CARs: Driving T-cell specificity to enhance anti-tumor immunity

PubMed Central

Kebriaei, Partow; Kelly, Susan S.; Manuri, Pallavi; Jena, Bipulendu; Jackson, Rineka; Shpall, Elizabeth; Champlin, Richard; Cooper, Laurence J. N.

2013-01-01

Adoptive transfer of antigen-specific T cells is a compelling tool to treat cancer. To overcome issues of immune tolerance which limits the endogenous adaptive immune response to tumor-associated antigens, robust systems for the genetic modification and characterization of T cells expressing chimeric antigen receptors (CARs) to redirect specificity have been produced. Refinements with regards to persistence and trafficking of the genetically modified T cells are underway to help improve the potency of genetically modified T cells. Clinical trials utilizing this technology demonstrate feasibility, and increasingly, antitumor activity, paving the way for multi-center trials to establish the efficacy of this novel T-cell therapy. PMID:22202074

A CRISPR New World: Attitudes in the Public toward Innovations in Human Genetic Modification

PubMed Central

Weisberg, Steven M.; Badgio, Daniel; Chatterjee, Anjan

2017-01-01

The potential to genetically modify human germlines has reached a critical tipping point with recent applications of CRISPR-Cas9. Even as researchers, clinicians, and ethicists weigh the scientific and ethical repercussions of these advances, we know virtually nothing about public attitudes on the topic. Understanding such attitudes will be critical to determining the degree of broad support there might be for any public policy or regulation developed for genetic modification research. To fill this gap, we gave an online survey to a large (2,493 subjects) and diverse sample of Americans. Respondents supported genetic modification research, although demographic variables influenced these attitudes—conservatives, women, African-Americans, and older respondents, while supportive, were more cautious than liberals, men, other ethnicities, and younger respondents. Support was also was slightly muted when the risks (unanticipated mutations and possibility of eugenics) were made explicit. The information about genetic modification was also presented as contrasting vignettes, using one of five frames: genetic editing, engineering, hacking, modification, or surgery. Despite the fact that the media and academic use of frames describing the technology varies, these frames did not influence people’s attitudes. These data contribute a current snapshot of public attitudes to inform policy with regard to human genetic modification. PMID:28589120

A CRISPR New World: Attitudes in the Public toward Innovations in Human Genetic Modification.

PubMed

Weisberg, Steven M; Badgio, Daniel; Chatterjee, Anjan

2017-01-01

The potential to genetically modify human germlines has reached a critical tipping point with recent applications of CRISPR-Cas9. Even as researchers, clinicians, and ethicists weigh the scientific and ethical repercussions of these advances, we know virtually nothing about public attitudes on the topic. Understanding such attitudes will be critical to determining the degree of broad support there might be for any public policy or regulation developed for genetic modification research. To fill this gap, we gave an online survey to a large (2,493 subjects) and diverse sample of Americans. Respondents supported genetic modification research, although demographic variables influenced these attitudes-conservatives, women, African-Americans, and older respondents, while supportive, were more cautious than liberals, men, other ethnicities, and younger respondents. Support was also was slightly muted when the risks (unanticipated mutations and possibility of eugenics) were made explicit. The information about genetic modification was also presented as contrasting vignettes, using one of five frames: genetic editing, engineering, hacking, modification, or surgery. Despite the fact that the media and academic use of frames describing the technology varies, these frames did not influence people's attitudes. These data contribute a current snapshot of public attitudes to inform policy with regard to human genetic modification.

Reproductive cloning combined with genetic modification.

PubMed

Strong, C

2005-11-01

Although there is widespread opposition to reproductive cloning, some have argued that its use by infertile couples to have genetically related children would be ethically justifiable. Others have suggested that lesbian or gay couples might wish to use cloning to have genetically related children. Most of the main objections to human reproductive cloning are based on the child's lack of unique nuclear DNA. In the future, it may be possible safely to create children using cloning combined with genetic modifications, so that they have unique nuclear DNA. The genetic modifications could be aimed at giving such children genetic characteristics of both members of the couple concerned. Thus, cloning combined with genetic modification could be appealing to infertile, lesbian, or gay couples who seek genetically related children who have genetic characteristics of both members. In such scenarios, the various objections to human reproductive cloning that are based on the lack of genetic uniqueness would no longer be applicable. The author argues that it would be ethically justifiable for such couples to create children in this manner, assuming these techniques could be used safely.

A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites

PubMed Central

Sajid, Mohammed; Chevalley-Maurel, Séverine; Ramesar, Jai; Klop, Onny; Franke-Fayard, Blandine M. D.; Janse, Chris J.; Khan, Shahid M.

2011-01-01

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research. PMID:22216235

Biomass Structure and Its Contributions to Recalcitrance During Consolidated Bioprocessing with Clostridium Thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akinosho, Hannah O.

Each study provided compelling reasoning to strongly consider lignin’s part in limiting CBP efficiencies at the laboratory and eventually the industrial scale. To make cellulosic ethanol production feasible with CBP, lignin structure and content must be manipulated with genetic modifications or carefully selected in natural variants to combat these difficulties.

From Precaution to Peril: Public Relations Across Forty Years of Genetic Engineering.

PubMed

Hogan, Andrew J

2016-12-01

The Asilomar conference on genetic engineering in 1975 has long been pointed to by scientists as a model for internal regulation and public engagement. In 2015, the organizers of the International Summit on Human Gene Editing in Washington, DC looked to Asilomar as they sought to address the implications of the new CRISPR gene editing technique. Like at Asilomar, the conveners chose to limit the discussion to a narrow set of potential CRISPR applications, involving inheritable human genome editing. The adoption by scientists in 2015 of an Asilomar-like script for discussing genetic engineering offers historians the opportunity to analyze the adjustments that have been made since 1975, and to identify the blind spots that remain in public engagement. Scientists did take important lessons from the fallout of their limited engagement with public concerns at Asilomar. Nonetheless, the scientific community has continued to overlook some of the longstanding public concerns about genetic engineering, in particular the broad and often covert genetic modification of food products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Harnessing epigenome modifications for better crops

USDA-ARS?s Scientific Manuscript database

Chemical DNA modifications such as methylation influence translation of the DNA code to specific genetic outcomes. While such modifications can be heritable, others are transient, and their overall contribution to plant genetic diversity remains intriguing but uncertain. The focus of this article is...

Lentiviral gene transduction of mouse and human hematopoietic stem cells.

PubMed

van Til, Niek P; Wagemaker, Gerard

2014-01-01

Lentiviral vectors can be used to genetically modify a broad range of cells. Hematopoietic stem cells (HSCs) are particularly suitable for lentiviral gene augmentation, because these cells can be enriched with relative ease from mouse bone marrow and human hematopoietic sources, and in principle require relatively limited cell numbers to completely reconstitute the hematopoietic system in vivo. Furthermore, lentiviral vectors are very efficient if pseudotyped with broad tropism envelope proteins. This chapter focuses on gene modification by the use of self-inactivating third-generation human immunodeficiency virus-derived lentiviral vectors for ex vivo HSC modification for both mouse and human application.

Genetic modification of Anopheles stephensi for resistance to multiple Plasmodium falciparum strains does not influence susceptibility to o'nyong'nyong virus or insecticides, or Wolbachia-mediated resistance to the malaria parasite.

PubMed

Pike, Andrew; Dimopoulos, George

2018-01-01

Mosquitoes that have been genetically engineered for resistance to human pathogens are a potential new tool for controlling vector-borne disease. However, genetic modification may have unintended off-target effects that could affect the mosquitoes' utility for disease control. We measured the resistance of five genetically modified Plasmodium-suppressing Anopheles stephensi lines to o'nyong'nyong virus, four classes of insecticides, and diverse Plasmodium falciparum field isolates and characterized the interactions between our genetic modifications and infection with the bacterium Wolbachia. The genetic modifications did not alter the mosquitoes' resistance to either o'nyong'nyong virus or insecticides, and the mosquitoes were equally resistant to all tested P. falciparum strains, regardless of Wolbachia infection status. These results indicate that mosquitoes can be genetically modified for resistance to malaria parasite infection and remain compatible with other vector-control measures without becoming better vectors for other pathogens.

Concise review: managing genotoxicity in the therapeutic modification of stem cells.

PubMed

Baum, Christopher; Modlich, Ute; Göhring, Gudrun; Schlegelberger, Brigitte

2011-10-01

The therapeutic use of procedures for genetic stem cell modification is limited by potential adverse events related to uncontrolled mutagenesis. Prominent findings have been made in hematopoietic gene therapy, demonstrating the risk of clonal, potentially malignant outgrowth on the basis of mutations acquired during or after therapeutic genome modification. The incidence and the growth rate of insertional mutants have been linked to the "stemness" of the target cells and vector-related features such as the integration pattern, the architecture, and the exact content of transgene cassettes. Milieu factors supporting the survival and expansion of mutants may eventually allow oncogenic progression. Similar concerns apply for medicinal products based on pluripotent stem cells. Focusing on the genetic stress induced by insertional mutagenesis and culture adaptation, we propose four conclusions. (a) Mutations occurring in the production of stem cell-based medicines may be unavoidable and need to be classified according to their risk to trigger the formation of clones that are sufficiently long-lived and mitotically active to acquire secondary transforming mutations. (b) The development of rational prevention strategies depends upon the identification of the specific mutations forming such "dominant clones" (which can also be addressed as cancer stem cell precursors) and a better knowledge of the mechanisms underlying their creation, expansion, and homeostatic control. (c) Quantitative assay systems are required to assess the practical value of preventive actions. (d) Improved approaches for the genetic modification of stem cells can address all critical steps in the origin and growth control of mutants. Copyright © 2011 AlphaMed Press.

When gene medication is also genetic modification--regulating DNA treatment.

PubMed

Foss, Grethe S; Rogne, Sissel

2007-07-26

The molecular methods used in DNA vaccination and gene therapy resemble in many ways the methods applied in genetic modification of organisms. In some regulatory regimes, this creates an overlap between 'gene medication' and genetic modification. In Norway, an animal injected with plasmid DNA, in the form of DNA vaccine or gene therapy, currently is viewed as being genetically modified for as long as the added DNA is present in the animal. However, regulating a DNA-vaccinated animal as genetically modified creates both regulatory and practical challenges. It is also counter-intuitive to many biologists. Since immune responses can be elicited also to alter traits, the borderline between vaccination and the modification of properties is no longer distinct. In this paper, we discuss the background for the Norwegian interpretation and ways in which the regulatory challenge can be handled.

Cell-based strategies to manage leukemia relapse: efficacy and feasibility of immunotherapy approaches.

PubMed

Rambaldi, A; Biagi, E; Bonini, C; Biondi, A; Introna, M

2015-01-01

When treatment fails, the clinical outcome of acute leukemia patients is usually very poor, particularly when failure occurs after transplantation. A second allogeneic stem cell transplant could be envisaged as an effective and feasible salvage option in younger patients having a late relapse and an available donor. Unmanipulated or minimally manipulated donor T cells may also be effective in a minority of patients but the main limit remains the induction of severe graft-versus-host disease. This clinical complication has brought about a huge research effort that led to the development of leukemia-specific T-cell therapy aiming at the direct recognition of leukemia-specific rather than minor histocompatibility antigens. Despite a great scientific interest, the clinical feasibility of such an approach has proven to be quite problematic. To overcome this limitation, more research has moved toward the choice of targeting commonly expressed hematopoietic specific antigens by the genetic modification of unselected T cells. The best example of this is represented by the anti-CD19 chimeric antigen receptor (CD19.CAR) T cells. As a possible alternative to the genetic manipulation of unselected T cells, specific T-cell subpopulations with in vivo favorable homing and long-term survival properties have been genetically modified by CAR molecules. Finally, the use of naturally cytotoxic effector cells such as natural killer and cytokine-induced killer cells has been proposed in several clinical trials. The clinical development of these latter cells could also be further expanded by additional genetic modifications using the CAR technology.

Moving receptor redirected adoptive cell therapy toward fine tuning of antitumor responses.

PubMed

Chicaybam, Leonardo; Bonamino, Martin Hernan

2014-10-01

Adoptive cell transfer (ACT) is emerging as a powerful modality of cancer treatment. While ACT has proved able to induce massive clinical responses, genetic modification of T lymphocytes further improved clinical responses obtained. One of the major current limitations of ACT is the inability to discern healthy from malignant cells, leading to on target/off tumor responses that can limit its application. We here discuss some of the approaches currently under development and potential solutions to circumvent these limitations and extend this potentially curative therapy to different tumors by targeting a variety of antigens.

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells.

PubMed

Wu, Yuxuan; Zhou, Hai; Fan, Xiaoying; Zhang, Ying; Zhang, Man; Wang, Yinghua; Xie, Zhenfei; Bai, Meizhu; Yin, Qi; Liang, Dan; Tang, Wei; Liao, Jiaoyang; Zhou, Chikai; Liu, Wujuan; Zhu, Ping; Guo, Hongshan; Pan, Hong; Wu, Chunlian; Shi, Huijuan; Wu, Ligang; Tang, Fuchou; Li, Jinsong

2015-01-01

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc(-/-)) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

Genetic Modification in Dedicated Bioenergy Crops and Strategies for Gene Confinement

USDA-ARS?s Scientific Manuscript database

Genetic modification of dedicated bioenergy crops is in its infancy; however, there are numerous advantages to the use of these tools to improve crops used for biofuels. Potential improved traits through genetic engineering (GE) include herbicide resistance, pest, drought, cold and salt tolerance, l...

Can the outcomes of mesenchymal stem cell-based therapy for myocardial infarction be improved? Providing weapons and armour to cells.

PubMed

Karpov, Andrey A; Udalova, Daria V; Pliss, Michael G; Galagudza, Michael M

2017-04-01

Use of mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI) has been found to have infarct-limiting effects in numerous experimental and clinical studies. However, recent meta-analyses of randomized clinical trials on MSC-based MI therapy have highlighted the need for improving its efficacy. There are two principal approaches for increasing therapeutic effect of MSCs: (i) preventing massive MSC death in ischaemic tissue and (ii) increasing production of cardioreparative growth factors and cytokines with transplanted MSCs. In this review, we aim to integrate our current understanding of genetic approaches that are used for modification of MSCs to enable their improved survival, engraftment, integration, proliferation and differentiation in the ischaemic heart. Genetic modification of MSCs resulting in increased secretion of paracrine factors has also been discussed. In addition, data on MSC preconditioning with physical, chemical and pharmacological factors prior to transplantation are summarized. MSC seeding on three-dimensional polymeric scaffolds facilitates formation of both intercellular connections and contacts between cells and the extracellular matrix, thereby enhancing cell viability and function. Use of genetic and non-genetic approaches to modify MSC function holds great promise for regenerative therapy of myocardial ischaemic injury. © 2016 John Wiley & Sons Ltd.

The effect of communicating the genetic risk of cardiometabolic disorders on motivation and actual engagement in preventative lifestyle modification and clinical outcome: a systematic review and meta-analysis of randomised controlled trials.

PubMed

Li, Sherly X; Ye, Zheng; Whelan, Kevin; Truby, Helen

2016-09-01

Genetic risk prediction of chronic conditions including obesity, diabetes and CVD currently has limited predictive power but its potential to engage healthy behaviour change has been of immense research interest. We aimed to understand whether the latter is indeed true by conducting a systematic review and meta-analysis investigating whether genetic risk communication affects motivation and actual behaviour change towards preventative lifestyle modification. We included all randomised controlled trials (RCT) since 2003 investigating the impact of genetic risk communication on health behaviour to prevent cardiometabolic disease, without restrictions on age, duration of intervention or language. We conducted random-effects meta-analyses for perceived motivation for behaviour change and clinical changes (weight loss) and a narrative analysis for other outcomes. Within the thirteen studies reviewed, five were vignette studies (hypothetical RCT) and seven were clinical RCT. There was no consistent effect of genetic risk on actual motivation for weight loss, perceived motivation for dietary change (control v. genetic risk group standardised mean difference (smd) -0·15; 95 % CI -1·03, 0·73, P=0·74) or actual change in dietary behaviour. Similar results were observed for actual weight loss (control v. high genetic risk SMD 0·29 kg; 95 % CI -0·74, 1·31, P=0·58). This review found no clear or consistent evidence that genetic risk communication alone either raises motivation or translates into actual change in dietary intake or physical activity to reduce the risk of cardiometabolic disorders in adults. Of thirteen studies, eight were at high or unclear risk of bias. Additional larger-scale, high-quality clinical RCT are warranted.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

2014-01-07

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Chemical modification of chitosan for efficient gene therapy.

PubMed

Jiang, Hu-Lin; Cui, Peng-Fei; Xie, Rong-Lin; Cho, Chong-Su

2014-01-01

Gene therapy involves the introduction of foreign genetic material into cells in order to exert a therapeutic effect. Successful gene therapy relies on effective vector system. Viral vectors are highly efficient in transfecting cells, but the undesirable complications limit their therapeutic applications. As a natural biopolymer, chitosan has been considered to be a good gene carrier candidate due to its ideal character which combines biocompatibility, low toxicity with high cationic density together. However, the low cell specificity and low transfection efficiency of chitosan as a gene carrier need to be overcome before undertaking clinical trials. This chapter is principally on those endeavors such as chemical modifications using cell-specific ligands and stimuli-response groups as well as penetrating modifications that have been done to increase the performances of chitosan in gene therapy. © 2014 Elsevier Inc. All rights reserved.

Bcl-xL Genetic Modification Enhanced the Therapeutic Efficacy of Mesenchymal Stem Cell Transplantation in the Treatment of Heart Infarction

PubMed Central

Xue, Xiaodong; Liu, Yu; Zhang, Jian; Liu, Tao; Yang, Zhonglu; Wang, Huishan

2015-01-01

Objectives. Low survival rate of mesenchymal stem cells (MSCs) severely limited the therapeutic efficacy of cell therapy in the treatment of myocardial infarction (MI). Bcl-xL genetic modification might enhance MSC survival after transplantation. Methods. Adult rat bone marrow MSCs were modified with human Bcl-xL gene (hBcl-xL-MSCs) or empty vector (vector-MSCs). MSC apoptosis and paracrine secretions were characterized using flow cytometry, TUNEL, and ELISA in vitro. In vivo, randomized adult rats with MI received myocardial injections of one of the three reagents: hBcl-xL-MSCs, vector-MSCs, or culture medium. Histochemistry, TUNEL, and echocardiography were carried out to evaluate cell engraftment, apoptosis, angiogenesis, scar formation, and cardiac functional recovery. Results. In vitro, cell apoptosis decreased 43%, and vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and plate-derived growth factor (PDGF) increased 1.5-, 0.7-, and 1.2-fold, respectively, in hBcl-xL-MSCs versus wild type and vector-MSCs. In vivo, cell apoptosis decreased 40% and 26% in hBcl-xL-MSC group versus medium and vector-MSC group, respectively. Similar results were observed in cell engraftment, angiogenesis, scar formation, and cardiac functional recovery. Conclusions. Genetic modification of MSCs with hBcl-xL gene could be an intriguing strategy to improve the therapeutic efficacy of cell therapy in the treatment of heart infarction. PMID:26074971

Bcl-xL Genetic Modification Enhanced the Therapeutic Efficacy of Mesenchymal Stem Cell Transplantation in the Treatment of Heart Infarction.

PubMed

Xue, Xiaodong; Liu, Yu; Zhang, Jian; Liu, Tao; Yang, Zhonglu; Wang, Huishan

2015-01-01

Objectives. Low survival rate of mesenchymal stem cells (MSCs) severely limited the therapeutic efficacy of cell therapy in the treatment of myocardial infarction (MI). Bcl-xL genetic modification might enhance MSC survival after transplantation. Methods. Adult rat bone marrow MSCs were modified with human Bcl-xL gene (hBcl-xL-MSCs) or empty vector (vector-MSCs). MSC apoptosis and paracrine secretions were characterized using flow cytometry, TUNEL, and ELISA in vitro. In vivo, randomized adult rats with MI received myocardial injections of one of the three reagents: hBcl-xL-MSCs, vector-MSCs, or culture medium. Histochemistry, TUNEL, and echocardiography were carried out to evaluate cell engraftment, apoptosis, angiogenesis, scar formation, and cardiac functional recovery. Results. In vitro, cell apoptosis decreased 43%, and vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and plate-derived growth factor (PDGF) increased 1.5-, 0.7-, and 1.2-fold, respectively, in hBcl-xL-MSCs versus wild type and vector-MSCs. In vivo, cell apoptosis decreased 40% and 26% in hBcl-xL-MSC group versus medium and vector-MSC group, respectively. Similar results were observed in cell engraftment, angiogenesis, scar formation, and cardiac functional recovery. Conclusions. Genetic modification of MSCs with hBcl-xL gene could be an intriguing strategy to improve the therapeutic efficacy of cell therapy in the treatment of heart infarction.

A Kantian argument against comparatively advantageous genetic modification.

PubMed

Jensen, David

2011-08-01

The genetic modification of children is becoming a more likely possibility given our rapid progress in medical technologies. I argue, from a broadly Kantian point of view, that at least one kind of such modification-modification by a parent for the sake of a child's comparative advantage-is not rationally justified. To argue this, I first characterize a necessary condition on reasons and rational justification: what is a reason for an agent to do an action in one set of circumstances must be a reason for any in those circumstances to do the action. I then show that comparatively advantageous genetic modification violates this principle since a child's "getting ahead" through genetic modification cannot be rationally justified unless other children also could receive the modification, thus rendering the advantage useless. Finally, I consider the major objection to this it seems to disallow all cases of a parent's helping a child get ahead, something that parents normally engage in with their children. I argue that typical practices of developing a comparative advantage in a child, as well as practices of societal competition in general, do not conflict because they involve circumstances that mitigate the universal character of reasons. Many ordinary cases of competitive advantage that we think of as unjust, in fact, can be explained by my argument.

Genetic interaction analysis of point mutations enables interrogation of gene function at a residue-level resolution

PubMed Central

Braberg, Hannes; Moehle, Erica A.; Shales, Michael; Guthrie, Christine; Krogan, Nevan J.

2014-01-01

We have achieved a residue-level resolution of genetic interaction mapping – a technique that measures how the function of one gene is affected by the alteration of a second gene – by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine. PMID:24842270

Genetics and epigenetics of rheumatoid arthritis

PubMed Central

Viatte, Sebastien; Plant, Darren; Raychaudhuri, Soumya

2013-01-01

Investigators have made key advances in rheumatoid arthritis (RA) genetics in the past 10 years. Although genetic studies have had limited influence on clinical practice and drug discovery, they are currently generating testable hypotheses to explain disease pathogenesis. Firstly, we review here the major advances in identifying RA genetic susceptibility markers both within and outside of the MHC. Understanding how genetic variants translate into pathogenic mechanisms and ultimately into phenotypes remains a mystery for most of the polymorphisms that confer susceptibility to RA, but functional data are emerging. Interplay between environmental and genetic factors is poorly understood and in need of further investigation. Secondly, we review current knowledge of the role of epigenetics in RA susceptibility. Differences in the epigenome could represent one of the ways in which environmental exposures translate into phenotypic outcomes. The best understood epigenetic phenomena include post-translational histone modifications and DNA methylation events, both of which have critical roles in gene regulation. Epigenetic studies in RA represent a new area of research with the potential to answer unsolved questions. PMID:23381558

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2013-05-14

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2017-09-12

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

2011-05-17

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications', include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetically modified yeast species, and fermentation processes using genetically modified yeast

DOEpatents

Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

2016-08-09

Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

Genetic diversity and structure related to expansion history and habitat isolation: stone marten populating rural-urban habitats.

PubMed

Wereszczuk, Anna; Leblois, Raphaël; Zalewski, Andrzej

2017-12-22

Population genetic diversity and structure are determined by past and current evolutionary processes, among which spatially limited dispersal, genetic drift, and shifts in species distribution boundaries have major effects. In most wildlife species, environmental modifications by humans often lead to contraction of species' ranges and/or limit their dispersal by acting as environmental barriers. However, in species well adapted to anthropogenic habitat or open landscapes, human induced environmental changes may facilitate dispersal and range expansions. In this study, we analysed whether isolation by distance and deforestation, among other environmental features, promotes or restricts dispersal and expansion in stone marten (Martes foina) populations. We genotyped 298 martens from eight sites at twenty-two microsatellite loci to characterize the genetic variability, population structure and demographic history of stone martens in Poland. At the landscape scale, limited genetic differentiation between sites in a mosaic of urban, rural and forest habitats was mostly influenced by isolation by distance. Statistical clustering and multivariate analyses showed weak genetic structuring with two to four clusters and a high rate of gene flow between them. Stronger genetic differentiation was detected for one stone marten population (NE1) located inside a large forest complex. Genetic differentiation between this site and all others was 20% higher than between other sites separated by similar distances. The genetic uniqueness index of NE1 was also twofold higher than in other sites. Past demographic history analyses showed recent expansion of this species in north-eastern Poland. A decrease in genetic diversity from south to north, and MIGRAINE analyses indicated the direction of expansion of stone marten. Our results showed that two processes, changes in species distribution boundaries and limited dispersal associated with landscape barriers, affect genetic diversity and structure in stone marten. Analysis of local barriers that reduced dispersal and large scale analyses of genetic structure and demographic history highlight the importance of isolation by distance and forest cover for the past colonization of central Europe by stone marten. This confirmed the hypothesis that human-landscape changes (deforestation) accelerated stone marten expansion, to which climate warming probably has also been contributing over the last few decades.

Germline Genetic Modification and Identity: the Mitochondrial and Nuclear Genomes.

PubMed

Scott, Rosamund; Wilkinson, Stephen

2017-12-01

In a legal 'first', the UK removed a prohibition against modifying embryos in human reproduction, to enable mitochondrial replacement techniques (MRTs), a move the Government distanced from 'germline genetic modification', which it aligned with modifying the nuclear genome. This paper (1) analyzes the uses and meanings of this term in UK/US legal and policy debates; and (2) evaluates related ethical concerns about identity. It shows that, with respect to identity, MRTs and nuclear genome editing techniques such as CRISPR/Cas-9 (now a policy topic), are not as different as has been supposed. While it does not follow that the two should be treated exactly alike, one of the central reasons offered for treating MRTs more permissively than nuclear genetic modification, and for not regarding MRTs as 'germline genetic modification', is thereby in doubt. Identity cannot, by itself, do the work thus far assigned to it, explicitly or otherwise, in law and policy.

Can GM sorghum impact Africa?

PubMed

Botha, Gerda M; Viljoen, Christopher D

2008-02-01

It is said that genetic modification (GM) of grain sorghum has the potential to alleviate hunger in Africa. To this end, millions of dollars have been committed to developing GM sorghum. Current developments in the genetic engineering of sorghum are similar to efforts to improve cassava and other traditional African crops, as well as rice in Asia. On closer analysis, GM sorghum is faced with the same limitations as 'Golden Rice' (GM rice) in the context of combating vitamin A deficiency (VAD) efficiently and sustainably. Thus, it is questionable whether the cost of developing GM sorghum can be justified when compared to the cost of investing in sustainable agricultural practice in Africa.

Attitudes Toward Genetic Modification Research: An Analysis of the Views of the Sputnik Generation.

ERIC Educational Resources Information Center

Miller, Jon D.

1982-01-01

Utilizing data from the 1977 National Assessment of Educational Progress (NAEP) survey of young adults, summarizes attitudes toward genetic modification research and the demographic, educational, and occupational correlates of these attitudes. (Author/SK)

Genetic analysis of the heparan modification network in Caenorhabditis elegans.

PubMed

Townley, Robert A; Bülow, Hannes E

2011-05-13

Heparan sulfates (HS) are highly modified sugar polymers in multicellular organisms that function in cell adhesion and cellular responses to protein signaling. Functionally distinct, cell type-dependent HS modification patterns arise as the result of a conserved network of enzymes that catalyze deacetylations, sulfations, and epimerizations in specific positions of the sugar residues. To understand the genetic interactions of the enzymes during the HS modification process, we have measured the composition of HS purified from mutant strains of Caenorhabditis elegans. From these measurements we have developed a genetic network model of HS modification. We find the interactions to be highly recursive positive feed-forward and negative feedback loops. Our genetic analyses show that the HS C-5 epimerase hse-5, the HS 2-O-sulfotransferase hst-2, or the HS 6-O-sulfotransferase hst-6 inhibit N-sulfation. In contrast, hse-5 stimulates both 2-O- and 6-O-sulfation and, hst-2 and hst-6 inhibit 6-O- and 2-O-sulfation, respectively. The effects of hst-2 and hst-6 on N-sulfation, 6-O-sulfation, and 2-O-sulfation appear largely dependent on hse-5 function. This core of regulatory interactions is further modulated by 6-O-endosulfatase activity (sul-1). 47% of all 6-O-sulfates get removed from HS and this editing process is dependent on hst-2, thereby providing additional negative feedback between 2-O- and 6-O-sulfation. These findings suggest that the modification patterns are highly sensitive to the relative composition of the HS modification enzymes. Our comprehensive genetic analysis forms the basis of understanding the HS modification network in metazoans.

Genetic engineering of mesenchymal stem cells and its application in human disease therapy.

PubMed

Hodgkinson, Conrad P; Gomez, José A; Mirotsou, Maria; Dzau, Victor J

2010-11-01

The use of stem cells for tissue regeneration and repair is advancing both at the bench and bedside. Stem cells isolated from bone marrow are currently being tested for their therapeutic potential in a variety of clinical conditions including cardiovascular injury, kidney failure, cancer, and neurological and bone disorders. Despite the advantages, stem cell therapy is still limited by low survival, engraftment, and homing to damage area as well as inefficiencies in differentiating into fully functional tissues. Genetic engineering of mesenchymal stem cells is being explored as a means to circumvent some of these problems. This review presents the current understanding of the use of genetically engineered mesenchymal stem cells in human disease therapy with emphasis on genetic modifications aimed to improve survival, homing, angiogenesis, and heart function after myocardial infarction. Advancements in other disease areas are also discussed.

Reinventing the ames test as a quantitative lab that connects classical and molecular genetics.

PubMed

Goodson-Gregg, Nathan; De Stasio, Elizabeth A

2009-01-01

While many institutions use a version of the Ames test in the undergraduate genetics laboratory, students typically are not exposed to techniques or procedures beyond qualitative analysis of phenotypic reversion, thereby seriously limiting the scope of learning. We have extended the Ames test to include both quantitative analysis of reversion frequency and molecular analysis of revertant gene sequences. By giving students a role in designing their quantitative methods and analyses, students practice and apply quantitative skills. To help students connect classical and molecular genetic concepts and techniques, we report here procedures for characterizing the molecular lesions that confer a revertant phenotype. We suggest undertaking reversion of both missense and frameshift mutants to allow a more sophisticated molecular genetic analysis. These modifications and additions broaden the educational content of the traditional Ames test teaching laboratory, while simultaneously enhancing students' skills in experimental design, quantitative analysis, and data interpretation.

Human germline genetic modification: scientific and bioethical perspectives.

PubMed

Smith, Kevin R; Chan, Sarah; Harris, John

2012-10-01

The latest mammalian genetic modification technology offers efficient and reliable targeting of genomic sequences, in the guise of designer genetic recombination tools. These and other improvements in genetic engineering technology suggest that human germline genetic modification (HGGM) will become a safe and effective prospect in the relatively near future. Several substantive ethical objections have been raised against HGGM including claims of unacceptably high levels of risk, damage to the status of future persons, and violations of justice and autonomy. This paper critically reviews the latest GM science and discusses the key ethical objections to HGGM. We conclude that major benefits are likely to accrue through the use of safe and effective HGGM and that it would thus be unethical to take a precautionary stance against HGGM. Copyright © 2012 IMSS. Published by Elsevier Inc. All rights reserved.

Efficient TALEN-mediated gene knockout in livestock

PubMed Central

Carlson, Daniel F.; Tan, Wenfang; Lillico, Simon G.; Stverakova, Dana; Proudfoot, Chris; Christian, Michelle; Voytas, Daniel F.; Long, Charles R.; Whitelaw, C. Bruce A.; Fahrenkrug, Scott C.

2012-01-01

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications. PMID:23027955

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes.

PubMed

Vargas-Tah, Alejandra; Gosset, Guillermo

2015-01-01

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement.

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes

PubMed Central

Vargas-Tah, Alejandra; Gosset, Guillermo

2015-01-01

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement. PMID:26347861

Genetic Analysis of the Heparan Modification Network in Caenorhabditis elegans*

PubMed Central

Townley, Robert A.; Bülow, Hannes E.

2011-01-01

Heparan sulfates (HS) are highly modified sugar polymers in multicellular organisms that function in cell adhesion and cellular responses to protein signaling. Functionally distinct, cell type-dependent HS modification patterns arise as the result of a conserved network of enzymes that catalyze deacetylations, sulfations, and epimerizations in specific positions of the sugar residues. To understand the genetic interactions of the enzymes during the HS modification process, we have measured the composition of HS purified from mutant strains of Caenorhabditis elegans. From these measurements we have developed a genetic network model of HS modification. We find the interactions to be highly recursive positive feed-forward and negative feedback loops. Our genetic analyses show that the HS C-5 epimerase hse-5, the HS 2-O-sulfotransferase hst-2, or the HS 6-O-sulfotransferase hst-6 inhibit N-sulfation. In contrast, hse-5 stimulates both 2-O- and 6-O-sulfation and, hst-2 and hst-6 inhibit 6-O- and 2-O-sulfation, respectively. The effects of hst-2 and hst-6 on N-sulfation, 6-O-sulfation, and 2-O-sulfation appear largely dependent on hse-5 function. This core of regulatory interactions is further modulated by 6-O-endosulfatase activity (sul-1). 47% of all 6-O-sulfates get removed from HS and this editing process is dependent on hst-2, thereby providing additional negative feedback between 2-O- and 6-O-sulfation. These findings suggest that the modification patterns are highly sensitive to the relative composition of the HS modification enzymes. Our comprehensive genetic analysis forms the basis of understanding the HS modification network in metazoans. PMID:21454666

Recent advances in genetic modification systems for Actinobacteria.

PubMed

Deng, Yu; Zhang, Xi; Zhang, Xiaojuan

2017-03-01

Actinobacteria are extremely important to human health, agriculture, and forests. Because of the vast differences of the characteristics of Actinobacteria, a lot of genetic tools have been developed for efficiently manipulating the genetics. Although there are a lot of successful examples of engineering Actinobacteria, they are still more difficult to be genetically manipulated than other model microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis etc. due to the diverse genomics and biochemical machinery. Here, we review the methods to introduce heterologous DNA into Actinobacteria and the available genetic modification tools. The trends and problems existing in engineering Actinobacteria are also covered.

Expanding the Scope of Site-Specific Recombinases for Genetic and Metabolic Engineering

PubMed Central

Gaj, Thomas; Sirk, Shannon J.; Barbas, Carlos F.

2014-01-01

Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study. PMID:23982993

Application of Carbon Nanotubes for Plant Genetic Transformation

NASA Astrophysics Data System (ADS)

Burlaka, Olga M.; Pirko, Yaroslav V.; Yemets, Alla I.; Blume, Yaroslav B.

In this chapter, the current state of using carbon nanotubes (CNTs; single- and multi-walled) that have attracted great interdisciplinary interest in recent decades due to their peculiar properties for genetic transformation of prokaryotic and eukaryotic cells will be enlightened. The covalent and non-covalent surface chemistry for the CNT functionalization with focus on the potential applications of surface modifications in design of biocompatible CNTs will be discussed. The properties of CNTs that are favorable for biotechnological use and current status of technical approaches that allow the increase in biocompatibility and lower nanotoxicity of engineered CNTs will be described. Decisions proposed by non-covalent surface modification of CNTs will be discussed. Existing data concerning mechanisms of CNT cell entry and factors governing toxicity, cellular uptake, intracellular traffic, and biodegradation of CNTs along with bioavailability of molecular cargoes of loaded CNTs will be discussed. Eco-friendly production of water dispersions of biologically functionalized multi-walled and single-walled CNTs for use as nano-vehicles for the DNA delivery in plant genetic transformation of plants will be described. The background, advantages, and problems of using CNTs in developing of novel methods of genetic transformation, including plant genetic transformation, will be highlighted. Special attention will be paid to the limitations of conventional gene transfer techniques and promising features of CNT-based strategies having improved efficacy, reproducibility, and accuracy along with less time consumption. Issues impeding manipulation of CNTs such as entangled bundle formation, low water solubility, inert properties of pristine CNTs, etc., and ways to solve arising tasks will be overviewed.

Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications.

PubMed

Telugu, Bhanu P; Park, Ki-Eun; Park, Chi-Hun

2017-08-01

Genetic modification of livestock has a longstanding and successful history, starting with domestication several thousand years ago. Modern animal breeding strategies predominantly based on marker-assisted and genomic selection, artificial insemination, and embryo transfer have led to significant improvement in the performance of domestic animals, and are the basis for regular supply of high quality animal derived food. However, the current strategy of breeding animals over multiple generations to introduce novel traits is not realistic in responding to the unprecedented challenges such as changing climate, pandemic diseases, and feeding an anticipated 3 billion increase in global population in the next three decades. Consequently, sophisticated genetic modifications that allow for seamless introgression of novel alleles or traits and introduction of precise modifications without affecting the overall genetic merit of the animal are required for addressing these pressing challenges. The requirement for precise modifications is especially important in the context of modeling human diseases for the development of therapeutic interventions. The animal science community envisions the genome editors as essential tools in addressing these critical priorities in agriculture and biomedicine, and for advancing livestock genetic engineering for agriculture, biomedical as well as "dual purpose" applications.

Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene.

