Mammalian Somatic Cells Research Articles

Creating Zinc Finger Nucleases Using a Modular-Assembly Approach

Homologous recombination is the most precise way to manipulate the genome. It has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines, but has not been available in other systems such as mammalian somatic cells. However, the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several-thousandfold in mammalian somatic cells. These double-strand breaks can be created in mammalian genomes by zinc finger nucleases (ZFNs), artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. This protocol describes how to use a polymerase chain reaction (PCR) technique to assemble new three-finger proteins and how to create a new ZFN.

Interchromatin granule clusters of the scorpionfly oocytes contain poly(A)+ RNA, heterogeneous ribonucleoproteins A/B and mRNA export factor NXF1

IGCs (interchromatin granule clusters), or nuclear speckles, are one of the most universal subnuclear organelles of eukaryotic cells. We have used insect oocytes to study the possible association of poly(A)+ RNA and some factors involved in mRNA export with IGCs. Oogenesis of the mecopteran, Panorpa communis, used as a model object, is characterized by a strict cessation of oocyte genome transcription activity towards the end of oocyte growth. Our previous studies on P. communis oocyte nuclei have shown that oocyte IGC counterparts in this species are very unusual, both in morphology and molecular composition, compared with the typical IGCs of mammalian somatic cells traditionally used as a model system. We have now used microinjections of 2'-O-Me(U)₂₂ probes conjugated with the fluorochrome TAMRA to localize poly(A)+ RNA in IGCs. RNA export proteins were also detected by immunofluorescent/confocal and immunogold labelling electron microscopy. We found that poly(A)+ RNA, hnRNPs A/B and NXF1 mRNA export factors are located in IGCs regardless of the transcriptional status of the nucleus. Our data support the idea of IGCs as universal and conserved nuclear domains that serve not only as splicing factor reservoirs, but also take part in mRNA retention and export.

Ionising radiation and genetic risks. XVI. A genome-based framework for risk estimation in the light of recent advances in genome research

Aims and objectives: The aim of this paper is to examine a framework for computational modelling of genetic risks of radiation using the human genome as the starting point. The convergence of insights gained from knowledge of repair of DNA double-strand breaks in mammalian somatic cells and of the architecture of the human genome makes this framework possible. We inquire whether the concepts underlying the framework remain valid in the light of advances in DNA repair studies and human genome research during the past five years.Materials and methods: We reviewed the advances in DNA repair studies and genome research, and in the latter, we focused on advances subsequent to the discovery of copy number variation in the genome which include an assessment of its nature, extent, mechanisms involved and its role in health and disease.Results: Our study shows that the concepts underlying our framework are valid. More specifically, the view that segmental duplications (which are abundant in the genome) can serve as entry points for modelling the origin of radiation-induced deletions via non-allelic homologous recombination in germ cells of human female remains robust.Conclusions: We posit that progress in genetic risk estimation in the 21st century will be driven mainly by the integration of genomic knowledge with that of DNA repair mechanisms, the latter involved in the origin of spontaneously-occurring deletions (which cause genomic disorders in humans) and of radiation-induced deletions in mammalian cells and extending the insights to irradiated human germ cells.

Development of Interspecies Somatic Cell Nuclear Transfer Embryos Reconstructed from Bactrian Camel (Camelus bactrianus) Fibroblasts and Dromedary Camel (Camelus dromedarius) Ooplasm.

