Transgenic Manipulation Research Articles

Development and characterization of a cell line from the embryos of half smooth tongue sole (Cynoglossus semilaevis)

A new cell line, CSEC, has been successfully established from embryos at gastrula stage of a cultured marine fish, half smooth tongue sole (Cynoglossus semilaevis). CSEC cells grow actively and stably more than 50 passages for over 200 d in DMEM medium supplemented with 15% FBS (fetal bovine serum), 2.5 ng/cm3 bFGF (basic fibroblast growth factor), 1 ng/cm3 LIF (leukemia inhibitory factor) and 50 mmol/dm3 2-ME (2-mecaptoethanol). The cells grew well in the temperature range of 24–30°C and the optimal growth temperature was 24°C. FBS and bFGF concentrations are the two key components for CSEC cells proliferation. Chromosome analysis reveals that CSEC cells have a normal diploid karyotype with 2n=42t. The significant fluorescent signals were observed in CSEC after transfection with the GFP reporter gene, suggesting that the CSEC cell line can be used as a useful tool for transgenic and genetic manipulation studies. CSEC cells showed the cytopathic effect (CPE) after infection with lymphosystis disease virus (LCDV) in 2 d. Moreover, the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy. It suggests that CSEC could be potentially used for the study of aquatic virus.

Transgenic zebrafish models of neurodegenerative diseases

Since the introduction of the zebrafish as a model for the study of vertebrate developmental biology, an extensive array of techniques for its experimental manipulation and analysis has been developed. Recently it has become apparent that these powerful methodologies might be deployed in order to elucidate the pathogenesis of human neurodegenerative diseases and to identify candidate therapeutic approaches. In this article, we consider evidence that the zebrafish central nervous system provides an appropriate setting in which to model human neurological disease and we review techniques and resources available for generating transgenic models. We then examine recent publications showing that appropriate phenotypes can be provoked in the zebrafish through transgenic manipulations analogous to genetic abnormalities known to cause human tauopathies, polyglutamine diseases or motor neuron degenerations. These studies show proof of concept that findings in zebrafish models can be applicable to the pathogenic mechanisms underlying human diseases. Consequently, the prospects for providing novel insights into neurodegenerative diseases by exploiting transgenic zebrafish models and discovery-driven approaches seem favorable.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat.

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Introductory Overview to the Proceedings of the XXth North American Testis Workshop

Introductory Overview to the Proceedings of the XXth North American Testis Workshop

Repetitive Transgenes in C. elegans Accumulate Heterochromatic Marks and Are Sequestered at the Nuclear Envelope in a Copy-Number- and Lamin-Dependent Manner

Chromatin is nonrandomly distributed in nuclear space, yet the functional significance of this remains unclear. Here, we make use of transgenes carrying developmentally regulated promoters to study subnuclear gene positioning during the development of Caenorhabditis elegans. We found that small transgenes (copy number ≤50) are randomly distributed in early embryonic nuclei, independent of promoter activity. However, in differentiated tissues, these same transgenes occupied specific subnuclear positions: When promoters are repressed, transgenes are found at the nuclear periphery, whereas active, developmentally regulated promoters are enriched in the nuclear core. The absence of specific transgene positioning in embryonic nuclei does not reflect an absence of proteins that mediate perinuclear sequestration: Embryonic nuclei are able to sequester much larger transgene arrays (copy number 300-500) at the periphery. This size-dependent peripheral positioning of gene arrays in early embryos correlates with the accumulation of heterochromatic marks (H3K9me3 and H3K27me3) on large arrays. Interestingly, depletion of nuclear lamina components caused release of arrays from the nuclear envelope and interfered with their efficient silencing. Our results suggest that developmentally silenced chromatin binds the nuclear lamina in a manner correlated with the deposition of heterochromatic marks. Peripheral sequestration of chromatin may, in turn, support the maintenance of silencing.

Contribution of animal models to the research of the causes of diabetes

In most publications, animal models of diabetes have mainly been investigated for their multiple etiologies as well as for changes leading to diabetes and their genetic derivation. Aspects which seem important and need a special research endeavor are the mechanism of the causes of diabetes and the lapse into complications in different species, their molecular basis and possible arrest and prevention. A concise list and and short discussion of the intensively studied rodents is presented of spontaneous or nutritional background causing Type 2 diabetes but omitting diabetes evoked by transgenic manipulations or gene knockout techniques.

