-15

    The high dose requirements of biopharmaceutical products led to the development of mammalian cell culture technologies that increase biomanufacturing capacity. The disposable Wave bioreactor is one of the most promising technologies, providing ease of operation and no cross-contamination, and using an innovative undulation movement that ensures good mixing and oxygen transfer without cell damage. However, its recentness demands further characterization. This study evaluated the residence time distribution (RTD) in Wave, allowing the characterization of mixing and flow and the comparison with ideal models and a Stirred tank reactor (STR) used for mammalian cell culture. RTD was determined using methylene blue with pulse input methodology, at three flow rates common in mammalian cell culture (3.3×10(-5)m(3)/h, 7.9×10(-5)m(3)/h, and 1.25×10(-4)m(3)/h) and one typical of microbial culture (5×10(-3)m(3)/h). Samples were taken periodically and the absorbance read at 660nm. It was observed that Wave behavior diverted from ideal models, but was similar to STR. Therefore, the deviations are not related to the particular Wave rocking mechanism, but could be associated with the inadequacy of these reactors to operate in continuous mode or to a possible inability of the theoretical models to properly describe the behavior of reactors designed for mammalian cell culture. Thus, the development of new theoretical models could better characterize the performance of these reactors.

  13. Antigen receptors and somatic hypermutation in B-cell chronic lymphocytic leukemia with Richter's transformation

    NARCIS (Netherlands)

    L.A. Smit; F. van Maldegem; A.W. Langerak; C.E. van der Schoot; M.J. de Wit; S. Bea; E. Campo; R.J. Bende; C.J.M. van Noesel

    2006-01-01

    Background and Objectives. Activation-induced cytidine deaminase is essential for somatic hypermutation and class switch recombination of the immunoglobulin genes in B cells. It has been proposed that aberrant targeting of the somatic hypermutation machinery is instrumental in initiation and progres

  14. Reconstituting mammalian spermatogenesis using stem cells

    Institute of Scientific and Technical Information of China (English)

    Paul J Turek

    2011-01-01

    While stock markets and economies are smoldering all over the world,stem cell science is on fire.The promise of curing many untreatable forms of human male infertility is now one small step closer based on a recent paper in Cell.1 In this work,scientists from Kyoto University in Japan created mature,fertile sperm from embryonic and induced pluripotent stem cells by grabbing the primitive germ cells called primordial germ cells just as they were being made from their pluripotent precursors in vitro and transplanting them back into genetically sterile mice.

  15. Propagation of elite rescue dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Oh, Hyun Ju; Choi, Jin; Kim, Min Jung; Kim, Geon A; Jo, Young Kwang; Choi, Yoo Bin; Lee, Byeong Chun

    2016-01-01

    The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6-dimethylaminopyridine (DMAP) (SOF-DMAP) or porcine zygote medium (PZM-5) with 1.9 mmol/L DMAP (PZM-DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF-DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6-DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.

  16. Somatic cell counts in bulk milk and their importance for milk processing

    Science.gov (United States)

    Savić, N. R.; Mikulec, D. P.; Radovanović, R. S.

    2017-09-01

    Bulk tank milk somatic cell counts are the indicator of the mammary gland health in the dairy herds and may be regarded as an indirect measure of milk quality. Elevated somatic cell counts are correlated with changes in milk composition The aim of this study was to assess the somatic cell counts that significantly affect the quality of milk and dairy products. We examined the somatic cell counts in bulk tank milk samples from 38 farms during the period of 6 months, from December to the May of the next year. The flow cytometry, Fossomatic was used for determination of somatic cell counts. In the same samples content of total proteins and lactose was determined by Milcoscan. Our results showed that average values for bulk tank milk samples were 273,605/ml from morning milking and 292,895/ml from evening milking. The average values for total proteins content from morning and evening milking are 3,31 and 3,34%, respectively. The average values for lactose content from morning and evening milking are 4,56 and 4,63%, respectively. The highest somatic cell count (516,000/ml) was detected in bulk tank milk sample from evening milk in the Winter and the lowest content of lactose was 4,46%. Our results showed that obtained values for bulk tank milk somatic cell counts did not significantly affected the content of total proteins and lactose.

  17. Complementation of mutant phenotypes and genotypes of cultured mammalian cells

    NARCIS (Netherlands)

    A.J.R. de Jonge

    1985-01-01

    textabstractThis dissertation describes experiments aimed at the complementation of a genetic mutation in cultured mammalian cells in order to investigate several aspects of the structure and functioning of the human genome. Complementation is indicated by the correction of a biochemical function in

  18. Current status and applications of somatic cell nuclear transfer in dogs.

    Science.gov (United States)

    Jang, Goo; Kim, Min Kyu; Lee, Byeong Chun

    2010-11-01

    Although somatic cell nuclear transfer (SCNT) technology and applications are well developed in most domesticated and laboratory animals, their use in dogs has advanced only slowly. Many technical difficulties had to be overcome before preliminary experiments could be conducted. First, due to the very low efficiency of dog oocyte maturation in vitro, in vivo matured oocytes were generally used. The nucleus of an in vivo matured oocyte was removed and a donor cell (from fetal or adult fibroblasts) was injected into the oocyte. Secondly, fusion of the reconstructed oocytes was problematic, and it was found that a higher electrical voltage was necessary, in comparison to other mammalian species. By transferring the resulting fused oocytes into surrogate females, several cloned offspring were born. SCNT was also used for producing cloned wolves, validating reproductive technologies for aiding conservation of endangered or extinct breeds. Although examples of transgenesis in canine species are very sparse, SCNT studies are increasing, and together with the new field of gene targeting technology, they have been applied in many fields of veterinary or bio-medical science. This review summarizes the current status of SCNT in dogs and evaluates its potential future applications.

  19. From cloned frogs to patient matched stem cells: induced pluripotency or somatic cell nuclear transfer?

    Science.gov (United States)

    Yamada, Mitsutoshi; Byrne, James; Egli, Dieter

    2015-10-01

    Nuclear transfer has seen a remarkable comeback in the past few years. Three groups have independently reported the derivation of stem cell lines by somatic cell nuclear transfer, from either adult, neonatal or fetal cells. Though the ability of human oocytes to reprogram somatic cells to stem cells had long been anticipated, success did not arrive on a straightforward path. Little was known about human oocyte biology, and nuclear transfer protocols developed in animals required key changes to become effective with human eggs. By overcoming these challenges, human nuclear transfer research has contributed to a greater understanding of oocyte biology, provided a point of reference for the comparison of induced pluripotent stem cells, and delivered a method for the generation of personalized stem cells with therapeutic potential.

  20. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific productivit

  1. Transcriptome Landscapes of Mammalian Embryonic Cells

    NARCIS (Netherlands)

    Brinkhof, B.

    2015-01-01

    This thesis describes research on gene expression profiles from different embryonic stages and cell types to identify genes involved in pluripotency or differentiation in bovine and porcine cells. The results are compared with data from other mammals. RNA expression profiles of morula and blastocyst

  2. Transcriptome Landscapes of Mammalian Embryonic Cells

    NARCIS (Netherlands)

    Brinkhof, B.

    2015-01-01

    This thesis describes research on gene expression profiles from different embryonic stages and cell types to identify genes involved in pluripotency or differentiation in bovine and porcine cells. The results are compared with data from other mammals. RNA expression profiles of morula and blastocyst

  3. Mammalian cell delivery via aerosol deposition.

    Science.gov (United States)

    Veazey, William S; Anusavice, Kenneth J; Moore, Karen

    2005-02-15

    The objective of this study was to test the hypothesis that bovine dermal fibroblasts can survive aerosol delivery via an airbrush with mean cell survival rates greater than 50%. This technology has great implications for burn and other wound therapies, for delivery of genetically altered cells in gene therapies, and for tissue engineering with tissue scaffolds. Bovine dermal fibroblasts were suspended at a concentration of 200,000 cells/mL in Hank's Balanced Salt Solution, and delivered into six-well tissue culture plates using a Badger 100G airbrush. Cells were delivered through three nozzle diameters (312, 484, and 746 microm) at five different air pressures (41, 55, 69, 96, and 124 kPa). Nine repetitions were performed for each treatment group, and cell viability was measured using trypan blue exclusion assay. Mean cell viability ranged from 37 to 94%, and depended on the combination of nozzle diameter and delivery pressure (p < 0.0001). Linear regression analysis was used to develop a stochastic model of cell delivery viability as a function of nozzle diameter and delivery air pressure. This study demonstrates the feasibility of using an airbrush to deliver viable cells in an aerosol to a substrate.

  4. Cryopreservation of Spin-Dried Mammalian Cells

    OpenAIRE

    Nilay Chakraborty; Menze, Michael A.; Jason Malsam; Alptekin Aksan; Hand, Steven C.; Mehmet Toner

    2011-01-01

    This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (

  5. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific

  6. Equipment for large-scale mammalian cell culture.

    Science.gov (United States)

    Ozturk, Sadettin S

    2014-01-01

    This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed.

  7. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

  8. Calcium Imaging of Sonoporation of Mammalian Cells

    Science.gov (United States)

    Sabens, David; Aehle, Matthew; Steyer, Grant; Kourennyi, Dmitri; Deng, Cheri X.

    2006-05-01

    Ultrasound mediated delivery of compounds is a relatively recent development in drug delivery and gene transfection techniques. Due to the lack of methods for real-time monitoring of sonoporation at the cellular level, the efficiency of drug/gene delivery and sonoporation associated side effects, such as the loss of cell viability and enhanced apoptosis, have been studied only through post US exposure analyses, requiring days for cell incubation. Furthermore, because microporation appears to be transient in nature, it was not possible to correlate transfection with microporation on an individual cellular basis. By studying the role of calcium in the cell and using fluorescent calcium imaging to study sonoporation it is possible to quantify both cell porosity and sonoporation side effects. Since both post sonoporation cell survival and delivery efficiency are related to the dynamic process of the cell membrane poration, calcium imaging of sonoporation will provide important knowledge to obtain improved understanding of sonoporation mechanism. Our experimental results demonstrated the feasibility of calcium imaging of sonoporation in Chinese Hamster Ovary (CHO) cells. We have measured the changes in the intracellular calcium concentration using Fura-2, a fluorescent probe, which indicate influx or flow of Calcium across the cell membrane. Analysis of data identified key aspects in the dynamic sonoporation process including the formation of pores in the cell membrane, and the relative temporal duration of the pores and their resealing. These observations are obtained through the analysis of the rate the calcium concentration changes within the cells, making it possible to visualize membrane opening and repair in real-time through such changes in the intracellular calcium concentration.

  9. Quantitative genetic-interaction mapping in mammalian cells

    Science.gov (United States)

    Roguev, Assen; Talbot, Dale; Negri, Gian Luca; Shales, Michael; Cagney, Gerard; Bandyopadhyay, Sourav; Panning, Barbara; Krogan, Nevan J

    2013-01-01

    Mapping genetic interactions (GIs) by simultaneously perturbing pairs of genes is a powerful tool for understanding complex biological phenomena. Here we describe an experimental platform for generating quantitative GI maps in mammalian cells using a combinatorial RNA interference strategy. We performed ~11,000 pairwise knockdowns in mouse fibroblasts, focusing on 130 factors involved in chromatin regulation to create a GI map. Comparison of the GI and protein-protein interaction (PPI) data revealed that pairs of genes exhibiting positive GIs and/or similar genetic profiles were predictive of the corresponding proteins being physically associated. The mammalian GI map identified pathways and complexes but also resolved functionally distinct submodules within larger protein complexes. By integrating GI and PPI data, we created a functional map of chromatin complexes in mouse fibroblasts, revealing that the PAF complex is a central player in the mammalian chromatin landscape. PMID:23407553

  10. Sex determination in mammalian germ cells

    OpenAIRE

    Spiller, Cassy M; Josephine Bowles

    2015-01-01

    Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatog...

  11. Cryopreservation of spin-dried mammalian cells.

    Directory of Open Access Journals (Sweden)

    Nilay Chakraborty

    Full Text Available This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1. Fourier Transform Infrared Spectroscopy (FTIR was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN(2 at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.

  12. Cryopreservation of spin-dried mammalian cells.

    Science.gov (United States)

    Chakraborty, Nilay; Menze, Michael A; Malsam, Jason; Aksan, Alptekin; Hand, Steven C; Toner, Mehmet

    2011-01-01

    This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN(2)) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.

  13. Protein diffusion in mammalian cell cytoplasm.

    Science.gov (United States)

    Kühn, Thomas; Ihalainen, Teemu O; Hyväluoma, Jari; Dross, Nicolas; Willman, Sami F; Langowski, Jörg; Vihinen-Ranta, Maija; Timonen, Jussi

    2011-01-01

    We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.

  14. Protein diffusion in mammalian cell cytoplasm.

    Directory of Open Access Journals (Sweden)

    Thomas Kühn

    Full Text Available We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS. A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.

  15. Occurrence of mastitis pathogens in relation to somatic cells

    Directory of Open Access Journals (Sweden)

    Marcela Vyletělová Klimešová

    2013-01-01

    Full Text Available There were examined 161 cows from 4 farms in total. The suspect animals were selected according to viscosity test results, clinical symptoms and somatic cell count (SCC. Milk samples were examined for the presence of pathogens and for SCC. 55 mastitis pathogens were identified. The most frequently isolated species was Enterococcus faecalis (n = 20, followed by Staphylococcus aureus (n = 6 and Streptococcus uberis (n = 5. The SCC ranged from 9 to 24 204 ths.ml−1. There was positive occurrence of bacteria genus Staphylococcus and Enterococcus at lower SCC (50 ths.ml−1 and at higher SCC numbers (> 300 ths. ml−1 bacteria genus Streptococcus, Enterobacter and Escherichia coli. Differences in SCC were significant (P < 0.001 in negative samples xg 131 SCC versus 491 for positive, 611 for staphylococci and 464 ths.ml−1 for other positive. SCC discrimination limit for practical likelihood of pathogen occurrence estimation in infectious sample groups was calculated. This limit for suspicion of infection is 159 for positive group, 113 for staphylococci and 174 ths.ml−1 for other positive. This could be possible to recommend the value 174 ths.ml−1 for practical use with target to apply preventive or curative measures.

  16. Cell therapy using induced pluripotent stem cells or somatic stem cells: this is the question.

    Science.gov (United States)

    Somoza, Rodrigo A; Rubio, Francisco J

    2012-05-01

    A lot of effort has been developed to bypass the use of embryonic stem cells (ES) in human therapies, because of several concerns and ethical issues. Some unsolved problems of using stem cells for human therapies, excluding the human embryonic origin, are: how to regulate cell plasticity and proliferation, immunological compatibility, potential adverse side-effects when stem cells are systemically administrated, and the in vivo signals to rule out a specific cell fate after transplantation. Currently, it is known that almost all tissues of an adult organism have somatic stem cells (SSC). Whereas ES are primary involved in the genesis of new tissues and organs, SSC are involved in regeneration processes, immuno-regulatory and homeostasis mechanisms. Although the differentiating potential of ES is higher than SSC, several studies suggest that some types of SSC, such as mesenchymal stem cells (MSC), can be induced epigenetically to differentiate into tissue-specific cells of different lineages. This unexpected pluripotency and the variety of sources that they come from, can make MSC-like cells suitable for the treatment of diverse pathologies and injuries. New hopes for cell therapy came from somatic/mature cells and the discovery that could be reprogrammed to a pluripotent stage similar to ES, thus generating induced pluripotent stem cells (iPS). For this, it is necessary to overexpress four main reprogramming factors, Sox2, Oct4, Klf4 and c-Myc. The aim of this review is to analyze the potential and requirements of cellular based tools in human therapy strategies, focusing on the advantage of using MSC over iPS.

  17. Live-cell imaging of mammalian RNAs with Spinach2

    Science.gov (United States)

    Strack, Rita L.; Jaffrey, Samie R.

    2015-01-01

    The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of “RNA mimics of GFP” or Spinach technology for tagging mammalian RNAs, and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag. PMID:25605384

  18. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

    Directory of Open Access Journals (Sweden)

    Sharma Shikhar

    2012-01-01

    Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

  19. Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

    Science.gov (United States)

    Hirsch, Calley L; Coban Akdemir, Zeynep; Wang, Li; Jayakumaran, Gowtham; Trcka, Dan; Weiss, Alexander; Hernandez, J Javier; Pan, Qun; Han, Hong; Xu, Xueping; Xia, Zheng; Salinger, Andrew P; Wilson, Marenda; Vizeacoumar, Frederick; Datti, Alessandro; Li, Wei; Cooney, Austin J; Barton, Michelle C; Blencowe, Benjamin J; Wrana, Jeffrey L; Dent, Sharon Y R

    2015-04-15

    Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming.

  20. Intracellular transport of cholesterol in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Brasaemle, D.L.

    1989-01-01

    The erythrocyte was selected as a simple cell for the study of transbilayer movement of cholesterol. Cholesterol oxidase was used to measure the distribution of ({sup 3}H)cholesterol across the erythrocyte membrane. Cholesterol oxidase was also used to estimate the rate of transport of low density lipoprotein (LDL) cholesterol to the plasma membrane of cultured Chinese hamster ovary (CHO) fibroblasts; the half-time of this process was 42 minutes. The rate of transport of LDL cholesterol to the plasma membrane was confirmed by a second procedure using amphotericin B. Amphotericin B was also used to estimate the rate of transport of endogenously synthesized cholesterol to the plasma membrane of CHO cells. New methodology was developed including improvements of the previously published cholesterol oxidase assay for plasma membrane cholesterol. A new method for detecting transport of cholesterol to the plasma membrane in cultured cells was developed using amphotericin B. Preliminary studies investigated the use of fluorescent polyenes, pimaricin and etruscomycin, as probes for plasma membrane cholesterol in transport studies. Finally, a modification of a previously published cell staining protocol yielded a simple, quantitative assay for cell growth.

  1. Crude subcellular fractionation of cultured mammalian cell lines

    OpenAIRE

    Holden Paul; Horton William A

    2009-01-01

    Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this...

  2. Universal area distributions in the monolayers of confluent mammalian cells

    Science.gov (United States)

    Wilk, Gary; Iwasa, Masatomo; Fuller, Patrick E.; Kandere-Grzybowska, Kristiana; Grzybowski, Bartosz A.

    2014-01-01

    When mammalian cells form confluent monolayers completely filling a plane, these apparently random “tilings” show regularity in the statistics of cell areas for various types of epithelial and endothelial cells. The observed distributions are reproduced by a model which accounts for cell growth and division, with the latter treated stochastically both in terms of the sizes of the dividing cells as well as the sizes of the “newborn” ones – remarkably, the modeled and experimental distributions fit well when all free parameters are estimated directly from experiments. PMID:24745461

  3. Space radiation effects on plant and mammalian cells

    Science.gov (United States)

    Arena, C.; De Micco, V.; Macaeva, E.; Quintens, R.

    2014-11-01

    The study of the effects of ionizing radiation on organisms is related to different research aims. The current review emphasizes the studies on the effects of different doses of sparsely and densely ionizing radiation on living organisms, with the final purpose of highlighting specific and common effects of space radiation in mammals and plants. This topic is extremely relevant in the context of radiation protection from space environment. The response of different organisms to ionizing radiation depends on the radiation quality/dose and/or the intrinsic characteristics of the living system. Macromolecules, in particular DNA, are the critical targets of radiation, even if there is a strong difference between damages encountered by plant and mammalian cells. The differences in structure and metabolism between the two cell types are responsible for the higher resistance of the plant cell compared with its animal counterpart. In this review, we report some recent findings from studies performed in Space or on Earth, simulating space-like levels of radiation with ground-based facilities, to understand the effect of ionizing radiation on mammalian and plant cells. In particular, our attention is focused on genetic alterations and repair mechanisms in mammalian cells and on structures and mechanisms conferring radioresistance to plant cells.

  4. Passive versus active local microrheology in mammalian cells and amoebae

    Science.gov (United States)

    Riviere, C.; Gazeau, F.; Marion, S.; Bacri, J.-C.; Wilhelm, C.

    2004-12-01

    We compare in this paper the rotational magnetic microrheology detailed by Marion et al [18] and Wilhelm et al [19] to the passive tracking microrheology. The rotational microrheology has been designed to explore, using magnetic rotating probes, the local intracellular microenvironment of living cells in terms of viscoelasticity. Passive microrheology techniques is based on the analysis of spontaneous diffusive motions of Brownian probes. The dependence of mean square displacement (MSD) with the time then directly reflects the type of movement (sub-, hyper- or diffusive motions). Using the same intracellular probes, we performed two types of measurements (active and passive). Based on the fluctuation-dissipation theorem, one should obtain the same information from the both techniques in a thermally equilibrium system. Interestingly, our measurements differ, and the discordances directly inform on active biological processes, which add to thermally activated fluctuations in our out-of equilibrium systems. In both cell models used, mammalian Hela cells and amoebae Entamoeba Histolytica, a hyper-diffusive regime at a short time is observed, which highlights the presence of an active non-thermal driving force, acting on the probe. However, the nature of this active force in mammalian cells and amoebae is different, according to their different phenotypes. In mammalian cells active processes are governed by the transport, via molecular motors, on the microtubule network. In amoebae, which are highly motile cells free of microtubule network, the active processes are dominated by strong fluxes of cytoplasm driven by extension of pseudopodia, in random directions, leading to an amplitude of motion one order of magnitude higher than for mammalian cells. Figs 7, Refs 32.

  5. Advances in mammalian spermatogonial stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xueming; LI Dexue; YUE Zhanpeng; LI Lingling; YU Jiaao

    2004-01-01

    Spermatogonial stem cell (SSC) transplantation is a novel technique by which testicular cells from normal, transgenic or mutant donor are introduced into the seminiferous tubules of recipient testes through microinjection. Subsequently, donor SSCs survive,migrate, anchor and proliferate in the recipient testis, furthermore, initiate spermatogenesis and even produce sperms capable of fertilization. This technique provides a new approach for the researches of spermatogenesis mechanism, regeneration of spermatogenesis in sterile individuals and reproduction of transgenic animals. This review focuses on the methodological breakthroughs and highlights the recent findings that have substantially increased understanding of SSC biology. The article provides a comprehensive overview of this technique and its multiple applications in basic science and medicine. And the perspective direction of this field in the near future is proposed.

  6. Bartonella entry mechanisms into mammalian host cells.

    Science.gov (United States)

    Eicher, Simone C; Dehio, Christoph

    2012-08-01

    The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment.

  7. A genetically encoded fluorescent probe in mammalian cells.

    Science.gov (United States)

    Chatterjee, Abhishek; Guo, Jiantao; Lee, Hyun Soo; Schultz, Peter G

    2013-08-28

    Fluorescent reporters are useful in vitro and in vivo probes of protein structure, function, and localization. Here we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be site-specifically incorporated into proteins in mammalian cells in response to the TAG codon with high efficiency using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. We further demonstrate that Anap can be used to image the subcellular localization of proteins in live mammalian cells. The small size of Anap, its environment-sensitive fluorescence, and the ability to introduce Anap at specific sites in the proteome by simple mutagenesis make it a unique and valuable tool in eukaryotic cell biology.

  8. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.

    1999-01-01

    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced......'s nutrient mixture F-10 and F-12. Fetal calf serum (5-20%) slightly decreased resazurin reduction during the first 2 days of incubation. The reduction of resazurin by mammalian cell culture media do not appear to be problematic under normal culture conditions, and it is primarily dependent upon the presence...

  9. Sensing the Heat Stress by Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Cates Jordan

    2011-08-01

    Full Text Available Abstract Background The heat-shock response network controls the adaptation and survival of the cell against environmental stress. This network is highly conserved and is connected with many other signaling pathways. A key element of the heat-shock network is the heat-shock transcription factor-1 (HSF, which is transiently activated by elevated temperatures. HSF translocates to the nucleus upon elevated temperatures, forming homotrimeric complexes. The HSF homotrimers bind to the heat shock element on the DNA and control the expression of the hsp70 gene. The Hsp70 proteins protect cells from thermal stress. Thermal stress causes the unfolding of proteins, perturbing thus the pathways under their control. By binding to these proteins, Hsp70 allows them to refold and prevents their aggregation. The modulation of the activity of the hsp70-promoter by the intensity of the input stress is thus critical for cell's survival. The promoter activity starts from a basal level and rapidly increases once the stress is applied, reaches a maximum level and attenuates slowely back to the basal level. This phenomenon is the hallmark of many experimental studies and of all computational network analysis. Results The molecular construct used as a measure of the response to thermal stress is a Hsp70-GFP fusion gene transfected in Chinese hamster ovary (CHO cells. The time profile of the GFP protein depends on the transient activity, Transient(t, of the heat shock system. The function Transient(t depends on hsp70 promoter activity, transcriptional regulation and the translation initiation effects elicited by the heat stress. The GFP time profile is recorded using flow cytometry measurements, a technique that allows a quantitative measurement of the fluorescence of a large number of cells (104. The GFP responses to one and two heat shocks were measured for 261 conditions of different temperatures and durations. We found that: (i the response of the cell to two

  10. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, D.J.C.; Stackhouse, M. [Los Alamos National Lab., NM (United States); Chen, D.S. [Rochester Univ., NY (United States). Dept. of Radiation Oncology

    1993-02-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  11. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, D.J.C.; Stackhouse, M. (Los Alamos National Lab., NM (United States)); Chen, D.S. (Rochester Univ., NY (United States). Dept. of Radiation Oncology)

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  12. Engineering considerations for process development in mammalian cell cultivation.