Interspecies somatic cell nucleus transfer could not only be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos but can also be used as a possible approach to conserve endangered species. It could provide a useful technique to preserve the wild Bactrian camels, which are threatened with extinction. In the present study embryos were reconstructed by using skin fibroblast cells from a Bactrian camel (Camelus bactrianus) as donor karyoplasts and dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for investigating the developmental potential of the these embryos. Mature oocytes were collected from super-stimulated dromedary camels by ultrasound guided transvaginal ovum pick-up, 26 h after GnRH administration. Serum-starved adult Bactrian camel skin fibroblast cells were injected in to the perivitalline space of enucleated dromedary oocytes. The fibroblast cells and recipient cytoplasm were fused by two DC pulses of 100 V/cm for 15 μs each using an Eppendorf electroporator at room temperature. Reconstructs were activated 1 h postfusion with 5μM ionomycine followed by exposure to 6-dimethylaminopurine for 4 h. The activated oocytes were then transferred to 500 μL of embryo culture medium I (modified potassium simplex optimization medium with essential and non-essential amino acids [KSOMaa] supplemented with 1% BSA) and cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. On Day 3 (Day 0 = day of activation) the cleaved embryos were transferred into 500μL of embryo culture medium II (modified KSOMaa supplemented with 10% FCS) and cultured under the same conditions until Day 7. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture. The embryos reconstructed from dromedary camel (Camelus dromedarius) fibroblasts and oocytes were treated in similar manner and worked as control. Out of 58 reconstructed embryos, in 3 replicates, 77.4 ± 2.1 couplets fused successfully and were cultured after activation. The embryos that cleaved (59.1 ± 5.7) and those developed to the blastocyst stage (34.4 ± 3.9) were not different from the control group. This study demonstrated, for the first time, that somatic cells from Bactrian camel can be reprogrammed in the dromedary camel ooplasm and such interspecies nuclear transfer embryos can develop to the blastocyst stage. Further studies are needed to see the feasibility of transferring these embryos into dromedary camel recipients and the possibility of producing Bactrian camel calves from dromedary recipient camels. (platform)

P71. Stem cells and gene expression during hemichordate head regeneration

Hemichordate acorn worms grow a new head on the anterior wound surface when the body is transected. In Ptychodera flava, a blastema is evident by 3 days and a new full-sized, functional head differentiates on the cut stump by 12 days. The hemichordate nervous system provides the most ancestral extant example of the dorsal hollow brain, formed by neurulation, characteristic of chordates and humans. Only in hemichordates does such a nervous system regenerate significantly. Animals throughout the phylogenetic scale share similar developmental gene regulatory networks, and our work, along with that of others, supports the conclusion that the same gene regulatory networks occur in hemichordate and chordate neural differentiation. Examination of the regeneration process by whole mount in situ hybridization (WISH) of sequences from Yamanaka pluripotency factors or “stem cell” genes, as defined by genes inducing pluripotency in mammalian somatic cells, and developmental organizer genes reveals a cascade of gene expression associated with the steps of regeneration. A subset of the stem cell genes produce the first WISH signals before a blastema is formed. The second set of stem cell genes begins to generate signal as the blastema is established and this signal persists in the blastema as organizer and early development gene signals appear as differentiation proceeds. These results suggest that activation of cells with regenerative capability involves the early expression of stem cell genes in the cells that will form the blastema. Signal from some stem cell genes continues as the expression of typical embryonic gene regulatory networks are activated for the differentiation of the tissues. In planarians, regeneration is based on mobilization of stem cells residing in the tissue of the animal to form a regeneration blastema. During regeneration of an amphibian limb, a blastema is also formed, apparently by dedifferentiation of tissue cells in the cut limb to stem cells which accumulate to establish the blastema. The nature of the cells and the tissue origin of the cells that participate in hemichordate blastema formation and head regeneration are being investigated. Supported by the PBRC Biomedical Fund UHF#12-209-04.

Genome-wide analysis of hepatic gene silencing in mammalian cell hybrids

Silencing of tissue-specific gene expression in mammalian somatic cell hybrids is a well-documented epigenetic phenomenon which is both profound (involving a large number of genes) and enigmatic. Our aim was to utilize whole-genome microarray analyses to determine the true extent of gene silencing on a genomic level. By comparing gene expression profiles of hepatoma × fibroblast cell hybrids with those of parental cells, we have identified over 300 liver-enriched genes that are repressed at least 5-fold in the cell hybrids, the majority of which are repressed at least 10-fold. Also, we identify nearly 200 fibroblast-enriched genes that are repressed at least 5-fold. Silenced hepatic genes include several that encode transcription factors and proteins involved in signal transduction pathways. These data suggest that extensive reprogramming occurs in cell hybrids, leading to a nearly global (although not complete) loss of tissue-specific gene expression.