PubMed

Welsch, Ralf; Arango, Jacobo; Bär, Cornelia; Salazar, Bertha; Al-Babili, Salim; Beltrán, Jesús; Chavarriaga, Paul; Ceballos, Hernan; Tohme, Joe; Beyer, Peter

2010-10-01

Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.

Provitamin A Accumulation in Cassava (Manihot esculenta) Roots Driven by a Single Nucleotide Polymorphism in a Phytoene Synthase Gene[W

PubMed Central

Welsch, Ralf; Arango, Jacobo; Bär, Cornelia; Salazar, Bertha; Al-Babili, Salim; Beltrán, Jesús; Chavarriaga, Paul; Ceballos, Hernan; Tohme, Joe; Beyer, Peter

2010-01-01

Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification. PMID:20889914

Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion

PubMed Central

Bohlke, Nina; Budisa, Nediljko

2014-01-01

One of the major challenges in contemporary synthetic biology is to find a route to engineer synthetic organisms with altered chemical constitution. In terms of core reaction types, nature uses an astonishingly limited repertoire of chemistries when compared with the exceptionally rich and diverse methods of organic chemistry. In this context, the most promising route to change and expand the fundamental chemistry of life is the inclusion of amino acid building blocks beyond the canonical 20 (i.e. expanding the genetic code). This strategy would allow the transfer of numerous chemical functionalities and reactions from the synthetic laboratory into the cellular environment. Due to limitations in terms of both efficiency and practical applicability, state-of-the-art nonsense suppression- or frameshift suppression-based methods are less suitable for such engineering. Consequently, we set out to achieve this goal by sense codon emancipation, that is, liberation from its natural decoding function – a prerequisite for the reassignment of degenerate sense codons to a new 21st amino acid. We have achieved this by redesigning of several features of the post-transcriptional modification machinery which are directly involved in the decoding process. In particular, we report first steps towards the reassignment of 5797 AUA isoleucine codons in Escherichia coli using efficient tools for tRNA nucleotide modification pathway engineering. PMID:24433543

A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation.

PubMed

Sato, Yuko; Kujirai, Tomoya; Arai, Ritsuko; Asakawa, Haruhiko; Ohtsuki, Chizuru; Horikoshi, Naoki; Yamagata, Kazuo; Ueda, Jun; Nagase, Takahiro; Haraguchi, Tokuko; Hiraoka, Yasushi; Kimura, Akatsuki; Kurumizaka, Hitoshi; Kimura, Hiroshi

2016-10-09

Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Does a medical history of hypertension influence disclosing genetic testing results of the risk for salt-sensitive hypertension, in primary care?

PubMed

Okayama, Masanobu; Takeshima, Taro; Harada, Masanori; Ae, Ryusuke; Kajii, Eiji

2016-01-01

Disclosing genetic testing results may contribute to the prevention and management of many common diseases. However, whether the presence of a disease influences these effects is unclear. This study aimed to clarify the difference in the effects of disclosing genetic testing results of the risk for developing salt-sensitive hypertension on the behavioral modifications with respect to salt intake in hypertensive and nonhypertensive patients. A cross-sectional study using a self-administered questionnaire was conducted for outpatients aged >20 years (N=2,237) at six primary care clinics and hospitals in Japan. The main factors assessed were medical histories of hypertension, salt preferences, reduced salt intakes, and behavior modifications for reducing salt intake. Behavioral modifications of participants were assessed using their behavior stages before and after disclosure of the hypothetical genetic testing results. Of the 2,237 participants, 1,644 (73.5%) responded to the survey. Of these respondents, 558 (33.9%) patients were hypertensive and 1,086 (66.1%) were nonhypertensive. After being notified of the result "If with genetic risk", the nonhypertensive participants were more likely to make positive behavioral modifications compared to the hypertensive patients among all participants and in those aged <65 years (adjusted relative ratio [ad-RR], 1.76; 95% confidence interval, 1.12-2.76 and ad-RR, 1.99; 1.11-3.57, respectively). In contrast, no difference in negative behavioral modifications between hypertensive and nonhypertensive patients was detected after being notified of the result "If without genetic risk" (ad-RR, 1.05; 95% confidence interval, 0.70-1.57). The behavior of modifying salt intake after disclosure of the genetic testing results differed between hypertensive and nonhypertensive patients. Disclosing a genetic risk for salt-sensitive hypertension was likely to cause nonhypertensive patients, especially those aged <65 years, to improve their behavior regarding salt intake. We conclude that disclosing genetic testing results could help prevent hypertension, and that the doctor should communicate the genetic testing results to those patients with a medical history of hypertension, or those who are at risk of developing hypertension.

Germline modification of domestic animals

PubMed Central

Tang, L.; González, R.; Dobrinski, I.

2016-01-01

Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation. As the genetic change is introduced into the male germ line just before the onset of spermatogenesis, the time required for the production of genetically modified sperm is significantly shorter using germ cell transplantation compared to cloning or embryonic stem (ES) cell based technology. Moreover, the GSC-mediated germline modification circumvents problems associated with embryo manipulation and nuclear reprogramming. Currently, engineering targeted mutations in domestic animals using GSCs remains a challenge as GSCs from those animals are difficult to maintain in vitro for an extended period of time. Recent advances in genome editing techniques such as Zinc-Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) greatly enhance the efficiency of engineering targeted genetic change in domestic animals as demonstrated by the generation of several gene knock-out pig and cattle models using those techniques. The potential of GSC-mediated germline modification in making targeted genetic modifications in domestic animal models will be maximized if those genome editing techniques can be applied in GSCs. PMID:27390591

Domesticated, Genetically Engineered, and Wild Plant Relatives Exhibit Unintended Phenotypic Differences: A Comparative Meta-Analysis Profiling Rice, Canola, Maize, Sunflower, and Pumpkin

PubMed Central

Hernández-Terán, Alejandra; Wegier, Ana; Benítez, Mariana; Lira, Rafael; Escalante, Ana E.

2017-01-01

Agronomic management of plants is a powerful evolutionary force acting on their populations. The management of cultivated plants is carried out by the traditional process of human selection or plant breeding and, more recently, by the technologies used in genetic engineering (GE). Even though crop modification through GE is aimed at specific traits, it is possible that other non-target traits can be affected by genetic modification due to the complex regulatory processes of plant metabolism and development. In this study, we conducted a meta-analysis profiling the phenotypic consequences of plant breeding and GE, and compared modified cultivars with wild relatives in five crops of global economic and cultural importance: rice, maize, canola, sunflower, and pumpkin. For these five species, we analyzed the literature with documentation of phenotypic traits that are potentially related to fitness for the same species in comparable conditions. The information was analyzed to evaluate whether the different processes of modification had influenced the phenotype in such a way as to cause statistical differences in the state of specific phenotypic traits or grouping of the organisms depending on their genetic origin [wild, domesticated with genetic engineering (domGE), and domesticated without genetic engineering (domNGE)]. In addition, we tested the hypothesis that, given that transgenic plants are a construct designed to impact, in many cases, a single trait of the plant (e.g., lepidopteran resistance), the phenotypic differences between domGE and domNGE would be either less (or inexistent) than between the wild and domesticated relatives (either domGE or domNGE). We conclude that (1) genetic modification (either by selective breeding or GE) can be traced phenotypically when comparing wild relatives with their domesticated relatives (domGE and domNGE) and (2) the existence and the magnitude of the phenotypic differences between domGE and domNGE of the same crop suggest consequences of genetic modification beyond the target trait(s). PMID:29259610

Targeting carbon for crop yield and drought resilience

PubMed Central

Griffiths, Cara A

2017-01-01

Abstract Current methods of crop improvement are not keeping pace with projected increases in population growth. Breeding, focused around key traits of stem height and disease resistance, delivered the step‐change yield improvements of the green revolution of the 1960s. However, subsequently, yield increases through conventional breeding have been below the projected requirement of 2.4% per year required by 2050. Genetic modification (GM) mainly for herbicide tolerance and insect resistance has been transformational, akin to a second green revolution, although GM has yet to make major inroads into intrinsic yield processes themselves. Drought imposes the major restriction on crop yields globally but, as yet, has not benefited substantially from genetic improvement and still presents a major challenge to agriculture. Much still has to be learnt about the complex process of how drought limits yield and what should be targeted. Mechanisms of drought adaptation from the natural environment cannot be taken into crops without significant modification for the agricultural environment because mechanisms of drought tolerance are often in contrast with mechanisms of high productivity required in agriculture. However, through convergence of fundamental and translational science, it would appear that a mechanism of sucrose allocation in crops can be modified for both productivity and resilience to drought and other stresses. Recent publications show how this mechanism can be targeted by GM, natural variation and a new chemical approach. Here, with an emphasis on drought, we highlight how understanding fundamental science about how crops grow, develop and what limits their growth and yield can be combined with targeted genetic selection and pioneering chemical intervention technology for transformational yield improvements. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:28653336

Quadratic genetic modifications: a streamlined route to cosmological simulations with controlled merger history

NASA Astrophysics Data System (ADS)

Rey, Martin P.; Pontzen, Andrew

2018-02-01

Recent work has studied the interplay between a galaxy's history and its observable properties using `genetically modified' cosmological zoom simulations. The approach systematically generates alternative histories for a halo, while keeping its cosmological environment fixed. Applications to date altered linear properties of the initial conditions, such as the mean overdensity of specified regions; we extend the formulation to include quadratic features, such as local variance, that determines the overall importance of smooth accretion relative to mergers in a galaxy's history. We introduce an efficient algorithm for this new class of modification and demonstrate its ability to control the variance of a region in a one-dimensional toy model. Outcomes of this work are twofold: (i) a clarification of the formulation of genetic modifications and (ii) a proof of concept for quadratic modifications leading the way to a forthcoming implementation in cosmological simulations.

Activated Dual Specificity Lymphocytes and Their Methods of Use | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the NCI have developed a method of using genetic modifications to generate leukocytes with multiple specificities. This technology represents a potential therapy for a wide variety of malignancies, and because of the genetic modification used, this therapy will be applicable to patients of any MHC type.

Gene Flow in Genetically Engineered Perennial Grasses: Lessons for Modification of Dedicated Bioenergy Crops

USDA-ARS?s Scientific Manuscript database

Genetic modification of dedicated bioenergy crops, such as switchgrass, will play a major role in crop improvement for a wide range of beneficial traits specific to biofuels. One obstacle that arises regarding transgenic improvement of perennials used for biofuels is the propensity of these plants t...

Characterization of unknown genetic modifications using high throughput sequencing and computational subtraction.

PubMed

Tengs, Torstein; Zhang, Haibo; Holst-Jensen, Arne; Bohlin, Jon; Butenko, Melinka A; Kristoffersen, Anja Bråthen; Sorteberg, Hilde-Gunn Opsahl; Berdal, Knut G

2009-10-08

When generating a genetically modified organism (GMO), the primary goal is to give a target organism one or several novel traits by using biotechnology techniques. A GMO will differ from its parental strain in that its pool of transcripts will be altered. Currently, there are no methods that are reliably able to determine if an organism has been genetically altered if the nature of the modification is unknown. We show that the concept of computational subtraction can be used to identify transgenic cDNA sequences from genetically modified plants. Our datasets include 454-type sequences from a transgenic line of Arabidopsis thaliana and published EST datasets from commercially relevant species (rice and papaya). We believe that computational subtraction represents a powerful new strategy for determining if an organism has been genetically modified as well as to define the nature of the modification. Fewer assumptions have to be made compared to methods currently in use and this is an advantage particularly when working with unknown GMOs.

"It just goes against the grain." Public understandings of genetically modified (GM) food in the UK.

PubMed

Shaw, Alison

2002-07-01

This paper reports on one aspect of qualitative research on public understandings of food risks, focusing on lay understandings of genetically modified (GM) food in the UK context. A range of theoretical, conceptual, and empirical literature on food, risk, and the public understanding of science are reviewed. The fieldwork methods are outlined and empirical data from a range of lay groups are presented. Major themes include: varying "technical" knowledge of science, the relationship between knowledge and acceptance of genetic modification, the uncertainty of scientific knowledge, genetic modification as inappropriate scientific intervention in "nature", the acceptability of animal and human applications of genetic modification, the appropriate boundaries of scientific innovation, the necessity for GM foods, the uncertainty of risks in GM food, fatalism about avoiding risks, and trust in "experts" to manage potential risks in GM food. Key discussion points relating to a sociological understanding of public attitudes to GM food are raised and some policy implications are highlighted.

Characterization of unknown genetic modifications using high throughput sequencing and computational subtraction

PubMed Central

Tengs, Torstein; Zhang, Haibo; Holst-Jensen, Arne; Bohlin, Jon; Butenko, Melinka A; Kristoffersen, Anja Bråthen; Sorteberg, Hilde-Gunn Opsahl; Berdal, Knut G

2009-01-01

Background When generating a genetically modified organism (GMO), the primary goal is to give a target organism one or several novel traits by using biotechnology techniques. A GMO will differ from its parental strain in that its pool of transcripts will be altered. Currently, there are no methods that are reliably able to determine if an organism has been genetically altered if the nature of the modification is unknown. Results We show that the concept of computational subtraction can be used to identify transgenic cDNA sequences from genetically modified plants. Our datasets include 454-type sequences from a transgenic line of Arabidopsis thaliana and published EST datasets from commercially relevant species (rice and papaya). Conclusion We believe that computational subtraction represents a powerful new strategy for determining if an organism has been genetically modified as well as to define the nature of the modification. Fewer assumptions have to be made compared to methods currently in use and this is an advantage particularly when working with unknown GMOs. PMID:19814792

CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations.

PubMed

Guernet, Alexis; Mungamuri, Sathish Kumar; Cartier, Dorthe; Sachidanandam, Ravi; Jayaprakash, Anitha; Adriouch, Sahil; Vezain, Myriam; Charbonnier, Françoise; Rohkin, Guy; Coutant, Sophie; Yao, Shen; Ainani, Hassan; Alexandre, David; Tournier, Isabelle; Boyer, Olivier; Aaronson, Stuart A; Anouar, Youssef; Grumolato, Luca

2016-08-04

Intratumor genetic heterogeneity underlies the ability of tumors to evolve and adapt to different environmental conditions. Using CRISPR/Cas9 technology and specific DNA barcodes, we devised a strategy to recapitulate and trace the emergence of subpopulations of cancer cells containing a mutation of interest. We used this approach to model different mechanisms of lung cancer cell resistance to EGFR inhibitors and to assess effects of combined drug therapies. By overcoming intrinsic limitations of current approaches, CRISPR-barcoding also enables investigation of most types of genetic modifications, including repair of oncogenic driver mutations. Finally, we used highly complex barcodes inserted at a specific genome location as a means of simultaneously tracing the fates of many thousands of genetically labeled cancer cells. CRISPR-barcoding is a straightforward and highly flexible method that should greatly facilitate the functional investigation of specific mutations, in a context that closely mimics the complexity of cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Chemical modulation of M13 bacteriophage and its functional opportunities for nanomedicine

PubMed Central

Chung, Woo-Jae; Lee, Doe-Young; Yoo, So Young

2014-01-01

M13 bacteriophage (phage) has emerged as an attractive bionanomaterial owing to its genetically tunable surface chemistry and its potential to self-assemble into hierarchical structures. Furthermore, because of its unique nanoscopic structure, phage has been proposed as a model system in soft condensed physics and as a biomimetic building block for structured functional materials. Genetic engineering of phage provides great opportunities to develop novel nanomaterials with functional surface peptide motifs; however, this biological approach is generally limited to peptides containing the 20 natural amino acids. To extend the scope of phage applications, strategies involving chemical modification have been employed to incorporate a wider range of functional groups, including synthetic chemical compounds. In this review, we introduce the design of chemoselective phage functionalization and discuss how such a strategy is combined with genetic engineering for a variety of medical applications, as reported in recent literature. PMID:25540583

Chemical modulation of M13 bacteriophage and its functional opportunities for nanomedicine.

PubMed

Chung, Woo-Jae; Lee, Doe-Young; Yoo, So Young

2014-01-01

M13 bacteriophage (phage) has emerged as an attractive bionanomaterial owing to its genetically tunable surface chemistry and its potential to self-assemble into hierarchical structures. Furthermore, because of its unique nanoscopic structure, phage has been proposed as a model system in soft condensed physics and as a biomimetic building block for structured functional materials. Genetic engineering of phage provides great opportunities to develop novel nanomaterials with functional surface peptide motifs; however, this biological approach is generally limited to peptides containing the 20 natural amino acids. To extend the scope of phage applications, strategies involving chemical modification have been employed to incorporate a wider range of functional groups, including synthetic chemical compounds. In this review, we introduce the design of chemoselective phage functionalization and discuss how such a strategy is combined with genetic engineering for a variety of medical applications, as reported in recent literature.

Unknown sequence amplification: Application to in vitro genome walking in Chlamydia trachomatis L2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Copley, C.G.; Boot, C.; Bundell, K.

1991-01-01

A recently described technique, Chemical Genetics' unknown sequence amplification method, which requires only one specific oligonucleotide, has broadened the applicability of the polymerase chain reaction to DNA of unknown sequence. The authors have adapted this technique to the study of the genome of Chlamydia trachomatis, an obligate intracellular bacterium, and describe modifications that significantly improve the utility of this approach. These techniques allow for rapid genomic analysis entirely in vitro, using DNA of limited quantity of purity.

Epigenome analysis of pluripotent stem cells

PubMed Central

Ricupero, Christopher L.; Swerdel, Mavis R.; Hart, Ronald P.

2015-01-01

Summary Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to isolate and enrich chromatin fragments using antibodies against specific chromatin modifications, such as DNA binding proteins or covalent histone modifications. Until recently, many ChIP protocols required millions of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis, that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain. PMID:23546758

Genome Editing of the Blood: Opportunities and Challenges

PubMed Central

Porteus, Matthew H.

2015-01-01

The ability to remove blood cells, including hematopoietic stem cells (HSCs), from a person and then re-transplant them (hematopoietic stem cell transplantation (HSCT) is a well-established treatment paradigm that can be used in both the autologous setting or in the allogeneic setting. Using allogeneic HSCT can cure different genetic diseases of the blood but has significant limitations. An alternative to allogeneic HSCT is to transplant genetically modified HSCs instead. A powerful approach to the precision modification of HSCs is to use genome editing whereby the genome is modified with spatial precision (at an exact location) in the genome and sometimes with nucleotide precision (the exact nucleotide changes are introduced). The progress and challenges of genome editing of blood are discussed. PMID:26029496

CARs in Chronic Lymphocytic Leukemia – Ready to Drive

PubMed Central

Wierda, William; Jena, Bipulendu; Cooper, Laurence J. N.; Shpall, Elizabeth

2013-01-01

Adoptive transfer of antigen-specific T cells has been adapted by investigators for treatment of chronic lymphocytic leukemia (CLL). To overcome issues of immune tolerance which limits the endogenous adaptive immune response to tumor-associated antigens (TAAs), robust systems for the genetic modification and characterization of T cells expressing chimeric antigen receptors (CARs) to redirect specificity have been produced. Refinements with regards to persistence and trafficking of the genetically modified T cells are underway to help improve potency. Clinical trials utilizing this technology demonstrate feasibility, and increasingly, these early-phase trials are demonstrating impressive anti-tumor effects, particularly for CLL patients, paving the way for multi-center trials to establish the efficacy of CAR+ T cell therapy. PMID:23225251

Expanding the scope of site-specific recombinases for genetic and metabolic engineering.

PubMed

Gaj, Thomas; Sirk, Shannon J; Barbas, Carlos F

2014-01-01

Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study. © 2013 Wiley Periodicals, Inc.

Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression.

PubMed

Ahmed, Nabil; Salsman, Vita S; Yvon, Eric; Louis, Chrystal U; Perlaky, Laszlo; Wels, Winfried S; Dishop, Meghan K; Kleinerman, Eugenie E; Pule, Martin; Rooney, Cliona M; Heslop, Helen E; Gottschalk, Stephen

2009-10-01

Human epidermal growth factor receptor 2 (HER2) is expressed by the majority of human osteosarcomas and is a risk factor for poor outcome. Unlike breast cancer, osteosarcoma cells express HER2 at too low, a level for patients to benefit from HER2 monoclonal antibodies. We reasoned that this limitation might be overcome by genetically modifying T cells with HER2-specific chimeric antigen receptors (CARs), because even a low frequency of receptor engagement could be sufficient to induce effector cell killing of the tumor. HER2-specific T cells were generated by retroviral transduction with a HER2-specific CAR containing a CD28.zeta signaling domain. HER2-specific T cells recognized HER2-positive osteosarcoma cells as judged by their ability to proliferate, produce immunostimulatory T helper 1 cytokines, and kill HER2-positive osteosarcoma cell lines in vitro. The adoptive transfer of HER2-specific T cells caused regression of established osteosarcoma xenografts in locoregional as well as metastatic mouse models. In contrast, delivery of nontransduced (NT) T cells did not change the tumor growth pattern. Genetic modification of T cells with CARs specific for target antigens, expressed at too low a level to be effectively recognized by monoclonal antibodies, may allow immunotherapy to be more broadly applicable for human cancer therapy.

Osteoblastic differentiation of human and equine adult bone marrow-derived mesenchymal stem cells when BMP-2 or BMP-7 homodimer genetic modification is compared to BMP-2/7 heterodimer genetic modification in the presence and absence of dexamethasone.

PubMed

Carpenter, Ryan S; Goodrich, Laurie R; Frisbie, David D; Kisiday, John D; Carbone, Beth; McIlwraith, C Wayne; Centeno, Christopher J; Hidaka, Chisa

2010-10-01

Bone marrow-derived mesenchymal stem cells (BMDMSCs) have been targeted for use in enhancement of bone healing; and their osteogenic potential may be further augmented by genes encoding bone morphogenetic proteins (BMP's). The purpose of this study was to compare the effect of genetic modification of human and equine BMDMSCs with BMP-2 or -7 or BMP-2 and -7 on their osteoblastogenic differentiation in the presence or absence of dexamethasone. The BMDMSCs were harvested from the iliac crest of three human donors and tuber coxae of three equine donors. Monolayer cells were genetically modified using adenovirus vectors encoding BMP-2, -7 or both and cultured in the presence or absence of dexamethasone. Expression of BMPs was confirmed by enzyme linked immunosorbent assay (ELISA). To evaluate osteoblastic differentiation, cellular morphology was assessed every other day and expression and secretion of alkaline phosphatase (ALP), as well as expression levels of osteonectin (OSTN), osteocalcin (OCN), and runt-related transcription factor-2 (Runx2) were measured for up to 14 days. Human and equine BMDMSCs showed a capacity for osteogenic differentiation regardless of genetic modification or dexamethasone supplementation. Dexamethasone supplementation was more important for osteoblastogenic differentiation of equine BMDMSCs than human BMDMSCs. Genetic modification of BMDMSCs increased ALP secretion with AdBMP-2 homodimer having the greatest effect in both human and equine cells compared to AdBMP 7 or AdBMP 2/7. BMP protein elution rates reached their maximal concentration between day 4 and 8 and remained relatively stable thereafter, suggesting that genetically modified BMDMSCs could be useful for cell-based delivery of BMPs to a site of bone formation. Published by Wiley Periodicals, Inc. J Orthop Res 28:1330-1337, 2010.

Acceptance of genetically modified foods: the relation between technology and evaluation.

PubMed

Tenbült, Petra; De Vries, Nanne K; van Breukelen, Gerard; Dreezens, Ellen; Martijn, Carolien

2008-07-01

This study investigates why consumers accept different genetically modified food products to different extents. The study shows that whether food products are genetically modified or not and whether they are processed or not are the two important features that affect the acceptance of food products and their evaluation (in terms of perceived healthiness, naturalness, necessity and tastiness). The extent to which these evaluation attributes and acceptance of a product are affected by genetic modification or processing depends on whether the product is negatively affected by the other technology: Any technological change to a 'natural' product (when nonprocessed products are genetically modified or when non-genetically modified products are processed) affect evaluation and acceptance stronger than a change to an technologically adapted product (when processed products are also genetically modified or vice versa). Furthermore, evaluation attributes appear to mediate the effects of genetic modification and processing on acceptance.

Genetic Control of the Secondary Modification of Deoxyribonucleic Acid in Escherichia coli1

PubMed Central

Mamelak, Linda; Boyer, Herbert W.

1970-01-01

The wild-type restriction and modification alleles of Escherichia coli K-12 and B were found to have no measurable effect on the patterns of methylated bases in the deoxyribonucleic acid (DNA) of these strains. The genetic region controlling the methylation of cytosine in E. coli K-12 was mapped close to his, and the presence or absence of this gene in E. coli B or E. coli K had no effect on the restriction and modification properties of these strains. Thus, only a few of the methylated bases in the DNA of these strains are involved in host modification, and the biological role of the remainder remains obscure. PMID:4919756

An exploratory study with preliminary results: The development and evaluation of a Genetics Concept Inventory

NASA Astrophysics Data System (ADS)

Hott, Adam M.

Modern science education reform includes the development of standards and recommendations for content as well as the development and evaluation of pedagogy, but demonstrates limited assessment of student knowledge. Student knowledge assessment is an important factor in measuring the scientific literacy of current students. Concept inventories have been developed and used for the past fourteen years to assess non-science major student conceptual understanding of a content area. Inventories have been developed in the fields of physics, astronomy, chemistry and biology. The development and evaluation of a Genetics Concept Inventory (GCI) is presented here. The reliability estimate of 0.62 is supported by a respected panel of genetics educators' revisions, no significant gender bias, and the ability of junior and senior biology majors to outperform the non-science majors. Pretest/Posttest comparisons show a significant increase in five of six genetics content areas as well as a 9% increase on the overall percent score for the instrument. Although the Genetics Concept Inventory presented here needs further modification and testing, it is the first step in the development of a quality assessment tool for genetics content.

Genetic Targeting of an Adenovirus Vector via Replacement of the Fiber Protein with the Phage T4 Fibritin

PubMed Central

Krasnykh, Victor; Belousova, Natalya; Korokhov, Nikolay; Mikheeva, Galina; Curiel, David T.

2001-01-01

The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues. This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue. Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand. Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand. Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules. Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner. PMID:11287567

Metabolic engineering of Cyanobacteria and microalgae for enhanced production of biofuels and high-value products.

PubMed

Gomaa, M A; Al-Haj, L; Abed, R M M

2016-10-01

A lot of research has been performed on Cyanobacteria and microalgae with the aim to produce numerous biotechnological products. However, native strains have a few shortcomings, like limitations in cultivation, harvesting and product extraction, which prevents reaching optimal production value at lowest costs. Such limitations require the intervention of genetic engineering to produce strains with superior properties. Promising advancements in the cultivation of Cyanobacteria and microalgae have been achieved by improving photosynthetic efficiency through increasing RuBisCO activity and truncation of light-harvesting antennae. Genetic engineering has also contributed to final product extraction by inducing autolysis and product secretory systems, to enable direct product recovery without going through costly extraction steps. In this review, we summarize the different enzymes and pathways that have been targeted thus far for improving cultivation aspects, harvesting and product extraction in Cyanobacteria and microalgae. With synthetic biology advancements, genetically engineered strains can be generated to resolve demanding process issues and achieve economic practicality. This comprehensive overview of gene modifications will be useful to researchers in the field to employ on their strains to increase their yields and improve the economic feasibility of the production process. © 2016 The Society for Applied Microbiology.

Conversing about Citrus Greening: Extension's Role in Educating about Genetic Modification Science as a Solution

ERIC Educational Resources Information Center

Ruth, Taylor K.; Lamm, Alexa J.; Rumble, Joy N.; Ellis, Jason D.

2017-01-01

Extension agents across the nation will need to facilitate difficult conversations with the public if genetic modification (GM) science is used to combat citrus greening disease. This study used the innovation characteristics described by Rogers to explore if using GM science as a solution to citrus greening had diffused amongst US residents. An…

Genetic modification of plant cell walls to enhance biomass yield and biofuel production in bioenergy crops.

PubMed

Wang, Yanting; Fan, Chunfen; Hu, Huizhen; Li, Ying; Sun, Dan; Wang, Youmei; Peng, Liangcai

2016-01-01

Plant cell walls represent an enormous biomass resource for the generation of biofuels and chemicals. As lignocellulose property principally determines biomass recalcitrance, the genetic modification of plant cell walls has been posed as a powerful solution. Here, we review recent progress in understanding the effects of distinct cell wall polymers (cellulose, hemicelluloses, lignin, pectin, wall proteins) on the enzymatic digestibility of biomass under various physical and chemical pretreatments in herbaceous grasses, major agronomic crops and fast-growing trees. We also compare the main factors of wall polymer features, including cellulose crystallinity (CrI), hemicellulosic Xyl/Ara ratio, monolignol proportion and uronic acid level. Furthermore, the review presents the main gene candidates, such as CesA, GH9, GH10, GT61, GT43 etc., for potential genetic cell wall modification towards enhancing both biomass yield and enzymatic saccharification in genetic mutants and transgenic plants. Regarding cell wall modification, it proposes a novel groove-like cell wall model that highlights to increase amorphous regions (density and depth) of the native cellulose microfibrils, providing a general strategy for bioenergy crop breeding and biofuel processing technology. Copyright © 2016 Elsevier Inc. All rights reserved.

[Ethical challenges of genetic manipulation and research with animals].

PubMed

Rodríguez Yunta, Eduardo

2012-01-01

Research with animals presents ethical questions both for being used as models of human diseases and for being a prerequisite for trials in humans, as in the introduction of genetic modifications. Some of these questions refer to the fact that, as models, they do not fully represent the human condition; that conducting toxicity tests causes great harm to animals; that their nature is altered by genetic modifications and that introducing genetically modified organisms is a risk. The use of animals in research for the benefit of humans imposes the moral responsibility to respect them, not making them suffer unnecessarily, since they are living beings capable of feeling.

Are You Ready for [a] Roundup?--What Chemistry Has to Do with Genetic Modifications

NASA Astrophysics Data System (ADS)

Pöpping, Bert

2001-06-01

Genetically modified crops are grown in most parts of the world nowadays. These transgenic plants have new properties such as herbicide tolerance or insect resistance that often cannot be introduced by conventional breeding. Using examples of very common transgenic varieties, the article explains how the knowledge of metabolic pathways and genetic information is used to design these plants and how the same knowledge is used to detect them. It reviews why detection of genetic modifications in plants has become necessary and describes the most common detection methods, from immunological assays to polymerase chain reaction and real-time detection.

Genetic transformation of fruit trees: current status and remaining challenges.

PubMed

Gambino, Giorgio; Gribaudo, Ivana

2012-12-01

Genetic transformation has emerged as a powerful tool for genetic improvement of fruit trees hindered by their reproductive biology and their high levels of heterozygosity. For years, genetic engineering of fruit trees has focussed principally on enhancing disease resistance (against viruses, fungi, and bacteria), although there are few examples of field cultivation and commercial application of these transgenic plants. In addition, over the years much work has been performed to enhance abiotic stress tolerance, to induce modifications of plant growth and habit, to produce marker-free transgenic plants and to improve fruit quality by modification of genes that are crucially important in the production of specific plant components. Recently, with the release of several genome sequences, studies of functional genomics are becoming increasingly important: by modification (overexpression or silencing) of genes involved in the production of specific plant components is possible to uncover regulatory mechanisms associated with the biosynthesis and catabolism of metabolites in plants. This review focuses on the main advances, in recent years, in genetic transformation of the most important species of fruit trees, devoting particular attention to functional genomics approaches and possible future challenges of genetic engineering for these species in the post-genomic era.

SIMILAR PATTERNS OF MITOCHONDRIAL VULNERABILITY AND RESCUE INDUCED BY GENETIC MODIFICATION OF α-SYNUCLEIN, PARKIN AND DJ-1 IN C. ELEGANS*

PubMed Central

Westlund, Beth; Perier, Celine; Burnam, Lucinda; Sluder, Anne; Hoener, Marius; Rodrigues, Cecilia MP; Alfonso, Aixa; Steer, Clifford; Liu, Leo; Przedborski, Serge; Wolozin, Benjamin

2014-01-01

How genetic and environmental factors interact in Parkinson’s disease is poorly understood. We have now compared the patterns of vulnerability and rescue of C. elegans with genetic modifications of three different genetic factors implicated in PD. We observed that expressing α-synuclein, deleting parkin (K08E3.7) or knocking down DJ-1 (B0432.2) or parkin, produces similar patterns of pharmacological vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than non-transgenic nematodes to mitochondrial complex I inhibitors, including rotenone, fenperoximate, pyridaben or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity to paraquat, sodium azide, divalent metal ions (FeII or CuII) or etoposide compared to non-transgenic nematodes. Each of the PD-related lines was also partially rescued by the anti-oxidant probucol, the mitochondrial complex II activator, D-β-hydroxybutyrate (DβHB) or the anti-apoptotic bile acid tauroursodeoxycholic acid (TUDCA). Complete protection in all lines was achieved by combining DβHB with TUDCA but not with probucol. These results show that diverse PD-related genetic modifications disrupt mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD. PMID:16239214

Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion.

PubMed

Bohlke, Nina; Budisa, Nediljko

2014-02-01

One of the major challenges in contemporary synthetic biology is to find a route to engineer synthetic organisms with altered chemical constitution. In terms of core reaction types, nature uses an astonishingly limited repertoire of chemistries when compared with the exceptionally rich and diverse methods of organic chemistry. In this context, the most promising route to change and expand the fundamental chemistry of life is the inclusion of amino acid building blocks beyond the canonical 20 (i.e. expanding the genetic code). This strategy would allow the transfer of numerous chemical functionalities and reactions from the synthetic laboratory into the cellular environment. Due to limitations in terms of both efficiency and practical applicability, state-of-the-art nonsense suppression- or frameshift suppression-based methods are less suitable for such engineering. Consequently, we set out to achieve this goal by sense codon emancipation, that is, liberation from its natural decoding function - a prerequisite for the reassignment of degenerate sense codons to a new 21st amino acid. We have achieved this by redesigning of several features of the post-transcriptional modification machinery which are directly involved in the decoding process. In particular, we report first steps towards the reassignment of 5797 AUA isoleucine codons in Escherichia coli using efficient tools for tRNA nucleotide modification pathway engineering. © 2014 The Authors. FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of the Federation of European Microbiological Societies.

Population-genetic models of sex-limited genomic imprinting.

PubMed

Kelly, S Thomas; Spencer, Hamish G

2017-06-01

Genomic imprinting is a form of epigenetic modification involving parent-of-origin-dependent gene expression, usually the inactivation of one gene copy in some tissues, at least, for some part of the diploid life cycle. Occurring at a number of loci in mammals and flowering plants, this mode of non-Mendelian expression can be viewed more generally as parentally-specific differential gene expression. The effects of natural selection on genetic variation at imprinted loci have previously been examined in a several population-genetic models. Here we expand the existing one-locus, two-allele population-genetic models of viability selection with genomic imprinting to include sex-limited imprinting, i.e., imprinted expression occurring only in one sex, and differential viability between the sexes. We first consider models of complete inactivation of either parental allele and these models are subsequently generalized to incorporate differential expression. Stable polymorphic equilibrium was possible without heterozygote advantage as observed in some prior models of imprinting in both sexes. In contrast to these latter models, in the sex-limited case it was critical whether the paternally inherited or maternally inherited allele was inactivated. The parental origin of inactivated alleles had a different impact on how the population responded to the different selection pressures between the sexes. Under the same fitness parameters, imprinting in the other sex altered the number of possible equilibrium states and their stability. When the parental origin of imprinted alleles and the sex in which they are inactive differ, an allele cannot be inactivated in consecutive generations. The system dynamics became more complex with more equilibrium points emerging. Our results show that selection can interact with epigenetic factors to maintain genetic variation in previously unanticipated ways. Copyright © 2017 Elsevier Inc. All rights reserved.

Epigenetic modification of the oxytocin and glucocorticoid receptor genes is linked to attachment avoidance in young adults.

PubMed

Ein-Dor, Tsachi; Verbeke, Willem J M I; Mokry, Michal; Vrtička, Pascal

2018-08-01

Attachment in the context of intimate pair bonds is most frequently studied in terms of the universal strategy to draw near, or away, from significant others at moments of personal distress. However, important interindividual differences in the quality of attachment exist, usually captured through secure versus insecure - anxious and/or avoidant - attachment orientations. Since Bowlby's pioneering writings on the theory of attachment, it has been assumed that attachment orientations are influenced by both genetic and social factors - what we would today describe and measure as gene by environment interaction mediated by epigenetic DNA modification - but research in humans on this topic remains extremely limited. We for the first time examined relations between intra-individual differences in attachment and epigenetic modification of the oxytocin receptor (OXTR) and glucocorticoid receptor (NR3C1) gene promoter in 109 young adult human participants. Our results revealed that attachment avoidance was significantly and specifically associated with increased OXTR and NR3C1 promoter methylation. These findings offer first tentative clues on the possible etiology of attachment avoidance in humans by showing epigenetic modification in genes related to both social stress regulation and HPA axis functioning.

m6ASNP: a tool for annotating genetic variants by m6A function.