    Science.gov (United States)

    Zhang, Hu; Wang, Weixiang; Quan, Chunshan; Fan, Shengdi

    2010-01-01

    Mammalian cell cultivation plays a great role in producing protein therapeutics in the last decades. Many engineering parameters are considered for optimization during process development in mammalian cell cultivation, only shear and mixing are especially highlighted in this paper. It is believed that shear stress due to agitation has been over-estimated to damage cells, but shear may result in nonlethal physiological responses. There is no cell damage in the regions where bubbles form, break up and coalescence, but shear stress becomes significant in the wake of rising bubbles and causes great damage to cells in bubble burst regions. Mixing is not sufficient to provide homogeneous dissolved oxygen tension, pH, CO2 and nutrients in large-scale bioreactors, which can bring severe problems for cell growth, product formation and process control. Scale-down reactors have been developed to address mixing and shear problems for parallel operations. Engineering characterization in conventional and recently developed scale-down bioreactors has been briefly introduced. Process challenges for cultivation of industrial cell lines in high cell densities as well as cultivation of stem cells and other human cells for regenerative medicine, tissue engineering and gene therapy are prospected. Important techniques, such as micromanipulation and nanomanipulation (optical tweezers) for single cell analysis, computational fluid dynamics (CFD) for shear and mixing characterization, and miniaturized bioreactors, are being developed to address those challenges.

  13. Guidelines for monitoring bulk tank milk somatic cell and bacterial counts.

    Science.gov (United States)

    Jayarao, B M; Pillai, S R; Sawant, A A; Wolfgang, D R; Hegde, N V

    2004-10-01

    This study was conducted to establish guidelines for monitoring bulk tank milk somatic cell count and bacterial counts, and to understand the relationship between different bacterial groups that occur in bulk tank milk. One hundred twenty-six dairy farms in 14 counties of Pennsylvania participated, each providing one bulk tank milk sample every 15 d for 2 mo. The 4 bulk tank milk samples from each farm were examined for bulk tank somatic cell count and bacterial counts including standard plate count, preliminary incubation count, laboratory pasteurization count, coagulase-negative staphylococcal count, environmental streptococcal count, coliform count, and gram-negative noncoliform count. The milk samples were also examined for presence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma. The bacterial counts of 4 bulk tank milk samples examined over an 8-wk period were averaged and expressed as mean bacterial count per milliliter. The study revealed that an increase in the frequency of isolation of Staphylococcus aureus and Streptococcus agalactiae was significantly associated with an increased bulk tank somatic cell count. Paired correlation analysis showed that there was low correlation between different bacterial counts. Bulk tank milk with low (standard plate count also had a significantly low level of mean bulk tank somatic cell count (count (count (counts (count (count was less likely to be associated with somatic cell or other bacterial counts. Herd size and farm management practices had considerable influence on somatic cell and bacterial counts in bulk tank milk. Dairy herds that used automatic milking detachers, sand as bedding material, dip cups for teat dipping instead of spraying, and practiced pre-and postdipping had significantly lower bulk tank somatic cell and/or bacterial counts. In conclusion, categorized bulk tank somatic cell and bacterial counts could serve as indicators and facilitate monitoring of herd udder health and milk

  14. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Athanasia D Panopoulos; Margaret Lutz; W Travis Berggren; Kun Zhang; Ronald M Evans; Gary Siuzdak; Juan Carlos Izpisua Belmonte; Oscar Yanes; SergioRuiz; Yasuyuki S Kida; Dinh Diep; Ralf Tautenhahn; Aida Herrerias; Erika M Batchelder; Nongluk Plongthongkum

    2012-01-01

    Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs)remains largely unexplored.Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration.Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency.Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies.We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming.Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence.

  15. Factors affecting somatic cell count in dairy goats: a review

    Energy Technology Data Exchange (ETDEWEB)

    Jimenez-Granda, R.; Sanchez-Rodriguez, M.; Arce, C.; Rodriguez-Estevez, V.

    2014-06-01

    Somatic cell count (SCC) in monitoring udder health has been described in numerous studies as a useful method for the diagnosis of intramammary infection (IMI), and it is considered in standards of quality and hygiene of cows milk in many countries. However, several authors have questioned the validity of SCC as a reliable IMI diagnosis tool in dairy goats. This review attempts to reflect the importance of different infectious and non-infectious factors that can modify SCC values in goat milk, and must, therefore, be taken into account when using the SCC as a tool in the improvement of udder health and the quality of milk in this species. In dairy goats, some investigations have shown that mammary bacterial infections are a major cause of increased SCC and loss of production. In goats however, the relationship between bacterial infections and SCC values is not as simple as in dairy cattle, since non-infectious factors also have a big impact on SCC. Intrinsic factors are those that depend directly on the animal: time and number of lactation (higher SCC late in lactation and in aged goats), prolificity (higher SCC in multiple births), milking time (higher SCC in evening compared to morning milking) and number of milkings per day, among others. Extrinsic factors include: milking routine (lower SCC in machine than in manual milking), seasonality and food. In addition, milk secretion in goats is mostly apocrine and therefore characterized by the presence of epithelial debris or cytoplasmic particles, which makes the use of DNA specific counters mandatory. All this information is of interest in order to correctly interpret the SCC in goat milk and to establish differential SCC standards. (Author)

  16. Factors affecting somatic cell count in dairy goats: a review

    Directory of Open Access Journals (Sweden)

    Rocío Jiménez-Granado

    2014-02-01

    Full Text Available Somatic cell count (SCC in monitoring udder health has been described in numerous studies as a useful method for the diagnosis of intramammary infection (IMI, and it is considered in standards of quality and hygiene of cow’s milk in many countries. However, several authors have questioned the validity of SCC as a reliable IMI diagnosis tool in dairy goats. This review attempts to reflect the importance of different infectious and non-infectious factors that can modify SCC values in goat milk, and must, therefore, be taken into account when using the SCC as a tool in the improvement of udder health and the quality of milk in this species. In dairy goats, some investigations have shown that mammary bacterial infections are a major cause of increased SCC and loss of production. In goats however, the relationship between bacterial infections and SCC values is not as simple as in dairy cattle, since non-infectious factors also have a big impact on SCC. Intrinsic factors are those that depend directly on the animal: time and number of lactation (higher SCC late in lactation and in aged goats, prolificity (higher SCC in multiple births, milking time (higher SCC in evening compared to morning milking and number of milkings per day, among others. Extrinsic factors include: milking routine (lower SCC in machine than in manual milking, seasonality and food. In addition, milk secretion in goats is mostly apocrine and therefore characterized by the presence of epithelial debris or cytoplasmic particles, which makes the use of DNA specific counters mandatory. All this information is of interest in order to correctly interpret the SCC in goat milk and to establish differential SCC standards.

  17. Mutation of mitochondria genome: trigger of somatic cell transforming to cancer cell

    OpenAIRE

    Jianping, Du

    2010-01-01

    Nearly 80 years ago, scientist Otto Warburg originated a hypothesis that the cause of cancer is primarily a defect in energy metabolism. Following studies showed that mitochondria impact carcinogenesis to remodel somatic cells to cancer cells through modifying the genome, through maintenance the tumorigenic phenotype, and through apoptosis. And the Endosymbiotic Theory explains the origin of mitochondria and eukaryotes, on the other hands, the mitochondria also can fall back. Compared to chro...

  18. Deletion of Dicer in Somatic Cells of the Female Reproductive Tract Causes Sterility

    Science.gov (United States)

    Nagaraja, Ankur K.; Andreu-Vieyra, Claudia; Franco, Heather L.; Ma, Lang; Chen, Ruihong; Han, Derek Y.; Zhu, Huifeng; Agno, Julio E.; Gunaratne, Preethi H.; DeMayo, Francesco J.; Matzuk, Martin M.

    2008-01-01

    Dicer is an evolutionarily conserved ribonuclease III that is necessary for microRNA (miRNA) processing and the synthesis of small interfering RNAs from long double-stranded RNA. Although it has been shown that Dicer plays important roles in the mammalian germline and early embryogenesis, the functions of Dicer-dependent pathways in the somatic cells of the female reproductive tract are unknown. Using a transgenic line in which Cre recombinase is driven by the anti-Müllerian hormone receptor type 2 promoter, we conditionally inactivated Dicer1 in the mesenchyme of the developing Müllerian ducts and postnatally in ovarian granulosa cells and mesenchyme-derived cells of the oviducts and uterus. Deletion of Dicer in these cell types results in female sterility and multiple reproductive defects including decreased ovulation rates, compromised oocyte and embryo integrity, prominent bilateral paratubal (oviductal) cysts, and shorter uterine horns. The paratubal cysts act as a reservoir for spermatozoa and oocytes and prevent embryos from transiting the oviductal isthmus and passing the uterotubal junction to enter the uterus for implantation. Deep sequencing of small RNAs in oviduct revealed down-regulation of specific miRNAs in Dicer conditional knockout females compared with wild type. The majority of these differentially expressed miRNAs are predicted to regulate genes important for Müllerian duct differentiation and mesenchyme-derived structures, and several of these putative target genes were significantly up-regulated upon conditional deletion of Dicer1. Thus, our findings reveal diverse and critical roles for Dicer and its miRNA products in the development and function of the female reproductive tract. PMID:18687735

  19. Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

    Science.gov (United States)

    Wu, Jun; Platero-Luengo, Aida; Sakurai, Masahiro; Sugawara, Atsushi; Gil, Maria Antonia; Yamauchi, Takayoshi; Suzuki, Keiichiro; Bogliotti, Yanina Soledad; Cuello, Cristina; Morales Valencia, Mariana; Okumura, Daiji; Luo, Jingping; Vilariño, Marcela; Parrilla, Inmaculada; Soto, Delia Alba; Martinez, Cristina A; Hishida, Tomoaki; Sánchez-Bautista, Sonia; Martinez-Martinez, M Llanos; Wang, Huili; Nohalez, Alicia; Aizawa, Emi; Martinez-Redondo, Paloma; Ocampo, Alejandro; Reddy, Pradeep; Roca, Jordi; Maga, Elizabeth A; Esteban, Concepcion Rodriguez; Berggren, W Travis; Nuñez Delicado, Estrella; Lajara, Jeronimo; Guillen, Isabel; Guillen, Pedro; Campistol, Josep M; Martinez, Emilio A; Ross, Pablo Juan; Izpisua Belmonte, Juan Carlos

    2017-01-26

    Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

  20. Biosynthetic labeling of hypusine in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, M.H.; Folk, J.E.

    1986-05-01

    Using a dual-label technique in which /sup 3/H - and /sup 14/C-labeled forms of putrescine and of spermidine were employed as biosynthetic precursors of hypusine, two -C-H bond cleavages were detected during production of this unique amino acid in Chinese hamster ovary cells. One of these cleavages occurs at the C-1 position of the 4-aminobutyl group during its transfer from the secondary amine nitrogen of spermidine to the nitrogen at the upsilon-position of a specific lysine residue in the polypeptide precursor of eukaryotic initiation factor 4D. Breakage of the other -C-H bond takes place at the C-2 position in this aminobutyl segment after it has been coupled to lysine to form the intermediate deoxyhypusine residue. Hydroxylation at this carbon atom, which constitutes the last step in hypusine biosynthesis, is the cause of bond cleavage. The data obtained are consistent with a notion that no additional -C-H bond fissions occur during hypusine biosynthesis. The authors findings permit a suggestion of a mechanism for enzymic aminobutyl group transfer in which 4-amino-butyraldehyde produced by oxidative cleavage of spermidine is coupled with the upsilon-amino group of a specific lysine residue to form an enzyme-bound imine intermediate.

  1. Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells.

    Science.gov (United States)

    Badal, Sujan; Her, Yeng F; Maher, L James

    2015-09-04

    Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Epigenetic reprogramming by somatic cell nuclear transfer: questions and potential solutions.

    Science.gov (United States)

    Huili, Ji; Haosheng, Lu; Dengke, Pan

    2014-12-01

    Somatic cell nuclear transfer (SCNT) is a technology by which a highly differentiated somatic nucleus is transferred into an enucleated oocyte to generate a reconstructed embryo that subsequently develops to an offspring. However, to date, the efficiency of cloned animal is still low. The major reason is incomplete nuclear reprogramming of donor cells after nuclear transfer, which results in abnormal epigenetic modifications, including DNA methylation, histone acetylation, gene imprinting, X-chromosome inactivation, and telomere length. Most improvements have been made in somatic epigenetic reprogramming with small molecules and manipulating expression of specific genes. It is expected that SCNT will soon have broad applications in both basic research and practical production. In this review, we summarize the recent progress in epigenetic reprogramming by somatic cell nuclear transfer; in particular, we focus on strategies for rescuing the epigenetic errors occurring during SCNT.

  3. Adult neural stem cells in the mammalian central nervous system

    Institute of Scientific and Technical Information of China (English)

    Dengke K Ma; Michael A Bonaguidi; Guo-li Ming; Hongjun Song

    2009-01-01

    Neural stem cells (NSCs) are present not only during the embryonic development but also in the adult brain of all mammalian species, including humans. Stem cell niche architecture in vivo enables adult NSCs to continuously generate functional neurons in specific brain regions throughout life. The adult neurogenesis process is subject to dynamic regulation by various physiological, pathological and pharmacological stimuli. Multipotent adult NSCs also appear to be intrinsically plastic, amenable to genetic programing during normal differentiation, and to epigenetic reprograming during de-differentiation into pluripotency. Increasing evidence suggests that adult NSCs significantly contribute to specialized neural functions under physiological and pathological conditions. Fully understanding the biology of adult NSCs will provide crucial insights into both the etiology and potential therapeutic interventions of major brain disorders. Here, we review recent progress on adult NSCs of the mammalian central nervous system, in-cluding topics on their identity, niche, function, plasticity, and emerging roles in cancer and regenerative medicine.

  4. Carbamazepine induces mitotic arrest in mammalian Vero cells

    Energy Technology Data Exchange (ETDEWEB)

    Perez Martin, J.M.; Fernandez Freire, P.; Labrador, V. [Departamento de Biologia, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Hazen, M.J. [Departamento de Biologia, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)], E-mail: mariajose.hazen@uam.es

    2008-01-01

    We reported recently that the anticonvulsant drug carbamazepine, at supratherapeutic concentrations, exerts antiproliferative effects in mammalian Vero cells, but the underlying mechanism has not been elucidated. This motivates us to examine rigorously whether growth arrest was associated with structural changes in cellular organization during mitosis. In the present work, we found that exposure of the cells to carbamazepine led to an increase in mitotic index, mainly due to the sustained block at the metaphase/anaphase boundary, with the consequent inhibition of cell proliferation. Indirect immunofluorescence, using antibodies directed against spindle apparatus proteins, revealed that mitotic arrest was associated with formation of monopolar spindles, caused by impairment of centrosome separation. The final consequence of the spindle defects induced by carbamazepine, depended on the duration of cell cycle arrest. Following the time course of accumulation of metaphase and apoptotic cells during carbamazepine treatments, we observed a causative relationship between mitotic arrest and induction of cell death. Conversely, cells released from the block of metaphase by removal of the drug, continued to progress through mitosis and resume normal proliferation. Our results show that carbamazepine shares a common antiproliferative mechanism with spindle-targeted drugs and contribute to a better understanding of the cytostatic activity previously described in Vero cells. Additional studies are in progress to extend these initial findings that define a novel mode of action of carbamazepine in cultured mammalian cells.

  5. Transcriptional landscape of ncRNA and Repeat elements in somatic cells

    KAUST Repository

    Ghosheh, Yanal

    2016-12-01

    The advancement of Nucleic acids (DNA and RNA) sequencing technology has enabled many projects targeted towards the identification of genome structure and transcriptome complexity of organisms. The first conclusions of the human and mouse projects have underscored two important, yet unexpected, findings. First, while almost the entire genome is transcribed, only 5% of it encodes for proteins. Thereby, most transcripts are noncoding RNA. This includes both short RNA (<200 nucleotides (nt)) comprising piRNAs; microRNAs (miRNAs); endogenous Short Interfering RNAs (siRNAs) among others, and includes lncRNA (>200nt). Second, a significant portion of the mammalian genome (45%) is composed of Repeat Elements (REs). RE are mostly relics of ancestral viruses that during evolution have invaded the host genome by producing thousands of copies. Their roles within their host genomes have yet to be fully explored considering that they sometimes produce lncRNA, and have been shown to influence expression at the transcriptional and post-transcriptional levels. Moreover, because some REs can still mobilize within host genomes, host genomes have evolved mechanisms, mainly epigenetic, to maintain REs under tight control. Recent reports indicate that REs activity is regulated in somatic cells, particularily in the brain, suggesting a physiological role of RE mobilization during normal development. In this thesis, I focus on the analysis of ncRNAs, specifically REs; piRNAs; lncRNAs in human and mouse post-mitotic somatic cells. The main aspects of this analysis are: Using sRNA-Seq, I show that piRNAs, a class of ncRNAs responsible for the silencing of Transposable elements (TEs) in testes, are present also in adult mouse brain. Furthermore, their regulation shows only a subset of testes piRNAs are expressed in the brain and may be controlled by known neurogenesis factors. To investigate the dynamics of the transcriptome during cellular differentiation, I examined deep RNA-Seq and Cap

  6. Somatic cell nuclear transfer-derived embryonic stem cell lines in humans: pros and cons.

    Science.gov (United States)

    Langerova, Alena; Fulka, Helena; Fulka, Josef

    2013-12-01

    The recent paper, published by Mitalipov's group in Cell (Tachibana et al., 2013 ), reporting the production of human somatic cell nuclear transfer (SCNT) embryonic stem cells (ESCs), opens again the debate if, in the era of induced pluripotent stem cells (iPSCs), the production of these cells is indeed necessary and, if so, whether they are different from ESCs produced from spare embryos and iPSCs. It is our opinion that these questions are very difficult to answer because it is still unclear whether and how normal ESCs differ from iPSCs.

  7. Engineering controlled mammalian type O-Glycosylation in plant cells

    DEFF Research Database (Denmark)

    Yang, Zhang; Drew, Damian Paul; Jørgensen, Bodil

    2011-01-01

    Human mucins are large heavily O-glycosylated glycoproteins (>200 kDa), which account for the majority of proteins in mucus layers that e.g. hydrate, lubricate and protect cells from proteases as well as from pathogens. O-linked mucin glycans are truncated in many cancers, yielding truncated cancer...... specific glyco-peptide epitopes, such as the Tn epitope (GalNAc sugar attached to either Serine or Threonine), which are antigenic to the immune system. In the present study, we have identified plant cells as the only eukaryotic cells without mammalian type O-glycosylation or competing (for sites) O...

  8. Self-renewal and pluripotency acquired through somatic reprogramming to human cancer stem cells.

    Directory of Open Access Journals (Sweden)

    Shogo Nagata

    Full Text Available Human induced pluripotent stem cells (iPSCs are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC lines through re-plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPARγ. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc-related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor.

  9. Self-renewal and pluripotency acquired through somatic reprogramming to human cancer stem cells.

    Science.gov (United States)

    Nagata, Shogo; Hirano, Kunio; Kanemori, Michele; Sun, Liang-Tso; Tada, Takashi

    2012-01-01

    Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re-plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPARγ. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc-related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor.

  10. Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.

    Science.gov (United States)

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Guo, Zhen-Hua; Zhu, Meng; Bai, Jing

    2016-01-01

    Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.

  11. Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

    Science.gov (United States)

    Secher, Jan O; Liu, Ying; Petkov, Stoyan; Luo, Yonglun; Li, Dong; Hall, Vanessa J; Schmidt, Mette; Callesen, Henrik; Bentzon, Jacob F; Sørensen, Charlotte B; Freude, Kristine K; Hyttel, Poul

    2017-03-01

    Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells.

  12. Genome-wide reprogramming in hybrids of somatic cells and embryonic stem cells.

    Science.gov (United States)

    Ambrosi, Dominic J; Tanasijevic, Borko; Kaur, Anupinder; Obergfell, Craig; O'Neill, Rachel J; Krueger, Winfried; Rasmussen, Theodore P

    2007-05-01

    Recent experiments demonstrate that somatic nuclei can be reprogrammed to a pluripotent state when fused to ESCs. The resulting hybrids are pluripotent as judged by developmental assays, but detailed analyses of the underlying molecular-genetic control of reprogrammed transcription in such hybrids are required to better understand fusion-mediated reprogramming. We produced hybrids of mouse ESCs and fibroblasts that, although nearly tetraploid, exhibit characteristics of normal ESCs, including apparent immortality in culture, ESC-like colony morphology, and pluripotency. Comprehensive analysis of the mouse embryonic fibroblast/ESC hybrid transcriptome revealed global patterns of gene expression reminiscent of ESCs. However, combined analysis of variance and hierarchical clustering analyses revealed at least seven distinct classes of differentially regulated genes in comparisons of hybrids, ESCs, and somatic cells. The largest class includes somatic genes that are silenced in hybrids and ESCs, but a smaller class includes genes that are expressed at nearly equivalent levels in hybrids and ESCs that contain many genes implicated in pluripotency and chromatin function. Reprogrammed genes are distributed throughout the genome. Reprogramming events include both transcriptional silencing and activation of genes residing on chromosomes of somatic origin. Somatic/ESC hybrid cell lines resemble their pre-fusion ESC partners in terms of behavior in culture and pluripotency. However, they contain unique expression profiles that are similar but not identical to normal ESCs. ESC fusion-mediated reprogramming provides a tractable system for the investigation of mechanisms of reprogramming. Disclosure of potential conflicts of interest is found at the end of this article.

  13. Dicer is associated with ribosomal DNA chromatin in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Lasse Sinkkonen

    Full Text Available BACKGROUND: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/- ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. CONCLUSION/SIGNIFICANCE: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.

  14. Baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Yu-kou LIU; Chih-chieh CHU; Tzong-yuan WU

    2006-01-01

    Aim:To identify a shuttle promoter that can mediate gene expression in both insect cells and mammalian cells to facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle.Methods:Recombinant baculoviruses carrying the β-galactosidase reporter gene under the control of an early to late(ETL)promoter of the Autographa califomica multiple nuclear polyhedrosis virus(AcMNPV)or a cytomegalovirus immediate early promoter (CMV promoter)were constructed.COS1,HeLa,CHO-K1,hFob1.19,and MCF-7 mammalian cells were tested for the expression of β-galactosidase.Results:ETL promoter activity was higher in bone-derived hFob1.19 than in COS1,HeLa,CHOK1,or MCF-7 mammalian cells.The transient plasmid transfection assay indicated that ETL promoter activity in mammalian cells was dependent on baculovirus gene expression.Conclusion:ETL promoter activity in mammalian cells is baculovirus gene expression-dependent,and the shuttle promoter will facilitate the application of baculovirus expression vectors in mammalian cell expression systems and for gene therapy.

  15. Transcriptional signatures of somatic neoblasts and germline cells in Macrostomum lignano.

    Science.gov (United States)

    Grudniewska, Magda; Mouton, Stijn; Simanov, Daniil; Beltman, Frank; Grelling, Margriet; de Mulder, Katrien; Arindrarto, Wibowo; Weissert, Philipp M; van der Elst, Stefan; Berezikov, Eugene

    2016-12-20

    The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.

  16. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  17. Mammalian target of rapamycin activity is required for expansion of CD34(+) hematopoietic progenitor cells

    NARCIS (Netherlands)

    Geest, Christian R.; Zwartkruis, Fried J.; Vellenga, Edo; Coffer, Paul J.; Buitenhuis, Miranda

    2009-01-01

    Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic sy

  18. Mammalian target of rapamycin activity is required for expansion of CD34+ hematopoietic progenitor cells

    NARCIS (Netherlands)

    Geest, C.R.; Zwartkruis, G.J.T.; Vellenga, E.; Coffer, P.J.; Buitenhuis, M.

    2009-01-01

    Background The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic sy

  19. Mammalian Cochlear Hair Cell Regeneration and Ribbon Synapse Reformation

    Directory of Open Access Journals (Sweden)

    Xiaoling Lu

    2016-01-01

    Full Text Available Hair cells (HCs are the sensory preceptor cells in the inner ear, which play an important role in hearing and balance. The HCs of organ of Corti are susceptible to noise, ototoxic drugs, and infections, thus resulting in permanent hearing loss. Recent approaches of HCs regeneration provide new directions for finding the treatment of sensor neural deafness. To have normal hearing function, the regenerated HCs must be reinnervated by nerve fibers and reform ribbon synapse with the dendrite of spiral ganglion neuron through nerve regeneration. In this review, we discuss the research progress in HC regeneration, the synaptic plasticity, and the reinnervation of new regenerated HCs in mammalian inner ear.

  20. Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

    Science.gov (United States)

    Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu

    2014-01-01

    Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

  1. Reprogramming somatic cells to pluripotency: a fresh look at Yamanaka's model.

    Science.gov (United States)

    Li, Yangxin; Shen, Zhenya; Shelat, Harnath; Geng, Yong-Jian

    2013-12-01

    In 2006, Dr Shinya Yamanaka succeeded to reprogram somatic cells into pluripotent stem cells (iPSC) by delivering the genes encoding Oct4, Sox2, Klf4, and c-Myc. This achievement represents a fundamental breakthrough in stem cell biology and opens up a new era in regenerative medicine. However, the molecular processes by which somatic cells are reprogrammed into iPSC remain poorly understood. In 2009, Yamanaka proposed the elite and stochastic models for reprogramming mechanisms. To date, many investigators in the field of iPSC research support the concept of stochastic model, i.e., somatic cell reprogramming is an event of epigenetic transformation. A mathematical model, f (Cd, k), has also been proposed to predict the stochastic process. Here we wish to revisit the Yamanaka model and summarize the recent advances in this research field.

  2. Identification of chikungunya virus interacting proteins in mammalian cells

    Indian Academy of Sciences (India)

    Mandar S Paingankar; Vidya A Arankalle

    2014-06-01

    Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.

  3. DNA Ligase I Is Not Essential for Mammalian Cell Viability

    Directory of Open Access Journals (Sweden)

    Li Han

    2014-04-01

    Full Text Available Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1 has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.