Behavioral assessments for pre-clinical pharmacovigilance

Want to try a fun social experiment? Mention the term behavioral research to any scientist and watch their reaction. Some find the idea of behavioral research daunting, while others think the field isn't really a science, just the mere act of watching a subject's performance while making a few checkmarks on a clipboard. For those that have run behavioral experiments, we understand the theory and importance behind the research, and have an appreciation for a thorough and coherent experimental study. In the last decade, the field of Neuroscience has been more accepting of behavior as a necessary part of the empirical studies that previously contained only cellular or histological findings. Science in general, is moving toward translational research (Zerhouni, 2005), so being able to show how the cellular or chemical changes affect the organism as a whole, has become a high priority. Transgenic mice have contributed enormous amounts of data to every sub category of neuroscience. The great minds of Biomedical Scientists are able to splice cells, taking out genes or introducing new ones in, but they need the expertise of a Behavioral Scientist in order to assess the changes to the organism that can not tell us what it is thinking or feeling. So let's explore this further and start by asking, where does behavioral testing begin, and why is it important? The answers to both depend on the study you are conducting, especially when you are considering adding a substance into the mix that can alter physiology. Genetically mutated mice are engineered to determine the effects of a specific gene, or more commonly, in an effort to mimic human symptoms of disease. Pharmaceutical companies and toxicologists find themselves needing to comprehensively assess drug efficacy in a high throughput fashion, something that can take years to do in both human and non-human primates over the course of the lifespan. The development of the transgenic mouse, allows pre-clinical screening, on a mass level in a comparatively short period of time. This has created a major concern for drug research studies in terms of accurately determining what changes can be attributed to the drug as opposed to the changes in behavior due to the transgene manipulation. Therefore, prior to using any transgenic mouse model of disease, the researcher needs to know what changes the gene modification has caused in the mouse model, and places a great emphasis on the art of behavioral phenotyping. If you know what behaviors are “normal” for your mouse model, then you can determine what tests to use in a battery of assessments, including what home cage behaviors are important in your selection of data points. Take a current Alzheimer's disease (AD) model as an example, the Tg 2576 for which the background strain is C57BL/6J. This particular AD mouse model carries the human gene for amyloid precursor protein (APP), which is responsible for the formation of AD like plaques in humans (Hsiao et al., 1996). Bred from a mouse that has the recessive gene Pdebrd1, severe visual impairments (Gimenez and Montoliu, 2001) and deafness (Johnson et al., 1997) are common in these animals after 9 months of age. Additionally, after a period of time the animals begin to lose their hair and whiskers, and barber (over groom) themselves. It is necessary that these key characteristics are known prior to designing/conducting any behavioral task that uses auditory or visual cues for the animal to complete, as results might imply cognitive deficits.

Carotenoid biosynthesis genes in rice: structural analysis, genome-wide expression profiling and phylogenetic analysis

Carotenoids, important lipid-soluble antioxidants in photosynthetic tissues, are known to be completely absent in rice endosperm. Many studies, involving transgenic manipulations of carotenoid biosynthesis genes, have been performed to get carotenoid-enriched rice grain. Study of genes involved in their biosynthesis can provide further information regarding the abundance/absence of carotenoids in different tissues. We have identified 16 and 34 carotenoid biosynthesis genes in rice and Populus genomes, respectively. A detailed analysis of the domain structure of carotenoid biosynthesis enzymes in rice, Populus and Arabidopsis has shown that highly conserved catalytic domains, along with other domains, are present in these proteins. Phylogenetic analysis of rice genes with Arabidopsis and other characterized carotenoid biosynthesis genes has revealed that homologous genes exist in these plants, and the duplicated gene copies probably adopt new functions. Expression of rice and Populus genes has been analyzed by full-length cDNA- and EST-based expression profiling. In rice, this analysis was complemented by real-time PCR, microarray and signature-based expression profiling, which reveal that carotenoid biosynthesis genes are highly expressed in light-grown tissues, have differential expression pattern during vegetative/reproductive development and are responsive to stress.