PubMed

Jiang, Shuai; Xie, Yubin; He, Zhihao; Zhang, Ya; Zhao, Yuli; Chen, Li; Zheng, Yueyuan; Miao, Yanyan; Zuo, Zhixiang; Ren, Jian

2018-05-01

Large-scale genome sequencing projects have identified many genetic variants for diverse diseases. A major goal of these projects is to characterize these genetic variants to provide insight into their function and roles in diseases. N6-methyladenosine (m6A) is one of the most abundant RNA modifications in eukaryotes. Recent studies have revealed that aberrant m6A modifications are involved in many diseases. In this study, we present a user-friendly web server called "m6ASNP" that is dedicated to the identification of genetic variants that target m6A modification sites. A random forest model was implemented in m6ASNP to predict whether the methylation status of an m6A site is altered by the variants that surround the site. In m6ASNP, genetic variants in a standard variant call format (VCF) are accepted as the input data, and the output includes an interactive table that contains the genetic variants annotated by m6A function. In addition, statistical diagrams and a genome browser are provided to visualize the characteristics and to annotate the genetic variants. We believe that m6ASNP is a very convenient tool that can be used to boost further functional studies investigating genetic variants. The web server "m6ASNP" is implemented in JAVA and PHP and is freely available at [60].

Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity

PubMed Central

López, Ana; Castelló, María José; Gil, María José; Zheng, Bo; Chen, Peng; Vera, Pablo

2015-01-01

tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response. PMID:26492405

Managing major data of genetically modified mice: from scientific demands to legal obligations.

PubMed

Staudt, Michael; Trauth, Jürgen; Hindi, Iris El; Galuschka, Claudia; Sitek, Dagmar; Schenkel, Johannes

2012-10-01

The number of genetically modified mice is increasing rapidly. Several limitations when working with these animals are to be considered: small colonies, the continued danger of loss, often a limited breeding-success, the need to keep those mutants in stock, difficult and costly import-procedures, and also a major (scientific) value of those mutants often available only with major restrictions. To gather relevant information about all active and archived genetically modified mouse lines available in-house (>1.500) and to deal with a unique resource for several, quite different purposes, a data base was developed enabling optimum knowledge management and easy access. The data base covers also legal restraints and is being linked with the institutional publication repository. To identify the lines available detailed information is provided for each line, as the international designation, a short name, the characterization/description, and the genetic modification including the technique used therefore. The origin of the mutation (gene-ID# and donor organism), the origin of regulatory elements and their donors are listed as well as the genetic background, back-cross generation, phenotype, possible publications, keywords, and some in-house information. Also aspects of animal welfare, obligations to record genetically modified organisms, and technology transfer are displayed; the latter to make licenses possible (if legally permitted). Material transfer agreements, patents, or legal restrictions are listed. This data base helps to avoid double-imports, saves animals and costs since a redundant generation or import can be omitted. However, this is a contribution to the 3R principles developed by Russell and Burch.

[Sporulation or competence development? A genetic regulatory network model of cell-fate determination in Bacillus subtilis].

PubMed

Lu, Zhenghui; Zhou, Yuling; Zhang, Xiaozhou; Zhang, Guimin

2015-11-01

Bacillus subtilis is a generally recognized as safe (GRAS) strain that has been widely used in industries including fodder, food, and biological control. In addition, B. subtilis expression system also plays a significant role in the production of industrial enzymes. However, its application is limited by its low sporulation frequency and transformation efficiency. Immense studies have been done on interpreting the molecular mechanisms of sporulation and competence development, whereas only few of them were focused on improving sporulation frequency and transformation efficiency of B. subtilis by genetic modification. The main challenge is that sporulation and competence development, as the two major developmental events in the stationary phase of B. subtilis, are regulated by the complicated intracellular genetic regulatory systems. In addition, mutual regulatory mechanisms also exist in these two developmental events. With the development of genetic and metabolic engineering, constructing genetic regulatory networks is currently one of the most attractive research fields, together with the genetic information of cell growth, metabolism, and development, to guide the industrial application. In this review, the mechanisms of sporulation and competence development of B. subtilis, their interactions, and the genetic regulation of cell growth were interpreted. In addition, the roles of these regulatory networks in guiding basic and applied research of B. subtilis and its related species were discussed.

Refining Intervention Targets in Family-Based Research: Lessons From Quantitative Behavioral Genetics

PubMed Central

Leve, Leslie D.; Harold, Gordon T.; Ge, Xiaojia; Neiderhiser, Jenae M.; Patterson, Gerald

2010-01-01

The results from a large body of family-based research studies indicate that modifying the environment (specifically dimensions of the social environment) through intervention is an effective mechanism for achieving positive outcomes. Parallel to this work is a growing body of evidence from genetically informed studies indicating that social environmental factors are central to enhancing or offsetting genetic influences. Increased precision in the understanding of the role of the social environment in offsetting genetic risk might provide new information about environmental mechanisms that could be applied to prevention science. However, at present, the multifaceted conceptualization of the environment in prevention science is mismatched with the more limited measurement of the environment in many genetically informed studies. A framework for translating quantitative behavioral genetic research to inform the development of preventive interventions is presented in this article. The measurement of environmental indices amenable to modification is discussed within the context of quantitative behavioral genetic studies. In particular, emphasis is placed on the necessary elements that lead to benefits in prevention science, specifically the development of evidence-based interventions. An example from an ongoing prospective adoption study is provided to illustrate the potential of this translational process to inform the selection of preventive intervention targets. PMID:21188273

Exercise-mediated changes in high-density lipoprotein: impact on form and function.

PubMed

Blazek, Alisa; Rutsky, Jessica; Osei, Kwame; Maiseyeu, Andrei; Rajagopalan, Sanjay

2013-09-01

The goal of this systematic review was to assess the current understanding of the effects of exercise intervention on high-density lipoprotein (HDL) cholesterol (HDL-C) and changes in HDL function as well as modification of these effects by genomic factors. The reviewed studies demonstrate that exercise has modest effects on HDL-C with limited data suggesting an effect on HDL function. Genetic polymorphisms in proteins associated with HDL metabolism play a role in modifying the HDL-C response to exercise and possibly its function. Exercise as an intervention for patients at risk for cardiovascular events can lead to small improvements in HDL-C and potential changes in HDL function. There is an important modifier effect of genetics in determining these changes. Copyright © 2013 Mosby, Inc. All rights reserved.

Combinatorial Roles of Heparan Sulfate Proteoglycans and Heparan Sulfates in Caenorhabditis elegans Neural Development

PubMed Central

Kinnunen, Tarja K.

2014-01-01

Heparan sulfate proteoglycans (HSPGs) play critical roles in the development and adult physiology of all metazoan organisms. Most of the known molecular interactions of HSPGs are attributed to the structurally highly complex heparan sulfate (HS) glycans. However, whether a specific HSPG (such as syndecan) contains HS modifications that differ from another HSPG (such as glypican) has remained largely unresolved. Here, a neural model in C. elegans is used to demonstrate for the first time the relationship between specific HSPGs and HS modifications in a defined biological process in vivo. HSPGs are critical for the migration of hermaphrodite specific neurons (HSNs) as genetic elimination of multiple HSPGs leads to 80% defect of HSN migration. The effects of genetic elimination of HSPGs are additive, suggesting that multiple HSPGs, present in the migrating neuron and in the matrix, act in parallel to support neuron migration. Genetic analyses suggest that syndecan/sdn-1 and HS 6-O-sulfotransferase, hst-6, function in a linear signaling pathway and glypican/lon-2 and HS 2-O-sulfotransferase, hst-2, function together in a pathway that is parallel to sdn-1 and hst-6. These results suggest core protein specific HS modifications that are critical for HSN migration. In C. elegans, the core protein specificity of distinct HS modifications may be in part regulated at the level of tissue specific expression of genes encoding for HSPGs and HS modifying enzymes. Genetic analysis reveals that there is a delicate balance of HS modifications and eliminating one HS modifying enzyme in a compromised genetic background leads to significant changes in the overall phenotype. These findings are of importance with the view of HS as a critical regulator of cell signaling in normal development and disease. PMID:25054285

Hypothetical link between infertility and genetically modified food.

PubMed

Gao, Mingxia; Li, Bin; Yuan, Wenzhen; Zhao, Lihui; Zhang, Xuehong

2014-01-01

It is speculated that genetically modified food (GMF)/genetically modified organism (GMO) is responsible for infertility development. The risk linked with a wide use of GMFs/GMOs offers the basic elements for social criticism. However, to date, it has not been justified whether the bad effects are directly resulted from products of genetic modifications or trans-genesis process. Extensive experience with the risk assessment of whole foods has been applied recently on the safety and nutritional testing of GMFs/GMOs. Investigations have tested the safety of GMFs including sub-acute, chronic, reproductive, multi-generation and carcinogenicity studies. We extrapolated the potential risks associated with GMFs/GMOs on reproduction, and analyzed the multi-aspect linked between infertility and GMFs/GMOs. It could be conjectured that GMFs/GMOs could be potential hazard on reproduction, linking to the development of infertility through influencing the endocrine metabolism, endometriosis. However, little evidence shows the impaction on embryo or reproductive related tumor due to the limited literatures, and needs further research. The article presents some related patents on GMFs/GMOs, and some methods for tracking GMOs.

Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

PubMed

Lee, Patrick C; Truong, Brian; Vega-Crespo, Agustin; Gilmore, W Blake; Hermann, Kip; Angarita, Stephanie Ak; Tang, Jonathan K; Chang, Katherine M; Wininger, Austin E; Lam, Alex K; Schoenberg, Benjamen E; Cederbaum, Stephen D; Pyle, April D; Byrne, James A; Lipshutz, Gerald S

2016-11-29

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

[Epigenetic alterations in acute lymphoblastic leukemia].

PubMed

Navarrete-Meneses, María Del Pilar; Pérez-Vera, Patricia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. It is well-known that genetic alterations constitute the basis for the etiology of ALL. However, genetic abnormalities are not enough for the complete development of the disease, and additional alterations such as epigenetic modifications are required. Such alterations, like DNA methylation, histone modifications, and noncoding RNA regulation have been identified in ALL. DNA hypermethylation in promoter regions is one of the most frequent epigenetic modifications observed in ALL. This modification frequently leads to gene silencing in tumor suppressor genes, and in consequence, contributes to leukemogenesis. Alterations in histone remodeling proteins have also been detected in ALL, such as the overexpression of histone deacetylases enzymes, and alteration of acetyltransferases and methyltransferases. ALL also shows alteration in the expression of miRNAs, and in consequence, the modification in the expression of their target genes. All of these epigenetic modifications are key events in the malignant transformation since they lead to the deregulation of oncogenes as BLK, WNT5B and WISP1, and tumor suppressors such as FHIT, CDKN2A, CDKN2B, and TP53, which alter fundamental cellular processes and potentially lead to the development of ALL. Both genetic and epigenetic alterations contribute to the development and evolution of ALL. Copyright © 2017 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola

PubMed Central

Arif, Rabia; Akram, Faiza; Jamil, Tazeen; Lee, Siu Fai

2017-01-01

Posttranslational modifications (PTMs) occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment on proteins or various amino acids pickings and their ultimate impact on consensus sequences present in mating type proteins using bioinformatics tools. Furthermore, we have also measured and walked through genomic DNA of various Sordaria strains to determine genetic diversity by genotyping the short sequence repeats (SSRs) of wild strains of S. fimicola collected from contrasting environments of two opposing slopes (harsh and xeric south facing slope and mild north facing slope) of Evolution Canyon (EC), Israel. Based on the whole genome sequence of S. macrospora, we targeted 20 genomic regions in S. fimicola which contain short sequence repeats (SSRs). Our data revealed genetic variations in strains from south facing slope and these findings assist in the hypothesis that genetic variations caused by stressful environments lead to evolution. PMID:28717646

Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola.

PubMed

Arif, Rabia; Akram, Faiza; Jamil, Tazeen; Mukhtar, Hamid; Lee, Siu Fai; Saleem, Muhammad

2017-01-01

Posttranslational modifications (PTMs) occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment on proteins or various amino acids pickings and their ultimate impact on consensus sequences present in mating type proteins using bioinformatics tools. Furthermore, we have also measured and walked through genomic DNA of various Sordaria strains to determine genetic diversity by genotyping the short sequence repeats (SSRs) of wild strains of S. fimicola collected from contrasting environments of two opposing slopes (harsh and xeric south facing slope and mild north facing slope) of Evolution Canyon (EC), Israel. Based on the whole genome sequence of S. macrospora , we targeted 20 genomic regions in S. fimicola which contain short sequence repeats (SSRs). Our data revealed genetic variations in strains from south facing slope and these findings assist in the hypothesis that genetic variations caused by stressful environments lead to evolution.

A Developmental-Genetic Model of Alcoholism: Implications for Genetic Research.

ERIC Educational Resources Information Center

Devor, Eric J.

1994-01-01

Research for biological-genetic markers of alcoholism is discussed in context of a multifactorial, heterogeneous, developmental model. Suggested that strategies used in linkage and association studies will require modification. Also suggested several extant associations of genetic markers represent true secondary interactive phenomena that alter…

Advances in genetic modification of pluripotent stem cells.

PubMed

Fontes, Andrew; Lakshmipathy, Uma

2013-11-15

Genetically engineered stem cells aid in dissecting basic cell function and are valuable tools for drug discovery, in vivo cell tracking, and gene therapy. Gene transfer into pluripotent stem cells has been a challenge due to their intrinsic feature of growing in clusters and hence not amenable to common gene delivery methods. Several advances have been made in the rapid assembly of DNA elements, optimization of culture conditions, and DNA delivery methods. This has lead to the development of viral and non-viral methods for transient or stable modification of cells, albeit with varying efficiencies. Most methods require selection and clonal expansion that demand prolonged culture and are not suited for cells with limited proliferative potential. Choosing the right platform based on preferred length, strength, and context of transgene expression is a critical step. Random integration of the transgene into the genome can be complicated due to silencing or altered regulation of expression due to genomic effects. An alternative to this are site-specific methods that target transgenes followed by screening to identify the genomic loci that support long-term expression with stem cell proliferation and differentiation. A highly precise and accurate editing of the genome driven by homology can be achieved using traditional methods as well as the newer technologies such as zinc finger nuclease, TAL effector nucleases and CRISPR. In this review, we summarize the different genetic engineering methods that have been successfully used to create modified embryonic and induced pluripotent stem cells. © 2013. Published by Elsevier Inc. All rights reserved.

Genetic and non-iodine-related factors in the aetiology of nodular goitre.

PubMed

Knudsen, Nils; Brix, Thomas Heiberg

2014-08-01

Genetic and a large number of environmental non-iodine-related factors play a role in the cause of nodular goitre. Most evidence for the influence of genetic and environmental factors in the cause of goitre is from cross-sectional, population-based studies. Only a few studies have included prospective data on risk factors for nodular goitre, although few prospective data are available on the effect of iodine and tobacco smoking on goitre development. Goitre is not one single phenotype. Many epidemiological studies do not distinguish diffuse from nodular goitre, as the investigated parameter is often thyroid volume or frequency with increased thyroid volume. Moreover, information on the presence and effect of gene-environment, gene-gene, and environment-environment effect modifications is limited. Thus, firm conclusions about the relative contributions and causality of the investigated risk factors should be made with caution. Smoking seems to be an established risk factor for nodular goitre, possibly with effect modification from iodine intake, as the risk associated with smoking is smaller or absent in areas with sufficient iodine intake. The use of oral contraceptives might have protective effects against goitre, and childbirth is an increased risk factor for goitre in areas with non-optimal iodine intake. Insulin resistance is a recently investigated risk factor, and the risk of goitre may be reversible with metformin treatment. Iodine remains the major environmental risk factor for nodular goitre. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chemical and Biological Tools for the Preparation of Modified Histone Proteins

PubMed Central

Howard, Cecil J.; Yu, Ruixuan R.; Gardner, Miranda L.; Shimko, John C.; Ottesen, Jennifer J.

2016-01-01

Eukaryotic chromatin is a complex and dynamic system in which the DNA double helix is organized and protected by interactions with histone proteins. This system is regulated through, a large network of dynamic post-translational modifications (PTMs) exists to ensure proper gene transcription, DNA repair, and other processes involving DNA. Homogenous protein samples with precisely characterized modification sites are necessary to better understand the functions of modified histone proteins. Here, we discuss sets of chemical and biological tools that have been developed for the preparation of modified histones, with a focus on the appropriate choice of tool for a given target. We start with genetic approaches for the creation of modified histones, including the incorporation of genetic mimics of histone modifications, chemical installation of modification analogs, and the use of the expanded genetic code to incorporate modified amino acids. Additionally, we will cover the chemical ligation techniques that have been invaluable in the generation of complex modified histones that are indistinguishable from the natural counterparts. Finally, we will end with a prospectus on future directions of synthetic chromatin in living systems. PMID:25863817

Ethics of genetic testing and research in sport: a position statement from the Australian Institute of Sport.

PubMed

Vlahovich, Nicole; Fricker, Peter A; Brown, Matthew A; Hughes, David

2017-01-01

As Australia's peak high-performance sport agency, the Australian Institute of Sport (AIS) has developed this position statement to address the implications of recent advances in the field of genetics and the ramifications for the health and well-being of athletes. Genetic testing has proven of value in the practice of clinical medicine. There are, however, currently no scientific grounds for the use of genetic testing for athletic performance improvement, sport selection or talent identification. Athletes and coaches should be discouraged from using direct-to-consumer genetic testing because of its lack of validation and replicability and the lack of involvement of a medical practitioner in the process. The transfer of genetic material or genetic modification of cells for performance enhancement is gene doping and should not be used on athletes. There are, however, valid roles for genetic research and the AIS supports genetic research which aims to enhance understanding of athlete susceptibility to injury or illness. Genetic research is only to be conducted after careful consideration of a range of ethical concerns which include the provision of adequate informed consent. The AIS is committed to providing leadership in delivering an ethical framework that protects the well-being of athletes and the integrity of sport, in the rapidly changing world of genomic science. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Covering Chemical Diversity of Genetically-Modified Tomatoes Using Metabolomics for Objective Substantial Equivalence Assessment

PubMed Central

Hirai, Tadayoshi; Oikawa, Akira; Matsuda, Fumio; Fukushima, Atsushi; Arita, Masanori; Watanabe, Shin; Yano, Megumu; Hiwasa-Tanase, Kyoko; Ezura, Hiroshi; Saito, Kazuki

2011-01-01

As metabolomics can provide a biochemical snapshot of an organism's phenotype it is a promising approach for charting the unintended effects of genetic modification. A critical obstacle for this application is the inherently limited metabolomic coverage of any single analytical platform. We propose using multiple analytical platforms for the direct acquisition of an interpretable data set of estimable chemical diversity. As an example, we report an application of our multi-platform approach that assesses the substantial equivalence of tomatoes over-expressing the taste-modifying protein miraculin. In combination, the chosen platforms detected compounds that represent 86% of the estimated chemical diversity of the metabolites listed in the LycoCyc database. Following a proof-of-safety approach, we show that % had an acceptable range of variation while simultaneously indicating a reproducible transformation-related metabolic signature. We conclude that multi-platform metabolomics is an approach that is both sensitive and robust and that it constitutes a good starting point for characterizing genetically modified organisms. PMID:21359231

CAR T-cell therapy for glioblastoma: ready for the next round of clinical testing?

PubMed

Prinzing, Brooke L; Gottschalk, Stephen M; Krenciute, Giedre

2018-05-01

The outcome for patients with glioblastoma (GBM) remains poor, and there is an urgent need to develop novel therapeutic approaches. T cells genetically modified with chimeric antigen receptors (CARs) hold the promise to improve outcomes since they recognize and kill cells through different mechanisms than conventional therapeutics. Areas covered: This article reviews CAR design, tumor associated antigens expressed by GBMs that can be targeted with CAR T cells, preclinical and clinical studies conducted with CAR T cells, and genetic approaches to enhance their effector function. Expert commentary: While preclinical studies have highlighted the potent anti-GBM activity of CAR T cells, the initial foray of CAR T-cell therapies into the clinic resulted only in limited benefits for GBM patients. Additional genetic modification of CAR T cells has resulted in a significant increase in their anti-GBM activity in preclinical models. We are optimistic that clinical testing of these enhanced CAR T cells will be safe and result in improved anti-glioma activity in GBM patients.

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

PubMed

White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

2008-12-01

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

Pesticides that inhibit the ubiquitin-proteasome system: effect measure modification by genetic variation in SKP1 in Parkinson׳s disease.

PubMed

Rhodes, Shannon L; Fitzmaurice, Arthur G; Cockburn, Myles; Bronstein, Jeff M; Sinsheimer, Janet S; Ritz, Beate

2013-10-01

Cytoplasmic inclusions known as Lewy bodies, a hallmark of Parkinson's disease (PD) pathology, may protect against cytotoxic proteins. Since the ubiquitin-proteasome system (UPS) degrades cytotoxic proteins, dysfunction in the UPS may contribute to PD etiology. Our goal in this study was to screen pesticides for proteasome inhibition and investigate (i) whether ambient exposures to pesticides that inhibit the UPS increase PD risk and (ii) whether genetic variation in candidate genes of the UPS pathway modify those increased risks. We assessed 26S UPS activity in SK-N-MC(u) cells by fluorescence. We recruited idiopathic PD cases (n=360) and population-based controls (n=816) from three counties in California with considerable commercial agriculture. We determined ambient pesticide exposure by our validated GIS-based model utilizing residential and workplace address histories. We limited effect measure modification assessment to Caucasians (287 cases, 453 controls). Eleven of 28 pesticides we screened inhibited 26S UPS activity at 10 µM. Benomyl, cyanazine, dieldrin, endosulfan, metam, propargite, triflumizole, and ziram were associated with increased PD risk. We estimated an odds ratio of 2.14 (95% CI: 1.42, 3.22) for subjects with ambient exposure to any UPS-inhibiting pesticide at both residential and workplace addresses; this association was modified by genetic variation in the s-phase kinase-associated protein 1 gene (SKP1; interaction p-value=0.005). Our results provide evidence that UPS-inhibiting pesticides play a role in the etiology of PD and suggest that genetic variation in candidate genes involved in the UPS pathway might exacerbate the toxic effects of pesticide exposures. © 2013 Published by Elsevier Inc.

Laboratory Course on "Streptomyces" Genetics and Secondary Metabolism

ERIC Educational Resources Information Center

Siitonen, Vilja; Räty, Kaj; Metsä-Ketelä, Mikko

2016-01-01

The "'Streptomyces' genetics and secondary metabolism" laboratory course gives an introduction to the versatile soil dwelling Gram-positive bacteria "Streptomyces" and their secondary metabolism. The course combines genetic modification of "Streptomyces"; growing of the strain and protoplast preparation, plasmid…

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2011 CFR

2011-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725...

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2012 CFR

2012-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725...

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2013 CFR

2013-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725...

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2014 CFR

2014-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725...

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.

PubMed

Biddle, Wil; Schmitt, Margaret A; Fisk, John D

2016-12-01

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNA Opt We suspected a modification of the tRNA Opt AUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNA Opt AUG is converted to inosine. We identified tRNA Opt AUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Irradiation influence on the detection of genetic-modified soybeans

NASA Astrophysics Data System (ADS)

Villavicencio, A. L. C. H.; Araújo, M. M.; Baldasso, J. G.; Aquino, S.; Konietzny, U.; Greiner, R.

2004-09-01

Three soybean varieties were analyzed to evaluate the irradiation influence on the detection of genetic modification. Samples were treated in a 60Co facility at dose levels of 0, 500, 800, and 1000Gy. The seeds were at first analyzed by Comet Assay as a rapid screening irradiation detection method. Secondly, germination test was performed to detect the viability of irradiated soybeans. Finally, because of its high sensitivity, its specificity and rapidity the polimerase chain reaction was the method applied for genetic modified organism detection. The analysis of DNA by the single technique of microgel electrophoresis of single cells (DNA Comet Assay) showed that DNA damage increased with increasing radiation doses. No negative influence of irradiation on the genetic modification detection was found.

Revisiting phage therapy: new applications for old resources.

PubMed

Nobrega, Franklin L; Costa, Ana Rita; Kluskens, Leon D; Azeredo, Joana

2015-04-01

The success of phage therapy is dependent on the development of strategies able to overcome the limitations of bacteriophages as therapeutic agents, the creation of an adequate regulatory framework, the implementation of safety protocols, and acceptance by the general public. Many approaches have been proposed to circumvent phages' intrinsic limitations but none have proved to be completely satisfactory. In this review we present the major hurdles of phage therapy and the solutions proposed to circumvent them. A thorough discussion of the advantages and drawbacks of these solutions is provided and special attention is given to the genetic modification of phages as an achievable strategy to shape bacteriophages to exhibit desirable biological properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Targeting carbon for crop yield and drought resilience.

PubMed

Griffiths, Cara A; Paul, Matthew J

2017-11-01

Current methods of crop improvement are not keeping pace with projected increases in population growth. Breeding, focused around key traits of stem height and disease resistance, delivered the step-change yield improvements of the green revolution of the 1960s. However, subsequently, yield increases through conventional breeding have been below the projected requirement of 2.4% per year required by 2050. Genetic modification (GM) mainly for herbicide tolerance and insect resistance has been transformational, akin to a second green revolution, although GM has yet to make major inroads into intrinsic yield processes themselves. Drought imposes the major restriction on crop yields globally but, as yet, has not benefited substantially from genetic improvement and still presents a major challenge to agriculture. Much still has to be learnt about the complex process of how drought limits yield and what should be targeted. Mechanisms of drought adaptation from the natural environment cannot be taken into crops without significant modification for the agricultural environment because mechanisms of drought tolerance are often in contrast with mechanisms of high productivity required in agriculture. However, through convergence of fundamental and translational science, it would appear that a mechanism of sucrose allocation in crops can be modified for both productivity and resilience to drought and other stresses. Recent publications show how this mechanism can be targeted by GM, natural variation and a new chemical approach. Here, with an emphasis on drought, we highlight how understanding fundamental science about how crops grow, develop and what limits their growth and yield can be combined with targeted genetic selection and pioneering chemical intervention technology for transformational yield improvements. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Enriching rice with Zn and Fe while minimizing Cd risk

PubMed Central

Slamet-Loedin, Inez H.; Johnson-Beebout, Sarah E.; Impa, Somayanda; Tsakirpaloglou, Nikolaos

2015-01-01

Enriching iron (Fe) and zinc (Zn) content in rice grains, while minimizing cadmium (Cd) levels, is important for human health and nutrition. Natural genetic variation in rice grain Zn enables Zn-biofortification through conventional breeding, but limited natural Fe variation has led to a need for genetic modification approaches, including over-expressing genes responsible for Fe storage, chelators, and transporters. Generally, Cd uptake and allocation is associated with divalent metal cations (including Fe and Zn) transporters, but the details of this process are still unknown in rice. In addition to genetic variation, metal uptake is sometimes limited by its bioavailability in the soil. The availability of Fe, Zn, and Cd for plant uptake varies widely depending on soil redox potential. The typical practice of flooding rice increases Fe while decreasing Zn and Cd availability. On the other hand, moderate soil drying improves Zn uptake but also increases Cd and decreases Fe uptake. Use of Zn- or Fe-containing fertilizers complements breeding efforts by providing sufficient metals for plant uptake. In addition, the timing of nitrogen fertilization has also been shown to affect metal accumulation in grains. The purpose of this mini-review is to identify knowledge gaps and prioritize strategies for improving the nutritional value and safety of rice. PMID:25814994

Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0.

PubMed

Zhang, Lei; Zhao, Xihua; Zhang, Guoxiu; Zhang, Jiajia; Wang, Xuedong; Zhang, Suping; Wang, Wei; Wei, Dongzhi

2016-02-09

Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes. In recent years, homologous recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. When introducing multiple gene deletions, we need a robust and convenient way to control selectable marker genes, especially when only a limited number of markers are available in filamentous fungi. Integration after transformation is predominantly nonhomologous in most fungi other than yeast. Fungal strains deficient in the non-homologous end-joining (NHEJ) pathway have limitations associated with gene function analyses despite they are excellent recipient strains for gene targets. We describe strategies and methods to address these challenges above and leverage the power of resilient NHEJ deficiency strains. We have established a foolproof light-inducible platform for one-step unmarked genetic modification in industrial eukaryotic microorganisms designated as 'LML 3.0', and an on-off control protocol of NHEJ pathway called 'OFN 1.0', using a synthetic light-switchable transactivation to control Cre recombinase-based excision and inversion. The methods provide a one-step strategy to sequentially modify genes without introducing selectable markers and NHEJ-deficiency. The strategies can be used to manipulate many biological processes in a wide range of eukaryotic cells.

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Yupeng; Gorkin, David U.; Dickel, Diane E.

Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulator y element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared withmore » existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types.« less

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

DOE PAGES

He, Yupeng; Gorkin, David U.; Dickel, Diane E.; ...

2017-02-13

Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulator y element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared withmore » existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types.« less

Ionome changes in Xylella fastidiosa-infected Nicotiana tabacum correlate with virulence and discriminate between subspecies of bacterial isolates.

PubMed

Oliver, J E; Sefick, S A; Parker, J K; Arnold, T; Cobine, P A; De La Fuente, L

2014-10-01

Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection.

The impact of genetic modification of human foods in the 21st century: a review.

PubMed

Uzogara, S G

2000-05-01

Genetic engineering of food is the science which involves deliberate modification of the genetic material of plants or animals. It is an old agricultural practice carried on by farmers since early historical times, but recently it has been improved by technology. Many foods consumed today are either genetically modified (GM) whole foods, or contain ingredients derived from gene modification technology. Billions of dollars in U.S. food exports are realized from sales of GM seeds and crops. Despite the potential benefits of genetic engineering of foods, the technology is surrounded by controversy. Critics of GM technology include consumer and health groups, grain importers from European Union (EU) countries, organic farmers, environmentalists, concerned scientists, ethicists, religious rights groups, food advocacy groups, some politicians and trade protectionists. Some of the specific fears expressed by opponents of GM technology include alteration in nutritional quality of foods, potential toxicity, possible antibiotic resistance from GM crops, potential allergenicity and carcinogenicity from consuming GM foods. In addition, some more general concerns include environmental pollution, unintentional gene transfer to wild plants, possible creation of new viruses and toxins, limited access to seeds due to patenting of GM food plants, threat to crop genetic diversity, religious, cultural and ethical concerns, as well as fear of the unknown. Supporters of GM technology include private industries, research scientists, some consumers, U.S. farmers and regulatory agencies. Benefits presented by proponents of GM technology include improvement in fruit and vegetable shelf-life and organoleptic quality, improved nutritional quality and health benefits in foods, improved protein and carbohydrate content of foods, improved fat quality, improved quality and quantity of meat, milk and livestock. Other potential benefits are: the use of GM livestock to grow organs for transplant into humans, increased crop yield, improvement in agriculture through breeding insect, pest, disease, and weather resistant crops and herbicide tolerant crops, use of GM plants as bio-factories to yield raw materials for industrial uses, use of GM organisms in drug manufacture, in recycling and/or removal of toxic industrial wastes. The potential risks and benefits of the new technology to man and the environment are reviewed. Ways of minimizing potential risks and maximizing the benefits of GM foods are suggested. Because the benefits of GM foods apparently far outweigh the risks, regulatory agencies and industries involved in GM food business should increase public awareness in this technology to enhance worldwide acceptability of GM foods. This can be achieved through openness, education, and research.

Genetic Modification of the Lung Directed Toward Treatment of Human Disease.

PubMed

Sondhi, Dolan; Stiles, Katie M; De, Bishnu P; Crystal, Ronald G

2017-01-01

Genetic modification therapy is a promising therapeutic strategy for many diseases of the lung intractable to other treatments. Lung gene therapy has been the subject of numerous preclinical animal experiments and human clinical trials, for targets including genetic diseases such as cystic fibrosis and α1-antitrypsin deficiency, complex disorders such as asthma, allergy, and lung cancer, infections such as respiratory syncytial virus (RSV) and Pseudomonas, as well as pulmonary arterial hypertension, transplant rejection, and lung injury. A variety of viral and non-viral vectors have been employed to overcome the many physical barriers to gene transfer imposed by lung anatomy and natural defenses. Beyond the treatment of lung diseases, the lung has the potential to be used as a metabolic factory for generating proteins for delivery to the circulation for treatment of systemic diseases. Although much has been learned through a myriad of experiments about the development of genetic modification of the lung, more work is still needed to improve the delivery vehicles and to overcome challenges such as entry barriers, persistent expression, specific cell targeting, and circumventing host anti-vector responses.

Engineering Large Animal Species to Model Human Diseases.

PubMed

Rogers, Christopher S

2016-07-01

Animal models are an important resource for studying human diseases. Genetically engineered mice are the most commonly used species and have made significant contributions to our understanding of basic biology, disease mechanisms, and drug development. However, they often fail to recreate important aspects of human diseases and thus can have limited utility as translational research tools. Developing disease models in species more similar to humans may provide a better setting in which to study disease pathogenesis and test new treatments. This unit provides an overview of the history of genetically engineered large animals and the techniques that have made their development possible. Factors to consider when planning a large animal model, including choice of species, type of modification and methodology, characterization, production methods, and regulatory compliance, are also covered. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation1

PubMed Central

Eichten, Steven R.; Schmitz, Robert J.; Springer, Nathan M.

2014-01-01

Chromatin modifications and epigenetics may play important roles in many plant processes, including developmental regulation, responses to environmental stimuli, and local adaptation. Chromatin modifications describe biochemical changes to chromatin state, such as alterations in the specific type or placement of histones, modifications of DNA or histones, or changes in the specific proteins or RNAs that associate with a genomic region. The term epigenetic is often used to describe a variety of unexpected patterns of gene regulation or inheritance. Here, we specifically define epigenetics to include the key aspects of heritability (stable transmission of gene expression states through mitotic or meiotic cell divisions) and independence from DNA sequence changes. We argue against generically equating chromatin and epigenetics; although many examples of epigenetics involve chromatin changes, those chromatin changes are not always heritable or may be influenced by genetic changes. Careful use of the terms chromatin modifications and epigenetics can help separate the biochemical mechanisms of regulation from the inheritance patterns of altered chromatin states. Here, we also highlight examples in which chromatin modifications and epigenetics affect important plant processes. PMID:24872382

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.

PubMed

Masiuk, Katelyn E; Brown, Devin; Laborada, Jennifer; Hollis, Roger P; Urbinati, Fabrizia; Kohn, Donald B

2017-09-06

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10 8 to 1 × 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 - cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 - cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 - cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 - cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Physiological significance of ghrelin revealed by studies using genetically engineered mouse models with modifications in the ghrelin system.

PubMed

Ariyasu, Hiroyuki; Akamizu, Takashi

2015-01-01

Ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R or ghrelin receptor), is a 28-amino acid acylated peptide mainly produced in the stomach. The pharmacological administration of ghrelin is known to exert diverse effects, such as stimulating GH secretion, promoting food intake, and increasing adiposity. In recent years, genetically engineered mouse models have provided important insights into the physiology of various hormones. In this review, we discuss current knowledge regarding the physiological significance of ghrelin on the basis of studies using genetically engineered mouse models with modifications in the ghrelin system.

Commodifying animals: ethical issues in genetic engineering of animals.

PubMed

Almond, B

2000-03-01

The genetic modification of living beings raises special ethical concerns which go beyond general discussion of animal rights or welfare. Although the goals may be similar, biotechnology has accelerated the process of modification of types traditionally carried out by cross-breeding. These changes are discussed in relation to two areas: biomedicine, and animal husbandry. Alternative ethical approaches are reviewed, and it is argued that the teleological thesis underlying virtue ethics has special relevance here. The case for and the case against genetic engineering and patenting of life-forms are examined, and conclusions are drawn which favour regulation, caution and respect for animals and animal species.

From engineering to editing the rat genome.

PubMed

Meek, Stephen; Mashimo, Tomoji; Burdon, Tom

2017-08-01

Since its domestication over 100 years ago, the laboratory rat has been the preferred experimental animal in many areas of biomedical research (Lindsey and Baker The laboratory rat. Academic, New York, pp 1-52, 2006). Its physiology, size, genetics, reproductive cycle, cognitive and behavioural characteristics have made it a particularly useful animal model for studying many human disorders and diseases. Indeed, through selective breeding programmes numerous strains have been derived that are now the mainstay of research on hypertension, obesity and neurobiology (Okamoto and Aoki Jpn Circ J 27:282-293, 1963; Zucker and Zucker J Hered 52(6):275-278, 1961). Despite this wealth of genetic and phenotypic diversity, the ability to manipulate and interrogate the genetic basis of existing phenotypes in rat strains and the methodology to generate new rat models has lagged significantly behind the advances made with its close cousin, the laboratory mouse. However, recent technical developments in stem cell biology and genetic engineering have again brought the rat to the forefront of biomedical studies and enabled researchers to exploit the increasingly accessible wealth of genome sequence information. In this review, we will describe how a breakthrough in understanding the molecular basis of self-renewal of the pluripotent founder cells of the mammalian embryo, embryonic stem (ES) cells, enabled the derivation of rat ES cells and their application in transgenesis. We will also describe the remarkable progress that has been made in the development of gene editing enzymes that enable the generation of transgenic rats directly through targeted genetic modifications in the genomes of zygotes. The simplicity, efficiency and cost-effectiveness of the CRISPR/Cas gene editing system, in particular, mean that the ability to engineer the rat genome is no longer a limiting factor. The selection of suitable targets and gene modifications will now become a priority: a challenge where ES culture and gene editing technologies can play complementary roles in generating accurate bespoke rat models for studying biological processes and modelling human disease.

Genome editing of crops: A renewed opportunity for food security.