  4. DNA ligase I is not essential for mammalian cell viability.

    Science.gov (United States)

    Han, Li; Masani, Shahnaz; Hsieh, Chih-lin; Yu, Kefei

    2014-04-24

    Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.

  5. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

    2011-01-01

    Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell...... intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body...... (NPB) morphology similar to the host ooplasm, which, together with detected posttranslational activity of somatic cell introduced into the bovine ooplasm, suggests a universal function of ooplasmic factors. However, the lack of distinct UBF localization in intergeneric embryos indicates failures...

  6. Labeling proteins on live mammalian cells using click chemistry.

    Science.gov (United States)

    Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

    2015-05-01

    We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

  7. Relationship between Somatic Cell Counts, Mastitis and Milk Quality in Ettawah Grade and PESA Goats

    Directory of Open Access Journals (Sweden)

    Molefe PETLANE

    2013-12-01

    Full Text Available Mastitis is a bacterial disease that leads to increased somatic cell counts and reduced milk quality in dairy goats. Reduction in quality is manifested through a reduction in fat, protein, lactose content and an increase in milk somatic cell counts and salts content. Thus mastitis affects productivity of animals and hence their economic value. The aim of this study was to analyze the effects of somatic cell counts (SCC and mastitis on milk quality in PE and PESA. On-Farm mastitis tests were performed on 38 lactating dairy goats and milk samples were collected from both mastitis positive and healthy animals from which quality parameters were measured using a milko tester while bacterial isolation and enumeration were done following standard protocols. Data was analyzed descriptively and the results showed that somatic cell counts and somatic cell score correlate positively with mastitis (P < 0.05. Lactose and fat content decreased with severity of mastitis in both breeds whereas in PESA protein content increased with mastitis. Salt content increases with mastitis in both breeds. S. aureus was the most isolated bacteria and associated with high SCC whereas E. coli was poorly isolated. The study concludes that mastitis leads to increased SCC and reduced milk quality in dairy goats.

  8. Role of Somatic Testicular Cells during Mouse Spermatogenesis in Three-Dimensional Collagen Gel Culture System.

    Science.gov (United States)

    Khajavi, Noushafarin; Akbari, Mohammad; Abdolsamadi, Hamid Reza; Abolhassani, Farid; Dehpour, Ahmad Reza; Koruji, Morteza; Habibi Roudkenar, Mehryar

    2014-02-03

    Spermatogonial stem cells (SSCs) are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional (3D) culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting (MACS) using GDNF family receptor alpha-1 (Gfrα-1) antibody. Two experimental designs were investigated. 1. Gfrα-1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfrα-1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 (SCP3)-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 (TTF1) as post meiotic markers. For statistical analysis student t test was performed. Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Collagen gel culture system supported by somatic testicular cells

  9. Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen.

    Science.gov (United States)

    Liu, Jie; Westhusin, Mark; Long, Charles; Johnson, Gregory; Burghardt, Robert; Kraemer, Duane

    2010-12-01

    Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important when an animal has died and the only viable genetic material available is frozen semen. Usefulness of somatic cells obtained from fresh (cultured) and frozen (isolated, not cultured) bovine semen for nuclear transfer was evaluated. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and crossbred Rodeo bull. All samples were processed immediately and cell growth was obtained from seven of the twelve ejaculates (58.3%). Cells from three bulls (with the best growth rates) were evaluated by optical microscopy and used in cloning experiments. In culture, these cells exhibited classic epithelial morphology and expressed cytokeratin and vimentin, indicating they were of epithelial origin. When cells from the three bulls were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively. Of the blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. Somatic cells isolated (not cultured) from frozen bovine semen were also used in the cloning experiments. Although cleavage occurred, no compact morulae or blastocysts were obtained. In conclusion, epithelial cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. Somatic cells from semen can be used as nucleus donors to produce cloned blastocyst-stage embryos.

  10. Transient transfection of mammalian cells using a violet diode laser

    Science.gov (United States)

    Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

    2010-07-01

    We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

  11. Shear stress induced stimulation of mammalian cell metabolism

    Science.gov (United States)

    Mcintire, L. V.; Frangos, J. A.; Eskin, S. G.

    1988-01-01

    A flow apparatus was developed for the study of the metabolic response of anchorage dependent cells to a wide range of steady and pulsatile shear stresses under well controlled conditions. Human umbilical vein endothelial cell monolayers were subjected to steady shear stresses of up to 24 dynes/sq cm, and the production of prostacyclin was determined. The onset of flow led to a burst in prostacyclin production which decayed to a long term steady state rate (SSR). The SSR of cells exposed to flow was greater than the basal release level, and increased linearly with increasing shear stress. It is demonstrated that shear stresses in certain ranges may not be detrimental to mammalian cell metabolism. In fact, throughout the range of shear stresses studied, metabolite production is maximized by maximizing shear stress.

  12. Genetic effects in somatic and germ cells, induced by ionizing radiations

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, B.; Benova, D.; Bajrakova, A.; Bulanova, M.; Vyglenov, A.; Rupova, I.; Georgieva, I. (Meditsinska Akademiya, Sofia (Bulgaria). Nauchen Inst. po Rentgenologiya i Radiobiologiya)

    1983-01-01

    Quantitative data are reported on injuries to hereditary structures in somatic and sex cells, induced by different types of ionizing radiation. The model systems used were human peripheral blood lymphocytes, in vitro irradiated with different doses: bone-marrow cells of mice and rats irradiated in vivo: mouse, rat, rabbit and hamster sex cells. To evaluate the dose-effect dependency after acute and chronic irradiation, the authors used mathematical models, describing the amount of chromosomal injuries. Attempt was made to estimate the somatic and genetic risk, following acute and chronic irradiation with different doses of ionizing radiations.

  13. The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells.

    Science.gov (United States)

    Brix, Jacob; Zhou, Yan; Luo, Yonglun

    2015-12-20

    Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprogramming, pluripotency, and differentiation capacity. Here, we review the epigenetic changes with a focus on histone modification (methylation and acetylation) and DNA modification (methylation) during iPSC induction. We look at changes in specific epigenetic signatures, aberrations and epigenetic memory during reprogramming and small molecules influencing the epigenetic reprogramming of somatic cells. Finally, we discuss how to improve iPSC generation and pluripotency through epigenetic manipulations.

  14. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  15. HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks.

    Directory of Open Access Journals (Sweden)

    Sandeep N Shah

    Full Text Available BACKGROUND: Although recent studies have identified genes expressed in human embryonic stem cells (hESCs that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1 gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. METHODS/PRINCIPAL FINDINGS: We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM. HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic

  16. Coupled transcription and translation within nuclei of mammalian cells.

    Science.gov (United States)

    Iborra, F J; Jackson, D A; Cook, P R

    2001-08-10

    It is widely assumed that the vital processes of transcription and translation are spatially separated in eukaryotes and that no translation occurs in nuclei. We localized translation sites by incubating permeabilized mammalian cells with [3H]lysine or lysyl-transfer RNA tagged with biotin or BODIPY; although most nascent polypeptides were cytoplasmic, some were found in discrete nuclear sites known as transcription "factories." Some of this nuclear translation also depends on concurrent transcription by RNA polymerase II. This coupling is simply explained if nuclear ribosomes translate nascent transcripts as those transcripts emerge from still-engaged RNA polymerases, much as they do in bacteria.

  17. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.

    Science.gov (United States)

    Abyzov, Alexej; Mariani, Jessica; Palejev, Dean; Zhang, Ying; Haney, Michael Seamus; Tomasini, Livia; Ferrandino, Anthony F; Rosenberg Belmaker, Lior A; Szekely, Anna; Wilson, Michael; Kocabas, Arif; Calixto, Nathaniel E; Grigorenko, Elena L; Huttner, Anita; Chawarska, Katarzyna; Weissman, Sherman; Urban, Alexander Eckehart; Gerstein, Mark; Vaccarino, Flora M

    2012-12-20

    Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

  18. Manufacture of biopharmaceutical proteins by mammalian cell culture systems.

    Science.gov (United States)

    Tolbert, W R

    1990-01-01

    In the last several years, dramatic advances have been in the development of new biopharmaceuticals including monoclonal antibodies for diagnosis and treatment and such genetically engineered proteins as tPA, Factor VIIIc, erythropoietin and soluble CD4, an anti-AIDS protein. Currently, there are several hundred such candidate drugs in human clinical trials. In most cases, these protein-based drugs will require manufacture by mammalian cell culture due to the inability of lower organisms to properly glycosylate, fold, make correct disulfide bonds and secrete active biomolecular forms. The need for large scale production from cell culture will greatly increase as more of the products in clinical trials are approved for commercial production. This will require significant reduction in manufacturing costs per gram, concomitant with increased capacity to hundreds or perhaps even thousands of kilograms annually. As an example, Invitron's multi-reactor manufacturing facility has operated at greater than one-half million liters per year and has experience with more than 250 mammalian cell lines for producing protein drug products.

  19. Phototransfection of mammalian cells using femtosecond laser pulses: optimization and applicability to stem cell differentiation

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-07-01

    Full Text Available Recently, femtosecond laser pulses have been utilized for the targeted introduction of genetic matter into mammalian cells. This rapidly expanding and developing novel optical technique using a tightly focused laser light beam is called...

  20. A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells.

    Science.gov (United States)

    Lackner, Andreas; Genta, Kathrin; Koppensteiner, Herwig; Herbacek, Irene; Holzmann, Klaus; Spiegl-Kreinecker, Sabine; Berger, Walter; Grusch, Michael

    2008-09-01

    Baculoviruses are widely used for protein production in insect cells, and their potential for gene transfer to mammalian cells is increasingly being recognized. Here we describe a baculovirus vector with a bicistronic mammalian expression cassette and demonstrate its suitability for efficient transient and stable protein expression in human glioblastoma cells. Bicistronic baculovirus vectors are safe, cost efficient, and easy to produce; thus, they represent an excellent gene transfer system for mammalian cells.

  1. Factors Affecting on Somatic Cells Count in Slovak Simmental Dairy Cows

    Directory of Open Access Journals (Sweden)

    Jozef Bujko

    2014-11-01

    Full Text Available The aim this work was to analyse factors affecting on the somatic cells count in Slovak Simmental dairy cows. Data were analysed using the SAS version 9.1.3. and linear model with fixed effects of herd, years and months controls, sire and breeding types. The analyses by the effect on somatic cells count was the highest effect of herd-years-months of control R2 = 0.151316 and effect of sire R2 = 0.054182. These effects were high statistical significant P<0.01. Correlation coefficients between milk in kg, fat, protein, lactose in % with somatic cells count were r= -0.25096, r= 0.02593, r= 0.22321and r=-0.39567.

  2. Piwi Is Required to Limit Exhaustion of Aging Somatic Stem Cells

    Directory of Open Access Journals (Sweden)

    Pedro Sousa-Victor

    2017-09-01

    Full Text Available Sophisticated mechanisms that preserve genome integrity are critical to ensure the maintenance of regenerative capacity while preventing transformation of somatic stem cells (SCs, yet little is known about mechanisms regulating genome maintenance in these cells. Here, we show that intestinal stem cells (ISCs induce the Argonaute family protein Piwi in response to JAK/STAT signaling during acute proliferative episodes. Piwi function is critical to ensure heterochromatin maintenance, suppress retrotransposon activation, and prevent DNA damage in homeostasis and under regenerative pressure. Accordingly, loss of Piwi results in the loss of actively dividing ISCs and their progenies by apoptosis. We further show that Piwi expression is sufficient to allay age-related retrotransposon expression, DNA damage, apoptosis, and mis-differentiation phenotypes in the ISC lineage, improving epithelial homeostasis. Our data identify a role for Piwi in the regulation of somatic SC function, and they highlight the importance of retrotransposon control in somatic SC maintenance.

  3. Somatic sex identity is cell-autonomous in the chicken

    OpenAIRE

    D. Zhao; McBride, D; Nandi, S; McQueen, HA; McGrew, MJ; Hocking, PM; Lewis, PD; Sang, HM; Clinton, M

    2010-01-01

    In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has yet to be established. We have examined three lateral gynandromorph chickens with the aim of investigating the nature of the sex-determining mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimeras...

  4. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

    Science.gov (United States)

    Klein, U.; Klein, G.; Ehlin-Henriksson, B.; Rajewsky, K.; Küppers, R.

    1995-01-01

    BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher. PMID:8529116

  5. The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells

    DEFF Research Database (Denmark)

    Brix, Jacob; Zhou, Yan; Luo, Yonglun

    2015-01-01

    Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprograming, pluripotency, and differentiation cap...

  6. Sex-specific DoublesexM expression in subsets of Drosophila somatic gonad cells

    Directory of Open Access Journals (Sweden)

    Oliver Brian

    2007-10-01

    Full Text Available Abstract Background In Drosophila melanogaster, a pre-mRNA splicing hierarchy controls sexual identity and ultimately leads to sex-specific Doublesex (DSX transcription factor isoforms. The male-specific DSXM represses genes involved in female development and activates genes involved in male development. Spatial and temporal control of dsx during embryogenesis is not well documented. Results Here we show that DSXM is specifically expressed in subsets of male somatic gonad cells during embryogenesis. Following testis formation, germ cells remain in contact with DSXM-expressing cells, including hub cells and premeiotic somatic cyst cells that surround germ cells during spermatogenesis in larval and adult testes. Conclusion We show that dsx is transcriptionally regulated in addition to being regulated at the pre-mRNA splicing level by the sex determination hierarchy. The dsx locus is spatially controlled by somatic gonad identity. The continuous expression of DSXM in cells contacting the germline suggests an ongoing short-range influence of the somatic sex determination pathway on germ cell development.

  7. Decreased somatic hypermutation induces an impaired peripheral B cell tolerance checkpoint

    NARCIS (Netherlands)

    T. Cantaert (Tineke); J.-N. Schickel (Jean-Nicolas); Bannock, J.M. (Jason M.); Ng, Y.-S. (Yen-Shing); Massad, C. (Christopher); Delmotte, F.R. (Fabien R.); Yamakawa, N. (Natsuko); Glauzy, S. (Salome); N. Chamberlain (Nicolas); Kinnunen, T. (Tuure); Menard, L. (Laurence); Lavoie, A. (Aubert); J.E. Walter (Jolan E.); L.D. Notarangelo (Luigi Daniele); Bruneau, J. (Julie); Al-Herz, W. (Waleed); S.S. Kilic; Ochs, H.D. (Hans D.); Cunningham-Rundles, C. (Charlotte); M. van der Burg (Mirjam); T.W. Kuijpers (Taco W.); S. Kracker (Sven); Kaneko, H. (Hideo); Sekinaka, Y. (Yujin); S. Nonoyama (Shigeaki); Durandy, A. (Anne); E. Meffre (Eric)

    2016-01-01

    textabstractPatients with mutations in AICDA, which encodes activation-induced cytidine deaminase (AID), display an impaired peripheral B cell tolerance. AID mediates class-switch recombination (CSR) and somatic hypermutation (SHM) in B cells, but the mechanism by which AID prevents the accumulation

  8. Polydimethylsiloxane SlipChip for mammalian cell culture applications.

    Science.gov (United States)

    Chang, Chia-Wen; Peng, Chien-Chung; Liao, Wei-Hao; Tung, Yi-Chung

    2015-11-07

    This paper reports a polydimethylsiloxane (PDMS) SlipChip for in vitro cell culture applications, multiple-treatment assays, cell co-cultures, and cytokine detection assays. The PDMS SlipChip is composed of two PDMS layers with microfluidic channels on each surface that are separated by a thin silicone fluid (Si-fluid) layer. The integration of Si-fluid enables the two PDMS layers to be slid to different positions; therefore, the channel patterns can be re-arranged for various applications. The SlipChip design significantly reduces the complexity of sample handling, transportation, and treatment processes. To apply the developed SlipChip for cell culture applications, human lung adenocarcinoma epithelial cells (A549) and lung fibroblasts (MRC-5) were cultured to examine the biocompatibility of the developed PDMS SlipChip. Moreover, embryonic pluripotent stem cells (ES-D3) were also cultured in the device to evaluate the retention of their stemness in the device. The experimental results show that cell morphology, viability and proliferation are not affected when the cells are cultured in the SlipChip, indicating that the device is highly compatible with mammalian cell culture. In addition, the stemness of the ES-D3 cells was highly retained after they were cultured in the device, suggesting the feasibility of using the SlipChip for stem cell research. Various cell experiments, such as simultaneous triple staining of cells and co-culture of MRC-5 with A549 cells, were also performed to demonstrate the functionalities of the PDMS SlipChip. Furthermore, we used a cytokine detection assay to evaluate the effect of endotoxin (lipopolysaccharides, LPS) treatment on the cytokine secretion of A549 cells using the SlipChip. The developed PDMS SlipChip provides a straightforward and effective platform for various on-chip in vitro cell cultures and consequent analysis, which is promising for a number of cell biology studies and biomedical applications.

  9. Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

    Full Text Available Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms, which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

  10. Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.

    Science.gov (United States)

    Yamada, Mitsutoshi; Johannesson, Bjarki; Sagi, Ido; Burnett, Lisa Cole; Kort, Daniel H; Prosser, Robert W; Paull, Daniel; Nestor, Michael W; Freeby, Matthew; Greenberg, Ellen; Goland, Robin S; Leibel, Rudolph L; Solomon, Susan L; Benvenisty, Nissim; Sauer, Mark V; Egli, Dieter

    2014-06-26

    The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

  11. Human endometrial side population cells exhibit genotypic, phenotypic and functional features of somatic stem cells.

    Directory of Open Access Journals (Sweden)

    Irene Cervelló

    Full Text Available During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC population. Here we explore the hypothesis that human endometrial side population (SP cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.

  12. Garcinielliptone FC: antiparasitic activity without cytotoxicity to mammalian cells.

    Science.gov (United States)

    Silva, Ana P; Silva, Marcos P; Oliveira, Cristiano G; Monteiro, Daniela C; Pinto, Pedro L; Mendonça, Ronaldo Z; Costa Júnior, Joaquim S; Freitas, Rivelilson M; de Moraes, Josué

    2015-06-01

    Garcinielliptone FC (GFC) is a natural prenylated benzophenone found in the seeds of Platonia insignis Mart. (Clusiaceae), a native Brazilian plant. It has been chemically characterized and it is known that GFC has several biological activities such as antioxidant and vasorelaxant properties. In this study, we report the in vitro effect of GFC against the blood fluke Schistosoma mansoni, the parasite responsible for schistosomiasis mansoni. The anti-S. mansoni activity and cytotoxicity toward mammalian cells were determined for the compound. GFC⩾6.25 μM showed antischistosomal activity and confocal laser scanning microscopy analysis demonstrated several morphological alterations on the tegument of worms, and a correlation between viability and tegumental damage was observed. In addition, at sub-lethal concentrations of GFC (⩽3.125 μM), the number of S. mansoni eggs was reduced. More importantly, GFC exhibited no activity toward mammalian cells and, therefore, there is an appreciable selectivity of this compound against the helminths. In conclusion, these findings indicate the potential of GFC as an antiparasitic agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. CRISPR Technology for Genome Activation and Repression in Mammalian Cells.

    Science.gov (United States)

    Du, Dan; Qi, Lei S

    2016-01-04

    Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems.

  14. RNAi pathway participates in chromosome segregation in mammalian cells.

    Science.gov (United States)

    Huang, Chuan; Wang, Xiaolin; Liu, Xu; Cao, Shuhuan; Shan, Ge

    2015-01-01

    The RNAi machinery is a mighty regulator in a myriad of life events. Despite lines of evidence that small RNAs and components of the RNAi pathway may be associated with structure and behavior of mitotic chromosomes in diverse organisms, a direct role of the RNAi pathway in mammalian mitotic chromosome segregation remains elusive. Here we report that Dicer and AGO2, two central components of the mammalian RNAi pathway, participate in the chromosome segregation. Knockdown of Dicer or AGO2 results in a higher incidence of chromosome lagging, and this effect is independent from microRNAs as examined with DGCR8 knockout cells. Further investigation has revealed that α-satellite RNA, a noncoding RNA derived from centromeric repeat region, is managed by AGO2 under the guidance of endogenous small interference RNAs (ASAT siRNAs) generated by Dicer. Furthermore, the slicer activity of AGO2 is essential for the chromosome segregation. Level and distribution of chromosome-associated α-satellite RNA have crucial regulatory effect on the localization of centromeric proteins such as centromere protein C1 (CENPC1). With these results, we also provide a paradigm in which the RNAi pathway participates in vital cellular events through the maintenance of level and distribution of noncoding RNAs in cells.

  15. Human somatic cell nuclear transfer and reproductive cloning: an Ethics Committee opinion.

    Science.gov (United States)

    2016-04-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer and cloning," last published in Fertil Steril 2012;98:804-7. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  17. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.

    1999-01-01

    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced...... by prolonged exposure to the Light in the laboratory. A pronounced reduction of resazurin was observed in the RPMI 1640 medium, which contains 1 mg/L of reduced glutathione. No significant differences in resazurin reduction were observed between McCoy's 5A medium, Dulbecco's Modified Essential medium, and Ham...

  18. PINK1/Parkin-mediated mitophagy in mammalian cells.

    Science.gov (United States)

    Eiyama, Akinori; Okamoto, Koji

    2015-04-01

    Mitochondria-specific autophagy (mitophagy) is a fundamental process critical for maintaining mitochondrial fitness in a myriad of cell types. Particularly, mitophagy contributes to mitochondrial quality control by selectively eliminating dysfunctional mitochondria. In mammalian cells, the Ser/Thr kinase PINK1 and the E3 ubiquitin ligase Parkin act cooperatively in sensing mitochondrial functional state and marking damaged mitochondria for disposal via the autophagy pathway. Notably, ubiquitin and deubiquitinases play vital roles in modulating Parkin activity and mitophagy efficiency. In this review, we highlight recent breakthroughs addressing the key issues of how PINK1 activates Parkin in response to mitochondrial malfunction, how Parkin localizes specifically to impaired mitochondria, and how ubiquitination and deubiquitination regulate PINK1/Parkin-mediated mitophagy.

  19. Computing in mammalian cells with nucleic acid strand exchange

    Science.gov (United States)

    Groves, Benjamin; Chen, Yuan-Jyue; Zurla, Chiara; Pochekailov, Sergii; Kirschman, Jonathan L.; Santangelo, Philip J.; Seelig, Georg

    2016-03-01

    DNA strand displacement has been widely used for the design of molecular circuits, motors, and sensors in cell-free settings. Recently, it has been shown that this technology can also operate in biological environments, but capabilities remain limited. Here, we look to adapt strand displacement and exchange reactions to mammalian cells and report DNA circuitry that can directly interact with a native mRNA. We began by optimizing the cellular performance of fluorescent reporters based on four-way strand exchange reactions and identified robust design principles by systematically varying the molecular structure, chemistry and delivery method. Next, we developed and tested AND and OR logic gates based on four-way strand exchange, demonstrating the feasibility of multi-input logic. Finally, we established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution.

  20. Depletion of mitochondria in mammalian cells through enforced mitophagy.

    Science.gov (United States)

    Correia-Melo, Clara; Ichim, Gabriel; Tait, Stephen W G; Passos, João F

    2017-01-01

    Mitochondria are not only the 'powerhouse' of the cell; they are also involved in a multitude of processes that include calcium storage, the cell cycle and cell death. Traditional means of investigating mitochondrial importance in a given cellular process have centered upon depletion of mtDNA through chemical or genetic means. Although these methods severely disrupt the mitochondrial electron transport chain, mtDNA-depleted cells still maintain mitochondria and many mitochondrial functions. Here we describe a straightforward protocol to generate mammalian cell populations with low to nondetectable levels of mitochondria. Ectopic expression of the ubiquitin E3 ligase Parkin, combined with short-term mitochondrial uncoupler treatment, stimulates widespread mitophagy and effectively eliminates mitochondria. In this protocol, we explain how to generate Parkin-expressing, mitochondria-depleted cells from scratch in 23 d, as well as offer a variety of methods for confirming mitochondrial clearance. Furthermore, we describe culture conditions to maintain mitochondrial-depleted cells for up to 30 d with minimal loss of viability, for longitudinal studies. This method should prove useful for investigating the importance of mitochondria in a variety of biological processes.

  1. Nourseothricin N-acetyl transferase: a positive selection marker for mammalian cells.

    Directory of Open Access Journals (Sweden)

    Bose S Kochupurakkal

    Full Text Available Development of Nourseothricin N-acetyl transferase (NAT as a selection marker for mammalian cells is described. Mammalian cells are acutely susceptible to Nourseothricin, similar to the widely used drug Puromycin, and NAT allows for quick and robust selection of transfected/transduced cells in the presence of Nourseothricin. NAT is compatible with other selection markers puromycin, hygromycin, neomycin, blasticidin, and is a valuable addition to the repertoire of mammalian selection markers.

  2. Genotoxicity of two arsenic compounds in germ cells and somatic cells of Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Ramos-Morales, P.; Rodriguez-Arnaiz, R. [Laboratorio de Genetica, Coyoacan (Mexico)

    1995-12-31

    Two arsenic compounds, sodium arsenite (NaAsO{sup 2}) and sodium arsenate (Na{sub 2}HasO{sub 4}), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanagaster. For germinal cells, the sex-linked recessive lethal test (SLRLT) and the sea chromosome loss test (SCLT) were used. In both tests, a broad scheme of 2-3-3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM used for Sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). The the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w{sup +}/w eye assay as well as in the mwh +/+ flr{sup 3} wing test, employing the standard and insecticide-resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds Solium and arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds. 75 refs., 4 figs., 4 tabs.