Dec2 Promotes Th2 Cell Differentiation by Enhancing IL-2R Signaling

Th cell differentiation is precisely regulated by thousands of genes at different stages. In the present study, we demonstrate that Dec2, a transcription factor belonging to the bHLH (basic helix-loop-helix) superfamily, is progressively induced during the course of Th2 differentiation, especially at the late stage. The up-regulated Dec2 can strongly promote Th2 development under Th2-inducing conditions, as evidenced by retrovirus-mediated gene transfer or transgenic manipulation. In addition, an enhancement of Th2 responses is also detectable in Dec2 transgenic mice in vivo. Conversely, RNA interference-mediated suppression of endogenous Dec2 could attenuate Th2 differentiation. Finally, we show that the enhanced Th2 development is at least in part due to substantial up-regulation of CD25 expression elicited by Dec2, thereby resulting in hyperresponsiveness to IL-2 stimulation.

Studying Populus Defenses Against Insect Herbivores in the Post-Genomic Era

Concurrent with the ever increasing demand for wood-based products, there is an obvious need to conserve forest ecosystems for their ecological value. Recent research suggests that these potentially competing objectives can be achieved by increasing or tailoring the productivity of planted forests through the use of biotechnology. Unfortunately, this strategy could be hindered by forest insect pests that pose a challenge to the sustainability of planted forests. While advancing genetic improvement or domestication of tree species for productivity in plantation forestry, it is critical that we also identify tree genes controlling resistance and/or tolerance mechanisms against insect pests. In this regard, Populus has emerged as the model angiosperm tree to investigate forest tree-insect interactions. In recent years a number of studies have harnessed the power of genomics to generate extensive inventories of Populus genes that may contribute to the defense response of Populus following insect attack. However, despite these advances, the challenge remains to link insect feeding-induced changes in gene expression with altered resistance or tolerance to insect attack. Following an introduction on the current state of knowledge concerning insect herbivores and Populus defenses, an overview is provided of emerging research strategies designed to identify and test the function of defense genes that directly mediate insect resistance. Topics discussed include: the use of transgenics to functionally characterize candidate defense genes, the identification of novel defense mechanisms through mutant population screens, and genetical genomic approaches to link gene expression changes with genotypic variation.

Specialized cells tag sexual and species identity in Drosophila melanogaster

Social interactions depend on individuals recognizing each other, and in this context many organisms use chemical signals to indicate species and sex. Cuticular hydrocarbon signals are used by insects, including Drosophila melanogaster, to distinguish conspecific individuals from others. These chemicals also contribute to intraspecific courtship and mating interactions. However, the possibility that sex and species identification are linked by common chemical signalling mechanisms has not been formally tested. Here we provide direct evidence that a single compound is used to communicate female identity among D. melanogaster, and to define a reproductive isolation barrier between D. melanogaster and sibling species. A transgenic manipulation eliminated cuticular hydrocarbons by ablating the oenocytes, specialized cells required for the expression of these chemical signals. The resulting oenocyte-less (oe(-)) females elicited the normal repertoire of courtship behaviours from males, but were actually preferred over wild-type females by courting males. In addition, wild-type males attempted to copulate with oe(-) males. Thus, flies lacking hydrocarbons are a sexual hyperstimulus. Treatment of virgin females with the aversive male pheromone cis-vaccenyl acetate (cVA) significantly delayed mating of oe(-) females compared to wild-type females. This difference was eliminated when oe(-) females were treated with a blend of cVA and the female aphrodisiac (7Z,11Z)-heptacosadiene (7,11-HD), showing that female aphrodisiac compounds can attenuate the effects of male aversive pheromones. 7,11-HD also was shown to have a crucial role in heterospecific encounters. Specifically, the species barrier was lost because males of other Drosophila species courted oe(-) D. melanogaster females, and D. simulans males consistently mated with them. Treatment of oe(-) females with 7,11-HD restored the species barrier, showing that a single compound can confer species identity. These results identify a common mechanism for sexual and species recognition regulated by cuticular hydrocarbons.

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and gamma-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra x maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and gamma-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.