CpG island clusters and pro-epigenetic selection for CpGs in protein-coding exons of HOX and other transcription factors

CpG dinucleotides contribute to epigenetic mechanisms by being the only site for DNA methylation in mammalian somatic cells. They are also mutation hotspots and approximately 5-fold depleted genome-wide. We report here a study focused on CpG sites in the coding regions of Hox and other transcription factor genes, comparing methylated genomes of Homo sapiens, Mus musculus, and Danio rerio with nonmethylated genomes of Drosophila melanogaster and Caenorhabditis elegans. We analyzed 4-fold degenerate, synonymous codons with the potential for CpG. That is, we studied "silent" changes that do not affect protein products but could damage epigenetic marking. We find that DNA-binding transcription factors and other developmentally relevant genes show, only in methylated genomes, a bimodal distribution of CpG usage. Several genetic code-based tests indicate, again for methylated genomes only, that the frequency of silent CpGs in Hox genes is much greater than expectation. Also informative are NCG-GNN and NCC-GNN codon doublets, for which an unusually high rate of G to C and C to G transversions was observed at the third (silent) position of the first codon. Together these results are interpreted as evidence for strong "pro-epigenetic" selection acting to preserve CpG sites in coding regions of many genes controlling development. We also report that DNA-binding transcription factors and developmentally important genes are dramatically overrepresented in or near clusters of three or more CpG islands, suggesting a possible relationship between evolutionary preservation of CpG dinucleotides in both coding regions and CpG islands.

Noninvasive optical laser technologies for the transplantation of mammalian nuclei

The results of pioneering studies on the development of radically new noninvasive methods for the transplantation of mammalian somatic cell nuclei with the use of optical laser manipulations are presented, and their comparison with traditional invasive methods is performed. It is shown that all the key steps, including the enucleation of a recipient cell, the transfer of a somatic cell (karyoplast), its bringing close together with the recipient cytoplast, and the fusion of the cytoplast with the somatic cell, can be effectively conducted using the laser only with the complete replacement by laser of traditional mechanical micromanipulators and other devices, including devices for electrofusion. The results indicate the unique potentialities of laser and the prospects of its application in modern cell engineering in a wide spectrum of studies on oocytes and early mammalian embryos, in particular in technologies of therapeutic and reproductive cloning.

Nuclear Bud Formation: A Novel Manifestation of Zidovudine Genotoxicity

Normal diploid somatic mammalian cell division generates 2 daughter cells as a result of a strict and well-controlled mitotic process. However, some defects during the progression of that process could generate an unbalanced distribution of chromosomes, aneuploidy and eventually, a malignant phenotype. Previous observations using a transgenic mouse model with diminished DNA repair capacity revealed the presence of nuclear buds (NBs) induced in vitro by the nucleoside analog zidovudine (Retrovir®, 3′-azido-3′-deoxythymidine, AZT). Here we used bone marrow mesenchymal cells, taken from mice with the Xpa^–/–Trp53^+/– genotype, that were cultured and exposed to 0 and 100 µM AZT for 24 hours. Fixed and denatured cells were processed by fluorescence in situ hybridization (FISH) with whole chromosome painting probes used to identify chromosomes in cells growing on glass chamber slides (2 probes/slide). A variety of sizes and shapes of NBs were observed. Some NBs had a large connection with the main nucleus (>¼ of the NB diameter), others hada smaller connection (<¼ of the NB diameter), some were circular and positioned close to the nucleus, while some resided in the cytoplasm separated from the nucleus or connected by a thin chromatin strand. We had hypothesized that NBs would progress in the process of budding until separation occurred, but this was not proven by time-lapse photography studies performed for 20 hours. From 1,126 cells scored in the unexposed cultures, 10.39 % of cells carried NBs, while from 1,108 cells scored in the AZT-exposed cultures 29.16% of cells carried NBs (p = 0.001). In AZT-exposed cells there were a total of 322 NBs scored; 46.6% or 150 NBs contained positive signals for one or both probes used, while 53% or 172 NBs had no probe signal. In addition, FISH analysis showed no preferential localization of any chromosome within the NBs. Among the NBs that carried no probe signal, the presence of positive signals with inversion of DAPI imaging demonstrated centromeric content. It has been hypothesized that NBs occur as a result of expulsion of amplified DNA from the main nucleus; however, this data demonstrates that NBs may contain any chromosome, suggesting that NBs do not consist of just amplified DNA.