  3. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

    Institute of Scientific and Technical Information of China (English)

    YING CHEN; QING ZHANG YANG; DA YUAN CHEN; MIN KANG WANG; JIN SONG LI; SHAO LIANG HUANG; XIANG YIN KONG; YAO ZHOU SHI; ZHI QIANG WANG; JIA HUI XIA; ZHI GAO LONG; ZHI XU HE; ZHI GANG XUE; WEN XIANG DING; HUI ZHEN SHENG; AILIAN LIU; KAI WANG; WEN WEI MAO; JIAN XIN CHU; YONG LU; ZHENG FU FANG; YING TANG SHI

    2003-01-01

    To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

  4. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

    Science.gov (United States)

    Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

    2015-07-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

  5. In vivo studies on chemically induced aneuploidy in mouse somatic and germinal cells.

    Science.gov (United States)

    Leopardi, P; Zijno, A; Bassani, B; Pacchierotti, F

    1993-05-01

    Within the context of a coordinated program to study aneuploidy induction sponsored by the European Community, nine chemicals were tested in mouse bone marrow and spermatocytes after intraperitoneal injection. In somatic cells, cell progression delay, hyperploidy, polyploidy induction and induction of micronucleated polychromatic erythrocyte (MnPCE) were studied. In germ cells hyperploidy induction was evaluated. The chemicals selected were: colchicine (COL), econazole (EZ), hydroquinone (HQ), thiabendazole (TB), diazepam (DZ), chloral hydrate (CH), cadmium chloride (CD), pyrimethamine (PY) and thimerosal (TM). Using literature data on c-mitotic effects in bone marrow as a reference, the same doses were tested in somatic and germ cells in order to compare the effects induced. Bone marrow cells were sampled 18 or 24 h after treatment. Germ cells were sampled 6, 8 or 18 h after treatment. Effects of COL and HQ in bone marrow have been reported elsewhere. Somatic effects were induced by CH (hyperploidy and cell cycle lengthening), TB (MnPCEs and cell cycle lengthening) and by PY (MnPCEs). EZ, DZ, CD and TM did not induce any kind of somatic effects. An increase in the incidence of hyperploid spermatocytes was induced by COL, at three dose levels, and by one dose of HQ and TB. All the other chemicals did not induce germinal aneuploidy at any dose or time tested. The hyperploidy control frequency ranged between 0.4 and 1.0% in somatic cells and from 0.3 to 0.9% in germ cells. In both somatic and germ cells, the maximum yield of induced hyperploidy did not exceed 3.5%. The time period of target cell sensitivity is probably restricted and this, associated with the heterogeneity and the asynchrony of cellular maturation processes, may account for our data. Under these circumstances, the negative data should be interpreted with some caution, particularly in germ cells, where additional indicators of chemical-cell interaction and cell cycle effects were not provided by

  6. Genetic aspects of somatic cell count and udder health in the Italian Valle del Belice dairy sheep

    NARCIS (Netherlands)

    Riggio, V.

    2012-01-01

    Mastitis is an inflammation of the udder, which leads to economic loss, mainly consisting of discarded milk, reduced milk production and quality, and increased health costs. Somatic cell count (SCC), and therefore somatic cell score (SCS), is widely used as indicator of mastitis. In this thesis, I

  7. A chemical genetics approach for specific differentiation of stem cells to somatic cells: a new promising therapeutical approach.

    Science.gov (United States)

    Sachinidis, Agapios; Sotiriadou, Isaia; Seelig, Bianca; Berkessel, Albrecht; Hescheler, Jürgen

    2008-01-01

    Cell replacement therapy of severe degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease through transplantation of somatic cells generated from embryonic stem (ES) cells is currently receiving considerable attention for the therapeutic applications. ES cells harvested from the inner cell mass (ICM) of the early embryo, can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells thereby providing an unlimited renewable source of somatic cells. In this context, identifying soluble factors, in particular chemically synthesized small molecules, and signal cascades involved in specific differentiation processes toward a defined tissue specific cell type are crucial for optimizing the generation of somatic cells in vitro for therapeutic approaches. However, experimental models are required allowing rapid and "easy-to-handle" parallel screening of chemical libraries to achieve this goal. Recently, the forward chemical genetic screening strategy has been postulated to screen small molecules in cellular systems for a specific desired phenotypic effect. The current review is focused on the progress of ES cell research in the context of the chemical genetics to identify small molecules promoting specific differentiation of ES cells to desired cell phenotype. Chemical genetics in the context of the cell ES-based cell replacement therapy remains a challenge for the near future for several scientific fields including chemistry, molecular biology, medicinal physics and robotic technologies.

  8. Research Progress on the Large-scale Culture Technology of Mammalian Cells

    Institute of Scientific and Technical Information of China (English)

    LI Chunyan; XIAO Jing; JIANG Yonghou

    2009-01-01

    The culture of mammalian cells is closely related to the development of biotechnology, which has been used extensively in the research and application fields of biology and medical science. In this article, various factors affecting cell cultivation and the application of microcarrier and bioreactor on large-scale culture of mammalian cells were reviewed.

  9. Studies of copper transport in mammalian cells using copper radioisotopes

    Energy Technology Data Exchange (ETDEWEB)

    Camakaris, J.; Voskoboinik, I.; Brooks, H.; Greenough, M. [University of Melbourne, Parkville, VIC (Australia). Department of Genetics; Smith, S. [Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW (Australia). Radiopharmaceuticals Division; Mercer, J. [Deakin University, Clayton, VIC (Australia). Centre of Cellular and Molecular Biology

    1998-12-31

    The trace element copper poses a major problem for all organisms. It is essential as a number of vital enzymes require it. Copper deficiency can lead to neurological disorders, osteoporosis and weakening of arteries. However Cu is also highly toxic and homeostatic mechanisms have evolved to maintain Cu at levels which satisfy requirements but do not cause toxicity. Toxicity is mediated by the oxidative capacity of Cu and its ability to generate toxic free radicals. There are several acquired and inherited diseases due to either Cu toxicity or Cu deficiency. The study of these diseases facilitates identification of genes and proteins involved in copper homeostasis, and this in turn will provide rational therapeutic approaches. Using the copper radioisotopes {sup 64}Cu (t1/2 = 12.8 hr) and {sup 67}Cu (t1/2 = 61 hr) we have developed a number of systems for studying copper transport in mammalian cells. These include investigation of copper uptake, copper efflux and ligand blot assays for Cu-binding proteins. Our studies have focused on Menkes disease which is an inherited and usually lethal copper deficiency disorder in humans. We have demonstrated that the Menkes protein is directly involved as a copper efflux pump in mammalian cells. Using cells overexpressing the Menkes protein we have provided the first biochemical evidence that this functions as a Cu translocating (across the membrane) P-type ATPase (Voskoboinik et al., FEBS Letters, in press). These studies were carried out using purified plasma membrane vesicles. We are now carrying out structure- function studies on this protein using targeted mutations and assaying using the radiocopper vesicle assay. Recently we have commenced studies on the role of amyloid precursor protein (APP) in copper transport and relationship of this to Alzheimers disease

  10. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    Science.gov (United States)

    Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

    2015-01-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  11. Gold nanoparticles enhanced electroporation for mammalian cell transfection.

    Science.gov (United States)

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2014-06-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e., better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials.

  12. Ultrastructure of germ cells and adjacent somatic cells correlated to initiation of meiosis in the fetal pig.

    Science.gov (United States)

    Byskov, A G; Høyer, P E; Björkman, N; Mørk, A B; Olsen, B; Grinsted, J

    1986-01-01

    The ultrastructure of female and male germ cells and associated somatic cells were studied in morphologically sex differentiated fetal pig gonads from day 27 to day 95 post insemination. Before meiosis starts in the ovary, the organelles of germ cells and somatic cells of both sexes are poorly developed. In oocytes in leptotene stage, the endoplasmic reticulum attains close proximity to the plasma membrane forming characteristic contact areas which in addition are only seen in male germ cells of the same age. As meiosis progresses, the organelles of the oocyte increase in number and degree of differentiation. In particular the ER is prominent in the diplotene stage. At midgestation the male germ cells become polarized, the organelles gathering at one side of the nucleus. In the granulosa cells the number and extension of organelles increase concomitantly with the oocytes proceeding through meiosis. The Sertoli cells grow progressively complex in shape, with numerous mitochondria and a prominent ER, whereas the Golgi complex remains poorly developed. Small dense bodies are present both in germ cells and somatic cells. They are electron dense, membrane bounded, rounded or elongated granules often connected with endoplasmic reticulum. No activity of peroxidase, catalase or acid phosphatase could be traced in the granules, making them improbable candidates as peroxisomes or lysosomes. Their number is high in all germ cells and somatic cells on day 27 post insemination and in germ cells in leptotene stage.

  13. A Comparative View on Human Somatic Cell Sources for iPSC Generation

    Directory of Open Access Journals (Sweden)

    Stefanie Raab

    2014-01-01

    Full Text Available The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs. Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types.

  14. The effect of ascetic acid on mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Mariana, Oana C [Los Alamos National Laboratory; Trujillo, Antoinette [Los Alamos National Laboratory; Sanders, Claire K [Los Alamos National Laboratory; Burnett, Kassidy S [Los Alamos National Laboratory; Freyer, James P [Los Alamos National Laboratory; Mourant, Judith R [Los Alamos National Laboratory

    2010-01-01

    Effects of the contrast agent, acetic acid, on mammalian cells are studied using light scattering measurements, viability and fluorescence pH assays. Results depend on whether cells are in PBS or are live and metabolizing. Acetic acid is a contrast agent used to aid the detection of cancerous and precancerous lesions of the uterine cervix. Typically 3% or 5% acetic acid is applied to the swface of the cervix and areas of the tissue that turn 'acetowhite' are considered more likely to be precancerous. The mechanism of action of acetic acid has never been understood in detail, although there are several hypotheses. One is that a decrease in pH causes cytokeratins in epithelial cells to polymerize. We will present data demonstrating that this is not the sole mechanism of acetowhitening. Another hypothesis is that a decrease in pH in the nucleus causes deacetylation of the histones which in turn results in a dense chromatin structure. Relevant to this hypothesis we have measured the internal pH of cells. Additional goals of this work are to understand what physical changes result in acetowhitening, to understand why there is variation in how cells respond to acetic acid, and to investigate how acetowhitening affects the light scatter properties measured by a fiber-optic probe we have developed for cervical cancer diagnostics.

  15. Probing Mammalian Cell Size Homeostasis by Channel-Assisted Cell Reshaping.

    Science.gov (United States)

    Varsano, Giulia; Wang, Yuedi; Wu, Min

    2017-07-11

    Cell size homeostasis can be achieved by size checkpoints that couple cell size to cell-cycle progression or by alternative mechanisms such as constant extension. In mammalian cells, the existence of strict size checkpoints remains controversial due to the technical limitations in determining cell size directly and accurately. We developed a microfabricated channel system that linearizes mammalian cell growth and facilitates cell size measurements. By tracking cell length, while directly visualizing cell-cycle progression in rat basophilic leukemia cells and RAW 264.7 macrophages, we examined the mechanisms of size homeostasis and the existence of a size checkpoint at the G1/S transition. Our analysis revealed a two-tier size homeostasis mechanism where a G1 "sizer" or "adder" could operate, depending on the birth size of the cells. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Longitudinal Analysis of Somatic Cell Count for Joint Genetic Evaluation of Mastitis and Recovery Liability

    DEFF Research Database (Denmark)

    Welderufael, Berihu Gebremedhin; de Koning, D J; Janss, Luc;

    Abstract Text: Better models of genetic evaluation for mastitis can be developed through longitudinal analysis of somatic cell count (SCC) which usually is used as a proxy for mastitis. Mastitis and recovery data with weekly observations of SCC were simulated for daughter groups of 60 and 240 per...

  17. Number and importance of somatic cells in goat’s milk

    Directory of Open Access Journals (Sweden)

    Lidija Kozačinski

    2001-04-01

    Full Text Available Goat’s milk samples were examined on mastitis using stable procedure (California-mastitis test. 427 of the examined milk samples (46.82% had positive reaction from 1 to 3 while other 485 samples (53.18% had negative reaction on the mastitis test, indicating that no illness of mammary gland occurred. Number of somatic cells, counted using “Fossomatic” counter, was 1.3x106/ml average. By comparing the results of mastitis-test evaluation (CMT with the number of somatic cells and findings of mastitis agents in milk showed that higher number of somatic cells is not the only indication of goat’s mammary gland illness. Mastitis-test is method that can exclude inflammation of goat’s mammary gland, but every positive reaction should be confirmed or eliminate with bacteriological examination. Based on the results of this research, it has been shown that the limit for somatic cells number in goat's milk can be over 1 000 000/ml.

  18. Somatic cell count assessment at the quarter or cow milking level

    NARCIS (Netherlands)

    Mollenhorst, H.; Tol, van der P.P.J.; Hogeveen, H.

    2010-01-01

    The aim was to investigate whether on-line somatic cell count (SCC) assessment, when combined with electrical conductivity (EC), should be implemented at the udder quarter or at the cow level. Data were collected from 3 farms with automatic milking systems, resulting in 3,191 quarter milkings used i

  19. Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem.

    Science.gov (United States)

    Oh, H J; Kim, M K; Jang, G; Kim, H J; Hong, S G; Park, J E; Park, K; Park, C; Sohn, S H; Kim, D Y; Shin, N S; Lee, B C

    2008-09-01

    The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.

  20. Correlations among somatic cell count, hygienic safety and quality of milk of primiparous cows

    Directory of Open Access Journals (Sweden)

    Adamov Nikola

    2009-11-01

    Full Text Available In this work we examined a total of 518 milk samples on the following parameters: somatic cell count (SCC, total bacteria count (CFU and IBC, fat, protein, lactose and dry matter non fat (DMNF contents, which were obtained from primiparous cows divided in three groups depending on the stage of lactation: the first group included the primiparous cows that were 10-100 days in lactation, the second group 101-200 days in lactation and the third group 201 and more days in lactation. The somatic cell count and the total bacterial count had highest values for the first group, intermediate for the third group, and lowest for the second group with these differences being statistically significant. Milk component contents varied among groups differently from previous two parameters but their differences were not significant in neither case. The somatic cell count of all three groups was positively and significantly correlated to the bacterial counts while these two parameters were generally in negative correlation with the milk component contents. No matter if the parameters that define the milk hygienic safety were positively or negatively correlated with the milk component contents, the correlation coefficients were not significant in neither case, which implies that significant reduction of milk components can be expected at somatic cell counts higher than the maximal obtained in this research of 236.000 SCC/ml.

  1. STUDY REGARDING THE CORELATION BETWEEN SOMATIC CELLS COUNT AND MAJOR CHEMICAL COMPOUNDS IN RAW MILK

    Directory of Open Access Journals (Sweden)

    S. ACATINCĂI

    2013-12-01

    Full Text Available This study approaches the dynamic of somatic cells number and chemical composition of milk during 13 months of control. The study also investigates the correlations between the number of somatic cells and some chemical parameters in milk. Studies were carried out on Romanian Black and White cows between March 2005 and March 2006 at the Didactical farm of the Banat University of Agricultural Sciences Timisoara. As quality indicator, the number of somatic cells has different values among the controls. Average values for the 13 months of control, with the exception of three controls, were below maximum limit admitted from 1th of January 2007 (600000 SCC/ml milk. There weren’t any significant differences for SCC between the two seasons. Chemical parameters in milk varied in close limits and the differences were not significant, with one exception for fat percent. Fat percent is higher (p<0.05 in the cold season 3.87% compared with 3.55% during the warm season. Somatic cells number is weak correlated with lactose and strong correlated with proteins.

  2. A proteomic perspective on the changes in milk proteins due to high somatic cell count

    NARCIS (Netherlands)

    Zhang, L.; Boeren, J.A.; Hooijdonk, van A.C.M.; Vervoort, J.J.M.; Hettinga, K.A.

    2015-01-01

    Although cows with subclinical mastitis have no difference in the appearance of their milk, milk composition and milk quality are altered because of the inflammation. To know the changes in milk quality with different somatic cell count (SCC) levels, 5 pooled bovine milk samples with SCC from 105 to

  3. Associations between somatic cell count patterns and the incidence of clinical mastitis

    NARCIS (Netherlands)

    Haas, de Y.; Barkema, H.W.; Schukken, Y.H.; Veerkamp, R.F.

    2005-01-01

    Associations between clinical mastitis (CM) and the proportional distribution of patterns in somatic cell count (SCC) on a herd level were determined in this study. Data on CM and SCC over a 12-month period from 274 Dutch herds were used. The dataset contained parts of 29,719 lactations from 22,955

  4. A matter of identity — Phenotype and differentiation potential of human somatic stem cells

    Directory of Open Access Journals (Sweden)

    S.E.P. New

    2015-07-01

    Full Text Available Human somatic stem cells with neural differentiation potential can be valuable for developing cell-based therapies, including treatment of birth-related defects, while avoiding issues associated with cell reprogramming. Precisely defining the “identity” and differentiation potential of somatic stem cells from different sources, has proven difficult, given differences in sets of specific markers, protocols used and lack of side-by-side characterization of these cells in different studies. Therefore, we set to compare expression of mesenchymal and neural markers in human umbilical cord-derived mesenchymal stem cells (UC-MSCs, pediatric adipose-derived stem cells (p-ADSCs in parallel with human neural stem cells (NSCs. We show that UC-MSCs at a basal level express mesenchymal and so-called “neural” markers, similar to that we previously reported for the p-ADSCs. All somatic stem cell populations studied, independently from tissue and patient of origin, displayed a remarkably similar expression of surface markers, with the main difference being the restricted expression of CD133 and CD34 to NSCs. Expression of certain surface and neural markers was affected by the expansion medium used. As predicted, UC-MSCs and p-ADSCs demonstrated tri-mesenchymal lineage differentiation potential, though p-ADSCs display superior chondrogenic differentiation capability. UC-MSCs and p-ADSCs responded also to neurogenic induction by up-regulating neuronal markers, but crucially they appeared morphologically immature when compared with differentiated NSCs. This highlights the need for further investigation into the use of these cells for neural therapies. Crucially, this study demonstrates the lack of simple means to distinguish between different cell types and the effect of culture conditions on their phenotype, and indicates that a more extensive set of markers should be used for somatic stem cell characterization, especially when developing therapeutic

  5. Gravity separation of fat, somatic cells, and bacteria in raw and pasteurized milks.

    Science.gov (United States)

    Caplan, Z; Melilli, C; Barbano, D M

    2013-04-01

    The objective of experiment 1 was to determine if the extent of gravity separation of milk fat, bacteria, and somatic cells is influenced by the time and temperature of gravity separation or the level of contaminating bacteria present in the raw milk. The objective of experiment 2 was to determine if different temperatures of milk heat treatment affected the gravity separation of milk fat, bacteria, and somatic cells. In raw milk, fat, bacteria, and somatic cells rose to the top of columns during gravity separation. About 50 to 80% of the fat and bacteria were present in the top 8% of the milk after gravity separation of raw milk. Gravity separation for 7h at 12°C or for 22h at 4°C produced equivalent separation of fat, bacteria, and somatic cells. The completeness of gravity separation of fat was influenced by the level of bacteria in the milk before separation. Milk with a high bacterial count had less (about 50 to 55%) gravity separation of fat than milk with low bacteria count (about 80%) in 22h at 4°C. Gravity separation caused fat, bacteria, and somatic cells to rise to the top of columns for raw whole milk and high temperature, short-time pasteurized (72.6°C, 25s) whole milk. Pasteurization at ≥76.9°C for 25s prevented all 3 components from rising, possibly due to denaturation of native bovine immunoglobulins that normally associate with fat, bacteria, and somatic cells during gravity separation. Gravity separation can be used to produce reduced-fat milk with decreased bacterial and somatic cell counts, and may be a critical factor in the history of safe and unique traditional Italian hard cheeses produced from gravity-separated raw milk. A better understanding of the mechanism of this natural process could lead to the development of new nonthermal thermal technology (that does not involve heating the milk to high temperatures) to remove bacteria and spores from milk or other liquids. Copyright © 2013 American Dairy Science Association. Published by

  6. Comparative reactivity of myeloperoxidase-derived oxidants with mammalian cells.

    Science.gov (United States)

    Rayner, Benjamin S; Love, Dominic T; Hawkins, Clare L

    2014-06-01

    Myeloperoxidase is an important heme enzyme released by activated leukocytes that catalyzes the reaction of hydrogen peroxide with halide and pseudo-halide ions to form various hypohalous acids. Hypohalous acids are chemical oxidants that have potent antibacterial, antiviral, and antifungal properties and, as such, play key roles in the human immune system. However, increasing evidence supports an alternative role for myeloperoxidase-derived oxidants in the development of disease. Excessive production of hypohalous acids, particularly during chronic inflammation, leads to the initiation and accumulation of cellular damage that has been implicated in many human pathologies including atherosclerosis, neurodegenerative disease, lung disease, arthritis, inflammatory cancers, and kidney disease. This has sparked a significant interest in developing a greater understanding of the mechanisms involved in myeloperoxidase-derived oxidant-induced mammalian cell damage. This article reviews recent developments in our understanding of the cellular reactivity of hypochlorous acid, hypobromous acid, and hypothiocyanous acid, the major oxidants produced by myeloperoxidase under physiological conditions.

  7. Bacterial xylanase expression in mammalian cells and transgenic mice.

    Science.gov (United States)

    Fontes, C M; Ali, S; Gilbert, H J; Hazlewood, G P; Hirst, B H; Hall, J

    1999-06-11

    The energy which simple-stomached livestock can derive from dietary plant material is limited by the lack of plant polysaccharide degrading enzymes in their gastro-intestinal (GI) tract and the inefficient microbial fermentation of such material in their hind-gut. In poultry the non-starch polysaccharides found in cereal grains can also impair normal digestive function as they form viscous gels in the GI tract inhibiting the breakdown and absorption of nutrients. The nutrition of such livestock could, therefore, be improved by the introduction of enzymes able to degrade plant polysaccharides in the small intestine. We describe the expression of a xylanase, XYLY', from the bacterium Clostridium thermocellum in mammalian cells and the exocrine pancreas of transgenic mice. The enzyme is synthesised, secreted and functionally active in the eukaryote system. This work demonstrates the feasibility of generating animals with the endogenous capacity to depolymerise the xylan component of hemi-cellulose.

  8. Epigenetic reprogramming in somatic cells induced by extract from germinal vesicle stage pig oocytes.

    Science.gov (United States)

    Bui, Hong-Thuy; Kwon, Deug-Nam; Kang, Min-Hui; Oh, Mi-Hye; Park, Mi-Ryung; Park, Woo-Jin; Paik, Seung-Sam; Van Thuan, Nguyen; Kim, Jin-Hoi

    2012-12-01

    Genomic reprogramming factors in the cytoplasm of germinal vesicle (GV) stage oocytes have been shown to improve the efficiency of producing cloned mouse offspring through the exposure of nuclei to a GV cytoplasmic extract prior to somatic cell nuclear transfer (SCNT) to enucleated oocytes. Here, we developed an extract of GV stage pig oocytes (GVcyto-extract) to investigate epigenetic reprogramming events in treated somatic cell nuclei. This extract induced differentiation-associated changes in fibroblasts, resulting in cells that exhibit pluripotent stem cell-like characteristics and that redifferentiate into three primary germ cell layers both in vivo and in vitro. The GVcyto-extract treatment induced large numbers of high-quality SCNT-generated blastocysts, with methylation and acetylation of H3-K9 and expression of Oct4 and Nanog at levels similar to in vitro fertilized embryos. Thus, GVcyto-extract could elicit differentiation plasticity in treated fibroblasts, and SCNT-mediated reprogramming reset the epigenetic state in treated cells more efficiently than in untreated cells. In summary, we provide evidence for the generation of stem-like cells from differentiated somatic cells by treatment with porcine GVcyto-extract.

  9. Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer.

    Science.gov (United States)

    Koo, Ok Jae; Hossein, Mohammad Shamim; Hong, So Gun; Martinez-Conejero, Jose A; Lee, Byeong Chun

    2009-02-01

    Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents - dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) - on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

  10. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masahito [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Umeyama, Kazuhiro [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); International Cluster for Bio-Resource Research, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Matsunari, Hitomi [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Takayanagi, Shuko [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Nakauchi, Hiromitsu [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, Tokyo University, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); and others

    2010-11-05

    Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  11. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies.

    Science.gov (United States)

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-07-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

  12. Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens. [Rats, hamsters

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Langenbach, R.

    1977-01-01

    We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro.

  13. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Wei SHEN; Lingjiang MIN; Qingyu PAN; Yujiang SUN; Jixian DENG; Qingjie PAN

    2008-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.

  14. Telomere length status of somatic cell sheep clones and their offspring.

    Science.gov (United States)

    Alexander, Basil; Coppola, Gianfranco; Perrault, Steven D; Peura, Teija T; Betts, Dean H; King, W Allan

    2007-12-01

    This study was carried out to determine the telomere length status of sheep clones and their offspring, and to examine telomere dynamics and chromosomal abnormalities in culture propagated donor cells. Skin samples were collected from somatic cell nuclear transfer-derived sheep clones, and three of their progeny generated by natural mating. Samples were collected from control animals (n = 35), spanning in age from 1 month to 36 months of age. Genomic DNA was extracted from cell/tissue samples and their telomere lengths were assessed by terminal restriction fragment (TRF) analysis. Results revealed: that (a) sheep clones derived from cultured somatic cells have shortened telomere lengths compared to age-matched controls; (b) the offspring derived from natural mating between clones had normal telomere lengths compared to their age-matched counterparts; and donor cell cultures beyond 20 population doublings had significantly (P < 0.05) shortened telomeres and exhibited a higher numerical and structural chromosomal abnormalities.