Obesity-Blocking Neurons in Drosophila

In mammals, fat store levels are communicated by leptin and insulin signaling to brain centers that regulate food intake and metabolism. By using transgenic manipulation of neural activity, we report the isolation of two distinct neuronal populations in flies that perform a similar function, the c673a-Gal4 and fruitless-Gal4 neurons. When either of these neuronal groups is silenced, fat store levels increase. This change is mediated through an increase in food intake and altered metabolism in c673a-Gal4-silenced flies, while silencing fruitless-Gal4 neurons alters only metabolism. Hyperactivation of either neuronal group causes depletion of fat stores by increasing metabolic rate and decreasing fatty acid synthesis. Altering the activities of these neurons causes changes in expression of genes known to regulate fat utilization. Our results show that the fly brain measures fat store levels and can induce changes in food intake and metabolism to maintain them within normal limits.

Production of Germline Transgenic Prairie Voles (Microtus ochrogaster) Using Lentiviral Vectors1

The study of alternative model organisms has yielded tremendous insights into the regulation of behavioral and physiological traits not displayed by more widely used animal models, such as laboratory rats and mice. In particular, comparative approaches often exploit species ideally suited for investigating specific phenomenon. For instance, comparative studies of socially monogamous prairie voles and polygamous meadow voles have been instrumental toward gaining an understanding of the genetic and neurobiological basis of social bonding. However, laboratory studies of less commonly used organisms, such as prairie voles, have been limited by a lack of genetic tools, including the ability to manipulate the genome. Here, we show that lentiviral vector-mediated transgenesis is a rapid and efficient approach for creating germline transgenics in alternative laboratory rodents. Injection of a green fluorescent protein (GFP)-expressing lentiviral vector into the perivitelline space of 23 single-cell embryos yielded three live offspring (13 %), one of which (33%) contained germline integration of a GFP transgene driven by the human ubiquitin-C promoter. In comparison, transfer of 23 uninjected embryos yielded six live offspring (26%). Green fluorescent protein is present in all tissues examined and is expressed widely in the brain. The GFP transgene is heritable and stably expressed until at least the F(2) generation. This technology has the potential to allow investigation of specific gene candidates in prairie voles and provides a general protocol to pursue germline transgenic manipulation in many different rodent species.

Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome

Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNAval promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

A new fibroblastic-like cell line from heart muscle of the Indian major carp ( Catla catla): Development and characterization

A cell line (SICH) was established from heart muscle of Indian major carp ( Catla catla), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 15% fetal bovine serum. These cells have been sub-cultured more than 130 times over a period of two years. The SICH cell line consists predominantly of fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 24 °C to 37 °C with an optimum temperature of 28 °C. The growth rate of heart cells increased as the FBS proportion increased from 2 to 20% at 28 °C with optimum growth at the concentrations of 10 or 15% FBS. PCR amplification of mitochondrial 12s rRNA using primers specific to C. catla confirmed the origin of this cell line from catla. The cells were successfully cryopreserved and revived at passage numbers 20, 35, 70 and 120. The cell cycle analysis by FACS (Fluorescence Activated Cell Sorter) revealed that most of the cells on the second day of culture were in S-phase, indicating a high growth rate. When the SICH cells were transfected with pEGFP using lipofectamine 2000 or chitosan nanoparticle, fluorescent signals were observed, suggesting that the SICH cell line could be a useful tool for transgenic and genetic manipulation studies. The bacterial extracellular products from Aeromonas sp., or Vibrio anguillarum were found to be toxic to this cell line. This cell line was further characterized by immunocytochemistry using confocal laser scanning microscopy. In addition, an attempt was also made to use this cell line for genotoxicity studies.

Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton)

Two new cell lines, designated RE and CB, were derived from the eye of rohu, Labeo rohita, and the brain of catla, Catla catla, respectively. The cell lines were maintained in Leibovitz's L-15 supplemented with 20% foetal bovine serum. The RE cell line was sub-cultured for more than 70 passages and the CB cell line for more than 35 passages. The RE cells are rounded and consist predominantly of epithelial cells. The CB cell line consists of predominantly fibroblastic-like cells. Both cell lines are able to grow at temperatures between 25 and 32 degrees C with an optimum of 28 degrees C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 degrees C, with optimum growth at concentrations of 15% or 20% foetal bovine serum. The cells were successfully cryopreserved and revived at different passage levels. The cell lines were not susceptible to four marine fish viruses. Extracellular products from Aeromonas sp. were toxic to the cell lines. When the cells were transfected with plasmid eukaryotic green fluorescent protein (pEGFP [Clontech, Carlsbad, CA, USA]) vector DNA, a significant fluorescent signal was observed suggesting that these cell lines could be a useful tool for transgenic and genetic manipulation studies. Polymerase chain reaction amplification of mitochondrial 12S rRNA from rohu and catla confirmed that the cell lines originated from these fish species. The cell lines were further characterized by immunocytochemistry using confocal laser scanning microscopy.