PubMed

Georges, Fawzy; Ray, Heather

2017-01-02

Genome editing of crop plants is a rapidly advancing technology whereby targeted mutations can be introduced into a plant genome in a highly specific manner and with great precision. For the most part, the technology does not incorporate transgenic modifications and is far superior to conventional chemical mutagenesis. In this study we bring into focus some of the underlying differences between the 3 existing technologies: classical plant breeding, genetic modification and genome editing. We discuss some of the main achievements from each area and highlight their common characteristics and individual limitations, while emphasizing the unique capabilities of genome editing. We subsequently examine the possible regulatory mechanisms which governments may be inclined to use in assessing the status of genome edited products. If assessed on the basis of their phenotype rather than the process by which they are obtained, these products will be categorized as equivalent to those produced by classical mutagenesis. This would mean that genome edited products will not be subject to the restrictions imposed on genetically modified products, except in some cases where the mutation involves a large sequence insertion into the genome. We conclude by examining the potential of societal acceptance of genome editing technology, reinforced by a scientific perspective on promoting such acceptance.

Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae.

PubMed

Deaner, Matthew; Holzman, Allison; Alper, Hal S

2018-04-16

Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts. Next, we devise and validate a scheme to allow modular construction of tRNA-sgRNA (TST) operons using an iterative Type IIs digestion/ligation extension approach, termed CRISPR-Ligation Extension of sgRNA Operons (LEGO). This approach enables facile construction of large TST operons. We demonstrate this utility by constructing a metabolic rewiring prototype for 2,3-butanediol production in 2 distinct yeast strain backgrounds. These results demonstrate that our approach can act as a surrogate for traditional genetic modification on a much shorter design-cycle timescale. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome editing of crops: A renewed opportunity for food security

PubMed Central

Georges, Fawzy

2017-01-01

ABSTRACT Genome editing of crop plants is a rapidly advancing technology whereby targeted mutations can be introduced into a plant genome in a highly specific manner and with great precision. For the most part, the technology does not incorporate transgenic modifications and is far superior to conventional chemical mutagenesis. In this study we bring into focus some of the underlying differences between the 3 existing technologies: classical plant breeding, genetic modification and genome editing. We discuss some of the main achievements from each area and highlight their common characteristics and individual limitations, while emphasizing the unique capabilities of genome editing. We subsequently examine the possible regulatory mechanisms which governments may be inclined to use in assessing the status of genome edited products. If assessed on the basis of their phenotype rather than the process by which they are obtained, these products will be categorized as equivalent to those produced by classical mutagenesis. This would mean that genome edited products will not be subject to the restrictions imposed on genetically modified products, except in some cases where the mutation involves a large sequence insertion into the genome. We conclude by examining the potential of societal acceptance of genome editing technology, reinforced by a scientific perspective on promoting such acceptance. PMID:28075688

[Differentiated perception of transgenic tomato sauce in the southern Chile].

PubMed

Schnettler Morales, B; Sepúlveda Bravo, O; Ruiz Fuentes, D; Denegri Coria, M

2008-03-01

The present study considers the debate generated in developed countries by genetically modified foods, the importance of this variable to consumers in Temuco (Araucanía Region, Chile) when purchasing tomato sauce and different market segments were studied through a personal survey administered to 400 people. Using conjoint analysis, it was determined that the presence of genetic modification in food was generally more important than the brand and purchase price. Using cluster analysis, three segments were distinguished, with the most numerous (49.3%) placing the greatest importance on the presence of genetic modification (GM) in food and rejecting the transgenic product. The second group (39.4%) gave the greatest importance to the brand and preferred tomato sauce with genetically modified ingredients. The smallest segment (11.3%) placed the greatest value on price and preferred transgenic tomato sauce. The three segments prefer the national brand, reject the store brand and react positively to lower prices. The segment sensitive to the presence of GM in food comprised mainly those younger than 35 years of age, single and with no children. The absence of GM in food of vegetable origin is desirable for young consumers in the Araucanía Region, but a significant proportion accepts genetic modification in food (50.7%).

Comparison of Allergenicity at Gly m 4 and Gly m Bd 30K of Soybean after Genetic Modification.

PubMed

Tsai, Jaw-Ji; Chang, Ching-Yun; Liao, En-Chih

2017-02-15

Despite rapid growth of genetically modified (GM) crops, effective evaluations of genetic modification on allergenicity are still lacking. Gly m Bd 30K is cross-reactive with cow's milk protein casein, Gly m 4, and with birch pollen allergen Bet v 1. Here we compared the allergenicity between GM and non-GM soybeans with respect to the foci Gly m 4 and Gly m Bd 30K. Recombinant allergens of Gly m Bd 30K and Gly m 4 were generated and polyclonal antibodies raised to identify these two allergenic components in soybeans. GM soybean was first PCR-confirmed using 35S promoter. A total of 20 soybeans (half GM, half non-GM) obtained from a food market were used to assess their allergenicity based on IgE-binding and histamine release. The concentrations of Gly m Bd 30K and Gly m 4 in soybeans were then determined. Most soybean-allergic patients (9 of 10) showed IgE-positive reactions to the allergen of 30 kDa in molecular weight. That allergen turned out to be Glycine max Gly m Bd 30K based on LC-MS/MS analyses. Gly m Bd 30K is therefore the major allergen in the soybean. An increase in the transcription of both the Gly m 4 (stress-induced protein SAM22) and Gly m Bd 28K (soybean allergen precursor) was found after genetic modification. The protein concentrations of Gly m 4 and Gly m Bd 30K were not statistically significant different between non-GM and GM soybeans. There were also no statistical significances between them in the tests of IgE binding and histamine release. In conclusion, soybeans showed similar concentrations of Gly m Bd 30K and Gly m 4 regardless of genetic modification or absence thereof. The allergenicity of both Gly m Bd 30K and Gly m 4 was therefore not altered after genetic modification. Patients showing hypersensitivity to soybeans and who had pre-existing allergy to birch pollen and cow's milk casein might not further increase their allergic reactions following exposures to the GM soybeans.

Transcript Isoform Variation Associated with Cytosine Modification in Human Lymphoblastoid Cell Lines.

PubMed

Zhang, Xu; Zhang, Wei

2016-06-01

Cytosine modification on DNA is variable among individuals, which could correlate with gene expression variation. The effect of cytosine modification on interindividual transcript isoform variation (TIV), however, remains unclear. In this study, we assessed the extent of cytosine modification-specific TIV in lymphoblastoid cell lines (LCLs) derived from unrelated individuals of European and African descent. Our study detected cytosine modification-specific TIVs for 17% of the analyzed genes at a 5% false discovery rate. Forty-five percent of the TIV-associated cytosine modifications correlated with the overall gene expression levels as well, with the corresponding CpG sites overrepresented in transcript initiation sites, transcription factor binding sites, and distinct histone modification peaks, suggesting that alternative isoform transcription underlies the TIVs. Our analysis also revealed 33% of the TIV-associated cytosine modifications that affected specific exons, with the corresponding CpG sites overrepresented in exon/intron junctions, splicing branching points, and transcript termination sites, implying that the TIVs are attributable to alternative splicing or transcription termination. Genetic and epigenetic regulation of TIV shared target preference but exerted independent effects on 61% of the common exon targets. Cytosine modification-specific TIVs detected from LCLs were differentially enriched in those detected from various tissues in The Cancer Genome Atlas, indicating their developmental dependency. Genes containing cytosine modification-specific TIVs were enriched in pathways of cancers and metabolic disorders. Our study demonstrated a prominent effect of cytosine modification variation on the transcript isoform spectrum over gross transcript abundance and revealed epigenetic contributions to diseases that were mediated through cytosine modification-specific TIV. Copyright © 2016 by the Genetics Society of America.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2013-03-12

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

PubMed

Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

2016-01-01

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

Which Individuals To Choose To Update the Reference Population? Minimizing the Loss of Genetic Diversity in Animal Genomic Selection Programs.

PubMed

Eynard, Sonia E; Croiseau, Pascal; Laloë, Denis; Fritz, Sebastien; Calus, Mario P L; Restoux, Gwendal

2018-01-04

Genomic selection (GS) is commonly used in livestock and increasingly in plant breeding. Relying on phenotypes and genotypes of a reference population, GS allows performance prediction for young individuals having only genotypes. This is expected to achieve fast high genetic gain but with a potential loss of genetic diversity. Existing methods to conserve genetic diversity depend mostly on the choice of the breeding individuals. In this study, we propose a modification of the reference population composition to mitigate diversity loss. Since the high cost of phenotyping is the limiting factor for GS, our findings are of major economic interest. This study aims to answer the following questions: how would decisions on the reference population affect the breeding population, and how to best select individuals to update the reference population and balance maximizing genetic gain and minimizing loss of genetic diversity? We investigated three updating strategies for the reference population: random, truncation, and optimal contribution (OC) strategies. OC maximizes genetic merit for a fixed loss of genetic diversity. A French Montbéliarde dairy cattle population with 50K SNP chip genotypes and simulations over 10 generations were used to compare these different strategies using milk production as the trait of interest. Candidates were selected to update the reference population. Prediction bias and both genetic merit and diversity were measured. Changes in the reference population composition slightly affected the breeding population. Optimal contribution strategy appeared to be an acceptable compromise to maintain both genetic gain and diversity in the reference and the breeding populations. Copyright © 2018 Eynard et al.

78 FR 3367 - Modification of Regulation Regarding the Extension of Time Limits

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-16

...-2747-01] RIN 0625-AA94 Modification of Regulation Regarding the Extension of Time Limits AGENCY: Import... the extension of time limits for submissions in antidumping (AD) and countervailing duty (CVD) proceedings. The modification, if adopted, will clarify that parties may request an extension of time limits...

Epigenetic Modifications in Essential Hypertension

PubMed Central

Wise, Ingrid A.; Charchar, Fadi J.

2016-01-01

Essential hypertension (EH) is a complex, polygenic condition with no single causative agent. Despite advances in our understanding of the pathophysiology of EH, hypertension remains one of the world’s leading public health problems. Furthermore, there is increasing evidence that epigenetic modifications are as important as genetic predisposition in the development of EH. Indeed, a complex and interactive genetic and environmental system exists to determine an individual’s risk of EH. Epigenetics refers to all heritable changes to the regulation of gene expression as well as chromatin remodelling, without involvement of nucleotide sequence changes. Epigenetic modification is recognized as an essential process in biology, but is now being investigated for its role in the development of specific pathologic conditions, including EH. Epigenetic research will provide insights into the pathogenesis of blood pressure regulation that cannot be explained by classic Mendelian inheritance. This review concentrates on epigenetic modifications to DNA structure, including the influence of non-coding RNAs on hypertension development. PMID:27023534

Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

NASA Astrophysics Data System (ADS)

Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo

An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.

GENETIC CONTROL OF RESTRICTION AND MODIFICATION IN ESCHERICHIA COLI1

PubMed Central

Boyer, Herbert

1964-01-01

Boyer, Herbert (Yale University, New Haven, Conn.). Genetic control of restriction and modification in Escherichia coli. J. Bacteriol. 88:1652–1660. 1964.—Bacterial crosses with K-12 strains of Escherichia coli as Hfr donors (Hfr Hayes, Hfr Cavalli, and Hfr P4X-6) and B/r strains of E. coli as F− recipients were found to differ from crosses between K-12 Hfr donors and K-12 F− recipients in two ways: (i) recombinants (leu, pro, lac, and gal) did not appear at discrete time intervals but did appear simultaneously 30 min after matings were initiated, and (ii) the linkage of unselected markers to selected markers was reduced. Integration of a genetic region linked to the threonine locus of K-12 into the B/r genome resulted in a hybrid which no longer gave anomalous results in conjugation experiments. A similar region of the B strain was introduced into the K-12 strain, which then behaved as a typical B F− recipient. These observations are interpreted as the manifestation of host-controlled modification and restriction on the E. coli chromosome. This was verified by experiments on the restriction and modification of the bacteriophage lambda, F-lac, F-gal, and sex-factor, F1. It was found that the genetic region that controlled the mating responses of the K-12 and B/r strains also controlled the modification and restriction properties of these two strains. The genes responsible for the restricting and modifying properties of the K-12 and B strains of E. coli were found to be allelic, linked to each other, and linked to the threonine locus. PMID:14240953

Selected topics from classical bacterial genetics.

PubMed

Raleigh, Elisabeth A; Elbing, Karen; Brent, Roger

2002-08-01

Current cloning technology exploits many facts learned from classical bacterial genetics. This unit covers those that are critical to understanding the techniques described in this book. Topics include antibiotics, the LAC operon, the F factor, nonsense suppressors, genetic markers, genotype and phenotype, DNA restriction, modification and methylation and recombination.

Riverine fish diversity varies according to geographical isolation and land use modification.

PubMed

Nicol, Elizabeth; Stevens, Jamie R; Jobling, Susan

2017-10-01

Understanding the environmental factors driving species-genetic diversity correlations (SGDCs) is critical for designing appropriate conservation and management strategies to protect biodiversity. Yet, few studies have explored the impact of changing land use patterns on SGDCs specifically in aquatic communities. This study examined patterns of genetic diversity in roach ( Rutilus rutilus L.) together with fish species composition across 19 locations in a large river catchment, spanning a gradient in land use. Our findings show significant correlations between some, but not all, species and genetic diversity end points. For example, genetic and species differentiation showed a weak but significant linear relationship across the Thames catchment, but additional diversity measures such as allelic richness and fish population abundance did not. Further examination of patterns in species and genetic diversity indicated that land use intensification has a modest effect on fish diversity compared to the combined influence of geographical isolation and land use intensification. These results indicate that environmental changes in riparian habitats have the potential to amplify shifts in the composition of stream fish communities in poorly connected river stretches. Conservation and management strategies for fish populations should, therefore, focus on enhancing connectivity between river stretches and limit conversion of nearby land to arable or urban use to maintain current levels of biodiversity.

Dealing with the genetic load in bacterial synthetic biology circuits: convergences with the Ohm's law

PubMed Central

Carbonell-Ballestero, M.; Garcia-Ramallo, E.; Montañez, R.; Rodriguez-Caso, C.; Macía, J.

2016-01-01

Synthetic biology seeks to envision living cells as a matter of engineering. However, increasing evidence suggests that the genetic load imposed by the incorporation of synthetic devices in a living organism introduces a sort of unpredictability in the design process. As a result, individual part characterization is not enough to predict the behavior of designed circuits and thus, a costly trial-error process is eventually required. In this work, we provide a new theoretical framework for the predictive treatment of the genetic load. We mathematically and experimentally demonstrate that dependences among genes follow a quantitatively predictable behavior. Our theory predicts the observed reduction of the expression of a given synthetic gene when an extra genetic load is introduced in the circuit. The theory also explains that such dependence qualitatively differs when the extra load is added either by transcriptional or translational modifications. We finally show that the limitation of the cellular resources for gene expression leads to a mathematical formulation that converges to an expression analogous to the Ohm's law for electric circuits. Similitudes and divergences with this law are outlined. Our work provides a suitable framework with predictive character for the design process of complex genetic devices in synthetic biology. PMID:26656950

Novel perspectives for the engineering of abiotic stress tolerance in plants.

PubMed

Cabello, Julieta V; Lodeyro, Anabella F; Zurbriggen, Matias D

2014-04-01

Adverse environmental conditions pose serious limitations to agricultural production. Classical biotechnological approaches towards increasing abiotic stress tolerance focus on boosting plant endogenous defence mechanisms. However, overexpression of regulatory elements or effectors is usually accompanied by growth handicap and yield penalties due to crosstalk between developmental and stress-response networks. Herein we offer an overview on novel strategies with the potential to overcome these limitations based on the engineering of regulatory systems involved in the fine-tuning of the plant response to environmental hardships, including post-translational modifications, small RNAs, epigenetic control of gene expression and hormonal networks. The development and application of plant synthetic biology tools and approaches will add new functionalities and perspectives to genetic engineering programs for enhancing abiotic stress tolerance. Copyright © 2013 Elsevier Ltd. All rights reserved.

Recent patents on genetic modification of plants and microbes for biomass conversion to biofuels.

PubMed

Lubieniechi, Simona; Peranantham, Thinesh; Levin, David B

2013-04-01

Development of sustainable energy systems based on renewable biomass feedstocks is now a global effort. Lignocellulosic biomass contains polymers of cellulose, hemicellulose, and lignin, bound together in a complex structure. Liquid biofuels, such as ethanol, can be made from biomass via fermentation of sugars derived from the cellulose and hemicellulose within lignocellulosic materials, but pre-treatment of the biomass to release sugars for microbial conversion is a significant barrier to commercial success of lignocellulosic biofuel production. Strategies to reduce the energy and cost inputs required for biomass pre-treatment include genetic modification of plant materials to reduce lignin content. Significant efforts are also underway to create recombinant microorganisms capable of converting sugars derived from lignocellulosic biomass to a variety of biofuels. An alternative strategy to reduce the costs of cellulosic biofuel production is the use of cellulolytic microorganisms capable of direct microbial conversion of ligno-cellulosic biomass to fuels. This paper reviews recent patents on genetic modification of plants and microbes for biomass conversion to biofuels.

Immune modulation by genetic modification of dendritic cells with lentiviral vectors.

PubMed

Liechtenstein, Therese; Perez-Janices, Noemi; Bricogne, Christopher; Lanna, Alessio; Dufait, Inès; Goyvaerts, Cleo; Laranga, Roberta; Padella, Antonella; Arce, Frederick; Baratchian, Mehdi; Ramirez, Natalia; Lopez, Natalia; Kochan, Grazyna; Blanco-Luquin, Idoia; Guerrero-Setas, David; Breckpot, Karine; Escors, David

2013-09-01

Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work. Copyright © 2013 Elsevier B.V. All rights reserved.

Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications.

PubMed

Porter, Joseph J; Mehl, Ryan A

2018-01-01

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis. Genetic code expansion (GCE) has emerged as a powerful tool to overcome this challenge as it allows for the site-specific incorporation of an Ox-PTM into translated protein. The resulting homogeneously modified protein products can then be rigorously characterized for the effects of individual Ox-PTMs. We outline the strengths and weaknesses of GCE as they relate to the field of oxidative stress and Ox-PTMs. An overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs, including antibody validation and therapeutic development, is described.

Nanoparticles for Site Specific Genome Editing

NASA Astrophysics Data System (ADS)

McNeer, Nicole Ali

Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to translate gene therapies for diseases of the blood and immune system to clinical practice. In addition, we have expanded the use of this technology to an additional nonhematopoietic model system: correction of the human cystic fibrosis transmembrane receptor gene in human bronchial epithelial cells. The work presented here represents (1) the first use of biodegradable nanoparticles for PNA delivery, (2) the first direct in vivo site-specific genome modification in human cells, and (3) the first use of triplex-PNA technology for site-specific genome editing in cystic fibrosis.

40 CFR 725.239 - Use of specific microorganisms in activities conducted outside a structure.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Bradyrhizobium japonicum. (2) Modification of traits. (i) The introduced genetic material must meet the criteria for poorly mobilizable listed in § 725.421(c). (ii) The introduced genetic material must consist only... sequences needed to move genetic material, including linkers, homopolymers, adaptors, transposons, insertion...

40 CFR 725.239 - Use of specific microorganisms in activities conducted outside a structure.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Bradyrhizobium japonicum. (2) Modification of traits. (i) The introduced genetic material must meet the criteria for poorly mobilizable listed in § 725.421(c). (ii) The introduced genetic material must consist only... sequences needed to move genetic material, including linkers, homopolymers, adaptors, transposons, insertion...

40 CFR 725.239 - Use of specific microorganisms in activities conducted outside a structure.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Bradyrhizobium japonicum. (2) Modification of traits. (i) The introduced genetic material must meet the criteria for poorly mobilizable listed in § 725.421(c). (ii) The introduced genetic material must consist only... sequences needed to move genetic material, including linkers, homopolymers, adaptors, transposons, insertion...

Chapter VIII. Contributions of propagation techniques and genetic modification to breeding - genetic engineering for disease resistance

USDA-ARS?s Scientific Manuscript database

Genetic engineering offers an opportunity to develop flower bulb crops with resistance to fungal, viral, and bacterial pathogens. Several of the flower bulb crops, Lilium spp., Gladiolus, Zantedeschia, Muscari, Hyacinthus, Narcissus, Ornithogalum, Iris, and Alstroemeria, have been transformed with t...

Genetic modification of lymphocytes by retrovirus-based vectors.

PubMed

Suerth, Julia D; Schambach, Axel; Baum, Christopher

2012-10-01

The genetic modification of lymphocytes is an important topic in the emerging field of gene therapy. Many clinical trials targeting immunodeficiency syndromes or cancer have shown therapeutic benefit; further applications address inflammatory and infectious disorders. Retroviral vector development requires a detailed understanding of the interactions with the host. Most researchers have used simple gammaretroviral vectors to modify lymphocytes, either directly or via hematopoietic stem and progenitor cells. Lentiviral, spumaviral (foamyviral) and alpharetroviral vectors were designed to reduce the necessity for cell stimulation and to utilize potentially safer integration properties. Novel surface modifications (pseudotyping) and transgenes, built using synthetic components, expand the retroviral toolbox, altogether promising increased specificity and potency. Product consistency will be an important criterion for routine clinical use. Copyright © 2012. Published by Elsevier Ltd.

Genetic modifications of pigs for medicine and agriculture

PubMed Central

Whyte, Jeffrey J.; Prather, Randall S.

2011-01-01

SUMMARY Genetically modified swine hold great promise in the fields of agriculture and medicine. Currently, these swine are being used to optimize production of quality meat, to improve our understanding of the biology of disease resistance, and to reduced waste. In the field of biomedicine, swine are anatomically and physiologically analogous to humans. Alterations of key swine genes in disease pathways provide model animals to improve our understanding of the causes and potential treatments of many human genetic disorders. The completed sequencing of the swine genome will significantly enhance the specificity of genetic modifications, and allow for more accurate representations of human disease based on syntenic genes between the two species. Improvements in both methods of gene alteration and efficiency of model animal production are key to enabling routine use of these swine models in medicine and agriculture. PMID:21671302

Tailored HIV-1 vectors for genetic modification of primary human dendritic cells and monocytes.

PubMed

Durand, Stéphanie; Nguyen, Xuan-Nhi; Turpin, Jocelyn; Cordeil, Stephanie; Nazaret, Nicolas; Croze, Séverine; Mahieux, Renaud; Lachuer, Joël; Legras-Lachuer, Catherine; Cimarelli, Andrea

2013-01-01

Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.

Top-down Mass Spectrometry of Cardiac Myofilament Proteins in Health and Disease

PubMed Central

Ying, Peng; Serife, Ayaz-Guner; Deyang, Yu; Ying, Ge

2014-01-01

Myofilaments are composed of thin and thick filaments which coordinate with each other to regulate muscle contraction and relaxation. Posttranslational modifications (PTMs) together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down mass spectrometry (MS) has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to study cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed. PMID:24945106

Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0

PubMed Central

Zhang, Lei; Zhao, Xihua; Zhang, Guoxiu; Zhang, Jiajia; Wang, Xuedong; Zhang, Suping; Wang, Wei; Wei, Dongzhi

2016-01-01

Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes. In recent years, homologous recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. When introducing multiple gene deletions, we need a robust and convenient way to control selectable marker genes, especially when only a limited number of markers are available in filamentous fungi. Integration after transformation is predominantly nonhomologous in most fungi other than yeast. Fungal strains deficient in the non-homologous end-joining (NHEJ) pathway have limitations associated with gene function analyses despite they are excellent recipient strains for gene targets. We describe strategies and methods to address these challenges above and leverage the power of resilient NHEJ deficiency strains. We have established a foolproof light-inducible platform for one-step unmarked genetic modification in industrial eukaryotic microorganisms designated as ‘LML 3.0’, and an on-off control protocol of NHEJ pathway called ‘OFN 1.0’, using a synthetic light-switchable transactivation to control Cre recombinase-based excision and inversion. The methods provide a one-step strategy to sequentially modify genes without introducing selectable markers and NHEJ-deficiency. The strategies can be used to manipulate many biological processes in a wide range of eukaryotic cells. PMID:26857594

Genetic Factors in Tendon Injury: A Systematic Review of the Literature

PubMed Central

Vaughn, Natalie H.; Stepanyan, Hayk; Gallo, Robert A.; Dhawan, Aman

2017-01-01

Background: Tendon injury such as tendinopathy or rupture is common and has multiple etiologies, including both intrinsic and extrinsic factors. The genetic influence on susceptibility to tendon injury is not well understood. Purpose: To analyze the published literature regarding genetic factors associated with tendon injury. Study Design: Systematic review; Level of evidence, 3. Methods: A systematic review of published literature was performed in concordance with the Preferred Reporting Items of Systematic Reviews and Meta-analysis (PRISMA) guidelines to identify current evidence for genetic predisposition to tendon injury. PubMed, Ovid, and ScienceDirect databases were searched. Studies were included for review if they specifically addressed genetic factors and tendon injuries in humans. Reviews, animal studies, or studies evaluating the influence of posttranscription factors and modifications (eg, proteins) were excluded. Results: Overall, 460 studies were available for initial review. After application of inclusion and exclusion criteria, 11 articles were ultimately included for qualitative synthesis. Upon screening of references of these 11 articles, an additional 15 studies were included in the final review, for a total of 26 studies. The genetic factors with the strongest evidence of association with tendon injury were those involving type V collagen A1, tenascin-C, matrix metalloproteinase–3, and estrogen-related receptor beta. Conclusion: The published literature is limited to relatively homogenous populations, with only level 3 and level 4 data. Additional research is needed to make further conclusions about the genetic factors involved in tendon injury. PMID:28856171

Overcoming substrate limitations for improved production of ethylene in E. coli.

PubMed

Lynch, Sean; Eckert, Carrie; Yu, Jianping; Gill, Ryan; Maness, Pin-Ching

2016-01-01

Ethylene is an important industrial compound for the production of a wide variety of plastics and chemicals. At present, ethylene production involves steam cracking of a fossil-based feedstock, representing the highest CO2-emitting process in the chemical industry. Biological ethylene production can be achieved via expression of a single protein, the ethylene-forming enzyme (EFE), found in some bacteria and fungi; it has the potential to provide a sustainable alternative to steam cracking, provided that significant increases in productivity can be achieved. A key barrier is determining factors that influence the availability of substrates for the EFE reaction in potential microbial hosts. In the presence of O2, EFE catalyzes ethylene formation from the substrates α-ketoglutarate (AKG) and arginine. The concentrations of AKG, a key TCA cycle intermediate, and arginine are tightly controlled by an intricate regulatory system that coordinates carbon and nitrogen metabolism. Therefore, reliably predicting which genetic changes will ultimately lead to increased AKG and arginine availability is challenging. We systematically explored the effects of media composition (rich versus defined), gene copy number, and the addition of exogenous substrates and other metabolites on the formation of ethylene in Escherichia coli expressing EFE. Guided by these results, we tested a number of genetic modifications predicted to improve substrate supply and ethylene production, including knockout of competing pathways and overexpression of key enzymes. Several such modifications led to higher AKG levels and higher ethylene productivity, with the best performing strain more than doubling ethylene productivity (from 81 ± 3 to 188 ± 13 nmol/OD600/mL). Both EFE activity and substrate supply can be limiting factors in ethylene production. Targeted modifications in central carbon metabolism, such as overexpression of isocitrate dehydrogenase, and deletion of glutamate synthase or the transcription regulator ArgR, can effectively enhance substrate supply and ethylene productivity. These results not only provide insight into the intricate regulatory network of the TCA cycle, but also guide future pathway and genome-scale engineering efforts to further boost ethylene productivity.

40 CFR 725.455 - Information to be included in the Tier II exemption request.

Code of Federal Regulations, 2010 CFR

2010-07-01

... identification. (2) Type of genetic modification and the function of the introduced genetic material. (3) Site of insertion. (4) Certification of compliance with the introduced genetic material criteria described in § 725... permits. (f) The certification statement required in § 725.25(b). Certification of submission of test data...

Scalable human ES culture for therapeutic use: propagation, differentiation, genetic modification and regulatory issues.

PubMed

Rao, M

2008-01-01

Embryonic stem cells unlike most adult stem cell populations can replicate indefinitely while preserving genetic, epigenetic, mitochondrial and functional profiles. ESCs are therefore an excellent candidate cell type for providing a bank of cells for allogenic therapy and for introducing targeted genetic modifications for therapeutic intervention. This ability of prolonged self-renewal of stem cells and the unique advantages that this offers for gene therapy, discovery efforts, cell replacement, personalized medicine and other more direct applications requires the resolution of several important manufacturing, gene targeting and regulatory issues. In this review, we assess some of the advance made in developing scalable culture systems, improvement in vector design and gene insertion technology and the changing regulatory landscape.

Germ line genome editing in clinics: the approaches, objectives and global society

PubMed Central

2017-01-01

Genome editing allows for the versatile genetic modification of somatic cells, germ cells and embryos. In particular, CRISPR/Cas9 is worldwide used in biomedical research. Although the first report on Cas9-mediated gene modification in human embryos focused on the prevention of a genetic disease in offspring, it raised profound ethical and social concerns over the safety of subsequent generations and the potential misuse of genome editing for human enhancement. The present article considers germ line genome editing approaches from various clinical and ethical viewpoints and explores its objectives. The risks and benefits of the following three likely objectives are assessed: the prevention of monogenic diseases, personalized assisted reproductive technology (ART) and genetic enhancement. Although genetic enhancement should be avoided, the international regulatory landscape suggests the inevitability of this misuse at ART centers. Under these circumstances, possible regulatory responses and the potential roles of public dialogue are discussed. PMID:26615180

Golden Rice and 'Golden' crops for human nutrition.

PubMed

Beyer, Peter

2010-11-30

Micronutrients are essential for a healthy life. Humans do not produce micronutrients, and hence they must obtain them through the foodchain. Staple crops are the predominant food source of mankind, but need to be complemented by other foodstuffs because they are generally deficient in one or the other micronutrient. Breeding for micronutrient-dense crops is not always a viable option because of the absence of genetic variability for the desired trait. Moreover, sterility issues and the complex genetic makeup of some crop plants make them unamenable to conventional breeding. In these cases, genetic modification remains the only viable option. The tools to produce a number of micronutrients in staple crops have recently become available thanks to the identification of the genes involved in the corresponding biochemical pathways at an unprecedented rate. Discarding genetic modification as a viable option is definitely not in the interest of human wellbeing. Copyright © 2010 Elsevier B.V. All rights reserved.

Evaluation of Gene Modification Strategies for the Development of Low-Alcohol-Wine Yeasts

PubMed Central

Kutyna, D. R.; Solomon, M. R.; Black, C. A.; Borneman, A.; Henschke, P. A.; Pretorius, I. S.; Chambers, P. J.

2012-01-01

Saccharomyces cerevisiae has evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO2. Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene, GPD1, was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites. PMID:22729542

The Use of Gene Modification and Advanced Molecular Structure Analyses towards Improving Alfalfa Forage.

PubMed

Lei, Yaogeng; Hannoufa, Abdelali; Yu, Peiqiang

2017-01-29

Alfalfa is one of the most important legume forage crops in the world. In spite of its agronomic and nutritive advantages, alfalfa has some limitations in the usage of pasture forage and hay supplement. High rapid degradation of protein in alfalfa poses a risk of rumen bloat to ruminants which could cause huge economic losses for farmers. Coupled with the relatively high lignin content, which impedes the degradation of carbohydrate in rumen, alfalfa has unbalanced and asynchronous degradation ratio of nitrogen to carbohydrate (N/CHO) in rumen. Genetic engineering approaches have been used to manipulate the expression of genes involved in important metabolic pathways for the purpose of improving the nutritive value, forage yield, and the ability to resist abiotic stress. Such gene modification could bring molecular structural changes in alfalfa that are detectable by advanced structural analytical techniques. These structural analyses have been employed in assessing alfalfa forage characteristics, allowing for rapid, convenient and cost-effective analysis of alfalfa forage quality. In this article, we review two major obstacles facing alfalfa utilization, namely poor protein utilization and relatively high lignin content, and highlight genetic studies that were performed to overcome these drawbacks, as well as to introduce other improvements to alfalfa quality. We also review the use of advanced molecular structural analysis in the assessment of alfalfa forage for its potential usage in quality selection in alfalfa breeding.

The Use of Gene Modification and Advanced Molecular Structure Analyses towards Improving Alfalfa Forage

PubMed Central

Lei, Yaogeng; Hannoufa, Abdelali; Yu, Peiqiang

2017-01-01

Alfalfa is one of the most important legume forage crops in the world. In spite of its agronomic and nutritive advantages, alfalfa has some limitations in the usage of pasture forage and hay supplement. High rapid degradation of protein in alfalfa poses a risk of rumen bloat to ruminants which could cause huge economic losses for farmers. Coupled with the relatively high lignin content, which impedes the degradation of carbohydrate in rumen, alfalfa has unbalanced and asynchronous degradation ratio of nitrogen to carbohydrate (N/CHO) in rumen. Genetic engineering approaches have been used to manipulate the expression of genes involved in important metabolic pathways for the purpose of improving the nutritive value, forage yield, and the ability to resist abiotic stress. Such gene modification could bring molecular structural changes in alfalfa that are detectable by advanced structural analytical techniques. These structural analyses have been employed in assessing alfalfa forage characteristics, allowing for rapid, convenient and cost-effective analysis of alfalfa forage quality. In this article, we review two major obstacles facing alfalfa utilization, namely poor protein utilization and relatively high lignin content, and highlight genetic studies that were performed to overcome these drawbacks, as well as to introduce other improvements to alfalfa quality. We also review the use of advanced molecular structural analysis in the assessment of alfalfa forage for its potential usage in quality selection in alfalfa breeding. PMID:28146083

CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii.

PubMed

Zhang, Cui; Gao, Han; Yang, Zhenke; Jiang, Yuanyuan; Li, Zhenkui; Wang, Xu; Xiao, Bo; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

2017-03-01

CRISPR/Cas9 has been successfully adapted for gene editing in malaria parasites including Plasmodium falciparum and Plasmodium yoelii. However, the reported methods were limited to editing one gene at a time. In practice, it is often desired to modify multiple genetic loci in a parasite genome. Here we describe a CRISPR/Cas9 mediated genome editing method that allows successive modification of more than one gene in the genome of P. yoelii using an improved single-vector system (pYCm) we developed previously. Drug resistant genes encoding human dihydrofolate reductase (hDHFR) and a yeast bifunctional protein (yFCU), with cytosine deaminase (CD) and uridyl phosphoribosyl transferase (UPRT) activities in the plasmid, allowed sequential positive (pyrimethamine, Pyr) and negative (5-fluorocytosine, 5FC) selections and generation of transgenic parasites free of the episomal plasmid after genetic modification. Using this system, we were able to efficiently tag a gene of interest (Pyp28) and subsequently disrupted two genes (Pyctrp and Pycdpk3) that are individually critical for ookinete motility. Disruption of the genes either eliminated (Pyctrp) or greatly reduced (Pycdpk3) ookinete forward motility in matrigel in vitro and completely blocked oocyst development in mosquito midgut. The method will greatly facilitate studies of parasite gene function, development, and disease pathogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

A Novel Tool for Microbial Genome Editing Using the Restriction-Modification System.

PubMed

Bai, Hua; Deng, Aihua; Liu, Shuwen; Cui, Di; Qiu, Qidi; Wang, Laiyou; Yang, Zhao; Wu, Jie; Shang, Xiuling; Zhang, Yun; Wen, Tingyi

2018-01-19

Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.

Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling.

PubMed

Pisarska, Margareta D; Akhlaghpour, Marzieh; Lee, Bora; Barlow, Gillian M; Xu, Ning; Wang, Erica T; Mackey, Aaron J; Farber, Charles R; Rich, Stephen S; Rotter, Jerome I; Chen, Yii-der I; Goodarzi, Mark O; Guller, Seth; Williams, John

2016-11-01

Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

PubMed Central

He, Yupeng; Gorkin, David U.; Dickel, Diane E.; Nery, Joseph R.; Castanon, Rosa G.; Lee, Ah Young; Shen, Yin; Visel, Axel; Pennacchio, Len A.; Ren, Bing; Ecker, Joseph R.

2017-01-01

Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulatory element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared with existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types. REPTILE is available at https://github.com/yupenghe/REPTILE/. PMID:28193886

Post-mortem re-cloning of a transgenic red fluorescent protein dog.

PubMed

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

2011-12-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

CAD-Based Aerodynamic Design of Complex Configurations using a Cartesian Method

NASA Technical Reports Server (NTRS)

Nemec, Marian; Aftosmis, Michael J.; Pulliam, Thomas H.

2003-01-01

A modular framework for aerodynamic optimization of complex geometries is developed. By working directly with a parametric CAD system, complex-geometry models are modified nnd tessellated in an automatic fashion. The use of a component-based Cartesian method significantly reduces the demands on the CAD system, and also provides for robust and efficient flowfield analysis. The optimization is controlled using either a genetic or quasi-Newton algorithm. Parallel efficiency of the framework is maintained even when subject to limited CAD resources by dynamically re-allocating the processors of the flow solver. Overall, the resulting framework can explore designs incorporating large shape modifications and changes in topology.

Improving Small Interfering RNA Delivery In Vivo Through Lipid Conjugation.

PubMed

Osborn, Maire F; Khvorova, Anastasia

2018-05-10

RNA interference (RNAi)-based therapeutics are approaching clinical approval for genetically defined diseases. Current clinical success is a result of significant innovations in the development of chemical architectures that support sustained, multi-month efficacy in vivo following a single administration. Conjugate-mediated delivery has established itself as the most promising platform for safe and targeted small interfering RNA (siRNA) delivery. Lipophilic conjugates represent a major class of modifications that improve siRNA pharmacokinetics and enable efficacy in a broad range of tissues. Here, we review current literature and define key features and limitations of this approach for in vivo modulation of gene expression.

Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host

PubMed Central

Timp, Winston; Feinberg, Andrew P.