  15. Re-engineering the mitochondrial genomes in mammalian cells

    Science.gov (United States)

    Koob, Michael D; Yoo, Young Hyun

    2010-01-01

    Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. They have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. The human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the significant advances in discovering the mtDNA defects, however, there are currently no effective therapies for these clinically devastating diseases due to the lack of technology for introducing specific modifications into the mitochondrial genomes and for generating accurate mtDNA disease models. The ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own efforts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation. PMID:21189990

  16. Antioxidation activities of pteridines in mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Y.; Shen, R. (Univ. of Texas, Galveston (United States))

    1991-03-11

    L-erythro-5,6,7,8-Tetrahydrobiopterin (BH{sub 4}), the cofactor for aromatic amino acid hydroxylases (AAA-H), is a predominant form of pteridines which occur ubiquitously in nature. When BH{sub 4} is oxidized to quinonoid dihydrobiopterin by AAA-H, it is regenerated by dihydropteridine reductase (DHPR) at the expense of NADH. The role of BH{sub 4} other than serving as the hydroxylase cofactor is not clear. The existence of BH{sub 4} and DHPR in tissues which are devoid of AAA-H suggests that BH{sub 4} may play an as yet undiscovered physiological function. This study demonstrates a BH{sub 4}-mediated antioxidation system, which consists of BH{sub 4}, DHPR, peroxidase and NADH in rat pheochromocytoma PC 12 cells and mouse macrophages J774A.1. This system was as effective as catalase and ascorbic acid in protecting cells against H{sub 2}O{sub 2} and xanthine/xanthine oxidase-induced toxicity and was more effective than catalase in defense against nitrofurantoin-induced toxicity. The antioxidation effect of this system was not due to peroxidase and was improved when synthetic pteridines were substituted for BH{sub 4}. Since BH{sub 4}, DHPR, peroxidases and NADH are widely distributed in major organs and blood cells, they may constitute an as yet little known antioxidation system in mammalian cells.

  17. A novel gene delivery system for mammalian cells.

    Science.gov (United States)

    Gibson, Brian; Duffy, Angela M; Gould Fogerite, Susan; Krause-Elsmore, Sara; Lu, Ruying; Shang, Gaofeng; Chen, Zi-Wei; Mannino, Raphael J; Bouchier-Hayes, David J; Harmey, Judith H

    2004-01-01

    Although gene therapy holds great promise for the treatment of both acquired and genetic diseases, its development has been limited by practical considerations. Non-viral efficacy of delivery remains quite poor. We are investigating the feasibility of a novel lipid-based delivery system, cochleates, to deliver transgenes to mammalian cells. Rhodamine-labelled empty cochleates were incubated with two cell-lines (4T1 adenocarcinoma and H36.12 macrophage hybridoma) and primary macrophages in vitro and in vivo. Cochleates containing green fluorescent protein (GFP) expression plasmid were incubated with 4T1 adenocarcinoma cells. Cellular uptake of labelled cochleates or transgene GFP expression were visualised with fluorescence microscopy. 4T1 and H36.12 lines showed 39% and 23.1% uptake of rhodamine-cochleates, respectively. Human monocyte-derived macrophages and mouse peritoneal macrophages had 48+/-5.38% and 51.46+/-15.6% uptake of rhodamine-cochleates in vitro. In vivo 25.69+/-0.127% of peritoneal macrophages were rhodamine-positive after intra-peritoneal injection of rhodamine-cochleates. 19.49+/-10.12% of 4T1 cells expressed GFP. Cochleates may therefore be an effective, non-toxic and non-immunogenic method to introduce transgenes in vitro and in vivo.

  18. Helium Ion Microscopy Visualizes Lipid Nanodomains in Mammalian Cells.

    Science.gov (United States)

    Schürmann, Matthias; Frese, Natalie; Beyer, André; Heimann, Peter; Widera, Darius; Mönkemöller, Viola; Huser, Thomas; Kaltschmidt, Barbara; Kaltschmidt, Christian; Gölzhäuser, Armin

    2015-11-18

    Cell membranes are composed of 2D bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, direct visualization of cell membrane nanodomains by helium ion microscopy (HIM) is presented. It is shown that HIM is capable to image biological specimens without any conductive coating and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ≈ 15 nm.

  19. Somatic (CSS and differential cell count (DCC during a lactation period in ass’milk

    Directory of Open Access Journals (Sweden)

    Paolo Polidori

    2010-01-01

    Full Text Available Hypoallergenic properties of ass’s milk protein fractions have been recently con- firmed, allowing ass’s milk to be considered as a valid substitute of the available hypoallergenic infant formulas. The objective of this study was to give a further contribution to the knowledge of ass’s milk safety and quality characteristics. A new procedure has been developed with a cytospin centrifuge in differential counts of milk somatic cells. Somatic cells count (SCC, differential somatic cells count (DCC and cultural examinations have been carried out in 62 milk samples collected from 11 asses at three different stages of lactation. Four major cells populations had been identified in ass’s milk too: lymphocytes (Ly, monocytes/macrophages (MA, polymorphonuclear neutrophils (PMNL, and epithelial cells (CE. The patterns of these cells have been discussed in comparison with cells found in dairy cows and ewes milk. In conclusion, a reproducible standard procedure has been developed to determine cell count of ass’s milk.

  20. discs large regulates somatic cyst cell survival and expansion in Drosophila testis

    Institute of Scientific and Technical Information of China (English)

    Fani Papagiannouli; Bernard M Mechler

    2009-01-01

    Gonad development requires a coordinated snma-germline interaction that ensures renewal and differentiation of germline and somatic stem cells to ultimately produce mature gametes. The Drosophila tumour suppressor gene discs sion, and formation of neuromuscular junctions. Here, we report the role of dig in testis development and its critical function in somatic cyst cells (SCCs). In these cells dig is primarily required for their survival and expansion, and contributes to spermatocyte cyst differentiation. Cell death primarily occurred in SCCs at the end of spermatogo-nial amplification at a time when Dig becomes restricted in wild-type (wt) testes to the distal somatic cells capping the growing spermatocyte cysts. RNAi depletion of dig transcripts in early SCCs fully prevented testis development, whereas depletion in late SCCs resulted in a breakdown of spermatocyte cyst structure and germ cell individualiza-tion. Specific dig expression in SCCs resulted in developmental rescue of dig mutant testes, whereas its expression in germ cells exerted no such effect, dig overexpression in wt testes led to spermatocyte cyst expansion at the expense of spermatogonial cysts. Our data demonstrate that dig is essentially required in SCCs for their survival, expansion, and differentiation, and for the encapsulation of the germline cells.

  1. Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; LI Rong; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Haiping; WANG Lili; FANG Changge; WAN Rong; LIU Ying

    2004-01-01

    The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.

  2. The influence of bisphenol A on mammalian cell cultivation.

    Science.gov (United States)

    Stiefel, Fabian; Paul, Albert Jesuran; Jacopo, Troisi; Sgueglia, Angelo; Stützle, Martina; Herold, Eva Maria; Hesse, Friedemann

    2016-01-01

    Bisphenol A (BPA) plays a substantial role in industry, as it is used for polycarbonate (PC) plastics and epoxy resins which are required for various plastic consumer products. However, BPA is known to be an endocrine disruptor, and its influence on humans, animals, and various cell lines was addressed in diverse studies. As the burden of BPA can be increased by using disposable plastic articles and single-use technologies for cultivation, it is essential to examine the consequences of BPA presence on mammalian cells, as they are a contributing factor in the production of complex pharmaceutical therapeutics. We selected three industrially relevant cell lines and analyzed systemic effects of BPA by comparing cell culture performance in BPA-free poly-ethylene terephthalate glycol (PETG) and in PC shaking flasks. We focused on the influence of BPA on cellular growth, viability, and several metabolic parameters. In addition, we determined the product concentration and aggregation behavior of the recombinant proteins expressed by these cell lines and the BPA concentration within the medium caused by leaching. Moreover, we performed EC50 studies to determine the toxic concentration of BPA. Our results indicated that leached BPA had no effect on specific growth rates and viability and toxicity appeared at about 10(4) times higher concentrations; however, it influenced the specific productivity rate and metabolic activity parameters of our Chinese hamster ovary (CHO) cell line. Consequently, one can neglect BPA from leaching in the culture as long as the selected cell line is BPA tolerant. Otherwise, BPA can be a hurdle for pharmaceutical production, as it can influence the specific productivity of recombinant proteins.

  3. Expression of a secreted protein in mammalian cells using baculovirus particles.

    Science.gov (United States)

    Jardin, Barbara Ann; Elias, Cynthia B; Prakash, Satya

    2012-01-01

    There are many methods presently available to produce recombinant proteins in mammalian systems. The BacMam system is a simple straightforward method which overlaps two well-established technologies, namely the BEVS insect cell system and the transduction of mammalian cells in vitro. This chapter describes a method for the study of gene expression in mammalian cells in a series of simple steps. Protocols outlined include the design and construction of the recombinant baculovirus, cell culture techniques required to maintain both insect and mammalian cells, generation of baculovirus stocks, and methods to obtain maximal and reproducible gene expression in mammalian cells. Currently available statistical techniques using factorial design of experiment to optimize conditions for recombinant protein in vitro are outlined. Then details with respect to process scale-up in disposable bioreactors are included.

  4. Epigenetic memory in somatic cell nuclear transfer and induced pluripotency: evidence and implications.

    Science.gov (United States)

    Firas, Jaber; Liu, Xiaodong; Polo, Jose M

    2014-07-01

    Six decades ago, seminal work conducted by John Gurdon on genome conservation resulted in major advancements towards nuclear reprogramming technologies such as somatic cell nuclear transfer (SCNT), cell fusion and transcription factor mediated reprogramming. This revolutionized our views regarding cell fate conversion and development. These technologies also shed light on the role of the epigenome in cellular identity, and how the memory of the cell of origin affects the reprogrammed cell. This review will discuss recent work on epigenetic memory retained in pluripotent cells derived by SCNT and transcription factor mediated reprogramming, and the challenges attached to it.

  5. Rotation of single live mammalian cells using dynamic holographic optical tweezers

    Science.gov (United States)

    Bin Cao; Kelbauskas, Laimonas; Chan, Samantha; Shetty, Rishabh M.; Smith, Dean; Meldrum, Deirdre R.

    2017-05-01

    We report on a method for rotating single mammalian cells about an axis perpendicular to the optical system axis through the imaging plane using dynamic holographic optical tweezers (HOTs). Two optical traps are created on the opposite edges of a mammalian cell and are continuously transitioned through the imaging plane along the circumference of the cell in opposite directions, thus providing the torque to rotate the cell in a controlled fashion. The method enables a complete 360° rotation of live single mammalian cells with spherical or near-to spherical shape in 3D space, and represents a useful tool suitable for the single cell analysis field, including tomographic imaging.

  6. DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Gao, Fei; Li, Shengting; Lin, Lin

    2011-01-01

    To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

  7. Production of transgenic7blastocyst of sheep by somatic cell cloning

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Five samples from primary cultures of five sheep ovarian granulosa cells were transfeeted by pEGFP N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucle ated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were ob tained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P>0.05) with the result ob tained with another cell line that had not gone through se rum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.

  8. Assessing Mitochondrial Unfolded Protein Response in Mammalian Cells.

    Science.gov (United States)

    Durand, Fiona; Hoogenraad, Nicholas

    2017-01-01

    Mitochondria serve a key role in the supply of energy to cells in the form of ATP, the supply of essential cellular components such as phospholipids and heme, in apoptosis and as a mediator of cellular signaling pathways. Mitochondria have their own DNA, consisting of a small number of genes, but the majority of the total protein complement is encoded in the nucleus, synthesized in the cytosol, and is imported into the mitochondria in a largely, if not completely unfolded form. These proteins need to be folded into their functional form within the organelle with the concomitant requirement that the organelle has its own suite of molecular chaperones and complexes to degrade damaged proteins to avoid stress arising from accumulation of unfolded proteins. This mitochondrial unfolded protein response can also be induced in cells and protein regulation can be determined using western blot, luciferase reporter assay, and sensitive mass spectrometry techniques. In this chapter, we describe a method to induce mtUPR in mammalian cells and the three methods to analyze components involved in it.

  9. Telomere homeostasis in mammalian germ cells: a review.

    Science.gov (United States)

    Reig-Viader, Rita; Garcia-Caldés, Montserrat; Ruiz-Herrera, Aurora

    2016-06-01

    Telomeres protect against genome instability and participate in chromosomal movements during gametogenesis, especially in meiosis. Thus, maintaining telomere structure and telomeric length is essential to both cell integrity and the production of germ cells. As a result, alteration of telomere homeostasis in the germ line may result in the generation of aneuploid gametes or gametogenesis disruption, triggering fertility problems. In this work, we provide an overview on fundamental aspects of the literature regarding the organization of telomeres in mammalian germ cells, paying special attention to telomere structure and function, as well as the maintenance of telomeric length during gametogenesis. Moreover, we discuss the different roles recently described for telomerase and TERRA in maintaining telomere functionality. Finally, we review how new findings in the field of reproductive biology underscore the role of telomere homeostasis as a potential biomarker for infertility. Overall, we anticipate that the study of telomere stability and equilibrium will contribute to improve diagnoses of patients; assess the risk of infertility in the offspring; and in turn, find new treatments.

  10. Xyloglucan Endotransglycosylase Activity in Carrot Cell Suspensions during cell Elongation and Somatic Embryogenesis.

    Science.gov (United States)

    Hetherington, P. R.; Fry, S. C.

    1993-11-01

    Xyloglucan endotransglycosylase (XET) has been proposed to contribute to cell elongation through wall loosening. To explore this relationship further, we assayed this enzyme activity in suspensions of carrot (Daucus carota L.) cells exhibiting various rates of cell elongation. In one cell line, elongation was induced by dilution into dichlorophenoxyacetic acid (2,4-D)-free medium. During this elongation, 93% of the XET activity was found in the culture medium; in nonelongating controls, by contrast, 68% was found in the cell extracts even though the specific activity of these extracts was lower than in the elongating cells. By far the highest rates of XET secretion per cell were in the elongating cells. A second cell line was induced to undergo somatic embryogenesis by dilution into 2,4-D-free medium. During the first 6 d, numerous globular embryoids composed of small, isodiametric cells were formed in the absence of cell elongation; extracellular XET activity was almost undetectable, and intracellular specific activity markedly declined. After 6 d, heart, torpedo, and cotyledonary embryoids began to appear (i.e. cell elongation resumed); the intracellular specific activity of XET rose rapidly and >80% of the XET activity accumulated in the medium. Thus, nonexpanding cell suspensions (whether or not they were rapidly dividing) produced and secreted less XET activity than did expanding cells. We propose that a XET molecule has an ephemeral wall-loosening role while it passes through the load-bearing layer of the wall on its way from the protoplast into the culture medium.

  11. Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

    OpenAIRE

    Domenico Iuso; Marta Czernik; Fiorella Di Egidio; Silvestre Sampino; Federica Zacchini; Michal Bochenek; Zdzislaw Smorag; Modlinski, Jacek A.; Grazyna Ptak; Pasqualino Loi

    2013-01-01

    The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA da...

  12. Genetic changes in Mammalian cells transformed by helium cells

    Energy Technology Data Exchange (ETDEWEB)

    Durante, M.; Grossi, G. (Naples Univ. (Italy). Dipt. di Scienze Fisiche); Yang, T.C.; Roots, R. (Lawrence Berkeley Lab., CA (USA))

    1990-11-01

    Midterm Syrian Hamster embryo (SHE) cells were employed to study high LET-radiation induced tumorigenesis. Normal SHE cells (secondary passage) were irradiated with accelerated helium ions at an incident energy of 22 MeV/u (9--10 keV/{mu}m). Transformed clones were isolated after growth in soft agar of cells obtained from the foci of the initial monolayer plated postirradiation. To study the progression process of malignant transformation, the transformed clones were followed by monolayer subculturing for prolonged periods of time. Subsequently, neoplasia tests in nude mice were done. In this work, however, we have focused on karyotypic changes in the banding patterns of the chromosomes during the early part of the progressive process of cell transformation for helium ion-induced transformed cells. 26 refs., 5 figs., 2 tabs.

  13. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    Energy Technology Data Exchange (ETDEWEB)

    Yue, Xiao-shan [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Fujishiro, Masako [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Toyoda, Masashi [Department of Reproductive Biology, National Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535 (Japan); Akaike, Toshihiro [Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Ito, Yoshihiro, E-mail: y-ito@riken.jp [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan)

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  14. UVC-induced stress granules in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Mohamed Taha Moutaoufik

    Full Text Available Stress granules (SGs are well characterized cytoplasmic RNA bodies that form under various stress conditions. We have observed that exposure of mammalian cells in culture to low doses of UVC induces the formation of discrete cytoplasmic RNA granules that were detected by immunofluorescence staining using antibodies to RNA-binding proteins. UVC-induced cytoplasmic granules are not Processing Bodies (P-bodies and are bone fide SGs as they contain TIA-1, TIA-1/R, Caprin1, FMRP, G3BP1, PABP1, well known markers, and mRNA. Concomitant with the accumulation of the granules in the cytoplasm, cells enter a quiescent state, as they are arrested in G1 phase of the cell cycle in order to repair DNA damages induced by UVC irradiation. This blockage persists as long as the granules are present. A tight correlation between their decay and re-entry into S-phase was observed. However the kinetics of their formation, their low number per cell, their absence of fusion into larger granules, their persistence over 48 hours and their slow decay, all differ from classical SGs induced by arsenite or heat treatment. The induction of these SGs does not correlate with major translation inhibition nor with phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α. We propose that a restricted subset of mRNAs coding for proteins implicated in cell cycling are removed from the translational apparatus and are sequestered in a repressed form in SGs.

  15. Somatic cell nuclear transfer in Oregon: expanding the pluripotent space and informing research ethics.

    Science.gov (United States)

    Lomax, Geoffrey P; DeWitt, Natalie D

    2013-12-01

    In May, Oregon Health and Science University (OHSU) announced the successful derivation, by the Mitalipov laboratory, of embryonic stem cells by somatic cell nuclear transfer. This experiment was recognized as a "formidable technical feat" and potentially a key step toward developing cell-based therapies. The OHSU report is also an example of how a scientific breakthrough can inform research ethics. This article suggests ways that nuclear transfer embryonic stem cell lines may contribute to research ethics by adding rigor to studies addressing pressing research questions important to the development of cell-based therapies.

  16. Optimization of somatic cell injection in the perspective of nuclear transfer in goldfish

    Directory of Open Access Journals (Sweden)

    Mahe Sophie

    2010-06-01

    Full Text Available Abstract Background Nuclear transfer has the potential to become one strategy for fish genetic resources management, by allowing fish reconstruction from cryopreserved somatic cells. Survival rates after nuclear transfer are still low however. The part played by unsuitable handling conditions is often questioned, but the different steps in the procedure are difficult to address separately. In this work led on goldfish (Carassius auratus, the step of somatic cells injection was explored. Non-enucleated metaphase II oocytes were used as a template to explore the toxicity of the injection medium, to estimate the best location where the cell should be injected, and to assess the delay necessary between cell injection and oocyte activation. Results Trout coelomic fluid was the most suitable medium to maintain freshly spawned oocytes at the metaphase II stage during oocyte manipulation. Oocytes were then injected with several media to test their toxicity on embryo development after fertilization. Trout coelomic fluid was the least toxic medium after injection, and the smallest injected volume (10 pL allowed the same hatching rates as the non injected controls (84.8% ± 23. In somatic cell transfer experiments using non enucleated metaphase II oocytes as recipient, cell plasma membrane was ruptured within one minute after injection. Cell injection at the top of the animal pole in the oocyte allowed higher development rates than cell injection deeper within the oocyte (respectively 59% and 23% at mid-blastula stage. Embryo development rates were also higher when oocyte activation was delayed for 30 min after cell injection than when activation was induced without delay (respectively 72% and 48% at mid-blastula stage. Conclusions The best ability of goldfish oocytes to sustain embryo development was obtained when the carrier medium was trout coelomic fluid, when the cell was injected close to the animal pole, and when oocyte activation was induced 30 min

  17. Control of mammalian germ cell entry into meiosis.

    Science.gov (United States)

    Feng, Chun-Wei; Bowles, Josephine; Koopman, Peter

    2014-01-25

    Germ cells are unique in undergoing meiosis to generate oocytes and sperm. In mammals, meiosis onset is before birth in females, or at puberty in males, and recent studies have uncovered several regulatory steps involved in initiating meiosis in each sex. Evidence suggests that retinoic acid (RA) induces expression of the critical pre-meiosis gene Stra8 in germ cells of the fetal ovary, pubertal testis and adult testis. In the fetal testis, CYP26B1 degrades RA, while FGF9 further antagonises RA signalling to suppress meiosis. Failsafe mechanisms involving Nanos2 may further suppress meiosis in the fetal testis. Here, we draw together the growing knowledge relating to these meiotic control mechanisms, and present evidence that they are co-ordinately regulated and that additional factors remain to be identified. Understanding this regulatory network will illuminate not only how the foundations of mammalian reproduction are laid, but also how mis-regulation of these steps can result in infertility or germline tumours. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  18. Roles of small molecules in somatic cell reprogramming.

    Science.gov (United States)

    Su, Jian-bin; Pei, Duan-qing; Qin, Bao-ming

    2013-06-01

    The Nobel Prize in Physiology and Medicine 2012 was awarded to Sir John B GURDON and Shinya YAMANAKA for their discovery that mature cells can be reprogrammed to become pluripotent. This event reaffirms the importance of research on cell fate plasticity and the technology progress in the stem cell field and regenerative medicine. Indeed, reprogramming technology has developed at a dazzling speed within the past 6 years, yet we are still at the early stages of understanding the mechanisms of cell fate identity. This is particularly true in the case of human induced pluripotent stem cells (iPSCs), which lack reliable standards in the evaluation of their fidelity and safety prior to their application. Along with the genetic approaches, small molecules nowadays become convenient tools for modulating endogenous protein functions and regulating key cellular processes, including the mesenchymal-to-epithelial transition, metabolism, signal transduction and epigenetics. Moreover, small molecules may affect not only the efficiency of clone formation but also the quality of the resulting cells. With increasing availability of such chemicals, we can better understand the biology of stems cells and further improve the technology of generation of stem cells.

  19. Reversal of informational entropy and the acquisition of germ-like immortality by somatic cells.

    Science.gov (United States)

    Kyriazis, Marios

    2014-01-01

    We live within an increasingly technological, information-laden environment for the first time in human evolution. This subjects us (and will continue to subject us in an accelerating fashion) to an unremitting exposure to 'meaningful information that requires action'. Directly dependent upon this new environment are novel evolutionary pressures, which can modify existing resource allocation mechanisms and may eventually favour the survival of somatic cells (particularly neurons) at the expense of germ line cells. In this theoretical paper I argue that persistent, structured information-sharing in both virtual and real domains, leads to increased biological complexity and functionality, which reflects upon human survival characteristics. Certain biological immortalisation mechanisms currently employed by germ cells may thus need to be downgraded in order to enable somatic cells to manage these new energy demands placed by our modern environment. Relevant concepts from a variety of disciplines such as the evolution of complex adaptive systems, information theory, digital hyper-connectivity, and cell immortalisation will be reviewed. Using logical, though sometimes speculative arguments, I will attempt to describe a new biology. A biology not driven by sex and reproduction but by information and somatic longevity.

  20. Sodium copper chlorophyllin (SCC) induces genetic damage in postmeiotic and somatic wing cells of Drosophila melanogaster.

    Science.gov (United States)

    Peñaloza, Emilio Pimentel; Cruces Martínez, Martha Patricia

    2013-01-01

    There is no apparent evidence to indicate that sodium copper chlorophyllin (SCC) is mutagenic. The aim of the present study was thus to determine the mutagenic effect of SCC, in postmeiotic germ cells of the adult male Drosophila. This investigation was based on the ability to examine whether SCC induced sex-linked recessive lethal mutations (SLRL), as well as the somatic mutation and recombination test (SMART). Four different SCC concentrations were used: 0, 45, 69, 80, and 100 mM. For SLRL, two broods were generated to test sperm and primarily spermatids. Results showed a significant frequency of recessive lethal mutations compared with control sperm cells with SCC at 69, 80, and 100 mM. In contrast, the frequency of somatic mutations rose by 0.21 only with 100 mM of SCC. These findings provide evidence that SCC is a weak mutagen in both cell lines. The differential response may be attributed to repair mechanisms that are active in somatic cells but almost absent in germ cells.

  1. Effects of herd management practices on somatic cell counts in an arid climate

    Directory of Open Access Journals (Sweden)

    Ali Sadeghi-Sefidmazgi

    2014-09-01

    Full Text Available The objective of this study was to evaluate associations between average lactation somatic cell counts (SCC and herd management practices in an arid climate. A total of 38,530 average lactation SCC records for 10,216 Holstein cows gathered on 25 dairy farms from January 2009 to October 2012 in Isfahan (Iran were analyzed. Average lactation SCC (cells × 1,000 was 250.79 ranging from 90.31 to 483.23 cells/mL across investigated farms. Herd-level management factors associated with average lactation SCC were determined separately using mixed linear models in the MIXED procedure with average lactation somatic cell score (SCS included as the dependent variable. Some of the management practices associated with low average lactation SCS included sawdust combined with sand bedding, using automatic cup removers, disinfection of the teats by dipping into disinfectant, using washable towels for teat cleaning, free-stall barns, wet disposable tissue for udder washing, wearing gloves during milking and the use of humidifiers and shade. Lower-production herds and larger-size herds had lower average lactation somatic cell counts. Most herd management practices associated with average lactation SCC in dairy herds in the arid region of Isfahan are in agreement with most previous studies. However, different results are found for use of humidifier, bedding materials and herd size.