Ecotoxicological effects of Methyl parathion on living things and environment

Methyl parathion; is an insecticide which is commonly used in our country in the aim of agriculture and it is known that some environmental problems arise due to the chronical usage of this pesticide. For this reason, it is very important to raise the awareness the public in terms of the risk factors because of the usage of methyl parathion. The researches showed that methyl parathion inhibits cholinesterase enzyme in the human brains, cause genotoxic effect in mammalian somatic cells in culture medium and increase the anormalities in chromosomal structure. It was determined that this insecticide causes a decrease in the body weights of experimental animals and an increase in the weight of liver, cellular damages in the livers, various neurological problems by affecting reproduction, nervous and cardiovascular systems. Since methyl parathion causes the formation of a toxic medium for human beings, birds, fresh water fish, other hydrophilous organisms as well as sea organisms by mixing to air, soil and water, it is well known that the usage of methyl parathion as an insecticide is a threatening factor for the ecosystem. The usage of methyl parathion has been forbidden in EU (European Union) countries since 2003 and some restrictions have been applied in United States of America. There is no limiting or forbidding enforcement related with the usage of methyl parathion which is in the 3rd order among commonly used pesticides in our country. To bring new arrangements for the usage of methyl parathion which constitutes various risk factors in terms of ecological factors may minimize the ecotoxicological effects of this pesticide. Key words: Methyl parathion, insecticide, ecotoxicological effect.

PiggyBac Transposon-mediated Long-term Gene Expression in Mice

Transposons are promising systems for somatic gene integration because they can not only integrate exogenous genes efficiently, but also be delivered to a variety of organs using a range of transfection methods. piggyBac (PB) transposon has a high transposability in mammalian cells in vitro, and has been used for genetic and preclinical studies. However, the transposability of PB in mammalian somatic cells in vivo has not been demonstrated yet. Here, we demonstrated PB-mediated sustained gene expression in adult mice. We constructed PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase (Gluc)] genes. Mice were transfected by injection of these pDNAs using a hydrodynamics-based procedure, and the conditions for high-level sustained gene expression were examined. Consequently, gene expressions were sustained over 2 months. Our results suggest that PB is useful for organ-selective somatic integration and sustained gene expression in mammals, and will contribute to basic genetic studies and gene therapies.

IPS cell technology in regenerative medicine

The promise of treating human genetic and degenerative diseases through the application of pluripotent cell-based tissue engineering and regenerative medicine has come significantly closer to realization since the isolation of human embryonic stem (ES) cells. While the study of ES cells has greatly increased our fundamental understanding of pluripotency, technical and ethical limitations have been seemingly insurmountable impediments to the application of these cells in the clinic. The recent discovery that somatic mammalian cells can be epigenetically reprogrammed to a pluripotent state through the exogenous expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC has yielded a new cell type for potential application in regenerative medicine, the induced pluripotent stem (iPS) cell. Here we discuss how advances in iPS cell technology have led to the generation of patient-specific cell lines that can potentially be used to model human diseases and ultimately act as therapeutic agents.

Characterization of somatic cell nuclear reprogramming by oocytes in which a linker histone is required for pluripotency gene reactivation

When transplanted into Xenopus oocytes, the nuclei of mammalian somatic cells are reprogrammed to express stem cell genes such as Oct4, Nanog, and Sox2. We now describe an experimental system in which the pluripotency genes Sox2 and Oct4 are repressed in retinoic acid-treated ES cells but are reprogrammed up to 100% within 24 h by injection of nuclei into the germinal vesicle (GV) of growing Xenopus oocytes. The isolation of GVs in nonaqueous medium allows the reprogramming of individual injected nuclei to be seen in real time. Analysis using fluorescence recovery after photobleaching shows that nuclear transfer is associated with an increase in linker histone mobility. A simultaneous loss of somatic H1 linker histone and incorporation of the oocyte-specific linker histone B4 precede transcriptional reprogramming. The loss of H1 is not required for gene reprogramming. We demonstrate both by antibody injection experiments and by dominant negative interference that the incorporation of B4 linker histone is required for pluripotency gene reactivation during nuclear reprogramming. We suggest that the binding of oocyte-specific B4 linker histone to chromatin is a key primary event in the reprogramming of somatic nuclei transplanted to amphibian oocytes.