2015-01-01

Although at the genetic level cancer is caused by diverse mutations, epigenetic modifications are characteristic of all cancers, from apparently normal precursor tissue to advanced metastatic disease, and these epigenetic modifications drive tumour cell heterogeneity. We propose a unifying model of cancer in which epigenetic dysregulation allows rapid selection for tumour cell survival at the expense of the host. Mechanisms involve both genetic mutations and epigenetic modifications that disrupt the function of genes that regulate the epigenome itself. Several exciting recent discoveries also point to a genome-scale disruption of the epigenome that involves large blocks of DNA hypomethylation, mutations of epigenetic modifier genes and alterations of heterochromatin in cancer (including large organized chromatin lysine modifications (LOCKs) and lamin-associated domains (LADs)), all of which increase epigenetic and gene expression plasticity. Our model suggests a new approach to cancer diagnosis and therapy that focuses on epigenetic dysregulation and has great potential for risk detection and chemoprevention. PMID:23760024

Selecting for Disabilities: Selection Versus Modification.

PubMed

Shaw, Joshua

2018-04-01

This essay considers one argument used to defend parents who use preimplantation genetic diagnosis (PGD) to select for deafness and other disabilities. Some bioethicists have argued that a distinction should be drawn between genetically modifying embryos to possess disabilities and using PGD to select embryos that already present markers of them, and that the former is unethical because it inflicts avoidable harms onto the resulting children, whereas the latter is permissible because it allows children with potentially impaired abilities to exist. This essay raises doubts about whether a meaningful moral distinction can be drawn between modification and selection. Arguments which distinguish modification from selection can be understood in two ways. One is to read them as presenting a No Harm, No Foul argument. Another is to read them as presenting a Harming Versus Letting Be argument. Neither succeeds, however, either in establishing a meaningful moral distinction between modification and selection, or in showing that the second is morally permissible in contradistinction to the first.

75 FR 30277 - Airworthiness Directives; Empresa Brasileira de Aeronautica S.A. (EMBRAER) Model ERJ 190-100 STD...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-01

... cracks resulted in modifications on the airplane Airworthiness Limitation Items (ALI), to include new... Limitation Items (ALI), to include new inspections tasks or modification of existing ones and its respective... modifications on the airplane Airworthiness Limitation Items (ALI), to include new inspections tasks or...

MULTIOBJECTIVE PARALLEL GENETIC ALGORITHM FOR WASTE MINIMIZATION

EPA Science Inventory

In this research we have developed an efficient multiobjective parallel genetic algorithm (MOPGA) for waste minimization problems. This MOPGA integrates PGAPack (Levine, 1996) and NSGA-II (Deb, 2000) with novel modifications. PGAPack is a master-slave parallel implementation of a...

Progress in the molecular and genetic modification breeding of beef cattle in China.

PubMed

Tong, Bin; Zhang, Li; Li, Guang-Peng

2017-11-20

The studies of beef cattle breeding in China have been greatly improved with the rapid development of the international beef cattle industrialization. The beef cattle breeding technologies have rapidly transformed from traditional breeding to molecular marker-assisted breeding, genomic selection and genetic modification breeding. Hundreds of candidate genes and molecular markers associated with growth, meat quality, reproduction performance and diseases resistance have been identified, and some of them have already been used in cattle breeding. Genes and molecular markers associated with growth and development are focused on the growth hormone, muscle regulatory factors, myostatin and insulin-like growth factors. Meat quality is mediated by fatty acid transport and deposition related signals, calpains and calpain system, muscle regulatory factors and muscle growth regulation pathways. Reproduction performance is regulated by GnRH-FSH-LH, growth differentiation factor 9, prolactin receptor and forkhead box protein O1. Disease resistance is modulated by the major histocompatibility complex gene family, toll-like receptors, mannose-binding lectin and interferon gene signals. In this review, we summarize the most recent progress in beef cattle breeding in marker-assisted selection, genome-wide selection and genetic modification breeding, aiming to provide a reference for further genetic breeding research of beef cattle in China.

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code

PubMed Central

Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

2016-01-01

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNALysUUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine–pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism. PMID:26791911

Modifications to the HIPAA Privacy, Security, Enforcement, and Breach Notification rules under the Health Information Technology for Economic and Clinical Health Act and the Genetic Information Nondiscrimination Act; other modifications to the HIPAA rules.

PubMed

2013-01-25

The Department of Health and Human Services (HHS or ``the Department'') is issuing this final rule to: Modify the Health Insurance Portability and Accountability Act (HIPAA) Privacy, Security, and Enforcement Rules to implement statutory amendments under the Health Information Technology for Economic and Clinical Health Act (``the HITECH Act'' or ``the Act'') to strengthen the privacy and security protection for individuals' health information; modify the rule for Breach Notification for Unsecured Protected Health Information (Breach Notification Rule) under the HITECH Act to address public comment received on the interim final rule; modify the HIPAA Privacy Rule to strengthen the privacy protections for genetic information by implementing section 105 of Title I of the Genetic Information Nondiscrimination Act of 2008 (GINA); and make certain other modifications to the HIPAA Privacy, Security, Breach Notification, and Enforcement Rules (the HIPAA Rules) to improve their workability and effectiveness and to increase flexibility for and decrease burden on the regulated entities.

Systematic prediction of control proteins and their DNA binding sites

PubMed Central

Sorokin, Valeriy; Severinov, Konstantin; Gelfand, Mikhail S.

2009-01-01

We present here the results of a systematic bioinformatics analysis of control (C) proteins, a class of DNA-binding regulators that control time-delayed transcription of their own genes as well as restriction endonuclease genes in many type II restriction-modification systems. More than 290 C protein homologs were identified and DNA-binding sites for ∼70% of new and previously known C proteins were predicted by a combination of phylogenetic footprinting and motif searches in DNA upstream of C protein genes. Additional analysis revealed that a large proportion of C protein genes are translated from leaderless RNA, which may contribute to time-delayed nature of genetic switches operated by these proteins. Analysis of genetic contexts of newly identified C protein genes revealed that they are not exclusively associated with restriction-modification genes; numerous instances of associations with genes originating from mobile genetic elements were observed. These instances might be vestiges of ancient horizontal transfers and indicate that during evolution ancestral restriction-modification system genes were the sites of mobile elements insertions. PMID:19056824

Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications

PubMed Central

2018-01-01

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis. Genetic code expansion (GCE) has emerged as a powerful tool to overcome this challenge as it allows for the site-specific incorporation of an Ox-PTM into translated protein. The resulting homogeneously modified protein products can then be rigorously characterized for the effects of individual Ox-PTMs. We outline the strengths and weaknesses of GCE as they relate to the field of oxidative stress and Ox-PTMs. An overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs, including antibody validation and therapeutic development, is described. PMID:29849913

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code.

PubMed

Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

2016-01-21

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNA(Lys)(UUU) with hypermodified 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine-pyrimidine mismatches. We show that mnm(5)s(2)U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism.

Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code

NASA Astrophysics Data System (ADS)

Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

2016-01-01

Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNALysUUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine-pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism.

Testing for Genetically Modified Foods Using PCR

ERIC Educational Resources Information Center

Taylor, Ann; Sajan, Samin

2005-01-01

The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

Variables Affecting Secondary School Students' Willingness to Eat Genetically Modified Food Crops

ERIC Educational Resources Information Center

Maes, Jasmien; Bourgonjon, Jeroen; Gheysen, Godelieve; Valcke, Martin

2018-01-01

A large-scale cross-sectional study (N = 4002) was set up to determine Flemish secondary school students' willingness to eat genetically modified food (WTE) and to link students' WTE to previously identified key variables from research on the acceptance of genetic modification (GM). These variables include subjective and objective knowledge about…

Generation of induced pluripotent stem cells from the pig

USDA-ARS?s Scientific Manuscript database

The value of stem cells has become increasingly evident in recent years with the advent of genetic engineering tools that allow site-specific modifications to the genome. The use of stem cells to induce modifications has several potential benefits for the livestock industry including improving anim...

Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.

PubMed

Xue, Haipeng; Wu, Jianbo; Li, Shenglan; Rao, Mahendra S; Liu, Ying

2016-01-01

Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability.

A century of landscape disturbance and urbanization of the San Francisco Bay region affects the present-day genetic diversity of the California Ridgway’s rail (Rallus obsoletus obsoletus)

USGS Publications Warehouse

Wood, Dustin A.; Bui, Thuy-Vy D.; Overton, Cory T.; Vandergast, Amy; Casazza, Michael L.; Hull, Joshua M.; Takekawa, John Y.

2016-01-01

Fragmentation and loss of natural habitat have important consequences for wild populations and can negatively affect long-term viability and resilience to environmental change. Salt marsh obligate species, such as those that occupy the San Francisco Bay Estuary in western North America, occupy already impaired habitats as result of human development and modifications and are highly susceptible to increased habitat loss and fragmentation due to global climate change. We examined the genetic variation of the California Ridgway’s rail (Rallus obsoletus obsoletus), a state and federally endangered species that occurs within the fragmented salt marsh of the San Francisco Bay Estuary. We genotyped 107 rails across 11 microsatellite loci and a single mitochondrial gene to estimate genetic diversity and population structure among seven salt marsh fragments and assessed demographic connectivity by inferring patterns of gene flow and migration rates. We found pronounced genetic structuring among four geographically separate genetic clusters across the San Francisco Bay. Gene flow analyses supported a stepping stone model of gene flow from south-to-north. However, contemporary gene flow among the regional embayments was low. Genetic diversity among occupied salt marshes and genetic clusters were not significantly different. We detected low effective population sizes and significantly high relatedness among individuals within salt marshes. Preserving genetic diversity and connectivity throughout the San Francisco Bay may require attention to salt marsh restoration in the Central Bay where habitat is both most limited and most fragmented. Incorporating periodic genetic sampling into the management regime may help evaluate population trends and guide long-term management priorities.

Transfer of plasmid DNA to clinical coagulase-negative staphylococcal pathogens by using a unique bacteriophage.

PubMed

Winstel, Volker; Kühner, Petra; Krismer, Bernhard; Peschel, Andreas; Rohde, Holger

2015-04-01

Genetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a unique Staphylococcus aureus strain via a specific S. aureus bacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinical Staphylococcus epidermidis isolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Modalities and future prospects of gene therapy in heart transplantation.

PubMed

Vassalli, Giuseppe; Roehrich, Marc-Estienne; Vogt, Pierre; Pedrazzini, Giovanni B; Siclari, Francesco; Moccetti, Tiziano; von Segesser, Ludwig K

2009-06-01

Heart transplantation is the treatment of choice for many patients with end-stage heart failure. Its success, however, is limited by organ shortage, side effects of immunosuppressive drugs, and chronic rejection. Gene therapy is conceptually appealing for applications in transplantation, as the donor organ is genetically manipulated ex vivo before transplantation. Localised expression of immunomodulatory genes aims to create a state of immune privilege within the graft, which could eliminate the need for systemic immunosuppression. In this review, recent advances in the development of gene therapy in heart transplantation are discussed. Studies in animal models have demonstrated that genetic modification of the donor heart with immunomodulatory genes attenuates ischaemia-reperfusion injury and rejection. Alternatively, bone marrow-derived cells genetically engineered with donor-type major histocompatibility complex (MHC) class I or II promote donor-specific hyporesponsiveness. Genetic engineering of naïve T cells or dendritic cells may induce regulatory T cells and regulatory dendritic cells. Despite encouraging results in animal models, however, clinical gene therapy trials in heart transplantation have not yet been started. The best vector and gene to be delivered remain to be identified. Pre-clinical studies in non-human primates are needed. Nonetheless, the potential of gene therapy as an adjunct therapy in transplantation is essentially intact.

Genetic modification of cerebral arterial wall: implications for prevention and treatment of cerebral vasospasm.

PubMed

Vijay, Anantha; Santhanam, R; Katusic, Zvonimir S

2006-10-01

Genetic modification of cerebral vessels represents a promising and novel approach for prevention and/or treatment of various cerebral vascular disorders, including cerebral vasospasm. In this review, we focus on the current understanding of the use of gene transfer to the cerebral arteries for prevention and/or treatment of cerebral vasospasm following subarachnoid hemorrhage (SAH). We also discuss the recent developments in vascular therapeutics, involving the autologous use of progenitor cells for repair of damaged vessels, as well as a cell-based gene delivery approach for the prevention and treatment of cerebral vasospasm.

Incidence and predictors of regimen-modification from first-line antiretroviral therapy in Thailand: a cohort study.

PubMed

Tsuchiya, Naho; Pathipvanich, Panita; Wichukchinda, Nuanjun; Rojanawiwat, Archawin; Auwanit, Wattana; Ariyoshi, Koya; Sawanpanyalert, Pathom

2014-10-30

Antiretroviral therapy markedly reduced mortality in HIV-infected individuals. However, in the previous studies, up to 50% of patients are compelled to modify their regimen in middle and low-income countries where salvage drug is still limited. This cohort study aimed to investigate the incidence and predictors of regimen modification from the first-line antiretroviral regimen in northern Thailand. All HIV-infected patients starting antiretroviral therapy (ART) with generic drug (GPOvir®; stavudine, lamivudine and nevirapine) at a governmental hospital in northern Thailand from 2002 to 2007 were recruited. Baseline characteristics and detailed information of regimen modification until the end of 2010 were ascertained from cohort database and medical charts. As a potential genetic predictor of regimen modification, HLA B allele was determined by bead-based array hybridization (WAKFlow® HLA typing kit). We investigated predictors of the regimen modification using Cox's proportional hazard models. Of 979 patients, 914 were eligible for the analysis. The observed events of regimen modification was 377, corresponding to an incidence 13.8/100 person-year-observation (95% CI:12.5-15.3) over 2,728 person years (PY) follow up. The main reasons for regimen modification were adverse effects (73.5%), especially lipodystrophy (63.2%) followed by rash (17.7%). Sixty three patients (17.1%) changed the regimen due to treatment failure. 2% and 19% of patients had HLA-B*35:05 and B*4001, respectively. HLA-B*35:05 was independently associated with rash-related regimen modification (aHR 7.73, 95% CI:3.16-18.9) while female gender was associated with lipodystrophy (aHR 2.11, 95% CI:1.51-2.95). Female gender (aHR 0.54, 95% CI: 0.30-0.96), elder age (aHR 0.56, 95% CI: 0.32-0.99) and having HLA-B*40:01 (aHR 0.29, 95% CI: 0.10-0.82) were protective for treatment failure related modification. HLA-B*35:05 and female gender were strong predictors of regimen modification due to rash and lipodystrophy, respectively. Female gender, elder age, and having HLA-B*40:01 had protective effects on treatment failure-related regimen modification. This study provides further information of regimen modification for future tailored ART in Asia.

Simultaneous quantification of oil and protein in cottonseed by low-field time-domain nuclear magnetic resonance

USDA-ARS?s Scientific Manuscript database

Modification of cottonseed quality traits is likely to be achieved through a combination of genetic modification, manipulation of nutrient allocation and selective breeding. Oil and protein stores comprise the majority of mass of cottonseed embryos. A more comprehensive understanding of the relation...

Non-destructive quantification of oil and protein in cottonseed by time-domain nuclear magnetic resonance

USDA-ARS?s Scientific Manuscript database

Modification of cotton seed quality traits is likely to be achieved through a combination of genetic modification, nutrient allocation, and selective breeding. Oil and protein stores comprise the majority of mass of cottonseed embryos. A more comprehensive understanding of the relationship between f...

Genetic engineering including superseding microinjection: new ways to make GM pigs.

PubMed

Galli, Cesare; Perota, Andrea; Brunetti, Dario; Lagutina, Irina; Lazzari, Giovanna; Lucchini, Franco

2010-01-01

Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.

Survival of Skin Graft between Transgenic Cloned Dogs and Non-Transgenic Cloned Dogs

PubMed Central

Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Park, Jung Eun; Park, Eun Jung; Lim, Sang Hyun; Yoon, Byung Il; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2014-01-01

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues. PMID:25372489

Physical characteristics of genetically-altered wheat related to technological protein separation

USDA-ARS?s Scientific Manuscript database

Wheat protein is a technologically challenging substrate for food and non-food applications because of its compositional diversity and susceptibility to denaturation. Genetic modification could be used to create cultivars capable of producing more uniform or focused and novel protein compositions t...

Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference

PubMed Central

Murphy, Katherine A.; Tabuloc, Christine A.; Cervantes, Kevin R.; Chiu, Joanna C.

2016-01-01

RNA interference has had major advances as a developing tool for pest management. In laboratory experiments, double-stranded RNA (dsRNA) is often administered to the insect by genetic modification of the crop, or synthesized in vitro and topically applied to the crop. Here, we engineered genetically modified yeast that express dsRNA targeting y-Tubulin in Drosophila suzukii. Our design takes advantage of the symbiotic interactions between Drosophila, yeast, and fruit crops. Yeast is naturally found growing on the surface of fruit crops, constitutes a major component of the Drosophila microbiome, and is highly attractive to Drosophila. Thus, this naturally attractive yeast biopesticide can deliver dsRNA to an insect pest without the need for genetic crop modification. We demonstrate that this biopesticide decreases larval survivorship, and reduces locomotor activity and reproductive fitness in adults, which are indicative of general health decline. To our knowledge, this is the first study to show that yeast can be used to deliver dsRNA to an insect pest. PMID:26931800

Germline Genetic Modification and Identity: the Mitochondrial and Nuclear Genomes

PubMed Central

Scott, Rosamund; Wilkinson, Stephen

2017-01-01

Abstract In a legal ‘first’, the UK removed a prohibition against modifying embryos in human reproduction, to enable mitochondrial replacement techniques (MRTs), a move the Government distanced from ‘germline genetic modification’, which it aligned with modifying the nuclear genome. This paper (1) analyzes the uses and meanings of this term in UK/US legal and policy debates; and (2) evaluates related ethical concerns about identity. It shows that, with respect to identity, MRTs and nuclear genome editing techniques such as CRISPR/Cas-9 (now a policy topic), are not as different as has been supposed. While it does not follow that the two should be treated exactly alike, one of the central reasons offered for treating MRTs more permissively than nuclear genetic modification, and for not regarding MRTs as ‘germline genetic modification’, is thereby in doubt. Identity cannot, by itself, do the work thus far assigned to it, explicitly or otherwise, in law and policy. PMID:29670305

Comparison of the physiological characteristics of transgenic insect-resistant cotton and conventional lines.

PubMed

Li, Xiaogang; Ding, Changfeng; Wang, Xingxiang; Liu, Biao

2015-03-04

The introduction of transgenic insect-resistant cotton into agricultural ecosystems has raised concerns regarding its ecological effects. Many studies have been conducted to compare the differences in characteristics between transgenic cotton and conventional counterparts. However, few studies have focused on the different responses of transgenic cotton to stress conditions, especially to the challenges of pathogens. The aim of this work is to determine the extent of variation in physiological characteristics between transgenic insect-resistant cotton and the conventional counterpart infected by cotton soil-borne pathogens. The results showed that the difference in genetic backgrounds is the main factor responsible for the effects on biochemical characteristics of transgenic cotton when incubating with cotton Fusarium oxysporum. However, genetic modification had a significantly greater influence on the stomatal structure of transgenic cotton than the effects of cotton genotypes. Our results highlight that the differences in genetic background and/or genetic modifications may introduce variations in physiological characteristics and should be considered to explore the potential unexpected ecological effects of transgenic cotton.

Germ line genome editing in clinics: the approaches, objectives and global society.

PubMed

Ishii, Tetsuya

2017-01-01

Genome editing allows for the versatile genetic modification of somatic cells, germ cells and embryos. In particular, CRISPR/Cas9 is worldwide used in biomedical research. Although the first report on Cas9-mediated gene modification in human embryos focused on the prevention of a genetic disease in offspring, it raised profound ethical and social concerns over the safety of subsequent generations and the potential misuse of genome editing for human enhancement. The present article considers germ line genome editing approaches from various clinical and ethical viewpoints and explores its objectives. The risks and benefits of the following three likely objectives are assessed: the prevention of monogenic diseases, personalized assisted reproductive technology (ART) and genetic enhancement. Although genetic enhancement should be avoided, the international regulatory landscape suggests the inevitability of this misuse at ART centers. Under these circumstances, possible regulatory responses and the potential roles of public dialogue are discussed. © The Author 2015. Published by Oxford University Press.

Genetic modification of human trabecular meshwork with lentiviral vectors.

PubMed

Loewen, N; Fautsch, M P; Peretz, M; Bahler, C K; Cameron, J D; Johnson, D H; Poeschla, E M

2001-11-20

Glaucoma, a group of optic neuropathies, is the leading cause of irreversible blindness. Neuronal apoptosis in glaucoma is primarily associated with high intraocular pressure caused by chronically impaired outflow of aqueous humor through the trabecular meshwork, a reticulum of mitotically inactive endothelial-like cells located in the angle of the anterior chamber. Anatomic, genetic, and expression profiling data suggest the possibility of using gene transfer to treat glaucomatous intraocular pressure dysregulation, but this approach will require stable genetic modification of the differentiated aqueous outflow tract. We injected transducing unit-normalized preparations of either of two lentiviral vectors or an oncoretroviral vector as a single bolus into the aqueous circulation of cultured human donor eyes, under perfusion conditions that mimicked natural anterior chamber flow and maintained viability ex vivo. Reporter gene expression was assessed in trabecular meshwork from 3 to 16 days after infusion of 1.0 x 10(8) transducing units of each vector. The oncoretroviral vector failed to transduce the trabecular meshwork. In contrast, feline immunodeficiency virus and human immunodeficiency virus vectors produced efficient, localized transduction of the trabecular meshwork in situ. The results demonstrate that lentiviral vectors permit efficient genetic modification of the human trabecular meshwork when delivered via the afferent aqueous circulation, a clinically accessible route. In addition, controlled comparisons in this study establish that feline and human immunodeficiency virus vectors are equivalently efficacious in delivering genes to this terminally differentiated human tissue.

Taking Another Look at Behavior Modification and Assertive Discipline.

ERIC Educational Resources Information Center

Palardy, J. Michael

1996-01-01

Behavior modification and assertive discipline approaches use rewards and negative consequences (extinction procedures) to deal with students' maladaptive behavior. Behavior modification has significant limitations, as it treats symptoms, not causes; yields only short-term benefits; has limited transfer value for other environments; and values…

Multidisciplinary approaches to stimulate wound healing.

PubMed

Businaro, Rita; Corsi, Mariangela; Di Raimo, Tania; Marasco, Sergio; Laskin, Debra L; Salvati, Bruno; Capoano, Raffaele; Ricci, Serafino; Siciliano, Camilla; Frati, Giacomo; De Falco, Elena

2016-08-01

New civil wars and waves of terrorism are causing crucial social changes, with consequences in all fields, including health care. In particular, skin injuries are evolving as an epidemic issue. From a physiological standpoint, although wound repair takes place more rapidly in the skin than in other tissues, it is still a complex organ to reconstruct. Genetic and clinical variables, such as diabetes, smoking, and inflammatory/immunological pathologies, are also important risk factors limiting the regenerative potential of many therapeutic applications. Therefore, optimization of current clinical strategies is critical. Here, we summarize the current state of the field by focusing on stem cell therapy applications in wound healing, with an emphasis on current clinical approaches being developed. These involve protocols for the ex vivo expansion of adipose tissue-derived mesenchymal stem cells by means of a patented Good Manufacturing Practice-compliant platelet lysate. Combinations of multiple strategies, including genetic modifications and stem cells, biomimetic scaffolds, and novel vehicles, such as nanoparticles, are also discussed as future approaches. © 2016 New York Academy of Sciences.

Genetic code translation displays a linear trade-off between efficiency and accuracy of tRNA selection.

PubMed

Johansson, Magnus; Zhang, Jingji; Ehrenberg, Måns

2012-01-03

Rapid and accurate translation of the genetic code into protein is fundamental to life. Yet due to lack of a suitable assay, little is known about the accuracy-determining parameters and their correlation with translational speed. Here, we develop such an assay, based on Mg(2+) concentration changes, to determine maximal accuracy limits for a complete set of single-mismatch codon-anticodon interactions. We found a simple, linear trade-off between efficiency of cognate codon reading and accuracy of tRNA selection. The maximal accuracy was highest for the second codon position and lowest for the third. The results rationalize the existence of proofreading in code reading and have implications for the understanding of tRNA modifications, as well as of translation error-modulating ribosomal mutations and antibiotics. Finally, the results bridge the gap between in vivo and in vitro translation and allow us to calibrate our test tube conditions to represent the environment inside the living cell.

A Comparative Study on Multifactor Dimensionality Reduction Methods for Detecting Gene-Gene Interactions with the Survival Phenotype

PubMed Central

Lee, Seungyeoun; Kim, Yongkang; Kwon, Min-Seok; Park, Taesung

2015-01-01

Genome-wide association studies (GWAS) have extensively analyzed single SNP effects on a wide variety of common and complex diseases and found many genetic variants associated with diseases. However, there is still a large portion of the genetic variants left unexplained. This missing heritability problem might be due to the analytical strategy that limits analyses to only single SNPs. One of possible approaches to the missing heritability problem is to consider identifying multi-SNP effects or gene-gene interactions. The multifactor dimensionality reduction method has been widely used to detect gene-gene interactions based on the constructive induction by classifying high-dimensional genotype combinations into one-dimensional variable with two attributes of high risk and low risk for the case-control study. Many modifications of MDR have been proposed and also extended to the survival phenotype. In this study, we propose several extensions of MDR for the survival phenotype and compare the proposed extensions with earlier MDR through comprehensive simulation studies. PMID:26339630

The potential of the combination of CRISPR/Cas9 and pluripotent stem cells to provide human organs from chimaeric pigs.

PubMed

Feng, Wanyou; Dai, Yifan; Mou, Lisha; Cooper, David K C; Shi, Deshun; Cai, Zhiming

2015-03-23

Clinical organ allotransplantation is limited by the availability of deceased human donors. However, the transplantation of human organs produced in other species would provide an unlimited number of organs. The pig has been identified as the most suitable source of organs for humans as organs of any size would be available. Genome editing by RNA-guided endonucleases, also known as clustered regularly interspaced short palindromic repeat (CRISPR/Cas9), in combination with induced pluripotent stem cells (iPSC), may have the potential to enable the creation of human organs from genetically-modified chimaeric pigs. These could potentially provide an unlimited supply of organs that would not be rejected by the recipient's immune system. However, substantial research is needed to prove that this approach will work. Genetic modification of chimaeric pigs could also provide useful models for developing therapies for various human diseases, especially in relation to drug development.

Production of marine plant biomass: Management, cultivation, and genetic modification of macrophytic algae

NASA Astrophysics Data System (ADS)

Vandermeer, J. P.

1982-12-01

Every second of every day, the Sun's fusion reactions convert thousands of tons of hydrogen into helium with the release of almost unimaginable amounts of energy. Through the photosynthetic activity of plants, both aquatic and terrestrial, a small fraction of this energy is trapped and stored as plant biomass. The oceans cover a greater fraction of the globe than do the land masses, making it appropriate to consider their contribution to the total biomass production, and their potential as a source of raw materials for the extraction of chemicals and fuels. A rather broad synthesis, convering the total seaweed resource and some of the constraints placed on harvesting these plants, attempts to farm the oceans to increase the supply of desirable species, attempts to cultivate seaweeds in enclosures where environmental parameters are controlled, and finally, the limited amount of genetic manipulation that was applied to these plants was presented. Only the larger red and brown seaweeds were considered because they represent the bulk of the biomass.

Reproductive medicine involving genome editing: clinical uncertainties and embryological needs.

PubMed

Ishii, Tetsuya

2017-01-01

Genome editing based on site-directed nucleases facilitated efficient and versatile genetic modifications in human cells. However, recent reports, demonstrating CRISPR/Cas9-mediated genome editing in human embryos have raised profound concerns worldwide. This commentary explores the clinical justification and feasibility of reproductive medicine using germline genome editing. Despite the perceived utility of reproductive medicine for treating intractable infertility, it is difficult to justify germline genome editing from the perspective of the prospective child. As suggested by the UK legalization regarding mitochondrial donation, the prevention of genetic disease in offspring by genome editing might be acceptable in limited cases of serious or life-threatening conditions, where no alternative medicine is available. Nonetheless, the mosaicism underlying human embryos as well as the off-target effect by artificial nucleases will likely hamper preimplantation genetic diagnosis prior to embryo transfer. Such considerations suggest that this type of reproductive medicine should not be developed toward a clinical application. However, the clinical uncertainties underscore the need for embryology that can address fundamental questions regarding germline aneuploidy and mosaicism using genome editing. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Developmental and genetic analysis of a short leaf mutant, a key resource for plant architecture modification in sorghum

USDA-ARS?s Scientific Manuscript database

Modification in plant architecture have been demonstrated as one of the major contributing factors that ushered in the Green Revolution resulting in achieving dramatic increases in grain yield for wheat and rice. For sorghum (Sorghum bicolor L. Moench.), possible alteration in plant architecture is ...

Improvement of physical properties of calcium phosphate cement by elastin-like polypeptide supplementation.

PubMed

Jang, Ji-Hyun; Shin, Sumi; Kim, Hyun-Jung; Jeong, Jinyoung; Jin, Hyo-Eon; Desai, Malav S; Lee, Seung-Wuk; Kim, Sun-Young

2018-03-26

Calcium phosphate cements (CPCs) are synthetic bioactive cements widely used as hard tissue substitutes. Critical limitations of use include their poor mechanical properties and poor anti-washout behaviour. To address those limitations, we combined CPC with genetically engineered elastin-like polypeptides (ELPs). We investigated the effect of the ELPs on the physical properties and biocompatibility of CPC by testing ELP/CPC composites with various liquid/powder ratios. Our results show that the addition of ELPs improved the mechanical properties of the CPC, including the microhardness, compressive strength, and washout resistance. The biocompatibility of ELP/CPC composites was also comparable to that of the CPC alone. However, supplementing CPC with ELPs functionalized with octaglutamate as a hydroxyapatite binding peptide increased the setting time of the cement. With further design and modification of our biomolecules and composites, our research will lead to products with diverse applications in biology and medicine.

A Simple And Rapid Minicircle DNA Vector Manufacturing System

PubMed Central

Kay, Mark A; He, Cheng-Yi; Chen, Zhi-Ying

2010-01-01

Minicircle DNA vectors consisting of a circular expression cassette devoid of the bacterial plasmid DNA backbone provides several advantages including sustained transgene expression in quiescent cells/tissues. Their use has been limited by labor-intensive production. We report on a strategy for making multiple genetic modifications in E.coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, the øC31-integrase and I-SceI homing-endonuclease. This bacterial strain is capable of producing highly purified minicircle yields in the same time frame as routine plasmid DNA. It is now feasible for minicircle DNA vectors to replace routine plasmids in mammalian transgene expression studies. PMID:21102455

Post-mortem re-cloning of a transgenic red fluorescent protein dog

PubMed Central

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo

2011-01-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908

40 CFR 62.15160 - What emission limits must I meet?

Code of Federal Regulations, 2010 CFR

2010-07-01

... modification after June 26, 1987, then you must comply with the dioxins/furans and mercury emission limits... operating permit, if a permit modification is required. Final compliance with the dioxins/furans limits must...

Disruption of FGF5 in Cashmere Goats Using CRISPR/Cas9 Results in More Secondary Hair Follicles and Longer Fibers

PubMed Central

Zhu, Haijing; Niu, Yiyuan; Ma, Baohua; Yu, Honghao; Lei, Anmin; Yan, Hailong; Shen, Qiaoyan; Shi, Lei; Zhao, Xiaoe; Hua, Jinlian; Huang, Xingxu; Qu, Lei; Chen, Yulin

2016-01-01

Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes. PMID:27755602

Targeted gene knockin in porcine somatic cells using CRISPR/Cas ribonucleoproteins

USDA-ARS?s Scientific Manuscript database

The domestic 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, physiolo...

Gene flow in genetically engineered perennial grasses: Lessons for modification of dedicated bioenergy crops

EPA Science Inventory

The potential ecological consequences of the commercialization of genetically engineered (GD) crops have been the subject of intense debate, particularly when the GE crops are perennial and capable of outcrossing to wild relatives. The essential ecological impact issues for engi...

Reasonable Foreseeability and Liability in Relation to Genetically Modified Organisms

ERIC Educational Resources Information Center

Khoury, Lara; Smyth, Stuart

2007-01-01

This article examines problems that may arise when addressing liability resulting from the genetic modification of microbes, animals, and plants. More specifically, it evaluates how uncertainties relating to the outcomes of these biotechnological innovations affect--or may affect--the courts' application of the reasonable foreseeability…

Epigenetic Contribution of the Myosin Light Chain Kinase Gene to the Risk for Acute Respiratory Distress Syndrome

PubMed Central

Szilágyi, Keely L.; Liu, Cong; Zhang, Xu; Wang, Ting; Fortman, Jeffrey D.; Zhang, Wei; Garcia, Joe G.N.

2016-01-01

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with a considerable case fatality rate (~30-40%). Health disparities exist with African descent subjects (ADs) exhibiting greater mortality than European descent individuals (EDs). Myosin light chain kinase (MLCK) is encoded by MYLK whose genetic variants are implicated in ARDS pathogenesis and may influence ARDS mortality. As baseline population-specific epigenetic changes, i.e. cytosine modifications, have been observed between AD and ED individuals, epigenetic variations in MYLK may provide insights into ARDS disparities. We compared methylation levels of MYLK CpGs between ARDS patients and ICU controls overall and by ethnicity in a nested case control study of 39 ARDS cases and 75 non-ARDS intensive care unit controls. Two MYLK CpG sites (cg03892735, cg23344121) were differentially modified between ARDS subjects and controls (p<0.05; q<0.25) in a logistic regression model, where no effect modification from ethnicity or age was found. One CpG site was associated with ARDS in patients less than 58 years old, cg19611163 (intron 19,20). Two CpG sites were associated with ARDS in EDs only, gene body CpG (cg01894985, intron 2,3) and CpG (cg16212219, intron 31,32), with higher modification levels exhibited in ARDS subjects than controls. Cis-acting mQTL (modified cytosine quantitative trait loci) were identified using linear regression between local genetic variants and modification levels for two ARDS-associated CpGs (cg23344121, cg16212219). In summary, these ARDS-associated MYLK CpGs with effect modification by ethnicity and local mQTL, suggest that MYLK epigenetic variation and local genetic background may contribute to health disparities observed in ARDS. PMID:27543902

The Use of Gene Modification and Advanced Molecular Structure Analyses towards Improving Alfalfa Forage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lei, Yaogeng; Hannoufa, Abdelali; Yu, Peiqiang

Alfalfa is one of the most important legume forage crops in the world. In spite of its agronomic and nutritive advantages, alfalfa has some limitations in the usage of pasture forage and hay supplement. High rapid degradation of protein in alfalfa poses a risk of rumen bloat to ruminants which could cause huge economic losses for farmers. Coupled with the relatively high lignin content, which impedes the degradation of carbohydrate in rumen, alfalfa has unbalanced and asynchronous degradation ratio of nitrogen to carbohydrate (N/CHO) in rumen. Genetic engineering approaches have been used to manipulate the expression of genes involved inmore » important metabolic pathways for the purpose of improving the nutritive value, forage yield, and the ability to resist abiotic stress. Such gene modification could bring molecular structural changes in alfalfa that are detectable by advanced structural analytical techniques. These structural analyses have been employed in assessing alfalfa forage characteristics, allowing for rapid, convenient and cost-effective analysis of alfalfa forage quality. In this article, we review two major obstacles facing alfalfa utilization, namely poor protein utilization and relatively high lignin content, and highlight genetic studies that were performed to overcome these drawbacks, as well as to introduce other improvements to alfalfa quality. We also review the use of advanced molecular structural analysis in the assessment of alfalfa forage for its potential usage in quality selection in alfalfa breeding.« less

Protein engineering and its applications in food industry.

PubMed

Kapoor, Swati; Rafiq, Aasima; Sharma, Savita

2017-07-24

Protein engineering is a young discipline that has been branched out from the field of genetic engineering. Protein engineering is based on the available knowledge about the proteins structure/function(s), tools/instruments, software, bioinformatics database, available cloned gene, knowledge about available protein, vectors, recombinant strains and other materials that could lead to change in the protein backbone. Protein produced properly from genetic engineering process means a protein that is able to fold correctly and to do particular function(s) efficiently even after being subjected to engineering practices. Protein is modified through its gene or chemically. However, modification of protein through gene is easier. There is no specific limitation of Protein Engineering tools; any technique that can lead to change the protein constituent of amino acid and result in the modification of protein structure/function is in the frame of Protein Engineering. Meanwhile, there are some common tools used to reach a specific target. More active industrial and pharmaceutical based proteins have been invented by the field of Protein Engineering to introduce new function as well as to change its interaction with surrounding environment. A variety of protein engineering applications have been reported in the literature. These applications range from biocatalysis for food and industry to environmental, medical and nanobiotechnology applications. Successful combinations of various protein engineering methods had led to successful results in food industries and have created a scope to maintain the quality of finished product after processing.

Sex determination strategies in 2012: towards a common regulatory model?