  2. Generation of embryonic stem cells from mouse adipose-tissue derived cells via somatic cell nuclear transfer.

    Science.gov (United States)

    Qin, Yiren; Qin, Jilong; Zhou, Chikai; Li, Jinsong; Gao, Wei-Qiang

    2015-01-01

    Somatic cells can be reprogrammed into embryonic stem cells (ESCs) by nuclear transfer (NT-ESCs), or into induced pluripotent stem cells (iPSCs) by the "Yamanaka method." However, recent studies have indicated that mouse and human iPSCs are prone to epigenetic and transcriptional aberrations, and that NT-ESCs correspond more closely to ESCs derived from in vitro fertilized embryos than iPSCs. In addition, the procedure of NT-ESCs does not involve gene modification. Demonstration of generation of NT-ESCs using an easily-accessible source of adult cell types would be very important. Adipose tissue is a source of readily accessible donor cells and can be isolated from both males and females at different ages. Here we report that NT-ESCs can be generated from adipose tissue-derived cells (ADCs). At morphological, mRNA and protein levels, these NT-ESCs show classic ESC colonies, exhibit alkaline phosphatase (AP) activity, and display normal diploid karyotypes. Importantly, these cells express pluripotent markers including Oct4, Sox2, Nanog and SSEA-1. Furthermore, they can differentiate in vivo into various types of cells from 3 germinal layers by teratoma formation assays. This study demonstrates for the first time that ESCs can be generated from the adipose tissue by somatic cell nuclear transfer (SCNT) and suggests that ADCs can be a new donor-cell type for potential therapeutic cloning.

  3. Roles of small molecules in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Jian-bin SU; Duan-qing PEI; Bao-ming QIN

    2013-01-01

    The Nobel Prize in Physiology and Medicine 2012 was awarded to Sir John B GURDON and Shinya YAMANAKA for their discovery that mature cells can be reprogrammed to become pluripotent.This event reaffirms the importance of research on cell fate plasticity and the technology progress in the stem cell field and regenerative medicine.Indeed,reprogramming technology has developed at a dazzling speed within the past 6 years,yet we are still at the early stages of understanding the mechanisms of cell fate identity.This is particularly true in the case of human induced pluripotent stem ceils (iPSCs),which lack reliable standards in the evaluation of their fidelity and safety prior to their application.Along with the genetic approaches,small molecules nowadays become convenient tools for modulating endogenous protein functions and regulating key cellular processes,including the mesenchymal-to-epithelial transition,metabolism,signal transduction and epigenetics.Moreover,small molecules may affect not only the efficiency of clone formation but also the quality of the resulting cells.With increasing availability of such chemicals,we can better understand the biology of stems cells and further improve the technology of generation of stem cells.

  4. Micropatterning of proteins and mammalian cells on indium tin oxide.

    Science.gov (United States)

    Shah, Sunny S; Howland, Michael C; Chen, Li-Jung; Silangcruz, Jaime; Verkhoturov, Stanislav V; Schweikert, Emile A; Parikh, Atul N; Revzin, Alexander

    2009-11-01

    This paper describes a novel surface engineering approach that combines oxygen plasma treatment and electrochemical activation to create micropatterned cocultures on indium tin oxide (ITO) substrates. In this approach, photoresist was patterned onto an ITO substrate modified with poly(ethylene) glycol (PEG) silane. The photoresist served as a stencil during exposure of the surface to oxygen plasma. Upon incubation with collagen (I) solution and removal of the photoresist, the ITO substrate contained collagen regions surrounded by nonfouling PEG silane. Chemical analysis carried out with time-of-flight secondary ion mass spectrometry (ToF-SIMS) at different stages in micropatterned construction verified removal of PEG-silane during oxygen plasma and presence of collagen and PEG molecules on the same surface. Imaging ellipsometry and atomic force microscopy (AFM) were employed to further investigate micropatterned ITO surfaces. Biological application of this micropatterning strategy was demonstrated through selective attachment of mammalian cells on the ITO substrate. Importantly, after seeding the first cell type, the ITO surfaces could be activated by applying negative voltage (-1.4 V vs Ag/AgCl). This resulted in removal of nonfouling PEG layer and allowed to attach another cell type onto the same surface and to create micropatterned cocultures. Micropatterned cocultures of primary hepatocytes and fibroblasts created by this strategy remained functional after 9 days as verified by analysis of hepatic albumin. The novel surface engineering strategy described here may be used to pattern multiple cell types on an optically transparent and conductive substrate and is envisioned to have applications in tissue engineering and biosensing.

  5. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K;

    2002-01-01

    will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells...... and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual......-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels....

  6. Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

    Directory of Open Access Journals (Sweden)

    Li-Ying Sung

    2014-12-01

    Full Text Available Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.

  7. Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells

    OpenAIRE

    2009-01-01

    The primary function of O6-alkylguanine-DNA alkyltransferase (AGT) is to maintain genomic integrity in the face of damage by both endogenous and exogenous alkylating agents. However, paradoxically, bacterial and mammalian AGTs have been shown to increase cytotoxicity and mutagenicity of dihaloalkanes and other bis-electrophiles when expressed in bacterial cells. We have extended these studies to mammalian cells using CHO cells that lack AGT expression and CHO cells stably transfected with a p...

  8. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    Directory of Open Access Journals (Sweden)

    Jakub Cieslak

    Full Text Available Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8 is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  9. Cigarette smoking during early pregnancy reduces the number of embryonic germ and somatic cells

    DEFF Research Database (Denmark)

    Mamsen, Linn; Lutterodt, M C; Andersen, Elisabeth Anne Wreford

    2010-01-01

    BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first-trimeste......BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first...... confounders such as alcohol and coffee consumption (P = 0.002). The number of germ cells in embryonic gonads, irrespective of gender, was also significantly reduced by 41% (95% CI 58-19%, P = 0.001) in exposed versus non-exposed embryonic gonads. CONCLUSIONS: Prenatal exposure to maternal cigarette smoke...... reduces the number of germ and somatic cells in embryonic male and female gonads. This effect may have long-term consequences on the future fertility of exposed offspring. These findings may provide one potential cause of the reduced fertility observed during recent years....

  10. Aberrant histone H4 acetylation in dead somatic cell-cloned calves

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Shaohua Wang; Qiang Li; Xiangdong Ding; Yunping Dai; Ning Li

    2008-01-01

    In somatic cell-cloned animals, inefficient epigenetic reprogramming can result in an inappropriate gene expression and histone H4 acetylation is one of the key epigenetic modifications regulating gene expression. In this study, we investigated the levels of histone H4 acetylation of 11 development-related genes and expression levels of 19 genes in lungs of three normal control calves and nine aber-rant somatic cell-cloned calves. The results showed that nine studied genes had decreased acetylation levels in aberrant clones (p 0.05). Whereas 13 genes had significantly decreased expression (p 0.05), and only one gene had higher expression level in clones (p < 0.05). Furthermore, FGFR, GHR, HGFR and IGF1 genes showed lowered levels of both histone H4 acetylation and expression in aberrant clones than in controls, and the level of histone H4 acetylation was even more lowered in aberrant clones than those in controls. It was suggested that the lower levels of histone H4 acetylation in aberrant clones caused by the previous memory of cell differentiation might not support enough chromatin reprogramming, thus affecting appropriate gene expressions, and growth and development of the cloned calves. To our knowledge, this is the first study on how histone H4 acetylation affects gene expression in organs of somatic cell-cloned calves.

  11. Total bacterial count and somatic cell count in refrigerated raw milk stored in communal tanks

    Directory of Open Access Journals (Sweden)

    Edmar da Costa Alves

    2014-09-01

    Full Text Available The current industry demand for dairy products with extended shelf life has resulted in new challenges for milk quality maintenance. The processing of milk with high bacterial counts compromises the quality and performance of industrial products. The study aimed to evaluate the total bacteria counts (TBC and somatic cell count (SCC in 768 samples of refrigerated raw milk, from 32 communal tanks. Samples were collected in the first quarter of 2010, 2011, 2012 and 2013 and analyzed by the Laboratory of Milk Quality - LQL. Results showed that 62.5%, 37.5%, 15.6% and 27.1% of the means for TBC in 2010, 2011, 2012 and 2013, respectively, were above the values established by legislation. However, we observed a significant reduction in the levels of total bacterial count (TBC in the studied periods. For somatic cell count, 100% of the means indicated values below 600.000 cells/mL, complying with the actual Brazilian legislation. The values found for the somatic cell count suggests the adoption of effective measures for the sanitary control of the herd. However, the results must be considered with caution as it highlights the need for quality improvements of the raw material until it achieves reliable results effectively.

  12. Transposable DNA elements and life history traits: II. Transposition of P DNA elements in somatic cells reduces fitness, mating activity, and locomotion of Drosophila melanogaster.

    Science.gov (United States)

    Woodruff, R C; Thompson, J N; Barker, J S; Huai, H

    1999-01-01

    Some transposable DNA elements in higher organisms are active in somatic cells, as well as in germinal cells. What effect does the movement of DNA elements in somatic cells have on life history traits? It has previously been reported that somatically active P and mariner elements in Drosophila induce genetic damage and significantly reduce lifespan. In this study, we report that the movement of P elements in somatic cells also significantly reduces fitness, mating activity, and locomotion of Drosophila melanogaster. If other elements cause similar changes in life history traits, it is doubtful if transposable DNA elements remain active for long in somatic cells in natural populations.

  13. Bacillus thuringiensis membrane-damaging toxins acting on mammalian cells.

    Science.gov (United States)

    Celandroni, Francesco; Salvetti, Sara; Senesi, Sonia; Ghelardi, Emilia

    2014-12-01

    Bacillus thuringiensis is widely used as a biopesticide in forestry and agriculture, being able to produce potent species-specific insecticidal toxins and considered nonpathogenic to other animals. More recently, however, repeated observations are documenting the association of this microorganism with various infectious diseases in humans, such as food-poisoning-associated diarrheas, periodontitis, bacteremia, as well as ocular, burn, and wound infections. Similar to B. cereus, B. thuringiensis produces an array of virulence factors acting against mammalian cells, such as phosphatidylcholine- and phosphatidylinositol-specific phospholipase C (PC-PLC and PI-PLC), hemolysins, in particular hemolysin BL (HBL), and various enterotoxins. The contribution of some of these toxins to B. thuringiensis pathogenicity has been studied in animal models of infection, following intravitreous, intranasal, or intratracheal inoculation. These studies lead to the speculation that the activities of PC-PLC, PI-PLC, and HBL are responsible for most of the pathogenic properties of B. thuringiensis in nongastrointestinal infections in mammals. This review summarizes data regarding the biological activity, the genetic basis, and the structural features of these membrane-damaging toxins.

  14. Effect of donor cell type on nuclear remodelling in rabbit somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Tian, J; Song, J; Li, H; Yang, D; Li, X; Ouyang, H; Lai, L

    2012-08-01

    Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos.

  15. MicroRNA-Mediated Reprogramming of Somatic Cells into Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Sandmaier, Shelley E S; Telugu, Bhanu Prakash V L

    2015-01-01

    MicroRNAs or miRNAs belong to a class of small noncoding RNAs that play a crucial role in posttranscriptional regulation of gene expression. Nascent miRNAs are expressed as a longer transcript, which are then processed into a smaller 18-23-nucleotide mature miRNAs that bind to the target transcripts and induce cleavage or inhibit translation. MiRNAs therefore represent another key regulator of gene expression in establishing and maintaining unique cellular fate. Several classes of miRNAs have been identified to be uniquely expressed in embryonic stem cells (ESC) and regulated by the core transcription factors Oct4, Sox2, and Klf4. One such class of miRNAs is the mir-302/367 cluster that is enriched in pluripotent cells in vivo and in vitro. Using the mir-302/367 either by themselves or in combination with the Yamanaka reprogramming factors (Oct4, Sox2, c-Myc, and Klf4) has resulted in the establishment of induced pluripotent stem cells (iPSC) with high efficiencies. In this chapter, we outline the methodologies for establishing and utilizing the miRNA-based tools for reprogramming somatic cells into iPSC.

  16. Mastitis diagnosis in dairy goats through somatic cell counts and California mastitis test. Preliminary results

    OpenAIRE

    Mendonça, Álvaro; Valentim, Ramiro; Nunes, Manuel; Correia, Teresa Montenegro; Trigo, Margarida; Maurício, Raimundo; Costa, Cristina; Coelho, Alípio

    2004-01-01

    The aim of this work was to evaluate somatic cell count (SCC) and Californian mastitis test (CMT) reliability as methods to survey mastitis in Serrana goats. Microbiological diagnosis, SCC and CTM were performed on 2028 samples, collected from individual glands during a lactation period. According to results CMT (predictive negative value = 69.5%) may be used as a cheap and practical method for sub clinical mastitis survey in Serrana goats. Decision on SCC use will depend on additional resear...

  17. Efficacy of clinoptilolite supplementation on milk yield and somatic cell count

    OpenAIRE

    Deniz Alic Ural

    2014-01-01

    ABSTRACTObjective. To determine the efficiency of clinoptilolite supplements on milk production and somatic cell count (SCC). Materials and methods. 80 Holstein–Friesian cows were used, between 2 and 4 years of age ad between their first and third lactation. Two groups made up of 40 animals were constituted, and one of the following treatments were assigned randomly: Control group (n=40) with a basal diet, and experimental group (Clinoptilolite; n=40) with a basal diet + 3% (p/p) of clinoptil...

  18. Genetic Analysis of Somatic Cell Score in Danish Holsteins Using a Liability-Normal Mixture Model

    DEFF Research Database (Denmark)

    Madsen, P; Shariati, M M; Ødegård, J

    2008-01-01

    Mixture models are appealing for identifying hidden structures affecting somatic cell score (SCS) data, such as unrecorded cases of subclinical mastitis. Thus, liability-normal mixture (LNM) models were used for genetic analysis of SCS data, with the aim of predicting breeding values for such cas...... categorizing only the most extreme SCS observations as mastitic, and such cases of subclinical infections may be the most closely related to clinical (treated) mastitis...

  19. Increased virus replication in mammalian cells by blocking intracellular innate defense responses

    NARCIS (Netherlands)

    Vries, W. de; Haasnoot, J.; Velden, J. van der; Montfort, T. van; Zorgdrager, F.; Paxton, W.; Cornelissen, M.; Kuppeveld, F.J.M. van; Haan, P. de; Berkhout, B.

    2008-01-01

    The mammalian innate immune system senses viral infection by recognizing viral signatures and activates potent antiviral responses. Besides the interferon (IFN) response, there is accumulating evidence that RNA silencing or RNA interference (RNAi) serves as an antiviral mechanism in mammalian cells.

  20. Perspectives for induced pluripotent stem cell technology: new insights into human physiology involved in somatic mosaicism.

    Science.gov (United States)

    Nagata, Naoki; Yamanaka, Shinya

    2014-01-31

    Induced pluripotent stem cell technology makes in vitro reprogramming of somatic cells from individuals with various genetic backgrounds possible. By applying this technology, it is possible to produce pluripotent stem cells from biopsy samples of arbitrarily selected individuals with various genetic backgrounds and to subsequently maintain, expand, and stock these cells. From these induced pluripotent stem cells, target cells and tissues can be generated after certain differentiation processes. These target cells/tissues are expected to be useful in regenerative medicine, disease modeling, drug screening, toxicology testing, and proof-of-concept studies in drug development. Therefore, the number of publications concerning induced pluripotent stem cells has recently been increasing rapidly, demonstrating that this technology has begun to infiltrate many aspects of stem cell biology and medical applications. In this review, we discuss the perspectives of induced pluripotent stem cell technology for modeling human diseases. In particular, we focus on the cloning event occurring through the reprogramming process and its ability to let us analyze the development of complex disease-harboring somatic mosaicism.

  1. Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention.

    Science.gov (United States)

    Tomasetti, Cristian; Li, Lu; Vogelstein, Bert

    2017-03-24

    Cancers are caused by mutations that may be inherited, induced by environmental factors, or result from DNA replication errors (R). We studied the relationship between the number of normal stem cell divisions and the risk of 17 cancer types in 69 countries throughout the world. The data revealed a strong correlation (median = 0.80) between cancer incidence and normal stem cell divisions in all countries, regardless of their environment. The major role of R mutations in cancer etiology was supported by an independent approach, based solely on cancer genome sequencing and epidemiological data, which suggested that R mutations are responsible for two-thirds of the mutations in human cancers. All of these results are consistent with epidemiological estimates of the fraction of cancers that can be prevented by changes in the environment. Moreover, they accentuate the importance of early detection and intervention to reduce deaths from the many cancers arising from unavoidable R mutations.

  2. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins

    Directory of Open Access Journals (Sweden)

    Ki-Eun Park

    2016-05-01

    Full Text Available The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT. By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.

  3. CHEMICAL COMPOSITION AND SOMATIC CELL EVOLUTION DURING LACTATION IN ROMANIAN BLACK AND WHITE COWS

    Directory of Open Access Journals (Sweden)

    L.T. CZISZTER

    2013-12-01

    Full Text Available The aim of the paper was to study the evolution of the chemical composition and somatic cell count during lactation in Romanian Black and White cows and effect of calving season on the shape of the lactation curve. Lactations form 125 multiparous cows were studied. Milk yield and sampling were carried out using the official performance control method A4. Milk was analyzed for composition in infrared spectrometry and for SCC using a viscosimeter. Results were modeled using Wood’s incomplete gamma function y=abxe(-cx, and season effect was assessed using ANOVA/MANOVA. A discussion was carried out regarding the shape of the lactation curves for milk yield, each milk component and SCC. The calving season had a significant effect (p<0.005 on the shape of the lactation curve for milk yield, milk chemical composition and milk somatic cell count. Summer calving cows had flatter lactation curves for milk yield and composition compared to winter calving cows. For somatic cell count spring calving cows had the flattest lactation curve while autumn calving cows has the steepest lactation curve.

  4. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.

    Science.gov (United States)

    Park, Ki-Eun; Park, Chi-Hun; Powell, Anne; Martin, Jessica; Donovan, David M; Telugu, Bhanu P

    2016-05-26

    The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.

  5. A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Petersen, Maja Borup Kjær

    2014-01-01

    A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments...... efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells......, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors....

  6. Modelling a tethered mammalian sperm cell undergoing hyperactivation

    KAUST Repository

    Curtis, M.P.

    2012-09-01

    The beat patterns of mammalian sperm flagella can be categorised into two different types. The first involves symmetric waves propagating down the flagellum with a net linear propulsion of the sperm cell. The second, hyperactive, waveform is classified by vigorous asymmetric waves of higher amplitude, lower wavenumber and frequency propagating down the flagellum resulting in highly curved trajectories. The latter beat pattern is part of the capacitation process whereby sperm prepare for the prospective penetration of the zona pellucida and fusion with the egg. Hyperactivation is often observed to initiate as sperm escape from epithelial and ciliary bindings formed within the isthmic regions of the female oviducts, leading to a conjecture in the literature that this waveform is mechanically important for sperm escape. Hence, we explore the mechanical effects of hyperactivation on a tethered sperm, focussing on a Newtonian fluid. Using a resistive force theory model we demonstrate that hyperactivation can indeed generate forces that pull the sperm away from a tethering point and consequently a hyperactivated sperm cell bound to an epithelial surface need not always be pushed by its flagellum. More generally, directions of the forces generated by tethered flagella are insensitive to reductions in beat frequency and the detailed flagellar responses depend on the nature of the binding at the tethering point. Furthermore, waveform asymmetry and amplitude increases enhance the tendency for a tethered flagellum to start tugging on its binding. The same is generally predicted to be true for reductions in the wavenumber of the flagellum beat, but not universally so, emphasising the dynamical complexity of flagellar force generation. Finally, qualitative observations drawn from experimental data of human sperm bound to excised female reproductive tract are also presented and are found to be consistent with the theoretical predictions. © 2012 Elsevier Ltd.

  7. Intra-hematopoietic cell fusion as a source of somatic variation in the hematopoietic system.

    Science.gov (United States)

    Skinner, Amy M; Grompe, Markus; Kurre, Peter

    2012-06-15

    Cell fusion plays a well-recognized, physiological role during development. Bone-marrow-derived hematopoietic cells have been shown to fuse with non-hematopoietic cells in a wide variety of tissues. Some organs appear to resolve the changes in ploidy status, generating functional and mitotically-competent events. However, cell fusion exclusively involving hematopoietic cells has not been reported. Indeed, genomic copy number variation in highly replicative hematopoietic cells is widely considered a hallmark of malignant transformation. Here we show that cell fusion occurs between cells of the hematopoietic system under injury as well as non-injury conditions. Experiments reveal the acquisition of genetic markers in fusion products, their tractable maintenance during hematopoietic differentiation and long-term persistence after serial transplantation. Fusion events were identified in clonogenic progenitors as well as differentiated myeloid and lymphoid cells. These observations provide a new experimental model for the study of non-pathogenic somatic diversity in the hematopoietic system.

  8. Effect of estrone on somatic and female gametophyte cell division and differentiation in Arabidospis thaliana cultured in vitro

    Directory of Open Access Journals (Sweden)

    Piotr Żabicki

    2014-04-01

    Full Text Available The aim of the study was to determine the effect of the mammalian female sex hormone estrone on differentiation of somatic tissues and on induction of autonomous endosperm in culture of female gametophyte cells of Arabidopsis thaliana ecotype Columbia (Col-0. In culture, estrone-stimulated development of autonomous endosperm (AE occurred in 14.7% of unpollinated pistils. The AE represented development stages similar to those of young endosperm after fertilization and AE of fis mutants in vivo. In the majority of ovules the AE was in a few-nucleate young stage. Some ovules showed more advanced stages of AE development, with nuclei and cytoplasm forming characteristic nuclear cytoplasmic domains (NCDs. Sporadically, AE was divided into regions characteristic for Arabidopsis endosperm formed after fertilization. Direct organogenesis (caulogenesis, rhizogenesis, callus proliferation and formation of trichome-like structures were observed during in vitro culture of hypocotyls and cotyledons of 3-day-old seedlings cultured on medium supplemented with estrone for 28 days. Histological analysis showed adventitious root formation and changes in explant anatomy caused by estrone.

  9. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

    Science.gov (United States)

    Jeong, Young-Hee; Lu, Hanlin; Park, Chi-Hun; Li, Meiyan; Luo, Huijuan; Kim, Joung Joo; Liu, Siyang; Ko, Kyeong Hee; Huang, Shujia; Hwang, In Sung; Kang, Mi Na; Gong, Desheng; Park, Kang Bae; Choi, Eun Ji; Park, Jung Hyun; Jeong, Yeon Woo; Moon, Changjong; Hyun, Sang-Hwan; Kim, Nam Hyung; Jeung, Eui-Bae; Yang, Huanming; Hwang, Woo Suk; Gao, Fei

    2016-08-09

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

  10. vasa is expressed in somatic cells of the embryonic gonad in a sex-specific manner in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Andrew D. Renault

    2012-08-01

    Vasa is a DEAD box helicase expressed in the Drosophila germline at all stages of development. vasa homologs are found widely in animals and vasa has become the gene of choice in identifying germ cells. I now show that Drosophila vasa expression is not restricted to the germline but is also expressed in a somatic lineage, the embryonic somatic gonadal precursor cells. This expression is sexually dimorphic, being maintained specifically in males, and is regulated post-transcriptionally. Although somatic Vasa expression is not required for gonad coalescence, these data support the notion that Vasa is not solely a germline factor.

  11. Two 5S genes are expressed in chicken somatic cells.

    OpenAIRE

    Lazar, E; Haendler, B.; Jacob, M

    1983-01-01

    Two 5S RNA species were detected in chicken cells. 5S I RNA has the nucleotide sequence of chicken 5S RNA previously published by Brownlee et al. (1) and 5S II RNA differs from it by 10 mutations. The secondary structure of both species is compatible with that proposed for other eukaryotic 5S RNAs. 5S II RNA represents 50-60% of 5S I RNA. Both species were found in total chicken liver and brain and were present in polysomes in the same relative proportions. Only one 5S RNA species could be de...

  12. Production of nuclear transfer embryos by using somatic cells isolated from milk in buffalo (Bubalus bubalis).

    Science.gov (United States)

    Golla, K; Selokar, N L; Saini, M; Chauhan, M S; Manik, R S; Palta, P; Singla, S K

    2012-10-01

    Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos.

  13. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

    Directory of Open Access Journals (Sweden)

    Richter Anne

    2012-11-01

    Full Text Available Abstract Background Somatic cell nuclear transfer (SCNT is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination. Results PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs and porcine ear fibroblasts (PEFs could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT. Conclusion The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

  14. Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells

    OpenAIRE

    Joyce Jose; Aaron B. Taylor; Kuhn, Richard J.; Dermody, Terence S.

    2017-01-01

    Sindbis virus (SINV [genus Alphavirus, family Togaviridae]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and ...

  15. Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells

    OpenAIRE

    Jose, Joyce; Aaron B. Taylor; Kuhn, Richard J.

    2017-01-01

    ABSTRACT Sindbis virus (SINV [genus Alphavirus, family Togaviridae]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replica...

  16. Mammary-Stem-Cell-Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation

    Directory of Open Access Journals (Sweden)

    Zheng Zhang

    2016-09-01

    Full Text Available Cancer genomics has provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC organoid-based approach for rapid generation of somatic genetically engineered mouse models (GEMMs. By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K-targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study has established a robust in vivo platform for functional cancer genomics and has discovered functional breast cancer mutations.

  17. Differentiation between antibodies to protamines and somatic nuclear antigens by means of a comparative fluorescence study on swollen nuclei of spermatozoa and somatic cells.