Mechanisms of double-strand break repair in somatic mammalian cells

Mechanisms of double-strand break repair in somatic mammalian cells

Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes

BackgroundWhen the nuclei of mammalian somatic cells are transplanted to amphibian oocytes in the first meiotic prophase, they are rapidly induced to begin transcribing several pluripotency genes, including Sox2 and Oct4. The more differentiated the donor cells of the nuclei, the longer it takes for the pluripotency genes to be activated after the nuclear transfer to oocytes. We have used this effect in order to investigate the role of histone modifications in this example of nuclear reprogramming.ResultsReverse transcription polymerase chain reaction analysis shows that the transcriptional reprogramming of pluripotency genes, such as Sox2 and Oct4, takes place in transplanted nuclei from C3H10T1/2 cells and from newly differentiated mouse embryonic stem cells. We find that the reprogramming of 10T1/2 nuclei is accompanied by an increased phosphorylation, an increased methylation and a rapidly reduced acetylation of several amino acids in H3 and other histones. These results are obtained by the immunofluorescent staining of transplanted nuclei and by Western blot analysis. We have also used chromatin immunoprecipitation analysis to define histone modifications associated with the regulatory or coding regions of pluripotency genes in transplanted nuclei. Histone phosphorylation is increased and histone acetylation is decreased in several regulatory and gene coding regions. An increase of histone H3 lysine 4 dimethylation (H3K4 me2) is seen in the regulatory regions and gene coding region of pluripotency genes in reprogrammed nuclei. Furthermore, histone H3 lysine 4 trimethylation (H3K4 me3) is observed more strongly in the regulatory regions of pluripotency genes in transplanted nuclei that are rapidly reprogrammed than in nuclei that are reprogrammed slowly and are not seen in β-globin, a gene that is not reprogrammed. When 10T1/2 nuclei are incubated in Xenopus oocyte extracts, histone H3 serine 10 (H3S10) is strongly phosphorylated within a few hours. Immunodepletion of Aurora B prevents this phosphorylation.ConclusionWe conclude that H3K4 me2 and me3 are likely to be important for the efficient reprogramming of pluripotency genes in somatic nuclei by amphibian oocytes and that Aurora B kinase is required for H3S10 phosphorylation which is induced in transplanted somatic cell nuclei.

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis.Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein.

P-Body Loss Is Concomitant with Formation of a Messenger RNA Storage Domain in Mouse Oocytes1

In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.

331. INVESTIGATING THE ROLE OF CYCLIN A2 DURING OOCYTE MEIOSIS

Aneuploidy is often derived from chromosomal segregation errors in oocytes, leading to Down Syndrome and early embryo loss. Thus it is important to understand the molecular control of female meiosis. Cyclins and cyclin-dependent kinases (CDKs), particular those contributing to Maturation Promoting Factor (MPF, or CDK1) activity, play key roles in regulating meiosis. While cyclin B1 is classically regarded as the regulatory component of MPF, cyclin A2 can also bind and activate CDK1, and in mammalian somatic cells it is known to promote both G1/S and G2/M transitions. There is an absolute requirement for cyclin A2 during development and differentiation as its deficiency results in early embryonic lethality. As such, cyclin A2 has not been extensively studied, particularly as to its role in meiosis. To examine the role cyclin A2 plays during mammalian female meiosis, we carried out knockdown experiments. Microinjection into mouse oocytes of cyclin A2 siRNA induced a ~70% knockdown. Cyclin A2 knockdown did not inhibit germinal vesicle breakdown but did act to delay it. Extrusion of the first polar body was significantly reduced (P = 0.002) in comparison to non-injected controls and those injected with a negative control siRNA. Furthermore, microinjection of cyclin A2 can stimulate entry into meiosis. Thus it seems possible that cyclin A2/CDK can exhibit MPF activity. In conclusion, our data suggest that cyclin A2 can regulate meiotic entry in oocytes and also plays an important role in successful passage through the first meiotic division.

A summary on stem cell research: A report on the 8th Annual Meeting of the International Society for Stem Cell Research

The 8th Annual Meeting of the International Society for Stem Cell Research (ISSCR) placed a particular emphasis on topics related to the engineering of induced pluripotent stem cells through reprogramming of mammalian somatic cells, embryonic and adult stem cells of different species, tissue regeneration, stem cell transplantation and cancer stem cells. This report provides an overview on the standpoint of stem cell research programs, which is based on the proceedings of the meeting.

Chk1–cyclin A/Cdk1 axis regulates origin firing programs in mammals

DNA replication is key to ensuring the complete duplication of genomic DNA prior to mitosis and is tightly regulated by both cell cycle machinery and checkpoint signals. Regulation of the S phase program occurs at several stages, affecting origin firing, replication fork elongation, fork velocity, and fork stability, all of which are dependent on S-phase-promoting kinase activity. Somatic mammalian cells use well-established origin programs by which specific regions of the genome are replicated at precise times. However, the mechanisms by which S phase kinases regulate origin firing in mammals are largely unknown. Here, we discuss recent advances in the understanding of how S phase programs are regulated in mammals at the correct regions and at the appropriate times.