PubMed Central

2012-01-01

Sex determination is a complicated process involving large-scale modifications in gene expression affecting virtually every tissue in the body. Although the evolutionary origin of sex remains controversial, there is little doubt that it has developed as a process of optimizing metabolic control, as well as developmental and reproductive functions within a given setting of limited resources and environmental pressure. Evidence from various model organisms supports the view that sex determination may occur as a result of direct environmental induction or genetic regulation. The first process has been well documented in reptiles and fish, while the second is the classic case for avian species and mammals. Both of the latter have developed a variety of sex-specific/sex-related genes, which ultimately form a complete chromosome pair (sex chromosomes/gonosomes). Interestingly, combinations of environmental and genetic mechanisms have been described among different classes of animals, thus rendering the possibility of a unidirectional continuous evolutionary process from the one type of mechanism to the other unlikely. On the other hand, common elements appear throughout the animal kingdom, with regard to a) conserved key genes and b) a central role of sex steroid control as a prerequisite for ultimately normal sex differentiation. Studies in invertebrates also indicate a role of epigenetic chromatin modification, particularly with regard to alternative splicing options. This review summarizes current evidence from research in this hot field and signifies the need for further study of both normal hormonal regulators of sexual phenotype and patterns of environmental disruption. PMID:22357269

Understanding RNA modifications: the promises and technological bottlenecks of the ‘epitranscriptome’

PubMed Central

Kapoor, Utkarsh

2017-01-01

The discovery of mechanisms that alter genetic information via RNA editing or introducing covalent RNA modifications points towards a complexity in gene expression that challenges long-standing concepts. Understanding the biology of RNA modifications represents one of the next frontiers in molecular biology. To this date, over 130 different RNA modifications have been identified, and improved mass spectrometry approaches are still adding to this list. However, only recently has it been possible to map selected RNA modifications at single-nucleotide resolution, which has created a number of exciting hypotheses about the biological function of RNA modifications, culminating in the proposition of the ‘epitranscriptome’. Here, we review some of the technological advances in this rapidly developing field, identify the conceptual challenges and discuss approaches that are needed to rigorously test the biological function of specific RNA modifications. PMID:28566301

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

PubMed Central

Carlile, Thomas M.; Rojas-Duran, Maria F.; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M.; Gilbert, Wendy V.

2014-01-01

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure2–8. mRNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function – it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding center9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as 100 novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease11–13. PMID:25192136

Constantly evolving safety assessment protocols for GM foods.

PubMed

Sesikeran, B; Vasanthi, Siruguri

2008-01-01

he introduction of GM foods has led to the evolution of a food safety assessment paradigm that establishes safety of the GM food relative to its conventional counterpart. The GM foods currently approved and marketed in several countries have undergone extensive safety testing under a structured safety assessment framework evolved by international organizations like FAO, WHO, Codex and OECD. The major elements of safety assessment include molecular characterization of inserted genes and stability of the trait, toxicity and allergenicity potential of the expressed substances, compositional analysis, potential for gene transfer to gut microflora and unintentional effects of the genetic modification. As more number and type of food crops are being brought under the genetic modification regime, the adequacy of existing safety assessment protocols for establishing safety of these foods has been questioned. Such crops comprise GM crops with higher agronomic vigour, nutritional or health benefit/ by modification of plant metabolic pathways and those expressing bioactive substances and pharmaceuticals. The safety assessment challenges of these foods are the potential of the methods to detect unintentional effects with higher sensitivity and rigor. Development of databases on food compositions, toxicants and allergens is currently seen as an important aid to development of safety protocols. With the changing global trends in genetic modification technology future challenge would be to develop GM crops with minimum amount of inserted foreign DNA so as to reduce the burden of complex safety assessments while ensuring safety and utility of the technology.

A pooling-based approach to mapping genetic variants associated with DNA methylation

PubMed Central

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; McEwen, Lisa M.; Kobor, Michael S.; Fraser, Hunter B.

2015-01-01

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a truly genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. We found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data. PMID:25910490

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

PubMed

Vinayak, Sumiti; Pawlowic, Mattie C; Sateriale, Adam; Brooks, Carrie F; Studstill, Caleb J; Bar-Peled, Yael; Cipriano, Michael J; Striepen, Boris

2015-07-23

Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

A pooling-based approach to mapping genetic variants associated with DNA methylation

DOE PAGES

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; ...

2015-04-24

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a trulymore » genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. Here we found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.« less

Catchment-Scale Conservation Units Identified for the Threatened Yarra Pygmy Perch (Nannoperca obscura) in Highly Modified River Systems

PubMed Central

Brauer, Chris J.; Unmack, Peter J.; Hammer, Michael P.; Adams, Mark; Beheregaray, Luciano B.

2013-01-01

Habitat fragmentation caused by human activities alters metapopulation dynamics and decreases biological connectivity through reduced migration and gene flow, leading to lowered levels of population genetic diversity and to local extinctions. The threatened Yarra pygmy perch, Nannoperca obscura, is a poor disperser found in small, isolated populations in wetlands and streams of southeastern Australia. Modifications to natural flow regimes in anthropogenically-impacted river systems have recently reduced the amount of habitat for this species and likely further limited its opportunity to disperse. We employed highly resolving microsatellite DNA markers to assess genetic variation, population structure and the spatial scale that dispersal takes place across the distribution of this freshwater fish and used this information to identify conservation units for management. The levels of genetic variation found for N. obscura are amongst the lowest reported for a fish species (mean heterozygosity of 0.318 and mean allelic richness of 1.92). We identified very strong population genetic structure, nil to little evidence of recent migration among demes and a minimum of 11 units for conservation management, hierarchically nested within four major genetic lineages. A combination of spatial analytical methods revealed hierarchical genetic structure corresponding with catchment boundaries and also demonstrated significant isolation by riverine distance. Our findings have implications for the national recovery plan of this species by demonstrating that N. obscura populations should be managed at a catchment level and highlighting the need to restore habitat and avoid further alteration of the natural hydrology. PMID:24349405

Reconciling genetic evolution and the associative learning account of mirror neurons through data-acquisition mechanisms.

PubMed

Lotem, Arnon; Kolodny, Oren

2014-04-01

An associative learning account of mirror neurons should not preclude genetic evolution of its underlying mechanisms. On the contrary, an associative learning framework for cognitive development should seek heritable variation in the learning rules and in the data-acquisition mechanisms that construct associative networks, demonstrating how small genetic modifications of associative elements can give rise to the evolution of complex cognition.

Research progress on bladder cancer molecular genetics.

PubMed

Kang, Zhengjun; Li, Yuhui; Yu, Yang; Guo, Zhan

2014-11-01

Bladder cancer is a common malignant urinary tumor with a high rate of recurrence and quick progression, which threats human health. With the research on bladder cancer molecular genetics, the knowledge of gene modification and the development of molecular detection methods, more tumor markers have been discovered, which may have potential for early diagnosis, clinical examination and prognosis. This article reviews the research progress on bladder cancer molecular genetics.

Administration Modifications on the WISC-R Performance Scale with Different Categories of Deaf Children.

ERIC Educational Resources Information Center

Sullivan, Patricia M.

1982-01-01

Two studies investigated the effects of administration modifications on subtest scaled scores of the Wechsler-Intelligence Scale for Children-Revised (WISC-R). Performance scale rated different groups of 57 severely/profoundly hearing-impaired children. Total communication was found to result in higher scores on all subtests in the genetic and…

78 FR 13476 - Modification of the Port Limits of Green Bay, WI

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-28

... limits is included in the docket as ``Attachment B: Green Bay (Proposed).'' Interested parties were given.... USCBP-2011-0031; CBP Dec. 13-2] Modification of the Port Limits of Green Bay, WI AGENCY: U.S. Customs... geographical limits of the port of Green Bay, Wisconsin. The port limits will be revised to refer to...

Genetically modified (GM) crops: milestones and new advances in crop improvement.

PubMed

Kamthan, Ayushi; Chaudhuri, Abira; Kamthan, Mohan; Datta, Asis

2016-09-01

New advances in crop genetic engineering can significantly pace up the development of genetically improved varieties with enhanced yield, nutrition and tolerance to biotic and abiotic stresses. Genetically modified (GM) crops can act as powerful complement to the crops produced by laborious and time consuming conventional breeding methods to meet the worldwide demand for quality foods. GM crops can help fight malnutrition due to enhanced yield, nutritional quality and increased resistance to various biotic and abiotic stresses. However, several biosafety issues and public concerns are associated with cultivation of GM crops developed by transgenesis, i.e., introduction of genes from distantly related organism. To meet these concerns, researchers have developed alternative concepts of cisgenesis and intragenesis which involve transformation of plants with genetic material derived from the species itself or from closely related species capable of sexual hybridization, respectively. Recombinase technology aimed at site-specific integration of transgene can help to overcome limitations of traditional genetic engineering methods based on random integration of multiple copy of transgene into plant genome leading to gene silencing and unpredictable expression pattern. Besides, recently developed technology of genome editing using engineered nucleases, permit the modification or mutation of genes of interest without involving foreign DNA, and as a result, plants developed with this technology might be considered as non-transgenic genetically altered plants. This would open the doors for the development and commercialization of transgenic plants with superior phenotypes even in countries where GM crops are poorly accepted. This review is an attempt to summarize various past achievements of GM technology in crop improvement, recent progress and new advances in the field to develop improved varieties aimed for better consumer acceptance.

Unexpected consequences of genetic selection in broilers and turkeys: problems and solutions.

PubMed

Hocking, P M

2014-02-01

1. Genetic theory leads to the expectation that unexpected consequences of genetic selection for production traits will inevitably occur and that these changes are likely to be undesirable. 2. Both artificial selection for production efficiency and "natural" selection for adaptation to the production environment result in selection sweeps that increase the frequencies of rare recessive alleles that have a negative effect on fitness. 3. Fitness is broadly defined as any trait that affects the ability to survive, reproduce and contribute to the next generation, such as musculoskeletal disease in growing broiler chickens and multiple ovulation in adult broiler parents. 4. Welfare concerns about the negative effects of genetic selection on bird welfare are sometimes exaggerated but are nevertheless real. Breeders have paid increasing attention to these traits over several decades and have demonstrated improvement in pedigree flocks. There is an urgent need to monitor changes in commercial flocks to ensure that genetic change is accompanied by improvements in that target population. 5. New technologies for trait measurement, whole genome selection and targeted genetic modification hold out the promise of efficient and rapid improvement of welfare traits in future breeding of broiler chickens and turkeys. The potential of targeted genetic modification for enhancing welfare traits is considerable, but the goal of achieving public acceptability for the progeny of transgenic poultry will be politically challenging.

Social and ethical issues in mitochondrial donation.

PubMed

Dimond, Rebecca

2015-09-01

The UK is at the forefront of mitochondrial science and is currently the only country in the world to legalize germ-line technologies involving mitochondrial donation. However, concerns have been raised about genetic modification and the 'slippery slope' to designer babies. This review uses academic articles, newspaper reports and public documents. Mitochondrial donation offers women with mitochondrial disease an opportunity to have healthy, genetically related children. Key areas of disagreement include safety, the creation of three-parent babies, impact on identity, implications for society, definitions of genetic modification and reproductive choice. The UK government legalized the techniques in March 2015. Scientific and medical communities across the world followed the developments with interest. It is expected that the first cohort of 'three parent' babies will be born in the UK in 2016. Their health and progress will be closely monitored. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular Mechanisms and Management of a Cutaneous Inflammatory Disorder: Psoriasis

PubMed Central

Cho, Dae Ho; Park, Hyun Jeong

2017-01-01

Psoriasis is a complex chronic inflammatory cutaneous disorder. To date, robust molecular mechanisms of psoriasis have been reported. Among diverse aberrant immunopathogenetic mechanisms, the current model emphasizes the role of Th1 and the IL-23/Th17 axis, skin-resident immune cells and major signal transduction pathways involved in psoriasis. The multiple genetic risk loci for psoriasis have been rapidly revealed with the advent of a novel technology. Moreover, identifying epigenetic modifications could bridge the gap between genetic and environmental risk factors in psoriasis. This review will provide a better understanding of the pathogenesis of psoriasis by unraveling the complicated interplay among immunological abnormalities, genetic risk foci, epigenetic modification and environmental factors of psoriasis. With advances in molecular biology, diverse new targets are under investigation to manage psoriasis. The recent advances in treatment modalities for psoriasis based on targeted molecules are also discussed. PMID:29232931

Genetic modification of stem cells for improved therapy of the infarcted myocardium.

PubMed

Haider, Husnain Kh; Mustafa, Anique; Feng, Yuliang; Ashraf, Muhammad

2011-10-03

The conventional treatment modalities for ischemic heart disease only provide symptomatic relief to the patient without repairing and regenerating the damaged myocardium. Stem cell transplantation has emerged as a promising alternative therapeutic approach for cardiovascular diseases. Stem cells possess the potential of differentiation to adopt morphofunctional cardiac and vasculogenic phenotypes to repopulate the scar tissue and restore regional blood flow in the ischemic myocardium. These beneficial therapeutic effects make stem cell transplantation the method of choice for the treatment of ischemic heart disease. The efficacy of stem cell transplantation may be augmented by genetic manipulation of the cells prior to transplantation. Not only will insertion of therapeutic transgene(s) into the stem cells support the survival and differentiation of cells in the unfavorable microenvironment of the ischemic myocardium, but also the genetically manipulated stem cells will serve as a source of the transgene expression product in the heart for therapeutic benefits. We provide an overview of the extensively studied stem cell types for cardiac regeneration, the various methods in which these cells have been genetically manipulated and rationale of genetic modification of stem cells for use in regenerative cardiovascular therapeutics.

Mobilome and genetic modification of bifidobacteria.

PubMed

Guglielmetti, S; Mayo, B; Álvarez-Martín, P

2013-06-01

Until recently, proper development of molecular studies in Bifidobacterium species has been hampered by growth difficulties, because of their exigent nutritive requirements, oxygen sensitivity and lack of efficient genetic tools. These studies, however, are critical to uncover the cross-talk between bifidobacteria and their hosts' cells and to prove unequivocally the supposed beneficial effects provided through the endogenous bifidobacterial populations or after ingestion as probiotics. The genome sequencing projects of different bifidobacterial strains have provided a wealth of genetic data that will be of much help in deciphering the molecular basis of the physiological properties of bifidobacteria. To this end, the purposeful development of stable cloning and expression vectors based on robust replicons - either from temperate phages or resident plasmids - is still needed. This review addresses the current knowledge on the mobile genetic elements of bifidobacteria (prophages, plasmids and transposons) and summarises the different types of vectors already available, together with the transformation procedures for introducing DNA into the cells. It also covers recent molecular studies performed with such vectors and incipient results on the genetic modification of these organisms, establishing the basis that would allow the use of bifidobacteria for future biotechnological applications.

75 FR 9814 - Airworthiness Directives; Empresa Brasileira de Aeronautica S.A. (EMBRAER) Model ERJ 190-100 STD...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-04

... airplane Airworthiness Limitation Items (ALI), to include new inspections tasks or modification of existing... (ALI), to include new inspections tasks or modification of existing ones and its respective thresholds... resulted in modifications on the airplane Airworthiness Limitation Items (ALI), to include new inspections...

Genetic structure in the southernmost populations of black-and-gold howler monkeys (Alouatta caraya) and its conservation implications

PubMed Central

Miño, Carolina Isabel; Fernández, Gabriela; Caputo, Mariela; Corach, Daniel

2017-01-01

Black-and-gold howler monkeys Alouatta caraya, are arboreal primates, inhabitants of Neotropical forests, highly susceptible to the yellow fever virus, considered early 'sentinels' of outbreaks, and thus, of major epidemiological importance. Currently, anthropogenic habitat loss and modifications threatens their survival. Habitat modification can prevent, reduce or change dispersal behavior, which, in turn, may influence patterns of gene flow. We explored past and contemporary levels of genetic diversity, elucidated genetic structure and identified its possible drivers, in ten populations (n = 138) located in the southernmost distribution range of the species in South America, in Argentina and Paraguay. Overall, genetic variability was moderate (ten microsatellites: 3.16 ± 0.18 alleles per locus, allelic richness of 2.93 ± 0.81, 0.443±0.025 unbiased expected heterozygosity; 22 haplotypes of 491-bp mitochondrial Control Region, haplotypic diversity of 0.930 ± 0.11, and nucleotide diversity of0.01± 0.007). Significant evidence of inbreeding was found in a population that was, later, decimated by yellow fever. Population-based gene flow measures (FST = 0.13; θST = 018), hierarchical analysis of molecular variance and Bayesian clustering methods revealed significant genetic structure, grouping individuals into four clusters. Shared haplotypes and lack of mitochondrial differentiation (non-significant θST) among some populations seem to support the hypothesis of historical dispersal via riparian forests. Current resistance analyses revealed a significant role of landscape features in modeling contemporary gene flow: continuous forest and riparian forests could promote genetic exchange, whereas disturbed forests or crop/grassland fields may restrict it. Estimates of effective population size allow anticipating that the studied populations will lose 75% of heterozygosity in less than 50 generations. Our findings suggest that anthropogenic modifications on native forests, increasingly ongoing in Northeastern Argentina, Southern Paraguay and Southeastern Brazil, might prevent the dispersal of howlers, leading to population isolation. To ensure long-term viability and maintain genetic connectivity of A. caraya remnant populations, we recommend preserving and restoring habitat continuity. To conserve the species genetic pool, as well, the four genetic clusters identified here should be considered separate Management Units and given high conservation priority. In light of our findings and considering complementary non-genetic information, we suggest upgrading the international conservation status of A. caraya to “Vulnerable”. PMID:28968440

Genetic engineering applied to agriculture has a long row to hoe.

PubMed

Miller, Henry I

2018-01-02

In spite of the lack of scientific justification for skepticism about crops modified with molecular techniques of genetic engineering, they have been the most scrutinized agricultural products in human history. The assumption that "genetically engineered" or "genetically modified" is a meaningful - and dangerous - classification has led to excessive and dilatory regulation. The modern molecular techniques are an extension, or refinement, of older, less precise, less predictable methods of genetic modification, but as long as today's activists and regulators remain convinced that so called "GMOs" represent a distinct and dangerous category of research and products, genetic engineering will fall short of its potential.

Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

PubMed Central

Lan, Feng; Liu, Junwei; Narsinh, Kazim H.; Hu, Shijun; Han, Leng; Lee, Andrew S.; Karow, Marisa; Nguyen, Patricia K.; Nag, Divya; Calos, Michele P.; Robbins, Robert C.; Wu, Joseph C.

2012-01-01

Background Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. Methods and Results We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. Conclusions The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types. PMID:22965984

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

PubMed

Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

2012-09-11

Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

Epigenetic Modifications of Major Depressive Disorder

PubMed Central

Saavedra, Kathleen; Molina-Márquez, Ana María; Saavedra, Nicolás; Zambrano, Tomás; Salazar, Luis A.

2016-01-01

Major depressive disorder (MDD) is a chronic disease whose neurological basis and pathophysiology remain poorly understood. Initially, it was proposed that genetic variations were responsible for the development of this disease. Nevertheless, several studies within the last decade have provided evidence suggesting that environmental factors play an important role in MDD pathophysiology. Alterations in epigenetics mechanism, such as DNA methylation, histone modification and microRNA expression could favor MDD advance in response to stressful experiences and environmental factors. The aim of this review is to describe genetic alterations, and particularly altered epigenetic mechanisms, that could be determinants for MDD progress, and how these alterations may arise as useful screening, diagnosis and treatment monitoring biomarkers of depressive disorders. PMID:27527165

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model.

PubMed

Goodrich, L R; Hidaka, C; Robbins, P D; Evans, C H; Nixon, A J

2007-05-01

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.

Investigating Novice and Expert Conceptions of Genetically Modified Organisms

ERIC Educational Resources Information Center

Potter, Lisa M.; Bissonnette, Sarah A.; Knight, Jonathan D.; Tanner, Kimberly D.

2017-01-01

The aspiration of biology education is to give students tools to apply knowledge learned in the classroom to everyday life. Genetic modification is a real-world biological concept that relies on an in-depth understanding of the molecular behavior of DNA and proteins. This study investigated undergraduate biology students' conceptions of…

A comparison of protein and phenolic compounds in seed from GMO and non-GMO soybean

USDA-ARS?s Scientific Manuscript database

Soybean protein is a valuable and important component in human and animal diets. Approximately 94% of the soybean planted in the US is genetically modified (GM) to enhance quality and productivity. Since value-added traits are continuously being developed by genetic modification, it is important t...

Genetically Modified Food: Knowledge and Attitude of Teachers and Students

ERIC Educational Resources Information Center

Mohapatra, Animesh K.; Priyadarshini, Deepika; Biswas, Antara

2010-01-01

The concepts behind the technology of genetic modification of organisms and its applications are complex. A diverse range of opinions, public concern and considerable media interest accompanies the subject. This study explores the knowledge and attitudes of science teachers and senior secondary biology students about the application of a rapidly…

Genetic engineering of stem cells for enhanced therapy.

PubMed

Nowakowski, Adam; Andrzejewska, Anna; Janowski, Miroslaw; Walczak, Piotr; Lukomska, Barbara

2013-01-01

Stem cell therapy is a promising strategy for overcoming the limitations of current treatment methods. The modification of stem cell properties may be necessary to fully exploit their potential. Genetic engineering, with an abundance of methodology to induce gene expression in a precise and well-controllable manner, is particularly attractive for this purpose. There are virus-based and non-viral methods of genetic manipulation. Genome-integrating viral vectors are usually characterized by highly efficient and long-term transgene expression, at a cost of safety. Non-integrating viruses are also highly efficient in transduction, and, while safer, offer only a limited duration of transgene expression. There is a great diversity of transfectable forms of nucleic acids; however, for efficient shuttling across cell membranes, additional manipulation is required. Both physical and chemical methods have been employed for this purpose. Stem cell engineering for clinical applications is still in its infancy and requires further research. There are two main strategies for inducing transgene expression in therapeutic cells: transient and permanent expression. In many cases, including stem cell trafficking and using cell therapy for the treatment of rapid-onset disease with a short healing process, transient transgene expression may be a sufficient and optimal approach. For that purpose, mRNA-based methods seem ideally suited, as they are characterized by a rapid, highly efficient transfection, with outstanding safety. Permanent transgene expression is primarily based on the application of viral vectors, and, due to safety concerns, these methods are more challenging. There is active, ongoing research toward the development of non-viral methods that would induce permanent expression, such as transposons and mammalian artificial chromosomes.

[The discussion of the infiltrative model of mathematical knowledge to genetics teaching].

PubMed

Liu, Jun; Luo, Pei-Gao

2011-11-01

Genetics, the core course of biological field, is an importance major-basic course in curriculum of many majors related with biology. Due to strong theoretical and practical as well as abstract of genetics, it is too difficult to study on genetics for many students. At the same time, mathematics is one of the basic courses in curriculum of the major related natural science, which has close relationship with the establishment, development and modification of genetics. In this paper, to establish the intrinsic logistic relationship and construct the integral knowledge network and to help students improving the analytic, comprehensive and logistic abilities, we applied some mathematical infiltrative model genetic knowledge in genetics teaching, which could help students more deeply learn and understand genetic knowledge.

Conservation and Divergence in the Candida Species Biofilm Matrix Mannan-Glucan Complex Structure, Function, and Genetic Control.

PubMed

Dominguez, Eddie; Zarnowski, Robert; Sanchez, Hiram; Covelli, Antonio S; Westler, William M; Azadi, Parastoo; Nett, Jeniel; Mitchell, Aaron P; Andes, David R

2018-04-03

Candida biofilms resist the effects of available antifungal therapies. Prior studies with Candida albicans biofilms show that an extracellular matrix mannan-glucan complex (MGCx) contributes to antifungal sequestration, leading to drug resistance. Here we implement biochemical, pharmacological, and genetic approaches to explore a similar mechanism of resistance for the three most common clinically encountered non- albicans Candida species (NAC). Our findings reveal that each Candida species biofilm synthesizes a mannan-glucan complex and that the antifungal-protective function of this complex is conserved. Structural similarities extended primarily to the polysaccharide backbone (α-1,6-mannan and β-1,6-glucan). Surprisingly, biochemical analysis uncovered stark differences in the branching side chains of the MGCx among the species. Consistent with the structural analysis, similarities in the genetic control of MGCx production for each Candida species also appeared limited to the synthesis of the polysaccharide backbone. Each species appears to employ a unique subset of modification enzymes for MGCx synthesis, likely accounting for the observed side chain diversity. Our results argue for the conservation of matrix function among Candida spp. While biogenesis is preserved at the level of the mannan-glucan complex backbone, divergence emerges for construction of branching side chains. Thus, the MGCx backbone represents an ideal drug target for effective pan- Candida species biofilm therapy. IMPORTANCE Candida species, the most common fungal pathogens, frequently grow as a biofilm. These adherent communities tolerate extremely high concentrations of antifungal agents, due in large part, to a protective extracellular matrix. The present studies define the structural, functional, and genetic similarities and differences in the biofilm matrix from the four most common Candida species. Each species synthesizes an extracellular mannan-glucan complex (MGCx) which contributes to sequestration of antifungal drug, shielding the fungus from this external assault. Synthesis of a common polysaccharide backbone appears conserved. However, subtle structural differences in the branching side chains likely rely upon unique modification enzymes, which are species specific. Our findings identify MGCx backbone synthesis as a potential pan- Candida biofilm therapeutic target. Copyright © 2018 Dominguez et al.

Dealing with an Unconventional Genetic Code in  Mitochondria: The Biogenesis and Pathogenic  Defects of the 5-Formylcytosine Modification in  Mitochondrial tRNAMet.

PubMed

Van Haute, Lindsey; Powell, Christopher A; Minczuk, Michal

2017-03-02

Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt-tRNAMet) also recognises AUA during translation initiation and elongation. Post-transcriptional modifications of tRNAs are important for structure, stability, correct folding and aminoacylation as well as decoding. The unique 5-formylcytosine (f5C) modification of position 34 in mt-tRNAMet has been long postulated to be crucial for decoding of unconventional methionine codons and efficient mitochondrial translation. However, the enzymes responsible for the formation of mitochondrial f5C have been identified only recently. The first step of the f5C pathway consists of methylation of cytosine by NSUN3. This is followed by further oxidation by ABH1. Here, we review the role of f5C, the latest breakthroughs in our understanding of the biogenesis of this unique mitochondrial tRNA modification and its involvement in human disease.

Epigenomics of Hypertension

PubMed Central

Liang, Mingyu; Cowley, Allen W.; Mattson, David L.; Kotchen, Theodore A.; Liu, Yong

2013-01-01

Multiple genes and pathways are involved in the pathogenesis of hypertension. Epigenomic studies of hypertension are beginning to emerge and hold great promise of providing novel insights into the mechanisms underlying hypertension. Epigenetic marks or mediators including DNA methylation, histone modifications, and non-coding RNA can be studied at a genome or near-genome scale using epigenomic approaches. At the single gene level, several studies have identified changes in epigenetic modifications in genes expressed in the kidney that correlate with the development of hypertension. Systematic analysis and integration of epigenetic marks at the genome scale, demonstration of cellular and physiological roles of specific epigenetic modifications, and investigation of inheritance are among the major challenges and opportunities for future epigenomic and epigenetic studies of hypertension. Essential hypertension is a multifactorial disease involving multiple genetic and environmental factors and mediated by alterations in multiple biological pathways. Because the non-genetic mechanisms may involve epigenetic modifications, epigenomics is one of the latest concepts and approaches brought to bear on hypertension research. In this article, we summarize briefly the concepts and techniques for epigenomics, discuss the rationale for applying epigenomic approaches to study hypertension, and review the current state of this research area. PMID:24011581

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

PubMed Central

Carroll, James A.; Olano, L. Rennee; Sturdevant, Daniel E.; Rosa, Patricia A.

2015-01-01

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. PMID:26655756

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

PubMed

Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

2016-02-15

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells

PubMed Central

Chicaybam, Leonardo; Barcelos, Camila; Peixoto, Barbara; Carneiro, Mayra; Limia, Cintia Gomez; Redondo, Patrícia; Lira, Carla; Paraguassú-Braga, Flávio; Vasconcelos, Zilton Farias Meira De; Barros, Luciana; Bonamino, Martin Hernán

2017-01-01

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology. PMID:28168187

Improving efficacy of cancer immunotherapy by genetic modification of natural killer cells.

PubMed

Burga, Rachel A; Nguyen, Tuongvan; Zulovich, Jane; Madonna, Sarah; Ylisastigui, Loyda; Fernandes, Rohan; Yvon, Eric

2016-11-01

Natural killer (NK) cells are members of the innate immune system that recognize target cells via activating and inhibitory signals received through cell receptors. Derived from the lymphoid lineage, NK cells are able to produce cytokines and exert a cytotoxic effect on viral infected and malignant cells. It is their unique ability to lyse target cells rapidly and without prior education that renders NK cells a promising effector cell for adoptive cell therapy. However, both viruses and tumors employ evasion strategies to avoid attack by NK cells, which represent biological challenges that need to be harnessed to fully exploit the cytolytic potential of NK cells. Using genetic modification, the function of NK cells can be enhanced to improve their homing, cytolytic activity, in vivo persistence and safety. Examples include gene modification to express chemokine, high-affinity Fc receptor and chimeric antigen receptors, suicide genes and the forced expression of cytokines such as interleukin (IL)-2 and IL-15. Preclinical studies have clearly demonstrated that such approaches are effective in improving NK-cell function, homing and safety. In this review, we summarize the recent advances in the genetic manipulations of NK cells and their application for cellular immunotherapeutic strategies. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Biosafety challenges for use of lentiviral vectors in gene therapy.

PubMed

Rothe, Michael; Modlich, Ute; Schambach, Axel

2013-12-01

Lentiviral vectors are promising tools for the genetic modification of cells in biomedical research and gene therapy. Their use in recent clinical trials for the treatment of adrenoleukodystrophy, β-thalassemia, Wiskott-Aldrich- Syndrome and metachromatic leukodystrophy underlined their efficacy for therapies especially in case of hereditary diseases. In comparison to gammaretroviral LTR-driven vectors, which were employed in the first clinical trials, lentiviral vectors present with some favorable features like the ability to transduce also non-dividing cells and a potentially safer insertion profile. However, genetic modification with viral vectors in general and stable integration of the therapeutic gene into the host cell genome bear concerns with respect to different levels of personal or environmental safety. Among them, insertional mutagenesis by enhancer mediated dysregulation of neighboring genes or aberrant splicing is still the biggest concern. However, also risks like immunogenicity of vector particles, the phenotoxicity of the transgene and potential vertical or horizontal transmission by replication competent retroviruses need to be taken into account. This review will give an overview on biosafety aspects that are relevant to the use of lentiviral vectors for genetic modification and gene therapy. Furthermore, assay systems aiming at evaluating biosafety in preclinical settings and recent promising clinical trials including efforts of monitoring of patients after gene therapy will be discussed.

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

PubMed Central

Ardui, Simon; Ameur, Adam; Vermeesch, Joris R; Hestand, Matthew S

2018-01-01

Abstract Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing. PMID:29401301

Cis-regulatory Elements and Human Evolution

PubMed Central

Siepel, Adam

2014-01-01

Modification of gene regulation has long been considered an important force in human evolution, particularly through changes to cis-regulatory elements (CREs) that function in transcriptional regulation. For decades, however, the study of cis-regulatory evolution was severely limited by the available data. New data sets describing the locations of CREs and genetic variation within and between species have now made it possible to study CRE evolution much more directly on a genome-wide scale. Here, we review recent research on the evolution of CREs in humans based on large-scale genomic data sets. We consider inferences based on primate divergence, human polymorphism, and combinations of divergence and polymorphism. We then consider “new frontiers” in this field stemming from recent research on transcriptional regulation. PMID:25218861

76 FR 75913 - Notice of Lodging of Modification of Consent Decree Under the Clean Water Act

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-05

... (``Regulated Bacteria'') and to comply with interim effluent limitations for those pollutants. The proposed Modification provides new, more stringent interim effluent limitations for Regulated Bacteria and requires... effluent limitations for Regulated Bacteria set forth in the Facility's National Pollutant Discharge...

Regulatory RNAs and chromatin modification in dosage compensation: a continuous path from flies to humans?

PubMed

Angelopoulou, Roxani; Lavranos, Giagkos; Manolakou, Panagiota

2008-03-20

Chromosomal sex determination is a widely distributed strategy in nature. In the most classic scenario, one sex is characterized by a homologue pair of sex chromosomes, while the other includes two morphologically and functionally distinct gonosomes. In mammalian diploid cells, the female is characterized by the presence of two identical X chromosomes, while the male features an XY pair, with the Y bearing the major genetic determinant of sex, i.e. the SRY gene. In other species, such as the fruitfly, sex is determined by the ratio of autosomes to X chromosomes. Regardless of the exact mechanism, however, all these animals would exhibit a sex-specific gene expression inequality, due to the different number of X chromosomes, a phenomenon inhibited by a series of genetic and epigenetic regulatory events described as "dosage compensation". Since adequate available data is currently restricted to worms, flies and mammals, while for other groups of animals, such as reptiles, fish and birds it is very limited, it is not yet clear whether this is an evolutionary conserved mechanism. However certain striking similarities have already been observed among evolutionary distant species, such as Drosophila melanogaster and Mus musculus. These mainly refer to a) the need for a counting mechanism, to determine the chromosomal content of the cell, i.e. the ratio of autosomes to gonosomes (a process well understood in flies, but still hypothesized in mammals), b) the implication of non-translated, sex-specific, regulatory RNAs (roX and Xist, respectively) as key elements in this process and the location of similar mediators in the Z chromosome of chicken c) the inclusion of a chromatin modification epigenetic final step, which ensures that gene expression remains stably regulated throughout the affected area of the gonosome. This review summarizes these points and proposes a possible role for comparative genetics, as they seem to constitute proof of maintained cell economy (by using the same basic regulatory elements in various different scenarios) throughout numerous centuries of evolutionary history.

Biopolymers Containing Unnatural Amino Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schultz, Peter

Although the main chain structure of polymers has a profound effect on their materials properties, the side groups can also have dramatic effects on their properties including conductivity, liquid crystallinity, hydrophobicity, elasticity and biodegradability. Unfortunately control over the side chain structure of polymers remains a challenge – it is difficult to control the sequence of chain elongation when mixtures of monomers are polymerized, and postpolymerization side chain modification is made difficult by polymer effects on side chain reactivity. In contrast, the mRNA templated synthesis of polypeptides on the ribosome affords absolute control over the primary sequence of the twenty aminomore » acid monomers. Moreover, the length of the biopolymer is precisely controlled as are sites of crosslinking. However, whereas synthetic polymers can be synthesized from monomers with a wide range of chemically defined structures, ribosomal biosynthesis is largely limited to the 20 canonical amino acids. For many applications in material sciences, additional building blocks would be desirable, for example, amino acids containing metallocene, photoactive, and halogenated side chains. To overcome this natural constraint we have developed a method that allows unnatural amino acids, beyond the common twenty, to be genetically encoded in response to nonsense or frameshift codons in bacteria, yeast and mammalian cells with high fidelity and good yields. Here we have developed methods that allow identical or distinct noncanonical amino acids to be incorporated at multiple sites in a polypeptide chain, potentially leading to a new class of templated biopolymers. We have also developed improved methods for genetically encoding unnatural amino acids. In addition, we have genetically encoded new amino acids with novel physical and chemical properties that allow selective modification of proteins with synthetic agents. Finally, we have evolved new metal-ion binding sites in proteins using a novel metal-ion binding amino acid, which may facilitate our ability to generate new protein based sensors and catalysts.« less

Biopolymers Containing Unnatural Building Blocks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schultz, Peter G.

2013-06-30

Although the main chain structure of polymers has a profound effect on their materials properties, the side groups can also have dramatic effects on their properties including conductivity, liquid crystallinity, hydrophobicity, elasticity and biodegradability. Unfortunately control over the side chain structure of polymers remains a challenge – it is difficult to control the sequence of chain elongation when mixtures of monomers are polymerized, and postpolymerization side chain modification is made difficult by polymer effects on side chain reactivity. In contrast, the mRNA templated synthesis of polypeptides on the ribosome affords absolute control over the primary sequence of the twenty aminomore » acid monomers. Moreover, the length of the biopolymer is precisely controlled as are sites of crosslinking. However, whereas synthetic polymers can be synthesized from monomers with a wide range of chemically defined structures, ribosomal biosynthesis is largely limited to the 20 canonical amino acids. For many applications in material sciences, additional building blocks would be desirable, for example, amino acids containing metallocene, photoactive, and halogenated side chains. To overcome this natural constraint we have developed a method that allows unnatural amino acids, beyond the common twenty, to be genetically encoded in response to nonsense or frameshift codons in bacteria, yeast and mammalian cells with high fidelity and good yields. Here we have developed methods that allow identical or distinct noncanonical amino acids to be incorporated at multiple sites in a polypeptide chain, potentially leading to a new class of templated biopolymers. We have also developed improved methods for genetically encoding unnatural amino acids. In addition, we have genetically encoded new amino acids with novel physical and chemical properties that allow selective modification of proteins with synthetic agents. Finally, we have evolved new metal-ion binding sites in proteins using a novel metal-ion binding amino acid, which may facilitate our ability to generate new protein based sensors and catalysts.« less

Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen.

PubMed

Biddle, Wil; Schmitt, Margaret A; Fisk, John D

2015-12-22

Understanding the interactions that drive the fidelity of the genetic code and the limits to which modifications can be made without breaking the translational system has practical implications for understanding the molecular mechanisms of evolution as well as expanding the set of encodable amino acids, particularly those with chemistries not provided by Nature. Because 61 sense codons encode 20 amino acids, reassigning the meaning of sense codons provides an avenue for biosynthetic modification of proteins, furthering both fundamental and applied biochemical research. We developed a simple screen that exploits the absolute requirement for fluorescence of an active site tyrosine in green fluorescent protein (GFP) to probe the pliability of the degeneracy of the genetic code. Our screen monitors the restoration of the fluorophore of GFP by incorporation of a tyrosine in response to a sense codon typically assigned another meaning in the genetic code. We evaluated sense codon reassignment at four of the 21 sense codons read through wobble interactions in Escherichia coli using the Methanocaldococcus jannaschii orthogonal tRNA/aminoacyl tRNA synthetase pair originally developed and commonly used for amber stop codon suppression. By changing only the anticodon of the orthogonal tRNA, we achieved sense codon reassignment efficiencies between 1% (Phe UUU) and 6% (Lys AAG). Each of the orthogonal tRNAs preferentially decoded the codon traditionally read via a wobble interaction in E. coli with the exception of the orthogonal tRNA with an AUG anticodon, which incorporated tyrosine in response to both the His CAU and His CAC codons with approximately equal frequencies. We applied our screen in a high-throughput manner to evaluate a 10(9)-member combined tRNA/aminoacyl tRNA synthetase library to identify improved sense codon reassigning variants for the Lys AAG codon. A single rapid screen with the ability to broadly evaluate reassignable codons will facilitate identification and improvement of the combinations of sense codons and orthogonal pairs that display efficient reassignment.