    Science.gov (United States)

    Samuel, T

    1978-05-01

    The indirect immunofluorescence test on swollen nuclei of rat thymocytes, chicken red blood cells and human and salmon spermatozoa was found to be an easy and satisfactory method for the discrimination between antibodies to sperm-specific nuclear antigens and somatic nuclear antigens. This study shows that nuclear antibodies present in the sera of vasectomized men and in rabbit antisera to human protamines are directed against the human sperm-specific nuclear antigens (protamines), and that they may cross-react with salmon protamine. These sera do not react with somatic nuclear antigens. This comparative fluorescence study and a complement fixation study, performed with sera from diabetic patients, proved that the administration of insulin retard (protamine-zinc-insulin) may lead to the formation of antibodies to the fish protamine. These antibodies may reveal a weak cross reaction with human protamines. The results obtained in this study also prove that the nuclei of chicken red blood cells and human sperm do not contain, or contain very small amounts of, histone fraction H1, and that salmon sperm nuclei do not contain any of the histone fractions, and suggest that the nuclei of mature human spermatozoa contain smaller amounts of histones in comparison to somatic cell nuclei.

  18. CRISPR mediated somatic cell genome engineering in the chicken.

    Science.gov (United States)

    Véron, Nadège; Qu, Zhengdong; Kipen, Phoebe A S; Hirst, Claire E; Marcelle, Christophe

    2015-11-01

    Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model.

  19. A comparison of somatic cell count and antimicrobial susceptibility of subclinical mastitis pathogens in organic and conventional dairy herds

    OpenAIRE

    Boutet, Philippe; Detilleux, Johann; Motkin, Michel; Deliege, M.; PIRAUX, E.; Depinois, A.; Debliquy, P.; Mainil, Jacques; Czaplicki, G.; Lekeux, Pierre

    2005-01-01

    A comparison of somatic cell count and antimicrobial susceptibility of subclinical mastitis pathogens in organic and conventional dairy herds.Bovine subclinical mastitis is the most important disease affecting dairy cows. The fluctuating increase in somatic cell count (SCC) that occurs causes major economic losses in dairy industry. This comparative study between conventional and organic dairy herds was conducted in the aim to better characterize which consequences might have different manage...

  20. Effect of season on milk temperature, milk growth hormone, prolactin, and somatic cell counts of lactating cattle

    Science.gov (United States)

    Igono, M. O.; Johnson, H. D.; Steevens, B. J.; Hainen, W. A.; Shanklin, M. D.

    1988-09-01

    Monthly fluctuations in milk temperature, somatic cell counts, milk growth hormone and prolactin of lactating cows were measured in milk samples over a 1 year period. The seasonal patterns in milk temperature, somatic cell count and milk prolactin concentration showed a positive trend with increasing environmental temperatures. Milk growth hormone concentration increased with lactation level and declined significantly during summer heat. Milk temperature and the measured hormonal levels may serve as indicators of the impact of the climatic environment on lactating cattle.

  1. Functional expression of mammalian receptors and membrane channels in different cells.

    Science.gov (United States)

    Eifler, Nora; Duckely, Myriam; Sumanovski, Lazar T; Egan, Terrance M; Oksche, Alexander; Konopka, James B; Lüthi, Anita; Engel, Andreas; Werten, Paul J L

    2007-08-01

    In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.

  2. Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (

    Directory of Open Access Journals (Sweden)

    Minghui Zhao

    2017-07-01

    Full Text Available Objective Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT. Methods Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3 in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR. Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Results Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Conclusion Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

  3. Bacteria-human somatic cell lateral gene transfer is enriched in cancer samples.

    Directory of Open Access Journals (Sweden)

    David R Riley

    Full Text Available There are 10× more bacterial cells in our bodies from the microbiome than human cells. Viral DNA is known to integrate in the human genome, but the integration of bacterial DNA has not been described. Using publicly available sequence data from the human genome project, the 1000 Genomes Project, and The Cancer Genome Atlas (TCGA, we examined bacterial DNA integration into the human somatic genome. Here we present evidence that bacterial DNA integrates into the human somatic genome through an RNA intermediate, and that such integrations are detected more frequently in (a tumors than normal samples, (b RNA than DNA samples, and (c the mitochondrial genome than the nuclear genome. Hundreds of thousands of paired reads support random integration of Acinetobacter-like DNA in the human mitochondrial genome in acute myeloid leukemia samples. Numerous read pairs across multiple stomach adenocarcinoma samples support specific integration of Pseudomonas-like DNA in the 5'-UTR and 3'-UTR of four proto-oncogenes that are up-regulated in their transcription, consistent with conversion to an oncogene. These data support our hypothesis that bacterial integrations occur in the human somatic genome and may play a role in carcinogenesis. We anticipate that the application of our approach to additional cancer genome projects will lead to the more frequent detection of bacterial DNA integrations in tumors that are in close proximity to the human microbiome.

  4. Analysis of in vivo somatic mutations in normal human cells

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, P.K.; Sahota, A.; Boyadjiev, S.A. [Indiana Univ. School of Medicine, Indianapolis, IN (United States)] [and others

    1994-09-01

    We have used the APRT locus located at 16q24.3 to study the nature of loss of heterozygosity (LOH) in human T lymphocytes in vivo. T lymphocytes were isolated from blood from APRT (+/{minus}) obligated heterozygotes with known germline mutations. The cells were immediatley placed in culture medium containing 100 {mu}M 2,6-diaminopurine (DAP) to select for drug-resistant clones ({minus}/{minus}) already present. These clones were first examined using polymorphic CA microsatellite repeat markers D16S303 and D16S305 that are distal and proximal to APRT, respectively. The retention of heterozygosity of these markers is suggestive of minor changes in the APRT gene, the exact nature of which were determined by DNA sequencing. Nineteen out of 70 DAP-resistant clones from one heterozygote showed APRT sequence changes. The loss of heterozygosity of markers D16S303 and D16S305 in the remaining clones suggests LOH involving multilocus chromosomal events. These clones were then sequentially typed using additional CA repeat markers proximal and distal to APRT. The extent of LOH in these clones was found to vary from <5 cM to almost the entire 16q arm. Preliminary results suggest that there are multiple sites along the chromosome from which LOH proceeds distally in these clones. Cytogenetic analysis of 10 clones suggested mitotic recombination in 9 and deletion in one. Studies are in progress to further characterize the molecular mechanisms of LOH.

  5. Preliminary assessment of somatic cell nuclear transfer in the dromedary (Camelus dromedarius).

    Science.gov (United States)

    Khatir, H; Anouassi, A

    2008-12-01

    Somatic cloning may enable the maintenance/expansion of the population of camels with the highest potential for milk production or the best racing performances. However, there have been no reports of embryonic or somatic nuclear transfer in camels. The aim of this study was to produce dromedary embryos by nuclear transfer using in vitro matured oocytes and two somatic cells from two sources (adult fibroblasts or granulosa cells). A total of 58 adult females were superstimulated by a single dose of eCG (3500 IU). Ten days later, their ovaries were collected postmortem. Cumulus-oocytes-complexes (COCs) were aspirated from stimulated follicles and were matured in vitro for 30 h. Fibroblasts (from live adult male) and granulosa cells (from slaughtered adult females) were used as donor karyoplasts and injected into mature enucleated dromedary oocytes. The cleavage rate was significantly higher (Pdromedary embryos from the two sources of donor cells (fibroblasts; n=5 vs. granulosa cells; n=7) was examined by transferring them to synchronized recipients. Two females (fibroblasts: 1/5; 20%, granulosa cells: 1/7; 14%) were confirmed pregnant by ultrasonography at 15 and 25 days following transfer. Later, the pregnancies were followed by pregnancy empirical-symptoms. These two pregnancies were lost between 25 and 60 days following transfer, respectively. In conclusion, the present study shows for the first time that the development of dromedary NT embryos derived from either adult fibroblasts or granulosa cells can occur in vitro and the transfer of these cloned embryos to recipients can result in pregnancies.

  6. THE EFFECT OF BLOOD AND MILK SERUM ZINC CONCENTRATION ON MILK SOMATIC CELL COUNT IN DAIRY COWS

    Directory of Open Access Journals (Sweden)

    Ivana Davidov

    2016-11-01

    Full Text Available The objective of this study was to evaluate the effect of blood and milk zinc concentration on somatic cell count and occurrence of subclinical mastitis cases. The study was performed on thirty Holstein cows approximate same body weight, ages 3 to 5 years, with equally milk production. Blood samples were taken after the morning milking from the caudal vein and milk from all four quarters was taken before morning milking. All samples of blood and milk were taken to determined zinc, using inductively coupled plasma mass spectrometry. 37.67% (11/30 cows have blood serum zinc concentration below 7µmol/l, and 63.33% or 19/30 cows have blood serum zinc concentration higher then 13µmol/l. Also 30% (9/30 cows have somatic cell count lower then 400.000/ml which indicate absence of subclinical mastitis, but 70% (21/30 cows have somatic cell count higher then 400.000/ml which indicate subclinical mastitis. Results indicate that cows with level of zinc in blood serum higher then 13 µmol/l have lower somatic cell count. Cows with lower zinc blood serum concentration then 7 µmol/l have high somatic cell count and high incidence of subclinical mastitis. According to results in this research there is no significant effect of milk serum zinc concentration on somatic cell count in dairy cows.

  7. Effect of Hypoxia on Ldh-c Expression in Somatic Cells of Plateau Pika.

    Science.gov (United States)

    Wei, Dengbang; Wei, Linna; Li, Xiao; Wang, Yang; Wei, Lian

    2016-08-01

    Sperm specific lactate dehydrogenases (LDH-C₄) is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, Ldh-c was originally thought to be expressed only in testes and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia-tolerant mammal living 3000-5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testes and sperm, but also in the somatic tissues of plateau pika. To reveal the effect of hypoxia on pika Ldh-c expression, we investigated the mRNA and protein level of Ldh-c as well as the biochemical index of anaerobic glycolysis in pika somatic tissues at the altitudes of 2200 m, 3200 m and 3900 m. Our results showed that mRNA and protein expression levels of Ldh-c in the tissues of pika's heart, liver, brain and skeletal muscle were increased significantly from 2200 m to 3200 m, but had no difference from 3200 m to 3900 m; the activities of LDH and the contents of lactate showed no difference from 2200 m to 3200 m, but were increased significantly from 3200 m to 3900 m. Hypoxia up-regulated and maintained the expression levels of Ldh-c in the pika somatic cells. Under the hypoxia condition, plateau pikas increased anaerobic glycolysis in somatic cells by LDH-C₄, and that may have reduced their dependence on oxygen and enhanced their adaptation to the hypoxic environment.

  8. A rapid and efficient method to express target genes in mammalian cells by baculovirus

    Institute of Scientific and Technical Information of China (English)

    Tong Cheng; Chen-Yu Xu; Ying-Bin Wang; Min Chen; Ting Wu; Jun Zhang; Ning-Shao Xia

    2004-01-01

    AIM: To investigate the modification of baculovirus vector and the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of Spodoptera frugiperta (Sf9) cells infected by recombinant baculoviruses.METHODS: Two recombinant baculoviruses (BacV-CMVEGFPA, BacV-CMV-EGFPB) containing CMV-EGFP expression cassette were constructed. HepG2 cells were directly incubated with the culture supernatant of Sf9 cells infected by recombinant baculoviruses, and reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM). The optimal transduction conditions were investigated by FCM assay in HepG2 cells. Gene-transfer and expression efficiencies in HepG2 or CV1 cells by baculovirus vectors were compared with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Twenty different mammalian cell lines were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the culture supernatant of infected Sf9 cells.RESULTS: CMV promoter could directly express reporter genes in Sf9 cells with a relatively low efficiency. Target cells incubated with the 1:1 diluted culture supernatant (moi=50) for 12 h at 37 ℃ could achieve the highest transduction and expression efficiencies with least impairment to cell viability. Under similar conditions the baculovirus vector could achieve the highest gene-transfer and expression efficiency than lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Most mammalian cell lines could be transduced with recombinant baculovirus. In primate adherent culture cells the recombinant baculovirus could arrive the highest infection and expression efficiencies, but it was not very satisfactory in the cell lines from mice and suspended culture cells.CONCLUSION: Mammalian cells incubated with the culture supernatant of infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign

  9. Analysis of protein-coding mutations in hiPSCs and their possible role during somatic cell reprogramming.

    Science.gov (United States)

    Ruiz, Sergio; Gore, Athurva; Li, Zhe; Panopoulos, Athanasia D; Montserrat, Nuria; Fung, Ho-Lim; Giorgetti, Alessandra; Bilic, Josipa; Batchelder, Erika M; Zaehres, Holm; Schöler, Hans R; Zhang, Kun; Izpisua Belmonte, Juan Carlos

    2013-01-01

    Recent studies indicate that human-induced pluripotent stem cells contain genomic structural variations and point mutations in coding regions. However, these studies have focused on fibroblast-derived human induced pluripotent stem cells, and it is currently unknown whether the use of alternative somatic cell sources with varying reprogramming efficiencies would result in different levels of genetic alterations. Here we characterize the genomic integrity of eight human induced pluripotent stem cell lines derived from five different non-fibroblast somatic cell types. We show that protein-coding mutations are a general feature of the human induced pluripotent stem cell state and are independent of somatic cell source. Furthermore, we analyse a total of 17 point mutations found in human induced pluripotent stem cells and demonstrate that they do not generally facilitate the acquisition of pluripotency and thus are not likely to provide a selective advantage for reprogramming.

  10. Molecular basis of mammalian cell invasion by Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Nobuko Yoshida

    2006-03-01

    Full Text Available Establishment of infection by Trypanosoma cruzi, the agent of Chagas' disease, depends on a series of events involving interactions of diverse parasite molecules with host components. Here we focus on the mechanisms of target cell invasion by metacyclic trypomastigotes (MT and mammalian tissue culture trypomastigotes (TCT. During MT or TCT internalization, signal transduction pathways are activated both in the parasite and the target cell, leading to Ca2+ mobilization. For cell adhesion, MT engage surface glycoproteins, such as gp82 and gp35/50, which are Ca2+ signal-inducing molecules. In T. cruzi isolates that enter host cells in gp82-mediated manner, parasite protein tyrosine kinase as well as phospholipase C are activated, and Ca2+ is released from I P3-sensitive stores, whereas in T. cruzi isolates that attach to target cells mainly through gp35/50, the signaling pathway involving adenylate cyclase appears to be stimulated, with Ca2+ release from acidocalciosomes. In addition, T. cruzi isolate-dependent inhibitory signals, mediated by MT-specific gp90, may be triggered both in the host cell and the parasite. The repertoire of TCT molecules implicated in cell invasion includes surface glycoproteins of gp85 family, with members containing binding sites for laminin and cytokeratin 18, enzymes such as cruzipain, trans-sialidase, and an oligopeptidase B that generates a Ca2+-agonist from a precursor molecule.O estabelecimento da infecção por Trypanosoma cruzi, o agente da doença de Chagas, depende de uma série de eventos envolvendo interações de diversas moléculas do parasita com componentes do hospedeiro. Focalizamos aqui os mecanismos de invasão celular por tripomastigotas metacíclicos (TM e por tripomastigotas de cultura de tecido (TCT. Durante a internalização de TM ou TCT, vias de transdução de sinal são ativadas tanto no parasita como na célula alvo, acarretando a mobilização de Ca2+. Para adesão, TM utiliza as glicoprote

  11. Droplet size influences division of mammalian cell factories in droplet microfluidic cultivation

    DEFF Research Database (Denmark)

    Periyannan Rajeswari, Prem Kumar; Joensson, Haakan N.; Svahn, Helene Andersson

    2017-01-01

    The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. Here, we report the influence of droplet size on mammalian cell division and viability during cultivation...... in droplets. Chinese Hamster Ovary (CHO) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pL droplets for 3 days. Periodic monitoring of the droplets during incubation showed that the cell divisions in 33 pL droplets stopped...... after 24 h, whereas continuous cell division was observed in 180 and 320 pL droplets for 72 h. The viability of the cells cultivated in the 33 pL droplets also dropped to about 50% in 72 h. In contrast, the viability of the cells in the larger droplets was above 90% even after 72 h of cultivation...

  12. Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells

    Directory of Open Access Journals (Sweden)

    Joyce Jose

    2017-02-01

    Full Text Available Sindbis virus (SINV [genus Alphavirus, family Togaviridae] is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels.

  13. Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells

    Science.gov (United States)

    Jose, Joyce; Taylor, Aaron B.

    2017-01-01

    ABSTRACT Sindbis virus (SINV [genus Alphavirus, family Togaviridae]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. PMID:28196962

  14. Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary.

    Science.gov (United States)

    Parte, Seema; Bhartiya, Deepa; Telang, Jyoti; Daithankar, Vinita; Salvi, Vinita; Zaveri, Kusum; Hinduja, Indira

    2011-08-01

    The present study was undertaken to detect, characterize, and study differentiation potential of stem cells in adult rabbit, sheep, monkey, and menopausal human ovarian surface epithelium (OSE). Two distinct populations of putative stem cells (PSCs) of variable size were detected in scraped OSE, one being smaller and other similar in size to the surrounding red blood cells in the scraped OSE. The smaller 1-3 μm very small embryonic-like PSCs were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4-7 μm cells with cytoplasmic localization of Oct-4 and minimal expression of SSEA-4 were possibly the tissue committed progenitor stem cells. Pluripotent gene transcripts of Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 in human and sheep OSE were detected by reverse transcriptase-polymerase chain reaction. The PSCs underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial-mesenchymal transition in 3 weeks of OSE culture. Germ cell markers like c-Kit, DAZL, GDF-9, VASA, and ZP4 were immuno-localized in oocyte-like structures. In conclusion, as opposed to the existing view of OSE being a bipotent source of oocytes and granulosa cells, mammalian ovaries harbor distinct very small embryonic-like PSCs and tissue committed progenitor stem cells population that have the potential to develop into oocyte-like structures in vitro, whereas mesenchymal fibroblasts appear to form supporting granulosa-like somatic cells. Research at the single-cell level, including complete gene expression profiling, is required to further confirm whether postnatal oogenesis is a conserved phenomenon in adult mammals.

  15. Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hong-Zhang [Institute of Biotechnology, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan, ROC (China); Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Wu, Carol P. [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Chao, Yu-Chan, E-mail: mbycchao@imb.sinica.edu.tw [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Catherine Yen-Yen, E-mail: liucat_2@yahoo.com [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)

    2011-02-11

    Research highlights: {yields} Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. {yields} Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. {yields} CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. {yields} Virus entry and gene expression can be separate events in different cell types. -- Abstract: The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

  16. Extracellular matrix of dental pulp stem cells: Applications in pulp tissue engineering using somatic MSCs

    Directory of Open Access Journals (Sweden)

    Sriram eRavindran

    2014-01-01

    Full Text Available Dental Caries affects approximately 90% of the world’s population. At present, the clinical treatment for dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality. Tissue engineering can potentially solve this problem by enabling regeneration of a functional pulp tissue. Dental pulp stem cells (DPSCs have been shown to be an excellent source for pulp regeneration. However, limited availability of these cells hinders its potential for clinical translation. We have investigated the possibility of using somatic mesenchymal stem cells from other sources for dental pulp tissue regeneration using a biomimetic dental pulp extracellular matrix (ECM incorporated scaffold. Human periodontal ligament stem cells (PDLSCs and human bone marrow stromal cells (HMSCs were investigated for their ability to differentiate towards an odontogenic lineage. In vitro real-time PCR results coupled with histological and immunohistochemical examination of the explanted tissues confirmed the ability of PDLSCs and HMSCs to form a vascularized pulp-like tissue. These findings indicate that the dental pulp stem derived ECM scaffold stimulated odontogenic differentiation of PDLSCs and HMSCs without the need for exogenous addition of growth and differentiation factors. This study represents a translational perspective toward possible therapeutic application of using a combination of somatic stem cells and extracellular matrix for pulp regeneration.

  17. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Directory of Open Access Journals (Sweden)

    Teruyuki Hayashi

    Full Text Available Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT in combination with fluorescence lifetime imaging microscopy (FLIM. Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  18. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Science.gov (United States)

    Hayashi, Teruyuki; Fukuda, Nanaho; Uchiyama, Seiichi; Inada, Noriko

    2015-01-01

    Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  19. Production of transgenic canine embryos using interspecies somatic cell nuclear transfer.

    Science.gov (United States)

    Hong, So Gun; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Koo, Ok Jae; Jang, Goo; Lee, Byeong Chun

    2012-02-01

    Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that

  20. A cloned toy poodle produced from somatic cells derived from an aged female dog.

    Science.gov (United States)

    Jang, G; Hong, S G; Oh, H J; Kim, M K; Park, J E; Kim, H J; Kim, D Y; Lee, B C

    2008-03-15

    To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.

  1. Droplet size influences division of mammalian cell factories in droplet microfluidic cultivation.

    Science.gov (United States)

    Periyannan Rajeswari, Prem Kumar; Joensson, Haakan N; Andersson-Svahn, Helene

    2017-01-01

    The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. Here, we report the influence of droplet size on mammalian cell division and viability during cultivation in droplets. Chinese Hamster Ovary (CHO) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pL droplets for 3 days. Periodic monitoring of the droplets during incubation showed that the cell divisions in 33 pL droplets stopped after 24 h, whereas continuous cell division was observed in 180 and 320 pL droplets for 72 h. The viability of the cells cultivated in the 33 pL droplets also dropped to about 50% in 72 h. In contrast, the viability of the cells in the larger droplets was above 90% even after 72 h of cultivation, making them a more suitable droplet size for 72-h cultivation. This study shows a direct correlation of microfluidic droplet size to the division and viability of mammalian cells. This highlights the importance of selecting suitable droplet size for mammalian cell factory screening assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. EVALUATION OF METHODS OF ANALYSIS TO DETERMINE THE SOMATIC CELL COUNT IN RAW MILK, KEPT IN THE COOLING TANK

    Directory of Open Access Journals (Sweden)

    Manoel Pereira Neto

    2014-06-01

    Full Text Available We analyzed the quality of raw milk from eight dairy property in Rio Grande do Norte, Brazil, stored in a cooling tank, in order to evaluate methods for determining somatic cell counts (SCC. The Somaticell® kit and a portable Direct Cell Counter (DCC were compared with each other and with the MilkoScanTM FT+ (FOSS Denmark, which uses Fourier Transform Infrared Spectroscopy (FTIS. Direct cell counter data were processed for somatic cell scores (log-transformed somatic cell count and analyzed with the SAS®, Statistical Analysis System. Comparison of means and correlation of somatic cell scores were conducted using Pearson’s correlation coefficient and the Tukey Test at 1%. No significant difference was observed for comparison of means. The correlation between somatic cell scores was significant, that is, 0.907 and 0.876 between the MilkoScanTM FT + and the Somaticell® kit and Direct Cell Count (DCC respectively, and 0.943 between the Somaticell® kit and Direct Cell Count (DCC. The methods can be recommended for monitoring the quality of raw milk kept in a cooling tank in the production unit.

  3. Relation Between the Cell Volume and the Cell Cycle Dynamics in Mammalian cell

    Science.gov (United States)

    Magno, A. C. G.; Oliveira, I. L.; Hauck, J. V. S.

    2016-08-01

    The main goal of this work is to add and analyze an equation that represents the volume in a dynamical model of the mammalian cell cycle proposed by Gérard and Goldbeter (2011) [1]. The cell division occurs when the cyclinB/Cdkl complex is totally degraded (Tyson and Novak, 2011)[2] and it reaches a minimum value. At this point, the cell is divided into two newborn daughter cells and each one will contain the half of the cytoplasmic content of the mother cell. The equations of our base model are only valid if the cell volume, where the reactions occur, is constant. Whether the cell volume is not constant, that is, the rate of change of its volume with respect to time is explicitly taken into account in the mathematical model, then the equations of the original model are no longer valid. Therefore, every equations were modified from the mass conservation principle for considering a volume that changes with time. Through this approach, the cell volume affects all model variables. Two different dynamic simulation methods were accomplished: deterministic and stochastic. In the stochastic simulation, the volume affects every model's parameters which have molar unit, whereas in the deterministic one, it is incorporated into the differential equations. In deterministic simulation, the biochemical species may be in concentration units, while in stochastic simulation such species must be converted to number of molecules which are directly proportional to the cell volume. In an effort to understand the influence of the new equation a stability analysis was performed. This elucidates how the growth factor impacts the stability of the model's limit cycles. In conclusion, a more precise model, in comparison to the base model, was created for the cell cycle as it now takes into consideration the cell volume variation

  4. Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer.

    Science.gov (United States)

    Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao; Zhao, Heng; Lv, Chaoxiang; Xu, Kaixiang; Cheng, Wenmin; Li, Hushan; Zhao, Hong-Ye; Wang, Wen; Wei, Hong-Jiang

    2016-09-22

    Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.

  5. Somatic cell nuclear transfer: infinite reproduction of a unique diploid genome.

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko

    2008-06-10

    In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the "Hayflick limit". However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to "passage" a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the "passage" of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.

  6. Computational analysis of mammalian cell division gated by a circadian clock: quantized cell cycles and cell size control.

    Science.gov (United States)

    Zámborszky, Judit; Hong, Christian I; Csikász Nagy, Attila

    2007-12-01

    Cell cycle and circadian rhythms are conserved from cyanobacteria to humans with robust cyclic features. Recently, molecular links between these two cyclic processes have been discovered. Core clock transcription factors, Bmal1 and Clock (Clk), directly regulate Wee1 kinase, which inhibits entry into the mitosis. We investigate the effect of this connection on the timing of mammalian cell cycle processes with computational modeling tools. We connect a minimal model of circadian rhythms, which consists of transcription-translation feedback loops, with a modified mammalian cell cycle model from Novak and Tyson (2004). As we vary the mass doubling time (MDT) of the cell cycle, stochastic simulations reveal quantized cell cycles when the activity of Wee1 is influenced by clock components. The quantized cell cycles disappear in the absence of coupling or when the strength of this link is reduced. More intriguingly, our simulations indicate that the circadian clock triggers critical size control in the mammalian cell cycle. A periodic brake on the cell cycle progress via Wee1 enforces size control when the MDT is quite different from the circadian period. No size control is observed in the absence of coupling. The issue of size control in the mammalian system is debatable, whereas it is well established in yeast. It is possible that the size control is more readily observed in cell lines that contain circadian rhythms, since not all cell types have a circadian clock. This would be analogous to an ultradian clock intertwined with quantized cell cycles (and possibly cell size control) in yeast. We present the first coupled model between the mammalian cell cycle and circadian rhythms that reveals quantized cell cycles and cell size control influenced by the clock.