The effects of prion protein expression on metal metabolism

The prion protein is a glycoprotein that binds metals such as copper and manganese. When converted to a proteinase resistant isoform it is associated with prion diseases such as Creutzfeldt–Jakob disease and bovine spongiform encephalopathy. Although, the co-ordination and metal affinity of the prion protein has been well studied, the association of the protein with cellular metal metabolism has been less well investigated. We used transgenic manipulation of prion protein expression and other recombinant techniques to alter expression of known copper binding proteins to investigate the role of the prion protein in copper metabolism. We found that changing the expression of the prion protein alters proteins associated with copper uptake, storage and export from the cell. In addition, alteration in the expression of superoxide dismutases increased prion protein expression dramatically. Reducing copper in the diet decreased expression of the prion protein in the brain while increased dietary manganese dramatically increased the protein's expression. Cellular prion infection also increased the expression of metal transporting proteins and increased cellular manganese concentrations. Overall our results show a close link between cellular resistance to oxidative stress and also copper metabolism. These findings are in line with previous data suggesting that the prion protein is an antioxidant and associated with copper uptake into cells. The disturbance to copper metabolism, as a result of altered prion protein expression clearly demonstrates the important role of the prion protein in copper metabolism. The implication is that prion protein expression has a homeostatic role in copper metabolism.

Murine cardiac mtDNA: effects of transgenic manipulation of nucleoside phosphorylation

Mitochondrial toxicity results from pyrimidine nucleoside reverse transcriptase inhibitors (NRTIs) for HIV/AIDS. In the heart, this can deplete mitochondrial (mt) DNA and cause cardiac dysfunction (eg, left ventricle hypertrophy, LVH). Four unique transgenic, cardiac-targeted overexpressors (TGs) were generated to determine their individual impact on native mitochondrial biogenesis and effects of NRTI administration on development of mitochondrial toxicity. TGs included cardiac-specific overexpression of native thymidine kinase 2 (TK2), two pathogenic TK2 mutants (H121N and I212N), and a mutant of mtDNA polymerase, pol-γ (Y955C). Each was treated with antiretrovirals (AZT-HAART, 3 or 10 weeks, zidovudine (AZT) + lamivudine (3TC) + indinavir, or vehicle control). Parameters included left ventricle (LV) performance (echocardiography), LV mtDNA abundance (real-time PCR), and mitochondrial fine structure (electron microscopy, EM) as a function of duration of treatment and presence of TG. mtDNA abundance significantly decreased in Y955C TG, increased in TK2 native and I212N TGs, and was unchanged in H121N TGs at 10 weeks regardless of treatment. Y955C and I212N TGs exhibited LVH during growth irrespective of treatment. Y955C TGs exhibited cardiomyopathy (CM) at 3 and 10 weeks irrespective of treatment, whereas H121N and I212N TGs exhibited CM only after 10 weeks AZT-HAART. EM features were consistent with cardiac dysfunction. mtDNA abundance and cardiac functional changes were related to TG expression of mitochondrially related genes, mutations thereof, and NRTIs.

Manipulating transgenes using a chromosome vector

Recent technological advances have enabled us to visualize the organization and dynamics of local chromatin structures; however, the comprehensive mechanisms by which chromatin organization modulates gene regulation are poorly understood. We designed a human artificial chromosome vector that allowed manipulation of transgenes using a method for delivering chromatin architectures into different cell lines from human to fish. This methodology enabled analysis of de novo construction, epigenetic maintenance and changes in the chromatin architecture of specific genes. Expressive and repressive architectures of human STAT3 were established from naked DNA in mouse embryonic stem cells and CHO cells, respectively. Delivery of STAT3 within repressive architecture to embryonic stem cells resulted in STAT3 activation, accompanied by changes in DNA methylation. This technology for manipulating a single gene with a specific chromatin architecture could be utilized in applied biology, including stem cell science and regeneration medicine.