A strategy for genetic modification of the spike-encoding segment of human reovirus T3D for reovirus targeting.

PubMed

van den Wollenberg, D J M; van den Hengel, S K; Dautzenberg, I J C; Cramer, S J; Kranenburg, O; Hoeben, R C

2008-12-01

Human Orthoreovirus Type 3 Dearing is not pathogenic to humans and has been evaluated clinically as an oncolytic agent. Its transduction efficiency and the tumor cell selectivity may be enhanced by incorporating ligands for alternative receptors. However, the genetic modification of reoviruses has been difficult, and genetic targeting of reoviruses has not been reported so far. Here we describe a technique for generating genetically targeted reoviruses. The propagation of wild-type reoviruses on cells expressing a modified sigma 1-encoding segment embedded in a conventional RNA polymerase II transcript leads to substitution of the wild-type genome segment by the modified version. This technique was used for generating reoviruses that are genetically targeted to an artificial receptor expressed on U118MG cells. These cells lack the junction adhesion molecule-1 and therefore resist infection by wild-type reoviruses. The targeted reoviruses were engineered to carry the ligand for this receptor at the C terminus of the sigma 1 spike protein. This demonstrates that the C terminus of the sigma 1 protein is a suitable locale for the insertion of oligopeptide ligands and that targeting of reoviruses is feasible. The genetically targeted viruses can be propagated using the modified U118MG cells as helper cells. This technique may be applicable for the improvement of human reoviruses as oncolytic agents.

GenInfoGuard--a robust and distortion-free watermarking technique for genetic data.

PubMed

Iftikhar, Saman; Khan, Sharifullah; Anwar, Zahid; Kamran, Muhammad

2015-01-01

Genetic data, in digital format, is used in different biological phenomena such as DNA translation, mRNA transcription and protein synthesis. The accuracy of these biological phenomena depend on genetic codes and all subsequent processes. To computerize the biological procedures, different domain experts are provided with the authorized access of the genetic codes; as a consequence, the ownership protection of such data is inevitable. For this purpose, watermarks serve as the proof of ownership of data. While protecting data, embedded hidden messages (watermarks) influence the genetic data; therefore, the accurate execution of the relevant processes and the overall result becomes questionable. Most of the DNA based watermarking techniques modify the genetic data and are therefore vulnerable to information loss. Distortion-free techniques make sure that no modifications occur during watermarking; however, they are fragile to malicious attacks and therefore cannot be used for ownership protection (particularly, in presence of a threat model). Therefore, there is a need for a technique that must be robust and should also prevent unwanted modifications. In this spirit, a watermarking technique with aforementioned characteristics has been proposed in this paper. The proposed technique makes sure that: (i) the ownership rights are protected by means of a robust watermark; and (ii) the integrity of genetic data is preserved. The proposed technique-GenInfoGuard-ensures its robustness through the "watermark encoding" in permuted values, and exhibits high decoding accuracy against various malicious attacks.

Drug Addiction and DNA Modifications.

PubMed

Brown, Amber N; Feng, Jian

2017-01-01

Drug addiction is a complex disorder which can be influenced by both genetic and environmental factors. Research has shown that epigenetic modifications can translate environmental signals into changes in gene expression, suggesting that epigenetic changes may underlie the causes and possibly treatment of substance use disorders. This chapter will focus on epigenetic modifications to DNA, which include DNA methylation and several recently defined additional DNA epigenetic changes. We will discuss the functions of DNA modifications and methods for detecting them, followed by a description of the research investigating the function and consequences of drug-induced changes in DNA methylation patterns. Understanding these epigenetic changes may provide us translational tools for the diagnosis and treatment of addiction in the future.

An Update on Genetic Modification of Chickpea for Increased Yield and Stress Tolerance.

PubMed

Kumar, Manoj; Yusuf, Mohd Aslam; Nigam, Manisha; Kumar, Manoj

2018-06-26

Chickpea is a highly nutritious grain legume crop, widely appreciated as a health food, especially in the Indian subcontinent. The major constraints on chickpea production are biotic (Helicoverpa, bruchid, aphid, ascochyta) and abiotic (drought, heat, salt, cold) stresses, which reduce the yield by up to 90%. Various strategies like conventional breeding, molecular breeding, and modern plant breeding have been used to overcome these problems. Conventionally, breeding programs aim at development of varieties that combine maximum number of traits through inter-specific hybridization, wide hybridization, and hybridization involving more than two parents. Breeding is difficult in this crop because of its self-pollinating nature and limited genetic variation. Recent advances in in vitro culture and gene technologies offer unique opportunities to realize the full potential of chickpea production. However, as of date, no transgenic chickpea variety has been approved for cultivation in the world. In this review, we provide an update on the development of genetically modified chickpea plants, including those resistant to Helicoverpa armigera, Callosobruchus maculatus, Aphis craccivora, as well as to drought and salt stress. The genes utilized for development of resistance against pod borer, bruchid, aphid, drought, and salt tolerance, namely, Bt, alpha amylase inhibitor, ASAL, P5CSF129A, and P5CS, respectively, are discussed.

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

PubMed Central

Siggs, Owen M.; Miosge, Lisa A.; Roots, Carla M.; Enders, Anselm; Bertram, Edward M.; Crockford, Tanya L.; Whittle, Belinda; Potter, Paul K.; Simon, Michelle M.; Mallon, Ann-Marie; Brown, Steve D. M.; Beutler, Bruce; Goodnow, Christopher C.; Lunter, Gerton; Cornall, Richard J.

2013-01-01

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis. PMID:23382690

Genetic Mutations and Epigenetic Modifications: Driving Cancer and Informing Precision Medicine

PubMed Central

Coyle, Krysta Mila; Boudreau, Jeanette E.

2017-01-01

Cancer treatment is undergoing a significant revolution from “one-size-fits-all” cytotoxic therapies to tailored approaches that precisely target molecular alterations. Precision strategies for drug development and patient stratification, based on the molecular features of tumors, are the next logical step in a long history of approaches to cancer therapy. In this review, we discuss the history of cancer treatment from generic natural extracts and radical surgical procedures to site-specific and combinatorial treatment regimens, which have incrementally improved patient outcomes. We discuss the related contributions of genetics and epigenetics to cancer progression and the response to targeted therapies and identify challenges and opportunities for the success of precision medicine. The identification of patients who will benefit from targeted therapies is more complex than simply identifying patients whose tumors harbour the targeted aberration, and intratumoral heterogeneity makes it difficult to determine if a precision therapy is successful during treatment. This heterogeneity enables tumors to develop resistance to targeted approaches; therefore, the rational combination of therapeutic agents will limit the threat of acquired resistance to therapeutic success. By incorporating the view of malignant transformation modulated by networks of genetic and epigenetic interactions, molecular strategies will enable precision medicine for effective treatment across cancer subtypes. PMID:28685150

Expanding the Symbiodinium (Dinophyceae, Suessiales) Toolkit Through Protoplast Technology.

PubMed

Levin, Rachel A; Suggett, David J; Nitschke, Matthew R; van Oppen, Madeleine J H; Steinberg, Peter D

2017-09-01

Dinoflagellates within the genus Symbiodinium are photosymbionts of many tropical reef invertebrates, including corals, making them central to the health of coral reefs. Symbiodinium have therefore gained significant research attention, though studies have been constrained by technical limitations. In particular, the generation of viable cells with their cell walls removed (termed protoplasts) has enabled a wide range of experimental techniques for bacteria, fungi, plants, and algae such as ultrastructure studies, virus infection studies, patch clamping, genetic transformation, and protoplast fusion. However, previous studies have struggled to remove the cell walls from armored dinoflagellates, potentially due to the internal placement of their cell walls. Here, we produce the first Symbiodinium protoplasts from three genetically and physiologically distinct strains via incubation with cellulase and osmotic agents. Digestion of the cell walls was verified by a lack of Calcofluor White fluorescence signal and by cell swelling in hypotonic culture medium. Fused protoplasts were also observed, motivating future investigation into intra- and inter-specific somatic hybridization of Symbiodinium. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Generation of Symbiodinium protoplasts opens exciting, new avenues for researching these crucial symbiotic dinoflagellates, including genetic modification. © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.

Sex determination strategies in 2012: towards a common regulatory model?

PubMed

Angelopoulou, Roxani; Lavranos, Giagkos; Manolakou, Panagiota

2012-02-22

Sex determination is a complicated process involving large-scale modifications in gene expression affecting virtually every tissue in the body. Although the evolutionary origin of sex remains controversial, there is little doubt that it has developed as a process of optimizing metabolic control, as well as developmental and reproductive functions within a given setting of limited resources and environmental pressure. Evidence from various model organisms supports the view that sex determination may occur as a result of direct environmental induction or genetic regulation. The first process has been well documented in reptiles and fish, while the second is the classic case for avian species and mammals. Both of the latter have developed a variety of sex-specific/sex-related genes, which ultimately form a complete chromosome pair (sex chromosomes/gonosomes). Interestingly, combinations of environmental and genetic mechanisms have been described among different classes of animals, thus rendering the possibility of a unidirectional continuous evolutionary process from the one type of mechanism to the other unlikely. On the other hand, common elements appear throughout the animal kingdom, with regard to a) conserved key genes and b) a central role of sex steroid control as a prerequisite for ultimately normal sex differentiation. Studies in invertebrates also indicate a role of epigenetic chromatin modification, particularly with regard to alternative splicing options. This review summarizes current evidence from research in this hot field and signifies the need for further study of both normal hormonal regulators of sexual phenotype and patterns of environmental disruption. © 2012 Angelopoulou et al; licensee BioMed Central Ltd.

Popular misconceptions: agricultural biotechnology.

PubMed

McHughen, Alan; Wager, Robert

2010-12-31

Agricultural biotechnology, especially genetic engineering or genetic modification (GM), is a topic of considerable controversy worldwide. The public debate is fraught with polarized views and opinions, some are held with religious zeal. Unfortunately, it is also marked with much ignorance and misinformation. Here we explore some popular misconceptions encountered in the public debate. Copyright © 2010 Elsevier B.V. All rights reserved.

An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

USDA-ARS?s Scientific Manuscript database

Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their rela...

Expanding the genetic code for site-specific labelling of tobacco mosaic virus coat protein and building biotin-functionalized virus-like particles.

PubMed

Wu, F C; Zhang, H; Zhou, Q; Wu, M; Ballard, Z; Tian, Y; Wang, J Y; Niu, Z W; Huang, Y

2014-04-18

A method for site-specific and high yield modification of tobacco mosaic virus coat protein (TMVCP) utilizing a genetic code expanding technology and copper free cycloaddition reaction has been established, and biotin-functionalized virus-like particles were built by the self-assembly of the protein monomers.

Longevity and aging: role of genes and of the extracellular matrix.

PubMed

Robert, L; Labat-Robert, J

2015-02-01

Longevity is different for every animal species as well as their genome, suggesting a correlation between genes and life-span. Estimates put the genetic effect from 5 to 35% approximately, suggesting that even genetic effects are dependent on environmental conditions. This contention is largely confirmed by the study of identical twins raised apart. They do not die at the same age and also for different reasons. Aging is not "genetically programmed", it is outside evolutionary constraint. Evolution favors early and efficient reproduction, but does not care for longevity. A number of mechanisms were shown to be involved in the age-dependent decline of vital functions, among them the Maillard reaction (non-enzymatic glycosylation) and the age-dependent upregulation of proteolytic activity. Aging of ECM is a complex process, comprising progressive modification of its macromolecular components and of cell-matrix interactions. An important process is the uncoupling with age of the elastin-receptor from its "young" transmission pathway loosing all physiological effects, but enhancing free radical and elastase release. These processes contribute to age-related ECM degradation, production of matrikins (ECM degradation products with biological activity) aggravating functional loss with age. Both genetic and post-genetic mechanisms are susceptible to be influenced by medical, pharmacological and dietary interventions. Among the genetic mechanisms, those attributed to Sirtuins (7 orthologs identified in the human genome) are especially important. Among the environmental effects, nutrition, hygiene and weather conditions play a role. These data justify some predictions on the evolution of life expectancy taking in account also socio-economic factors. Biological constraints become evident by the comparison of centenarians and supercentenarians (less than 1% of the centenarians) putting an upper limit to the attainable human lifespan.

Readiness of adolescents to use genetically modified organisms according to their knowledge and emotional attitude towards GMOs.

PubMed

Lachowski, Stanisław; Jurkiewicz, Anna; Choina, Piotr; Florek-Łuszczki, Magdalena; Buczaj, Agnieszka; Goździewska, Małgorzata

2017-06-07

Agriculture based on genetically modified organisms plays an increasingly important role in feeding the world population, which is evidenced by a considerable growth in the size of land under genetically modified crops (GM). Uncertainty and controversy around GM products are mainly due to the lack of accurate and reliable information, and lack of knowledge concerning the essence of genetic modifications, and the effect of GM food on the human organism, and consequently, a negative emotional attitude towards what is unknown. The objective of the presented study was to discover to what extent knowledge and the emotional attitude of adolescents towards genetically modified organisms is related with acceptance of growing genetically modified plants or breeding GM animals on own farm or allotment garden, and the purchase and consumption of GM food, as well as the use of GMOs in medicine. The study was conducted by the method of a diagnostic survey using a questionnaire designed by the author, which covered a group of 500 adolescents completing secondary school on the level of maturity examination. The collected material was subjected to statistical analysis. Research hypotheses were verified using chi-square test (χ ² ), t-Student test, and stepwise regression analysis. Stepwise regression analysis showed that the readiness of adolescents to use genetically modified organisms as food or for the production of pharmaceuticals, the production of GM plants or animals on own farm, depends on an emotional-evaluative attitude towards GMOs, and the level of knowledge concerning the essence of genetic modifications.

Field and microcosm experiments to evaluate the effects of agricultural Cu treatment on the density and genetic structure of microbial communities in two different soils.

PubMed

Ranjard, Lionel; Echairi, Abdelwahad; Nowak, Virginie; Lejon, David P H; Nouaïm, Rachida; Chaussod, Rémi

2006-11-01

The effects of Cu amendment on indigenous soil microorganisms were investigated in two soils, a calcareous silty clay (Ep) and a sandy soil (Au), by means of a 1-year field experiment and a two-month microcosm incubation. Cu was added as 'Bordeaux mixture' [CuSO(4), Ca(OH)(2)] at the standard rate used in viticulture (B1=16 kg Cu kg(-1) soil) and at a higher level of contamination (B3=48 kg Cu ha(-1) soil). More extractable Cu was observed in sandy soil (Au) than in silty soil (Ep). Furthermore, total Cu and Cu-EDTA declined with time in Au soil, whereas they remained stable in Ep soil. Quantitative modifications of the microflora were assessed by C-biomass measurements and qualitative modifications were assessed by the characterization of the genetic structure of bacterial and fungal communities from DNA directly extracted from the soil, using B- and F-ARISA (bacterial and fungal automated ribosomal intergenic spacer analysis). In the field study, no significant modifications were observed in C-biomass whereas microcosm incubation showed a decrease in B3 contamination only. ARISA fingerprinting showed slight but significant modifications of bacterial and fungal communities in field and microcosm incubation. These modifications were transient in all cases, suggesting a short-term effect of Cu stress. Microcosm experiments detected the microbial community modifications with greater precision in the short-term, while field experiments showed that the biological effects of Cu contamination may be overcome or hidden by pedo-climatic variations.

Deoxyribonucleic acid restriction and modification systems in Salmonella: chromosomally located systems of different serotypes.

PubMed Central

Bullas, L R; Colson, C; Neufeld, B

1980-01-01

With the use of four different phages, Salmonella strains representing 85 different serotypes were examined to determine their restriction-modification phenotype. They fell into one of three groups on this basis: group 1, those which lacked the common LT system; group 2, those in which only the LT system could be recognized; and group 3. those which possessed the LT system and at least one other system shown with some serotypes to be closely linked to serB. The specificity of the serB-linked restriction-modification system was unique for each serotype, but different strains of the same serotype expressed the same specificity. Two of the systems were shown to behave in genetic crosses as functional alleles of the S. typhimurium SB system. It is possible that these serB-linked restriction-modification systems constitute a large multiallelic series of genes extending throughout the Salmonella genus and Escherichia coli. We suggest that the division of the Salmonella into the three restriction-modification groups may be significant in defining a "biological grouping" of the different serotypes within the genus which may ultimately be useful in describing the Salmonella species. From the genetic relatedness between the genes of some of the Salmonella restriction-modification systems with those of the E. coli systems, we deduce that the restriction endonuclases produced by the Salmonella serB-linked systems are of type 1. Determination of the nucleotide sequences of the recognition sites of the restriction endonucleases of selected Salmonella systems should further our understanding of specificity with these enzymes. PMID:6243623

PLMD: An updated data resource of protein lysine modifications.

PubMed

Xu, Haodong; Zhou, Jiaqi; Lin, Shaofeng; Deng, Wankun; Zhang, Ying; Xue, Yu

2017-05-20

Post-translational modifications (PTMs) occurring at protein lysine residues, or protein lysine modifications (PLMs), play critical roles in regulating biological processes. Due to the explosive expansion of the amount of PLM substrates and the discovery of novel PLM types, here we greatly updated our previous studies, and presented a much more integrative resource of protein lysine modification database (PLMD). In PLMD, we totally collected and integrated 284,780 modification events in 53,501 proteins across 176 eukaryotes and prokaryotes for up to 20 types of PLMs, including ubiquitination, acetylation, sumoylation, methylation, succinylation, malonylation, glutarylation, glycation, formylation, hydroxylation, butyrylation, propionylation, crotonylation, pupylation, neddylation, 2-hydroxyisobutyrylation, phosphoglycerylation, carboxylation, lipoylation and biotinylation. Using the data set, a motif-based analysis was performed for each PLM type, and the results demonstrated that different PLM types preferentially recognize distinct sequence motifs for the modifications. Moreover, various PLMs synergistically orchestrate specific cellular biological processes by mutual crosstalks with each other, and we totally found 65,297 PLM events involved in 90 types of PLM co-occurrences on the same lysine residues. Finally, various options were provided for accessing the data, while original references and other annotations were also present for each PLM substrate. Taken together, we anticipated the PLMD database can serve as a useful resource for further researches of PLMs. PLMD 3.0 was implemented in PHP + MySQL and freely available at http://plmd.biocuckoo.org. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Genetically modified pigs produced with a nonviral episomal vector

PubMed Central

Manzini, Stefano; Vargiolu, Alessia; Stehle, Isa M; Bacci, Maria Laura; Cerrito, Maria Grazia; Giovannoni, Roberto; Zannoni, Augusta; Bianco, Maria Rosaria; Forni, Monica; Donini, Pierluigi; Papa, Michele; Lipps, Hans J; Lavitrano, Marialuisa

2006-01-01

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy. PMID:17101993

Gene doping: the hype and the reality

PubMed Central

Wells, D J

2008-01-01

Some spectacular results from genetic manipulation of laboratory rodents and increasing developments in human gene therapy raise the spectre of genetic modification or ‘gene doping' in sports. Candidate targets include the induction of muscle hypertrophy through overexpression of specific splice variants of insulin-like growth factor-1 or blockade of the action of myostatin, increasing oxygen delivery by raising the hematocrit through the use of erythropoietin, induction of angiogenesis with vascular endothelial growth factors or related molecules and changes in muscle phenotype through expression of peroxisome-proliferator-activated receptor- delta and associated molecules. Some of these potential genetic enhancements, particularly where the genetic modification and its action are confined to the muscles, may be undetectable using current tests. This had lead to exaggerated predictions that gene doping in athletics will be common within the next few years. However, a review of the methods of gene transfer and the current ‘state of the art' in development of genetic treatments for human disease show that the prospects for gene doping remain essentially theoretical at present. Despite this conclusion, it will be important to continue to monitor improvements in the technology and to develop methods of detection, particularly those based on identifying patterns of changes in response to doping as opposed to the detection of specific agents. PMID:18500383

Gene doping: the hype and the reality.

PubMed

Wells, D J

2008-06-01

Some spectacular results from genetic manipulation of laboratory rodents and increasing developments in human gene therapy raise the spectre of genetic modification or 'gene doping' in sports. Candidate targets include the induction of muscle hypertrophy through overexpression of specific splice variants of insulin-like growth factor-1 or blockade of the action of myostatin, increasing oxygen delivery by raising the hematocrit through the use of erythropoietin, induction of angiogenesis with vascular endothelial growth factors or related molecules and changes in muscle phenotype through expression of peroxisome-proliferator-activated receptor- delta and associated molecules. Some of these potential genetic enhancements, particularly where the genetic modification and its action are confined to the muscles, may be undetectable using current tests. This had lead to exaggerated predictions that gene doping in athletics will be common within the next few years. However, a review of the methods of gene transfer and the current 'state of the art' in development of genetic treatments for human disease show that the prospects for gene doping remain essentially theoretical at present. Despite this conclusion, it will be important to continue to monitor improvements in the technology and to develop methods of detection, particularly those based on identifying patterns of changes in response to doping as opposed to the detection of specific agents.

In Genes We Trust: Germline Engineering, Eugenics, and the Future of the Human Genome.

PubMed

Powell, Russell

2015-12-01

Liberal proponents of genetic engineering maintain that developing human germline modification technologies is morally desirable because it will result in a net improvement in human health and well-being. Skeptics of germline modification, in contrast, fear evolutionary harms that could flow from intervening in the human germline, and worry that such programs, even if well intentioned, could lead to a recapitulation of the scientifically and morally discredited projects of the old eugenics. Some bioconservatives have appealed as well to the value of retaining our "given" human biological nature as a reason for restraining the development and use of human genetic modification technologies even where they would tend to increase well-being. In this article, I argue that germline intervention will be necessary merely to sustain the levels of genetic health that we presently enjoy for future generations-a goal that should appeal to bioliberals and bioconservatives alike. This is due to the population-genetic consequences of relaxed selection pressures in human populations caused by the increasing efficacy and availability of conventional medicine. This heterodox conclusion, which I present as a problem of intergenerational justice, has been overlooked in medicine and bioethics due to certain misconceptions about human evolution, which I attempt to rectify, as well as the sordid history of Darwinian approaches to medicine and social policy, which I distinguish from the present argument. © The Author 2015. Published by Oxford University Press, on behalf of the Journal of Medicine and Philosophy Inc. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Epigenetic contribution of the myosin light chain kinase gene to the risk for acute respiratory distress syndrome.

PubMed

Szilágyi, Keely L; Liu, Cong; Zhang, Xu; Wang, Ting; Fortman, Jeffrey D; Zhang, Wei; Garcia, Joe G N

2017-02-01

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with a considerable case fatality rate (∼30%-40%). Health disparities exist with African descent (AD) subjects exhibiting greater mortality than European descent (ED) individuals. Myosin light chain kinase is encoded by MYLK, whose genetic variants are implicated in ARDS pathogenesis and may influence ARDS mortality. As baseline population-specific epigenetic changes, that is, cytosine modifications, have been observed between AD and ED individuals, epigenetic variations in MYLK may provide insights into ARDS disparities. We compared methylation levels of MYLK cytosine-guanine dinucleotides (CpGs) between ARDS patients and intensive care unit (ICU) controls overall and by ethnicity in a nested case-control study of 39 ARDS cases and 75 non-ARDS ICU controls. Two MYLK CpG sites (cg03892735 and cg23344121) were differentially modified between ARDS subjects and controls (P < 0.05; q < 0.25) in a logistic regression model, where no effect modification by ethnicity or age was found. One CpG site was associated with ARDS in patients aged <58 years, cg19611163 (intron 19, 20). Two CpG sites were associated with ARDS in EDs only, gene body CpG (cg01894985, intron 2, 3) and CpG (cg16212219, intron 31, 32), with higher modification levels exhibited in ARDS subjects than controls. Cis-acting modified cytosine quantitative trait loci (mQTL) were identified using linear regression between local genetic variants and modification levels for 2 ARDS-associated CpGs (cg23344121 and cg16212219). In summary, these ARDS-associated MYLK CpGs with effect modification by ethnicity and local mQTL suggest that MYLK epigenetic variation and local genetic background may contribute to health disparities observed in ARDS. Copyright © 2016 Elsevier Inc. All rights reserved.

Biofortified Crops Generated by Breeding, Agronomy, and Transgenic Approaches Are Improving Lives of Millions of People around the World

PubMed Central

Garg, Monika; Sharma, Natasha; Sharma, Saloni; Kapoor, Payal; Kumar, Aman; Chunduri, Venkatesh; Arora, Priya

2018-01-01

Biofortification is an upcoming, promising, cost-effective, and sustainable technique of delivering micronutrients to a population that has limited access to diverse diets and other micronutrient interventions. Unfortunately, major food crops are poor sources of micronutrients required for normal human growth. The manuscript deals in all aspects of crop biofortification which includes—breeding, agronomy, and genetic modification. It tries to summarize all the biofortification research that has been conducted on different crops. Success stories of biofortification include lysine and tryptophan rich quality protein maize (World food prize 2000), Vitamin A rich orange sweet potato (World food prize 2016); generated by crop breeding, oleic acid, and stearidonic acid soybean enrichment; through genetic transformation and selenium, iodine, and zinc supplementation. The biofortified food crops, especially cereals, legumes, vegetables, and fruits, are providing sufficient levels of micronutrients to targeted populations. Although a greater emphasis is being laid on transgenic research, the success rate and acceptability of breeding is much higher. Besides the challenges biofortified crops hold a bright future to address the malnutrition challenge. PMID:29492405

Biofortified Crops Generated by Breeding, Agronomy, and Transgenic Approaches Are Improving Lives of Millions of People around the World.

PubMed

Garg, Monika; Sharma, Natasha; Sharma, Saloni; Kapoor, Payal; Kumar, Aman; Chunduri, Venkatesh; Arora, Priya

2018-01-01

Biofortification is an upcoming, promising, cost-effective, and sustainable technique of delivering micronutrients to a population that has limited access to diverse diets and other micronutrient interventions. Unfortunately, major food crops are poor sources of micronutrients required for normal human growth. The manuscript deals in all aspects of crop biofortification which includes-breeding, agronomy, and genetic modification. It tries to summarize all the biofortification research that has been conducted on different crops. Success stories of biofortification include lysine and tryptophan rich quality protein maize (World food prize 2000), Vitamin A rich orange sweet potato (World food prize 2016); generated by crop breeding, oleic acid, and stearidonic acid soybean enrichment; through genetic transformation and selenium, iodine, and zinc supplementation. The biofortified food crops, especially cereals, legumes, vegetables, and fruits, are providing sufficient levels of micronutrients to targeted populations. Although a greater emphasis is being laid on transgenic research, the success rate and acceptability of breeding is much higher. Besides the challenges biofortified crops hold a bright future to address the malnutrition challenge.

Electrochemical genosensors in food safety assessment.

PubMed

Martín-Fernández, Begoña; Manzanares-Palenzuela, C Lorena; Sánchez-Paniagua López, Marta; de-Los-Santos-Álvarez, Noemí; López-Ruiz, Beatriz

2017-09-02

The main goal of food safety assessment is to provide reliable information on the identity and composition of food and reduce the presence of harmful components. Nowadays, there are many countries where rather than the presence of pathogens, common public concerns are focused on the presence of hidden allergens, fraudulent practices, and genetic modifications in food. Accordingly, food regulations attempt to offer a high level of protection and to guarantee transparent information to the consumers. The availability of analytical methods is essential to comply these requirements. Protein-based strategies are usually employed for this purpose, but present some limitations. Because DNA is a more stable molecule, present in most tissues, and can be amplified, there has been an increasing interest in developing DNA-based approaches (polymerase chain reaction, microarrays, and genosensors). In this regard, electrochemical genosensors may play a major role in fulfilling the needs of food industry, such as reliable, portable, and affordable devices. This work reviews the achievements of this technology applied to allergen detection, species identification, and genetically modified organisms testing. We summarized the legislative framework, current design strategies in sensor development, their analytical characteristics, and future prospects.

Effects of plants genetically modified for insect resistance on nontarget organisms.

PubMed

O'Callaghan, Maureen; Glare, Travis R; Burgess, Elisabeth P J; Malone, Louise A

2005-01-01

Insect resistance, based on Bacillus thuringiensis (Bt) endotoxins, is the second most widely used trait (after herbicide resistance) in commercial genetically modified (GM) crops. Other modifications for insect resistance, such as proteinase inhibitors and lectins, are also being used in many experimental crops. The extensive testing on nontarget plant-feeding insects and beneficial species that has accompanied the long-term and wide-scale use of Bt plants has not detected significant adverse effects. GM plants expressing other insect-resistant proteins that have a broader spectrum of activity have been tested on only a limited number of nontarget species. Little is known about the persistence of transgene-derived proteins in soil, with the exception of Bt endotoxins, which can persist in soil for several months. Bt plants appear to have little impact on soil biota such as earthworms, collembolans, and general soil microflora. Further research is required on the effects of GM plants on soil processes such as decomposition. Assessment of nontarget impacts is an essential part of the risk assessment process for insect-resistant GM plants.

Microgravity as a research tool to improve US agriculture

NASA Astrophysics Data System (ADS)

Bula, R. J.; Stankovic, Bratislav

2000-01-01

Crop production and utilization are undergoing significant modifications and improvements that emanate from adaptation of recently developed plant biotechnologies. Several innovative technologies will impact US agriculture in the next century. One of these is the transfer of desirable genes from organisms to economically important crop species in a way that cannot be accomplished with traditional plant breeding techniques. Such plant genetic engineering offers opportunities to improve crop species for a number of characteristics as well as use as source materials for specific medical and industrial applications. Although plant genetic engineering is having an impact on development of new crop cultivars, several major constraints limit the application of this technology to selected crop species and genotypes. Consequently, gene transfer systems that overcome these constraints would greatly enhance development of new crop materials. If results of a recent gene transfer experiment conducted in microgravity during a Space Shuttle mission are confirmed, and with the availability of the International Space Station as a permanent space facility, commercial plant transformation activity in microgravity could become a new research tool to improve US agriculture. .

40 CFR 60.1705 - What emission limits must I meet? By when?

Code of Federal Regulations, 2010 CFR

2010-07-01

... modification after June 26, 1987, then you must comply with the dioxins/furans and mercury emission limits... construction or operating permit, if a permit modification is required. Final compliance with the dioxins...

Resistant starch and other dietary fiber components in tubers from a high-amylose potato.

PubMed

Zhao, Xue; Andersson, Mariette; Andersson, Roger

2018-06-15

Tubers from a genetically modified high-amylose line T-2012 and its parental potato cultivar Dinamo were analyzed for resistant starch (RS) and dietary fiber (DF) after cooking and cold storage. For uncooked potatoes, the high-amylose tubers (30% of dry matter, DM) had much lower RS than the parent tubers (56% of DM). However, after cooking, the high-amylose tubers gave more RS (13% of DM) than the parent (4% of DM), and the RS level increased further to about 20% of DM after 1 day of cold storage. The altered RS content was attributable to changes in amylose content, starch granule structure, and amylopectin structure induced by the genetic modification. The high-amylose tubers also contained more DF (10-14% of DM) than the parent (5-7% of DM). Furthermore, cell wall composition was indirectly affected by the genetic modification, giving more cellulose and less pectin in the high-amylose tubers than the parent. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

[Observation on twins of cosmic radiation influence as a risk factor].

PubMed

Vişănescu, Magdalena

2009-01-01

During flight, cosmic radiation is capable to penetrate the walls of airplanes and produce mutations in human chromosomes. To evaluate the correlations between cosmic radiation and the pathology of flying personnel. Two twin patients, one with 15 years work as a flight crew member, and the other with regular work on the ground. The patients were examined clinically, biochemically and with imaging methods. The genetic and environmental contribution to their pathology was noted. The patient, working in flight, received additional genetic tests to refute or to confirm the presence of chromosome alterations induced by the cosmic radiation. The patient, member of the flight crew, presented hypercholesterolemia, more evident structure modifications of the mammary gland, but chromosome alterations were not present. Having a first grade relative with dyslipidemia and uterine cancer, the structural or blood homeostasis modifications of the flight crew member can be either due to a high, genetically transmitted, susceptibility to the effects of the plane's microclimate and possibly to effects of lifestyle.

Adaptation to the Local Environment by Modifications of the Photoperiod Response in Crops

PubMed Central

Nakamichi, Norihito

2015-01-01

Flowering plants produce a meristem at the shoot tip where specialized tissue generates shoot apical meristems at the appropriate time to differentiate into reproductive structures, pollinate and efficiently generate seeds. The complex set of molecular and phenological events culminating in development of a flowering meristem is referred to as ‘flowering time’. Flowering time affects plant productivity because plants dedicate energy to produce flowers and seeds rather than vegetative tissue once the molecular decision to initiate flowering has been taken. Thus, initiation of flowering time is an important decision in plants, especially in annual plants including crops. Humans have introduced crops into latitudes and climate areas far from their origin or natural ecosystem, requiring in many cases modification of native flowering times. Recent molecular–genetic studies shed light on the genetic basis related to such introductions. In this review, recent progress regarding crop introductions and their genetic bases are summarized, as well as the potential of other agricultural plants to be introduced into different climatic zones. PMID:25432974

Social and ethical issues in mitochondrial donation

PubMed Central

Dimond, Rebecca

2015-01-01

Introduction or background The UK is at the forefront of mitochondrial science and is currently the only country in the world to legalize germ-line technologies involving mitochondrial donation. However, concerns have been raised about genetic modification and the ‘slippery slope’ to designer babies. Sources of data This review uses academic articles, newspaper reports and public documents. Areas of agreement Mitochondrial donation offers women with mitochondrial disease an opportunity to have healthy, genetically related children. Areas of controversy Key areas of disagreement include safety, the creation of three-parent babies, impact on identity, implications for society, definitions of genetic modification and reproductive choice. Growing points The UK government legalized the techniques in March 2015. Scientific and medical communities across the world followed the developments with interest. Areas timely for developing research It is expected that the first cohort of ‘three parent’ babies will be born in the UK in 2016. Their health and progress will be closely monitored. PMID:26351372

Folate Biofortification in Hydroponically Cultivated Spinach by the Addition of Phenylalanine.

PubMed

Watanabe, Sho; Ohtani, Yuta; Tatsukami, Yohei; Aoki, Wataru; Amemiya, Takashi; Sukekiyo, Yasunori; Kubokawa, Seiichi; Ueda, Mitsuyoshi

2017-06-14

Folate is an important vitamin mainly ingested from vegetables, and folate deficiency causes various health problems. Recently, several studies demonstrated folate biofortification in plants or food crops by metabolic engineering through genetic modifications. However, the production and sales of genetically modified foods are under strict regulation. Here, we developed a new approach to achieve folate biofortification in spinach (Spinacia oleracea) without genetic modification. We hydroponically cultivated spinach with the addition of three candidate compounds expected to fortify folate. As a result of liquid chromatography tandem mass spectrometry analysis, we found that the addition of phenylalanine increased the folate content up to 2.0-fold (306 μg in 100 g of fresh spinach), representing 76.5% of the recommended daily allowance for adults. By measuring the intermediates of folate biosynthesis, we revealed that phenylalanine activated folate biosynthesis in spinach by increasing the levels of pteridine and p-aminobenzoic acid. Our approach is a promising and practical approach to cultivate nutrient-enriched vegetables.

Progress and biotechnological prospects in fish transgenesis.

PubMed

Tonelli, Fernanda M P; Lacerda, Samyra M S N; Tonelli, Flávia C P; Costa, Guilherme M J; de França, Luiz Renato; Resende, Rodrigo R

2017-11-01

The history of transgenesis is marked by milestones such as the development of cellular transdifferentiation, recombinant DNA, genetic modification of target cells, and finally, the generation of simpler genetically modified organisms (e.g. bacteria and mice). The first transgenic fish was developed in 1984, and since then, continuing technological advancements to improve gene transfer have led to more rapid, accurate, and efficient generation of transgenic animals. Among the established methods are microinjection, electroporation, lipofection, viral vectors, and gene targeting. Here, we review the history of animal transgenesis, with an emphasis on fish, in conjunction with major developments in genetic engineering over the past few decades. Importantly, spermatogonial stem cell modification and transplantation are two common techniques capable of revolutionizing the generation of transgenic fish. Furthermore, we discuss recent progress and future biotechnological prospects of fish transgenesis, which has strong applications for the aquaculture industry. Indeed, some transgenic fish are already available in the current market, validating continued efforts to improve economically important species with biotechnological advancements. Copyright © 2017. Published by Elsevier Inc.

Tolerance to MHC class II disparate allografts through genetic modification of bone marrow

PubMed Central

Jindra, Peter T.; Tripathi, Sudipta; Tian, Chaorui; Iacomini, John; Bagley, Jessamyn

2012-01-01

Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-Ab (I-Ab) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction. PMID:22833118

Virus-Derived Gene Expression and RNA Interference Vector for Grapevine

PubMed Central

Kurth, Elizabeth G.; Peremyslov, Valera V.; Prokhnevsky, Alexey I.; Kasschau, Kristin D.; Miller, Marilyn; Carrington, James C.