  7. Regulation of taurine transport systems by protein kinase CK2 in mammalian cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry; Hansen, Daniel Bloch

    2011-01-01

    Maintaining cell volume is critical for cellular function yet shift in cell volume is a prerequisite for mitosis and apoptosis. The ubiquitously and evolutionary conserved serine/threonine kinase CK2 promotes cell survival and suppresses apoptosis. The present review describes how mammalian cells...

  8. Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Park, JungEun; Oh, HyunJu; Hong, SoGun; Kim, MinJung; Kim, GeonA; Koo, OkJae; Kang, SungKeun; Jang, Goo; Lee, ByeongChun

    2011-03-01

    As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment.

  9. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  10. Effects of pigment glands andgossypol on somatic cell cul-ture of upland cotton (Gos-sypium hirsutum L.)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The effects of pigment glands and gossypol on the somatic cell culture of upland cotton were studied, using the materials as follows: three pairs of glanded and glandless upland cotton near isogenic lines, TM-1, and Coker 312. The results showed that the pigment glands and gossypol contents in the explants had great inhibiting effect on the induction and growth of callus in somatic cell culture of upland cotton, and the induction rate of callus and the single callus weight of glandless cotton were much higher than those of their glanded near isogenic lines. It was easier to obtain regeneration plants from glandless cotton than from their glanded near isogenic lines. There was a significant inverse correlation between the gossypol contents in the explants and callus induction rate, with the correlation coefficient of ?0.84. The vitro gossypol in the medium had some inhibiting effect on the induction and growth of callus, especially for the glandless cotton. However, a certain concentration of vitro gossypol in the medium (0.1 mg/L) was an aid to the steadiness growth of callus in glandless cotton somatic cell culture, with a high rate of embryogenic cells which was in favor of plant regeneration, and it was also relatively easy to obtain regeneration plants when they were transferred into differentiation medium with 0.1 mg/L of vitro gossypol, even for some cultivars which are difficult in somatic cell culture. In addition, the gossypol content and its variation in the seedlings and callus during culture of Coker 312 were discussed, as well as the relationship between gossypol variation in the explants and its somatic cell culture. The probability of vitro gossypol used in cotton somatic cell culture for the improvement of somatic cell culture was suggested.

  11. Auxin induces cell proliferation in an experimental model of mammalian renal tubular epithelial cells.

    Science.gov (United States)

    Cernaro, Valeria; Medici, Maria Antonietta; Leonello, Giuseppa; Buemi, Antoine; Kohnke, Franz Heinrich; Villari, Antonino; Santoro, Domenico; Buemi, Michele

    2015-06-01

    Indole-3-acetic acid is the main auxin produced by plants and plays a key role in the plant growth and development. This hormone is also present in humans where it is considered as a uremic toxin deriving from tryptophan metabolism. However, beyond this peculiar aspect, the involvement of auxin in human pathophysiology has not been further investigated. Since it is a growth hormone, we evaluated its proliferative properties in an in vitro model of mammalian renal tubular epithelial cells. We employed an experimental model of renal tubular epithelial cells belonging to the LLC-PK1 cell line that is derived from the kidney of healthy male pig. Growth effects of auxin against LLC-PK1 cell lines were determined by a rapid colorimetric assay. Increasing concentrations of auxin (to give a final concentration from 1 to 1000 ng/mL) were added and microplates were incubated for 72 h. Each auxin concentration was assayed in four wells and repeated four times. Cell proliferation significantly increased, compared to control cells, 72 h after addition of auxin to cultured LLC-PK1 cells. Statistically significant values were observed when 100 ng/mL (p auxin influences cell growth not only in plants, where its role is well documented, but also in mammalian cell lines. This observation opens new scenarios in the field of tissue regeneration and may stimulate a novel line of research aiming at investigating whether this hormone really influences human physiology and pathophysiology and in particular, kidney regeneration.

  12. Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer.

    Science.gov (United States)

    Wu, Zhenfang; Xu, Zhiqian; Zou, Xian; Zeng, Fang; Shi, Junsong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Li, Zicong

    2013-12-01

    The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.

  13. An Epigenetic Modifier Results in Improved In Vitro Blastocyst production after Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Zhang, Yunhai; Li, Juan; Villemoes, Klaus

    2007-01-01

    were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production......The present study was designed to examine the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on development of porcine cloned embryos. Our results showed that treatment of cloned embryos derived from sow oocytes with 50 nM TSA for up to 24 h after the onset of activation could...... significantly improve blastocyst yield compared to the control (46.4 ± 4.6% vs 17.7 ± 4.9% for treated and untreated embryos, respectively; p cell number per blastocyst were observed. In order to assess if the improvement is cell line specific, three cell lines...

  14. Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells.

    Science.gov (United States)

    Shipony, Zohar; Mukamel, Zohar; Cohen, Netta Mendelson; Landan, Gilad; Chomsky, Elad; Zeliger, Shlomit Reich; Fried, Yael Chagit; Ainbinder, Elena; Friedman, Nir; Tanay, Amos

    2014-09-04

    Stable maintenance of gene regulatory programs is essential for normal function in multicellular organisms. Epigenetic mechanisms, and DNA methylation in particular, are hypothesized to facilitate such maintenance by creating cellular memory that can be written during embryonic development and then guide cell-type-specific gene expression. Here we develop new methods for quantitative inference of DNA methylation turnover rates, and show that human embryonic stem cells preserve their epigenetic state by balancing antagonistic processes that add and remove methylation marks rather than by copying epigenetic information from mother to daughter cells. In contrast, somatic cells transmit considerable epigenetic information to progenies. Paradoxically, the persistence of the somatic epigenome makes it more vulnerable to noise, since random epimutations can accumulate to massively perturb the epigenomic ground state. The rate of epigenetic perturbation depends on the genomic context, and, in particular, DNA methylation loss is coupled to late DNA replication dynamics. Epigenetic perturbation is not observed in the pluripotent state, because the rapid turnover-based equilibrium continuously reinforces the canonical state. This dynamic epigenetic equilibrium also explains how the epigenome can be reprogrammed quickly and to near perfection after induced pluripotency.

  15. Farm management factors associated with bulk tank somatic cell count in Irish dairy herds

    Directory of Open Access Journals (Sweden)

    Kelly PT

    2009-04-01

    Full Text Available Abstract The relationship between bulk tank somatic cell count (SCC and farm management and infrastructure was examined using data from 398 randomly selected, yet representative, Irish dairy farms where the basal diet is grazed grass. Median bulk tank SCC for the farms was 282,887 cells/ml ranging from 82,209 to 773,028 cells/ml. Two questionnaires were administered through face-to-face contact with each farmer. Herd-level factors associated with bulk tank SCC were determined using linear models with annual somatic cell score (i.e., arithmetic mean of the natural logarithm of bulk tank SCC included as the dependent variable. All herd level factors were analysed individually in separate regression models, which included an adjustment for geographical location of the farm; a multiple regression model was subsequently developed. Management practices associated with low SCC included the use of dry cow therapy, participation in a milk recording scheme and the use of teat disinfection post-milking. There was an association between low SCC and an increased level of hygiene and frequency of cleaning of the holding yard, passageways and cubicles. Herd management factors associated with bulk tank SCC in Irish grazing herds are generally in agreement with most previous studies from confinement systems of milk production.

  16. Farm management factors associated with bulk tank somatic cell count in Irish dairy herds

    Science.gov (United States)

    2009-01-01

    The relationship between bulk tank somatic cell count (SCC) and farm management and infrastructure was examined using data from 398 randomly selected, yet representative, Irish dairy farms where the basal diet is grazed grass. Median bulk tank SCC for the farms was 282,887 cells/ml ranging from 82,209 to 773,028 cells/ml. Two questionnaires were administered through face-to-face contact with each farmer. Herd-level factors associated with bulk tank SCC were determined using linear models with annual somatic cell score (i.e., arithmetic mean of the natural logarithm of bulk tank SCC) included as the dependent variable. All herd level factors were analysed individually in separate regression models, which included an adjustment for geographical location of the farm; a multiple regression model was subsequently developed. Management practices associated with low SCC included the use of dry cow therapy, participation in a milk recording scheme and the use of teat disinfection post-milking. There was an association between low SCC and an increased level of hygiene and frequency of cleaning of the holding yard, passageways and cubicles. Herd management factors associated with bulk tank SCC in Irish grazing herds are generally in agreement with most previous studies from confinement systems of milk production. PMID:22081962

  17. Mechanism for multiplicity of steady states with distinct cell concentration in continuous culture of mammalian cells.

    Science.gov (United States)

    Yongky, Andrew; Lee, Jongchan; Le, Tung; Mulukutla, Bhanu Chandra; Daoutidis, Prodromos; Hu, Wei-Shou

    2015-07-01

    Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells.

  18. Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells.

    Science.gov (United States)

    Jose, Joyce; Taylor, Aaron B; Kuhn, Richard J

    2017-02-14

    Sindbis virus (SINV [genus Alphavirus, family Togaviridae]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels.IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and

  19. M1 AFLATOXIN, TOTAL BACTERIAL COUNT AND SOMATIC CELL COUNT IN ORGANIC AND CONVENTIONAL MILK

    Directory of Open Access Journals (Sweden)

    M. Trevisani

    2009-09-01

    Full Text Available Comparative quality evaluation of organic and conventional milk produced in similar environmental condition was performed. Bulk-tank milk was sampled once a week during 30 weeks from 10 organic and 10 conventional dairy farms where aflatoxin M1 level was previous tested during 11 months on bulk-tank milk from tanker at the processing plant. Somatic Cells and Total Microbial Counts did not show differences that can be related to the organic production system, suggesting an effect induced by farm size and technical factors. Higher level of Aflatoxin M1 was found in organic than conventional milk.

  20. NUCLEAR REPROGRAMMING AND ITS EPIGENETIC CONSEQUENCES IN SOMATIC CELL NUCLEAR TRANSFER DERIVED ANIMALS

    Directory of Open Access Journals (Sweden)

    Meirelles FV

    2015-12-01

    Full Text Available Somatic cell nuclear transfer (SCNT is a powerful tool to understand the mechanisms driving the cellular reprogramming, as well as addressing fundamental questions on developmental biology. Hundreds of animals have already been generated through this technology, however its large-scale use is still hampered by the low efficiency and disrupted phenotypes reported in SCNT derived newborns. Therefore, this review aims to expose some of the consequences often seen in cloned animals, as well as to discuss and propose some possibilities and perspectives to avoid epigenetic failures during reprogramming.

  1. RESEARCHES REGARDING THE SOMATIC CELLS NUMBER FROM RAW MILK USED IN TELEMEA CHEESE TECHNOLOGICAL PROCESS

    Directory of Open Access Journals (Sweden)

    ANDRA SULER

    2013-12-01

    Full Text Available It is known that by milk production hygiene must be assure: milk microbiological security, increase the sensorial and nutritive properties, increase term of availability and consumption. The milk hygienic national strategies involved: raw material risk contamination avoiding and reducing as can is possible and the microorganisms destroying or stopping development of those. In this paper it is presented the results of somatic cells number determination by raw milk used in Telemea cheese technological processes within 5 research stations. Determinations were effectuated on 2 series with 57 samples each of them, prelevated in reception phase in summer and winter seasons.

  2. Microarray analysis of siberian ginseng cyclic somatic embryogenesis culture systems provides insight into molecular mechanisms of embryogenic cell cluster generation.

    Directory of Open Access Journals (Sweden)

    Chenguang Zhou

    Full Text Available Four systems of cyclic somatic embryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI, generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC, induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS and DEC cultured by bioreactor (ECB, respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE. Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters.

  3. Plant cell electrophysiology: applications in growth enhancement, somatic hybridisation and gene transfer.

    Science.gov (United States)

    Ochatt, Sergio

    2013-12-01

    The use and exploitation of electrophysiology with plant cells have witnessed a slow but steady increase for a number of purposes in recent years. First envisaged only as a tool for the recovery of somatic hybrid plants following protoplast electrofusion, or for transient and/or stable genetic transformation following electroporation-mediated entry of foreign genes into protoplasts and cells, electrophysiological studies with plant cells and tissues have since spanned into other areas, and particularly for the assessment of the possible effects of electric and electromagnetic fields on the subsequent growth and differentiation competences of the electro-treated cells. This review will critically discuss these various applications of electrophysiology and will also aim at analysing the fundamental physiological and physico-chemical mechanisms underlying them. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development.

    Science.gov (United States)

    Lee, W-J; Lee, J-H; Jeon, R-H; Jang, S-J; Lee, S-C; Park, J-S; Lee, S-L; King, W-A; Rho, G-J

    2017-02-13

    Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

  5. Exogenous melatonin reduces somatic cell count of milk in Holstein cows

    Science.gov (United States)

    Yang, Minghui; Shi, Jianmin; Tian, Jianhua; Tao, Jingli; Chai, Menglong; Wang, Jing; Xu, Zhiyuan; Song, Yukun; Zhu, Kuanfeng; Ji, Pengyun; Liu, Guoshi

    2017-01-01

    High somatic cell counts in milk caused by mastitis significantly influence the quality of milk and result in substantial annual economic loss. This study evaluated the beneficial effects of melatonin (MT) on milk somatic cell count (SCC) in cows. To examine the effects of melatonin on SCC, one hundred twenty cows were divided into four groups based on milk SCC. In each group, half of the cows were treated with melatonin (S.C.). Melatonin treatment significantly reduced milk SCC. To explore the potential mechanism, 20 cows with relatively high SCC were selected to evaluate the biochemical and immunological profiles of their blood after melatonin treatment. Treatment with MT significantly reduced SCC in milk, lowered serum cortisol concentrations and increased the levels of albumin, alanine transaminase and lactate dehydrogenase. Following treatment with MT, the concentration of IgG and IgM rose transiently then decreased significantly, similar to changes observed for white blood cells and lymphocytes. In conclusion, MT treatment improved the quality of milk by reducing SCC. This may be due to melatonin improving immune activity in cows. PMID:28240296

  6. Risk factors associated with clinical mastitis in low somatic cell count British dairy herds.

    Science.gov (United States)

    Peeler, E J; Green, M J; Fitzpatrick, J L; Morgan, K L; Green, L E

    2000-11-01

    A cross-sectional survey of dairy farms with low bulk milk somatic cell counts was carried out to assess the level of clinical mastitis and to quantify risk factors associated with the incidence rate of clinical mastitis. Questionnaires were sent to 3009 milk operations with an annual mean bulk milk somatic cell count of less than 100,000 cells/ml during 1997. A response rate was 61%. The mean incidence of clinical mastitis reported was 22.8 cases per 100 cows/yr. Negative binomial regression models were used to assess statistically significant risk factors associated with the incidence of clinical mastitis. The incidence increased when farmers reported that they had straw yard housing for milking cows (compared with cubicle housing), mucked out the calving area less frequently than once per month, kept cows standing in a yard after milking, always practiced postmilking teat disinfection, had greater than 50% replacement rate, had some cows that leaked milk on entry to the parlor, had some cows that leaked milk at other times, and foremilked before cluster attachment. The incidence of clinical mastitis was lower on farms when the gathering yard used before milking was scraped at least twice a day, cows were offered feed after both milkings, rubber gloves were not worn during milking, teat liners were changed after 6000 milkings, and the average dry period was less than 40 d. The study has identified areas of the environment in which efforts to improve hygiene should be focused.

  7. Rakkyo fructan as a cryoprotectant for serum-free cryopreservation of mammalian cells.

    Science.gov (United States)

    Ogawa, Akiko; Mizui, Shinya; Chida, Yasuhito; Shimizu, Masafumi; Terada, Satoshi; Ohura, Takeshi; Kobayashi, Kyo-Ichi; Yasukawa, Saori; Moriyama, Nobuyuki

    2014-07-01

    Cryopreservation refers to the long-term storage of mammalian cells. Mammalian serum is generally used as a cryoprotectant, but is associated with problems including the risk of contamination by pathogens and quality control issues. Therefore, a serum-free cryopreservation method needs to be established. In this study, we focused on rakkyo fructan, a fructose polymer, derived from the Japanese shallot as an alternative factor to serum. Fructan contributes to tolerance to frost and dehydration in plants by stabilizing the plant membrane. However, whether fructan protects mammalian cells against freezing stress remains unknown. The ability of rakkyo fructan to be an alternative cryoprotectant to fetal bovine serum (FBS) was examined in the present study. 2E3-O, a mouse hybridoma, was preserved in rakkyo fructan, was highly viable after being defrosted, and then proliferated rapidly. When rakkyo fructan was combined with dimethylsulfoxide (DMSO), its ability to protect the hybridoma against freezing stress was improved. The rakkyo fructan and DMSO mixture was used in the cryopreservation of the mammalian cell lines CHO-DP12, a producer of recombinant antibodies, and HepG2, human hepatoma cells frequently tested in bio-artificial livers. Following the freezing and thawing processes, CHO-DP12 cells retained their ability to produce recombinant antibodies and as did HepG2 cells for albumin and mRNA expression of cytochrome P450 enzymes. These results indicate that rakkyo fructan is a promising cryoprotectant that prevents mammalian cells from freezing stress similar to FBS.

  8. Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal.

    Science.gov (United States)

    Lee, Eunsong; Song, Kilyoung

    2007-12-01

    The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.

  9. Frequent somatic mosaicism of NEMO in T cells of patients with X-linked anhidrotic ectodermal dysplasia with immunodeficiency.

    Science.gov (United States)

    Kawai, Tomoki; Nishikomori, Ryuta; Izawa, Kazushi; Murata, Yuuki; Tanaka, Naoko; Sakai, Hidemasa; Saito, Megumu; Yasumi, Takahiro; Takaoka, Yuki; Nakahata, Tatsutoshi; Mizukami, Tomoyuki; Nunoi, Hiroyuki; Kiyohara, Yuki; Yoden, Atsushi; Murata, Takuji; Sasaki, Shinya; Ito, Etsuro; Akutagawa, Hiroshi; Kawai, Toshinao; Imai, Chihaya; Okada, Satoshi; Kobayashi, Masao; Heike, Toshio

    2012-06-07

    Somatic mosaicism has been described in several primary immunodeficiency diseases and causes modified phenotypes in affected patients. X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the NF-κB essential modulator (NEMO) gene and manifests clinically in various ways. We have previously reported a case of XL-EDA-ID with somatic mosaicism caused by a duplication mutation of the NEMO gene, but the frequency of somatic mosaicism of NEMO and its clinical impact on XL-EDA-ID is not fully understood. In this study, somatic mosaicism of NEMO was evaluated in XL-EDA-ID patients in Japan. Cells expressing wild-type NEMO, most of which were derived from the T-cell lineage, were detected in 9 of 10 XL-EDA-ID patients. These data indicate that the frequency of somatic mosaicism of NEMO is high in XL-ED-ID patients and that the presence of somatic mosaicism of NEMO could have an impact on the diagnosis and treatment of XL-ED-ID patients.

  10. The yeast I-Sce I meganuclease induces site-directed chromosomal recombination in mammalian cells.

    Science.gov (United States)

    Choulika, A; Perrin, A; Dujon, B; Nicolas, J F

    1994-11-01

    Double-strand breaks in genomic DNA stimulate recombination. Until now it was not possible to induce in vivo site-directed double-strand breaks in a mammalian chromosomal target. In this article we describe the use of I-Sce I meganuclease, a very rare cutter yeast endonuclease, to induce site-directed double-strand breaks mediated recombination. The results demonstrate the potential of the I-Sce I system for chromosome manipulation in mammalian cells.

  11. Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells

    Directory of Open Access Journals (Sweden)

    Mazel-Sanchez Beryl

    2010-03-01

    Full Text Available Abstract In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.

  12. Will brain cells derived from induced pluripotent stem cells or directly converted from somatic cells (iNs) be useful for schizophrenia research?

    Science.gov (United States)

    Filippich, Cheryl; Wolvetang, Ernst J; Mowry, Bryan J

    2013-09-01

    The reprogramming of nonneuronal somatic cells to induced pluripotent stem cells and their derivation to functional brain cells as well as the related methods for direct conversion of somatic cells to neurons have opened up the possibility of conducting research on cellular disease models from living schizophrenia patients. We review the published literature on schizophrenia that has used this rapidly developing technology, highlighting the need for specific aims and reproducibility. The key issues for consideration for future schizophrenia research in this field are discussed and potential investigations using this technology are put forward for critical assessment by the reader.

  13. Silver-doped calcium phosphate nanoparticles: synthesis, characterization, and toxic effects toward mammalian and prokaryotic cells.

    Science.gov (United States)

    Peetsch, Alexander; Greulich, Christina; Braun, Dieter; Stroetges, Christian; Rehage, Heinz; Siebers, Bettina; Köller, Manfred; Epple, Matthias

    2013-02-01

    Spherical silver-doped calcium phosphate nanoparticles were synthesized in a co-precipitation route from calcium nitrate/silver nitrate and ammonium phosphate in a continuous process and colloidally stabilized by carboxymethyl cellulose. Nanoparticles with 0.39 wt% silver content and a diameter of about 50-60 nm were obtained. The toxic effects toward mammalian and prokaryotic cells were determined by viability tests and determination of the minimal inhibitory and minimal bactericidal concentrations (MIC and MBC). Three mammalian cells lines, i.e. human mesenchymal stem cells (hMSC) and blood peripheral mononuclear cells (PBMC, monocytes and T-lymphocytes), and two prokaryotic strains, i.e. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used. Silver-doped calcium phosphate nanoparticles and silver acetate showed similar effect toward mammalian and prokaryotic cells with toxic silver concentrations in the range of 1-3 μg mL(-1).

  14. Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test

    Energy Technology Data Exchange (ETDEWEB)

    Tomkins, D.J.; Wei, L.; Laurie, K.E.

    1985-01-01

    It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dip/sup r/) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinese hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis (TH-thymidine, autoradiography) or protein synthesis (TVS-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dip/sup r/ may not be a suitable marker for the somatic cell mutation test.

  15. Transcription factor Oct1 is a somatic and cancer stem cell determinant.

    Directory of Open Access Journals (Sweden)

    Jessica Maddox

    Full Text Available Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1(HI cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24(LOCD44(HI cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDH(HI and dye efflux(HI cells, and increasing Oct1 increases the proportion of ALDH(HI cells. Normal ALDH(HI cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.

  16. Making cardiomyocytes with your chemistry set:Small molecule-induced cardiogenesis in somatic cells

    Institute of Scientific and Technical Information of China (English)

    Woong-Hee; Kim; Da-Woon; Jung; Darren; Reece; Williams

    2015-01-01

    Cell transplantation is an attractive potential therapy for heart diseases. For example, myocardial infarction(MI) is a leading cause of mortality in many countries. Numerous medical interventions have been developed to stabilize patients with MI and, although this has increased survival rates, there is currently no clinically approved method to reverse the loss of cardiac muscle cells(cardiomyocytes) that accompanies this disease. Cell transplantation has been proposed as a method to replace cardiomyocytes, but a safe and reliable source of cardiogenic cells is required. An ideal source would be the patients’ own somatic tissue cells, which could be converted into cardiogenic cells and transplanted into the site of MI. However, these are difficult to produce in large quantities and standardized protocols to produce cardiac cells would be advantageous for the research community. To achieve these research goals, small molecules represent attractive tools to control cell behavior. In this editorial, we introduce the use of small molecules in stem cell research and summarize their application to the induction of cardiogenesis in noncardiac cells. Exciting new developments in this field are discussed, which we hope will encourage cardiac stem cell biologists to further consider employing small molecules in their culture protocols.

  17. Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors.

    Directory of Open Access Journals (Sweden)

    Yan Jiang

    Full Text Available Interspecies somatic cell nuclear transfer (iSCNT involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%; and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%. Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 µM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38% compared to embryos generated from non-supplemented oocytes (P<0.01. They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They

  18. Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique

    DEFF Research Database (Denmark)

    Booth, P J; Tan, S J; Reipurth, R

    2001-01-01

    Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods...... embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1 recipients pregnant at 5.......8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1 blastocysts per successfully fused and surviving reconstructed...

  19. Genotoxicity effects of Flusilazole on the somatic cells of Allium cepa.

    Science.gov (United States)

    Ozakca, Dilek Unal; Silah, Hulya

    2013-09-01

    The aim of this study was to evaluate the effects of the fungicide flusilazole on somatic cells of Allium cepa. For evaluation of cytogenetic effects, root meristem cells of A. cepa were treated with 10, 20, 30 and 45 ppm (EC50 concentration) for 24, 48 and 72 h. The mitotic index and different types of chromosomal abnormalities such as bridges, stickiness and laggards were determined in both control and test groups. Acridine orange/Ethidium bromide double staining and fluorescence microscope was used to determine the stability of chromosome structure. Data obtained from staining process indicated that ratio of necrotic cells significantly increased by the flusilazole presoaking. The RAPD-PCR method was used and the higher doses treated-group (45 ppm) was more distant to the control group compare with others.

  20. Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

    DEFF Research Database (Denmark)

    Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga;

    2015-01-01

    genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree......Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated...... of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required...