2012-01-01

The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests. PMID:22438553

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a trulymore » genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. Here we found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.« less

RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

PubMed Central

Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

2016-01-01

Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation. PMID:26392149

DNA Mapping Made Simple: An Intellectual Activity about the Genetic Modification of Organisms

ERIC Educational Resources Information Center

Marques, Miguel; Arrabaca, Joao; Chagas, Isabel

2004-01-01

Since the discovery of the DNA double helix (in 1953 by Watson and Crick), technologies have been developed that allow scientists to manipulate the genome of bacteria to produce human hormones, as well as the genome of crop plants to achieve high yield and enhanced flavor. The universality of the genetic code has allowed DNA isolated from a…

Allozyme-Specific Modification of a Maize Seed Chitinase by a Protein Secreted by the Fungal Pathogen Stenocarpella maydis

USDA-ARS?s Scientific Manuscript database

Stenocarpella maydis causes both dry-ear rot and stalk rot of maize. Maize inbreds have varying levels of resistance to S. maydis ear rot. The genetic basis of resistance appears to rely on multiple genetic factors, none of which are known. The commonly used stiff stalk inbred B73 has been shown ...

Mudskippers and Their Genetic Adaptations to an Amphibious Lifestyle

PubMed Central

You, Xinxin; Sun, Min; Li, Jia; Bian, Chao; Chen, Jieming; Yu, Hui; Shi, Qiong

2018-01-01

Simple Summary Mudskippers are an interesting group of goggle-eyed amphibious fish that can live both in water and on land. They are a useful model for obtaining insights into the genetic mechanisms underlying the terrestrial adaptations of amphibious fish. This review summarizes the morphological and physiological modifications of representative mudskippers, and focuses on the recent advancement of genomic studies on their genetic adaptations to the amphibious lifestyle. Abstract Mudskippers are the largest group of amphibious teleost fish that are uniquely adapted to live on mudflats. During their successful transition from aqueous life to terrestrial living, these fish have evolved morphological and physiological modifications of aerial vision and olfaction, higher ammonia tolerance, aerial respiration, improved immunological defense against terrestrial pathogens, and terrestrial locomotion using protruded pectoral fins. Comparative genomic and transcriptomic data have been accumulated and analyzed for understanding molecular mechanisms of the terrestrial adaptations. Our current review provides a general introduction to mudskippers and recent research advances of their genetic adaptations to the amphibious lifestyle, which will be helpful for understanding the evolutionary transition of vertebrates from water to land. Our insights into the genomes and transcriptomes will also support molecular breeding, functional identification, and natural compound screening. PMID:29414871

Investigating Novice and Expert Conceptions of Genetically Modified Organisms

PubMed Central

Potter, Lisa M.; Bissonnette, Sarah A.; Knight, Jonathan D.; Tanner, Kimberly D.

2017-01-01

The aspiration of biology education is to give students tools to apply knowledge learned in the classroom to everyday life. Genetic modification is a real-world biological concept that relies on an in-depth understanding of the molecular behavior of DNA and proteins. This study investigated undergraduate biology students’ conceptions of genetically modified organisms (GMOs) when probed with real-world, molecular and cellular, and essentialist cues, and how those conceptions compared across biology expertise. We developed a novel written assessment tool and administered it to 120 non–biology majors, 154 entering biology majors, 120 advanced biology majors (ABM), and nine biology faculty. Results indicated that undergraduate biology majors rarely included molecular and cellular rationales in their initial explanations of GMOs. Despite ABM demonstrating that they have much of the biology knowledge necessary to understand genetic modification, they did not appear to apply this knowledge to explaining GMOs. Further, this study showed that all undergraduate student populations exhibited evidence of essentialist thinking while explaining GMOs, regardless of their level of biology training. Finally, our results suggest an association between scientifically accurate ideas and the application of molecular and cellular rationales, as well as an association between misconceptions and essentialist rationales. PMID:28821537

PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications. PMID:23688076

RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation

PubMed Central

Kim, J-H; Jang, J-W; Lee, Y-S; Lee, J-W; Chi, X-Z; Li, Y-H; Kim, M-K; Kim, D-M; Choi, B-S; Kim, J; Kim, H-M; van Wijnen, A; Park, IlY; Bae, S-C

2014-01-01

Transcription factors of the RUNX family (RUNXs), which play pivotal roles in normal development and neoplasia, are regulated by various post-translational modifications. To understand the molecular mechanisms underlying the regulation of RUNXs, we performed a large-scale functional genetic screen of a fly mutant library. The screen identified dPias (the fly ortholog of mammalian PIASs), an E3 ligase for the SUMO (small ubiquitin-like modifier) modification, as a novel genetic modifier of lz (the fly ortholog of mammalian RUNX3). Molecular biological analysis revealed that lz/RUNXs are sumoylated by dPias/PIAS1 at an evolutionarily conserved lysine residue (K372 of lz, K144 of RUNX1, K181 of RUNX2 and K148 of RUNX3). PIAS1-mediated sumoylation inhibited RUNX3 transactivation activity, and this modification was promoted by the AKT1 kinase. Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer. In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3. Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins. PMID:24777122

Heritability of Measures of Kidney Disease Among Zuni Indians: The Zuni Kidney Project

PubMed Central

MacCluer, Jean W.; Scavini, Marina; Shah, Vallabh O.; Cole, Shelley A.; Laston, Sandra L.; Voruganti, V. Saroja; Paine, Susan S.; Eaton, Alfred J.; Comuzzie, Anthony G.; Tentori, Francesca; Pathak, Dorothy R.; Bobelu, Arlene; Bobelu, Jeanette; Ghahate, Donica; Waikaniwa, Mildred; Zager, Philip G.

2010-01-01

Background The long-term goal of the GKDZI (Genetics of Kidney Disease in Zuni Indians) Study is to identify genes, environmental factors, and genetic-environmental interactions that modulate susceptibility to renal disease and intermediate phenotypes. Study Design A community-based participatory research approach was used to recruit family members of individuals with kidney disease. Setting & Participants The study was conducted in the Zuni Indians, a small endogamous tribe located in rural New Mexico. We recruited members of extended families, ascertained through a proband with kidney disease and at least 1 sibling with kidney disease. 821 participants were recruited, comprising 7,702 relative pairs. Predictor Outcomes & Measurements Urine albumin-creatinine ratio (UACR) and hematuria were determined in 3 urine samples and expressed as a true ratio. Glomerular filtration rate (GFR) was estimated using the Modification of Diet in Renal Disease (MDRD) Study equation modified for American Indians. Probands were considered to have kidney disease if UACR was ≥0.2 in 2 or more of 3 spot urine samples or estimated GFR was decreased according to the CRIC (Chronic Renal Insufficiency Cohort) Study criteria. Results Kidney disease was identified in 192 participants (23.4%). There were significant heritabilities for estimated GFR, UACR, serum creatinine, serum urea nitrogen, and uric acid and a variety of phenotypes related to obesity, diabetes, and cardiovascular disease. There were significant genetic correlations of some kidney-related phenotypes with these other phenotypes. Limitations Limitations include absence of renal biopsy, possible misclassification bias, lack of direct GFR measurements, and failure to include all possible environmental interactions. Conclusions Many phenotypes related to kidney disease showed significant heritabilities in Zuni Indians, and there were significant genetic correlations with phenotypes related to obesity, diabetes, and cardiovascular disease. The study design serves as a paradigm for the conduct of research in relatively isolated, endogamous, underserved populations. PMID:20646805

Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an amazingly versatile tool for the study of mammalian biology.

PubMed

Babinet, C; Cohen-Tannoudji, M

2001-09-01

The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene "targeting"). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions, etc.) and chromosomal rearrangements such as large deletions, duplications and translocations. Finally, methods have been devised which permit the creation of conditional mutations, allowing the study of gene function throughout the life of an animal, when gene inactivation entails embryonic lethality. In this paper, we present an overview of the methods and scenarios used for the programmed modification of mouse genome, and we underline their enormous interest for the study of mammalian biology.

Rejuvenation of human cardiac progenitor cells with Pim-1 kinase.

PubMed

Mohsin, Sadia; Khan, Mohsin; Nguyen, Jonathan; Alkatib, Monique; Siddiqi, Sailay; Hariharan, Nirmala; Wallach, Kathleen; Monsanto, Megan; Gude, Natalie; Dembitsky, Walter; Sussman, Mark A

2013-10-25

Myocardial function is enhanced by adoptive transfer of human cardiac progenitor cells (hCPCs) into a pathologically challenged heart. However, advanced age, comorbidities, and myocardial injury in patients with heart failure constrain the proliferation, survival, and regenerative capacity of hCPCs. Rejuvenation of senescent hCPCs will improve the outcome of regenerative therapy for a substantial patient population possessing functionally impaired stem cells. Reverse phenotypic and functional senescence of hCPCs by ex vivo modification with Pim-1. C-kit-positive hCPCs were isolated from heart biopsy samples of patients undergoing left ventricular assist device implantation. Growth kinetics, telomere lengths, and expression of cell cycle regulators showed significant variation between hCPC isolated from multiple patients. Telomere length was significantly decreased in hCPC with slow-growth kinetics concomitant with decreased proliferation and upregulation of senescent markers compared with hCPC with fast-growth kinetics. Desirable youthful characteristics were conferred on hCPCs by genetic modification using Pim-1 kinase, including increases in proliferation, telomere length, survival, and decreased expression of senescence markers. Senescence characteristics of hCPCs are ameliorated by Pim-1 kinase resulting in rejuvenation of phenotypic and functional properties. hCPCs show improved cellular properties resulting from Pim-1 modification, but benefits were more pronounced in hCPC with slow-growth kinetics relative to hCPC with fast-growth kinetics. With the majority of patients with heart failure presenting advanced age, infirmity, and impaired regenerative capacity, the use of Pim-1 modification should be incorporated into cell-based therapeutic approaches to broaden inclusion criteria and address limitations associated with the senescent phenotype of aged hCPC.

Rejuvenation of Human Cardiac Progenitor Cells With Pim-1 Kinase

PubMed Central

Mohsin, Sadia; Khan, Mohsin; Nguyen, Jonathan; Alkatib, Monique; Siddiqi, Sailay; Hariharan, Nirmala; Wallach, Kathleen; Monsanto, Megan; Gude, Natalie; Dembitsky, Walter; Sussman, Mark A.

2014-01-01

Rationale Myocardial function is enhanced by adoptive transfer of human cardiac progenitor cells (hCPCs) into a pathologically challenged heart. However, advanced age, comorbidities, and myocardial injury in patients with heart failure constrain the proliferation, survival, and regenerative capacity of hCPCs. Rejuvenation of senescent hCPCs will improve the outcome of regenerative therapy for a substantial patient population possessing functionally impaired stem cells. Objective Reverse phenotypic and functional senescence of hCPCs by ex vivo modification with Pim-1. Methods and Results C-kit–positive hCPCs were isolated from heart biopsy samples of patients undergoing left ventricular assist device implantation. Growth kinetics, telomere lengths, and expression of cell cycle regulators showed significant variation between hCPC isolated from multiple patients. Telomere length was significantly decreased in hCPC with slow-growth kinetics concomitant with decreased proliferation and upregulation of senescent markers compared with hCPC with fast-growth kinetics. Desirable youthful characteristics were conferred on hCPCs by genetic modification using Pim-1 kinase, including increases in proliferation, telomere length, survival, and decreased expression of senescence markers. Conclusions Senescence characteristics of hCPCs are ameliorated by Pim-1 kinase resulting in rejuvenation of phenotypic and functional properties. hCPCs show improved cellular properties resulting from Pim-1 modification, but benefits were more pronounced in hCPC with slow-growth kinetics relative to hCPC with fast-growth kinetics. With the majority of patients with heart failure presenting advanced age, infirmity, and impaired regenerative capacity, the use of Pim-1 modification should be incorporated into cell-based therapeutic approaches to broaden inclusion criteria and address limitations associated with the senescent phenotype of aged hCPC. PMID:24044948

Genetic engineering and chemical conjugation of potato virus X.

PubMed

Lee, Karin L; Uhde-Holzem, Kerstin; Fischer, Rainer; Commandeur, Ulrich; Steinmetz, Nicole F

2014-01-01

Here we report the genetic engineering and chemical modification of potato virus X (PVX) for the presentation of various peptides, proteins, and fluorescent dyes, or other chemical modifiers. Three different ways of genetic engineering are described and by these means, peptides are successfully expressed not only when the foot and mouth disease virus (FMDV) 2A sequence or a flexible glycine-serine linker is included, but also when the peptide is fused directly to the PVX coat protein. When larger proteins or unfavorable peptide sequences are presented, a partial fusion via the FMDV 2A sequence is preferable. When these PVX chimeras retain the ability to assemble into viral particles and are thus able to infect plants systemically, they can be utilized to inoculate susceptible plants for isolation of sufficient amounts of virus particles for subsequent chemical modification. Chemical modification is required for the display of nonbiological ligands such as fluorophores, polymers, and small drug compounds. We present three methods of chemical bioconjugation. For direct conjugation of small chemical modifiers to solvent exposed lysines, N-hydroxysuccinimide chemistry can be applied. Bio-orthogonal reactions such as copper-catalyzed azide-alkyne cycloaddition or hydrazone ligation are alternatives to achieve more efficient conjugation (e.g., when working with high molecular weight or insoluble ligands). Furthermore, hydrazone ligation offers an attractive route for the introduction of pH-cleavable cargos (e.g., therapeutic molecules).

Modifying plants for biofuel and biomaterial production.

PubMed

Furtado, Agnelo; Lupoi, Jason S; Hoang, Nam V; Healey, Adam; Singh, Seema; Simmons, Blake A; Henry, Robert J

2014-12-01

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost-effective option for biochemical conversion to biofuel. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Epigenetic modification of OXT and human sociability.

PubMed

Haas, Brian W; Filkowski, Megan M; Cochran, R Nick; Denison, Lydia; Ishak, Alexandra; Nishitani, Shota; Smith, Alicia K

2016-07-05

Across many mammalian species there exist genetic and biological systems that facilitate the tendency to be social. Oxytocin is a neuropeptide involved in social-approach behaviors in humans and others mammals. Although there exists a large, mounting body of evidence showing that oxytocin signaling genes are associated with human sociability, very little is currently known regarding the way the structural gene for oxytocin (OXT) confers individual differences in human sociability. In this study, we undertook a comprehensive approach to investigate the association between epigenetic modification of OXT via DNA methylation, and overt measures of social processing, including self-report, behavior, and brain function and structure. Genetic data were collected via saliva samples and analyzed to target and quantify DNA methylation across the promoter region of OXT We observed a consistent pattern of results across sociability measures. People that exhibit lower OXT DNA methylation (presumably linked to higher OXT expression) display more secure attachment styles, improved ability to recognize emotional facial expressions, greater superior temporal sulcus activity during two social-cognitive functional MRI tasks, and larger fusiform gyrus gray matter volume than people that exhibit higher OXT DNA methylation. These findings provide empirical evidence that epigenetic modification of OXT is linked to several overt measures of sociability in humans and serve to advance progress in translational social neuroscience research toward a better understanding of the evolutionary and genetic basis of normal and abnormal human sociability.

Modified Phasemeter for a Heterodyne Laser Interferometer

NASA Technical Reports Server (NTRS)

Loya, Frank M.

2010-01-01

Modifications have been made in the design of instruments of the type described in "Digital Averaging Phasemeter for Heterodyne Interferometry". A phasemeter of this type measures the difference between the phases of the unknown and reference heterodyne signals in a heterodyne laser interferometer. The phasemeter design lacked immunity to drift of the heterodyne frequency, was bandwidth-limited by computer bus architectures then in use, and was resolution-limited by the nature of field-programmable gate arrays (FPGAs) then available. The modifications have overcome these limitations and have afforded additional improvements in accuracy, speed, and modularity. The modifications are summarized.

The genesis and correction of unprofessional behavior in surgeons: The role of society, education and genetics.

PubMed

Talati, Jamsheer Jehangir

2016-05-01

Most surgeons are ethical. Increasingly, however, a variety of unprofessional behaviors are surfacing. Awareness of these behaviors and their causation is required to plan their eradication. To (i) identify the prevalent causes of unprofessional behaviors amongst surgeons; and (ii) suggest corrective interventions. Literature was searched and models constructed to interpret interrelationships between causes. Unprofessional behaviors extend beyond those frequently discussed, necessitating the term 'dysprofessionalism'. Causal influences arise from (i) an overpowering society; (ii) limited education and (iii) the underdeveloped state of human nature at birth. Societies corrupt by role-modeling avarice and encouraging industry-despite consequent pollution. Society brooks no interference. Surgeons are loath to oppose, resulting in an unprofessional silence. Surgical education based on best evidence is an indoctrination, with little opportunity to deploy alternatives. Evidence based guidelines increasingly risk errors, as publication fraud increases. Effective interaction with government/legislation is not taught. Human nature and our brain remain arrested in a stage of strongly stabilized evolutionary selection. Humans are born with larval brains requiring intense educational interventions. Genetic modification holds promise as it can circumvent birth in undeveloped states, and facilitate trans-generational transfer of knowledge. CRISPR/Cas-9 techniques make this possible, necessitating ethical discussion-an urgent issue. Reforming society would otherwise be an impossible task as behaviors cannot be taught in classrooms. Instances of dysprofessionalism are unlikely to diminish using current approaches. Discussion of the ethics of genetically modifying embryos is urgently needed, as this could provide a possible shortcut to positive changes in human behavior, but risks unwanted changes and misuse. Copyright © 2016 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

Crowdsourcing the Moral Limits of Human Gene Editing?

PubMed

Juengst, Eric T

2017-05-01

In 2015, a flourish of "alarums and excursions" by the scientific community propelled CRISPR/Cas9 and other new gene-editing techniques into public attention. At issue were two kinds of potential gene-editing experiments in humans: those making inheritable germ-line modifications and those designed to enhance human traits beyond what is necessary for health and healing. The scientific consensus seemed to be that while research to develop safe and effective human gene editing should continue, society's moral uncertainties about these two kinds of experiments needed to be better resolved before clinical trials of either type should be attempted. In the United States, the National Academies of Science, Engineering and Medicine (NASEM) convened the Committee on Human Gene Editing: Scientific, Medical and Ethical Considerations to pursue that resolution. The committee's 2017 consensus report has been widely interpreted as "opening the door" to inheritable human genetic modification and holding a line against enhancement interventions. But on a close reading it does neither. There are two reasons for this eccentric conclusion, both of which depend upon the strength of the committee's commitment to engaging diverse public voices in the gene-editing policy-making process. © 2017 The Hastings Center.

Inhibition of oxidative metabolism leads to p53 genetic inactivation and transformation in neural stem cells

PubMed Central

Bartesaghi, Stefano; Graziano, Vincenzo; Galavotti, Sara; Henriquez, Nick V.; Betts, Joanne; Saxena, Jayeta; Minieri, Valentina; A, Deli; Karlsson, Anna; Martins, L. Miguel; Capasso, Melania; Nicotera, Pierluigi; Brandner, Sebastian; De Laurenzi, Vincenzo; Salomoni, Paolo

2015-01-01

Alterations of mitochondrial metabolism and genomic instability have been implicated in tumorigenesis in multiple tissues. High-grade glioma (HGG), one of the most lethal human neoplasms, displays genetic modifications of Krebs cycle components as well as electron transport chain (ETC) alterations. Furthermore, the p53 tumor suppressor, which has emerged as a key regulator of mitochondrial respiration at the expense of glycolysis, is genetically inactivated in a large proportion of HGG cases. Therefore, it is becoming evident that genetic modifications can affect cell metabolism in HGG; however, it is currently unclear whether mitochondrial metabolism alterations could vice versa promote genomic instability as a mechanism for neoplastic transformation. Here, we show that, in neural progenitor/stem cells (NPCs), which can act as HGG cell of origin, inhibition of mitochondrial metabolism leads to p53 genetic inactivation. Impairment of respiration via inhibition of complex I or decreased mitochondrial DNA copy number leads to p53 genetic loss and a glycolytic switch. p53 genetic inactivation in ETC-impaired neural stem cells is caused by increased reactive oxygen species and associated oxidative DNA damage. ETC-impaired cells display a marked growth advantage in the presence or absence of oncogenic RAS, and form undifferentiated tumors when transplanted into the mouse brain. Finally, p53 mutations correlated with alterations in ETC subunit composition and activity in primary glioma-initiating neural stem cells. Together, these findings provide previously unidentified insights into the relationship between mitochondria, genomic stability, and tumor suppressive control, with implications for our understanding of brain cancer pathogenesis. PMID:25583481

Why using genetics to address welfare may not be a good idea.

PubMed

Thompson, P B

2010-04-01

Welfare of animals in livestock production systems is now widely defined in terms of 3 classes of measures: veterinary health, mental well-being (or feelings), and natural behaviors. Several well-documented points of tension exist among welfare indicators in these 3 classes. Strategies that aim to improve welfare using genetics can increase resistance to disease and may also be able to relieve stress or injury. One strategy is to reduce the genetic proclivity of the bird to engage in behaviors that are frustrated in modern production systems. Another is to develop strains less prone to behaviors hurtful to other hens. Yet another is to make overall temperament a goal for genetic adjustments. These genetic approaches may score well in terms of veterinary and psychological well-being. Yet they also involve changes in behavioral repertoire and tendencies of the resulting bird. Although it has seemed reasonable to argue that such animals are better off than frustrated or injured animals reflecting more species-typical behaviors, there is a point of view that holds that modification of a species-typical trait is ipso facto a decline in the well-being of the animal. Additionally, a significant amount of anecdotal evidence has been accumulated that suggests that many animal advocates and members of the public find manipulation of genetics to be an ethically unacceptable approach to animal welfare, especially when modifications in the environment could also be a response to welfare problems. Hence, though promising from one perspective, genetic strategies to improve welfare may not be acceptable to the public.

Most Colorful Example of Genetic Assimilation? Exploring the Evolutionary Destiny of Recurrent Phenotypic Accommodation.

PubMed

Badyaev, Alexander V; Potticary, Ahva L; Morrison, Erin S

2017-08-01

Evolution of adaptation requires both generation of novel phenotypic variation and retention of a locally beneficial subset of this variation. Such retention can be facilitated by genetic assimilation, the accumulation of genetic and molecular mechanisms that stabilize induced phenotypes and assume progressively greater control over their reliable production. A particularly strong inference into genetic assimilation as an evolutionary process requires a system where it is possible to directly evaluate the extent to which an induced phenotype is progressively incorporated into preexisting developmental pathways. Evolution of diet-dependent pigmentation in birds-where external carotenoids are coopted into internal metabolism to a variable degree before being integrated with a feather's developmental processes-provides such an opportunity. Here we combine a metabolic network view of carotenoid evolution with detailed empirical study of feather modifications to show that the effect of physical properties of carotenoids on feather structure depends on their metabolic modification, their environmental recurrence, and biochemical redundancy, as predicted by the genetic assimilation hypothesis. Metabolized carotenoids caused less stochastic variation in feather structure and were more closely integrated with feather growth than were dietary carotenoids of the same molecular weight. These patterns were driven by the recurrence of organism-carotenoid associations: commonly used dietary carotenoids and biochemically redundant derived carotenoids caused less stochastic variation in feather structure than did rarely used or biochemically unique compounds. We discuss implications of genetic assimilation processes for the evolutionary diversification of diet-dependent animal coloration.

[Detection of genetically modified soy (Roundup-Ready) in processed food products].

PubMed

Hagen, M; Beneke, B

2000-01-01

In this study, the application of a qualitative and a quantitative method of analysis to detect genetically modified RR-Soy (Roundup-Ready Soy) in processed foods is described. A total of 179 various products containing soy such as baby food and diet products, soy drinks and desserts, tofu and tofu products, soy based meat substitutes, soy protein, breads, flour, granules, cereals, noodles, soy bean sprouts, fats and oils as well as condiments were investigated following the pattern of the section 35 LMBG-method L 23.01.22-1. The DNA was extracted from the samples and analysed using a soybean specific lectin gene PCR as well as a PCR, specific for the genetic modification. Additional, by means of PCR in combination with fluorescence-detection (TaqMan 5'-Nuclease Assay), suspicious samples were subjected to a real-time quantification of the percentage of genetically modified RR-Soy. The methods of analysis proved to be extremely sensitive and specific in regard to the food groups checked. The fats and oils, as well as the condiments were the exceptions in which amplifiable soy DNA could not be detected. The genetic modification of RR-Soy was detected in 34 samples. Eight of these samples contained more than 1% of RR-Soy. It is necessary to determine the percentage of transgenic soy in order to assess whether genetically modified ingredients were deliberately added, or whether they were caused by technically unavoidable contamination (for example during transportation and processing).

Novel Functional Genomics Approaches: A Promising Future in the Combat Against Plant Viruses.

PubMed

Fondong, Vincent N; Nagalakshmi, Ugrappa; Dinesh-Kumar, Savithramma P

2016-10-01

Advances in functional genomics and genome editing approaches have provided new opportunities and potential to accelerate plant virus control efforts through modification of host and viral genomes in a precise and predictable manner. Here, we discuss application of RNA-based technologies, including artificial micro RNA, transacting small interfering RNA, and Cas9 (clustered regularly interspaced short palindromic repeat-associated protein 9), which are currently being successfully deployed in generating virus-resistant plants. We further discuss the reverse genetics approach, targeting induced local lesions in genomes (TILLING) and its variant, known as EcoTILLING, that are used in the identification of plant virus recessive resistance gene alleles. In addition to describing specific applications of these technologies in plant virus control, this review discusses their advantages and limitations.

Systems biology solutions for biochemical production challenges.

PubMed

Hansen, Anne Sofie Lærke; Lennen, Rebecca M; Sonnenschein, Nikolaus; Herrgård, Markus J

2017-06-01

There is an urgent need to significantly accelerate the development of microbial cell factories to produce fuels and chemicals from renewable feedstocks in order to facilitate the transition to a biobased society. Methods commonly used within the field of systems biology including omics characterization, genome-scale metabolic modeling, and adaptive laboratory evolution can be readily deployed in metabolic engineering projects. However, high performance strains usually carry tens of genetic modifications and need to operate in challenging environmental conditions. This additional complexity compared to basic science research requires pushing systems biology strategies to their limits and often spurs innovative developments that benefit fields outside metabolic engineering. Here we survey recent advanced applications of systems biology methods in engineering microbial production strains for biofuels and -chemicals. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Engineering Delivery Vehicles for Genome Editing.

PubMed

Nelson, Christopher E; Gersbach, Charles A

2016-06-07

The field of genome engineering has created new possibilities for gene therapy, including improved animal models of disease, engineered cell therapies, and in vivo gene repair. The most significant challenge for the clinical translation of genome engineering is the development of safe and effective delivery vehicles. A large body of work has applied genome engineering to genetic modification in vitro, and clinical trials have begun using cells modified by genome editing. Now, promising preclinical work is beginning to apply these tools in vivo. This article summarizes the development of genome engineering platforms, including meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas9, and their flexibility for precise genetic modifications. The prospects for the development of safe and effective viral and nonviral delivery vehicles for genome editing are reviewed, and promising advances in particular therapeutic applications are discussed.

Seventeen years of statin pharmacogenetics: a systematic review.

PubMed

Leusink, Maarten; Onland-Moret, N Charlotte; de Bakker, Paul I W; de Boer, Anthonius; Maitland-van der Zee, Anke H

2016-01-01

We evaluated the evidence of pharmacogenetic associations with statins in a systematic review. Two separate outcomes were considered of interest: modification of low-density lipoprotein cholesterol (LDL-C) response and modification of risk for cardiovascular events. In candidate gene studies, 141 loci were claimed to be associated with LDL-C response. Only 5% of these associations were positively replicated. In addition, six genome-wide association studies of LDL-C response identified common SNPs in APOE, LPA, SLCO1B1, SORT1 and ABCG2 at genome-wide significance. None of the investigated SNPs consistently affected the risk reduction for cardiovascular events. Only five genetic loci were consistently associated with LDL-C response. However, as effect sizes are modest, there is no evidence for the value of genetic testing in clinical practice.

The social and economic impact of biofortification through genetic modification.

PubMed

De Steur, Hans; Demont, Matty; Gellynck, Xavier; Stein, Alexander J

2017-04-01

Genetic modification (GM) has been advocated as an alternative or complement to micronutrient interventions such as supplementation, fortification or dietary diversification. While proof-of-concept of various GM biofortified crops looks promising, the decision tree of policy makers is much more complex, and requires insight on their socio-economic impacts: Will it actually work? Is it financially sound? Will people accept it? Can it be implemented in a globalized world? This review shows that GM biofortification could effectively reduce the burden of micronutrient deficiencies, in an economically viable way, and is generally well received by target beneficiaries, despite some resistance and uncertainty. Practically, however, protectionist and/or unscientific regulations in some developed countries raise the (perceived) bar for implementation in target countries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature-triggered hierarchical self-assembly

NASA Astrophysics Data System (ADS)

Mozhdehi, Davoud; Luginbuhl, Kelli M.; Simon, Joseph R.; Dzuricky, Michael; Berger, Rüdiger; Varol, H. Samet; Huang, Fred C.; Buehne, Kristen L.; Mayne, Nicholas R.; Weitzhandler, Isaac; Bonn, Mischa; Parekh, Sapun H.; Chilkoti, Ashutosh

2018-05-01

Post-translational modification of proteins is a strategy widely used in biological systems. It expands the diversity of the proteome and allows for tailoring of both the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the potential of post-translational modifications in biomaterials synthesis has remained largely untapped. As a proof of concept to demonstrate the feasibility of creating a genetically encoded biohybrid material through post-translational modification, we report here the generation of a family of three stimulus-responsive hybrid materials—fatty-acid-modified elastin-like polypeptides—using a one-pot recombinant expression and post-translational lipidation methodology. These hybrid biomaterials contain an amphiphilic domain, composed of a β-sheet-forming peptide that is post-translationally functionalized with a C14 alkyl chain, fused to a thermally responsive elastin-like polypeptide. They exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be controlled at the sequence level.

An improved genetic algorithm for designing optimal temporal patterns of neural stimulation

NASA Astrophysics Data System (ADS)

Cassar, Isaac R.; Titus, Nathan D.; Grill, Warren M.

2017-12-01

Objective. Electrical neuromodulation therapies typically apply constant frequency stimulation, but non-regular temporal patterns of stimulation may be more effective and more efficient. However, the design space for temporal patterns is exceedingly large, and model-based optimization is required for pattern design. We designed and implemented a modified genetic algorithm (GA) intended for design optimal temporal patterns of electrical neuromodulation. Approach. We tested and modified standard GA methods for application to designing temporal patterns of neural stimulation. We evaluated each modification individually and all modifications collectively by comparing performance to the standard GA across three test functions and two biophysically-based models of neural stimulation. Main results. The proposed modifications of the GA significantly improved performance across the test functions and performed best when all were used collectively. The standard GA found patterns that outperformed fixed-frequency, clinically-standard patterns in biophysically-based models of neural stimulation, but the modified GA, in many fewer iterations, consistently converged to higher-scoring, non-regular patterns of stimulation. Significance. The proposed improvements to standard GA methodology reduced the number of iterations required for convergence and identified superior solutions.

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen.

PubMed

Levin, Rebecca S; Hertz, Nicholas T; Burlingame, Alma L; Shokat, Kevan M; Mukherjee, Shaeri

2016-08-16

TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPase-activating protein (GAP) enzymes, and is exclusive to membrane-localized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection.

The Effect of Modified Control Limits on the Performance of a Generic Commercial Aircraft Engine

NASA Technical Reports Server (NTRS)

Csank, Jeffrey T.; May, Ryan D.; Gou, Ten-Huei; Litt, Jonathan S.

2012-01-01

This paper studies the effect of modifying the control limits of an aircraft engine to obtain additional performance. In an emergency situation, the ability to operate an engine above its normal operating limits and thereby gain additional performance may aid in the recovery of a distressed aircraft. However, the modification of an engine s limits is complex due to the risk of an engine failure. This paper focuses on the tradeoff between enhanced performance and risk of either incurring a mechanical engine failure or compromising engine operability. The ultimate goal is to increase the engine performance, without a large increase in risk of an engine failure, in order to increase the probability of recovering the distressed aircraft. The control limit modifications proposed are to extend the rotor speeds, temperatures, and pressures to allow more thrust to be produced by the engine, or to increase the rotor accelerations and allow the engine to follow a fast transient. These modifications do result in increased performance; however this study indicates that these modifications also lead to an increased risk of engine failure.

Population-specific genetic modification of Huntington's disease in Venezuela.

PubMed

Chao, Michael J; Kim, Kyung-Hee; Shin, Jun Wan; Lucente, Diane; Wheeler, Vanessa C; Li, Hong; Roach, Jared C; Hood, Leroy; Wexler, Nancy S; Jardim, Laura B; Holmans, Peter; Jones, Lesley; Orth, Michael; Kwak, Seung; MacDonald, Marcy E; Gusella, James F; Lee, Jong-Min

2018-05-01

Modifiers of Mendelian disorders can provide insights into disease mechanisms and guide therapeutic strategies. A recent genome-wide association (GWA) study discovered genetic modifiers of Huntington's disease (HD) onset in Europeans. Here, we performed whole genome sequencing and GWA analysis of a Venezuelan HD cluster whose families were crucial for the original mapping of the HD gene defect. The Venezuelan HD subjects develop motor symptoms earlier than their European counterparts, implying the potential for population-specific modifiers. The main Venezuelan HD family inherits HTT haplotype hap.03, which differs subtly at the sequence level from European HD hap.03, suggesting a different ancestral origin but not explaining the earlier age at onset in these Venezuelans. GWA analysis of the Venezuelan HD cluster suggests both population-specific and population-shared genetic modifiers. Genome-wide significant signals at 7p21.2-21.1 and suggestive association signals at 4p14 and 17q21.2 are evident only in Venezuelan HD, but genome-wide significant association signals at the established European chromosome 15 modifier locus are improved when Venezuelan HD data are included in the meta-analysis. Venezuelan-specific association signals on chromosome 7 center on SOSTDC1, which encodes a bone morphogenetic protein antagonist. The corresponding SNPs are associated with reduced expression of SOSTDC1 in non-Venezuelan tissue samples, suggesting that interaction of reduced SOSTDC1 expression with a population-specific genetic or environmental factor may be responsible for modification of HD onset in Venezuela. Detection of population-specific modification in Venezuelan HD supports the value of distinct disease populations in revealing novel aspects of a disease and population-relevant therapeutic strategies.

Population-specific genetic modification of Huntington's disease in Venezuela

PubMed Central

Chao, Michael J.; Kim, Kyung-Hee; Shin, Jun Wan; Lucente, Diane; Wheeler, Vanessa C.; Li, Hong; Roach, Jared C.; Hood, Leroy; Jardim, Laura B.; Jones, Lesley; Orth, Michael; Kwak, Seung; MacDonald, Marcy E.; Gusella, James F.

2018-01-01

Modifiers of Mendelian disorders can provide insights into disease mechanisms and guide therapeutic strategies. A recent genome-wide association (GWA) study discovered genetic modifiers of Huntington's disease (HD) onset in Europeans. Here, we performed whole genome sequencing and GWA analysis of a Venezuelan HD cluster whose families were crucial for the original mapping of the HD gene defect. The Venezuelan HD subjects develop motor symptoms earlier than their European counterparts, implying the potential for population-specific modifiers. The main Venezuelan HD family inherits HTT haplotype hap.03, which differs subtly at the sequence level from European HD hap.03, suggesting a different ancestral origin but not explaining the earlier age at onset in these Venezuelans. GWA analysis of the Venezuelan HD cluster suggests both population-specific and population-shared genetic modifiers. Genome-wide significant signals at 7p21.2–21.1 and suggestive association signals at 4p14 and 17q21.2 are evident only in Venezuelan HD, but genome-wide significant association signals at the established European chromosome 15 modifier locus are improved when Venezuelan HD data are included in the meta-analysis. Venezuelan-specific association signals on chromosome 7 center on SOSTDC1, which encodes a bone morphogenetic protein antagonist. The corresponding SNPs are associated with reduced expression of SOSTDC1 in non-Venezuelan tissue samples, suggesting that interaction of reduced SOSTDC1 expression with a population-specific genetic or environmental factor may be responsible for modification of HD onset in Venezuela. Detection of population-specific modification in Venezuelan HD supports the value of distinct disease populations in revealing novel aspects of a disease and population-relevant therapeutic strategies. PMID:29750799

Evaluating maturation and genetic modification of human dendritic cells in a new polyolefin cell culture bag system.

PubMed

Macke, Lars; Garritsen, Henk S P; Meyring, Wilhelm; Hannig, Horst; Pägelow, Ute; Wörmann, Bernhard; Piechaczek, Christoph; Geffers, Robert; Rohde, Manfred; Lindenmaier, Werner; Dittmar, Kurt E J

2010-04-01

Dendritic cells (DCs) are applied worldwide in several clinical studies of immune therapy of malignancies, autoimmune diseases, and transplantations. Most legislative bodies are demanding high standards for cultivation and transduction of cells. Closed-cell cultivating systems like cell culture bags would simplify and greatly improve the ability to reach these cultivation standards. We investigated if a new polyolefin cell culture bag enables maturation and adenoviral modification of human DCs in a closed system and compare the results with standard polystyrene flasks. Mononuclear cells were isolated from HLA-A*0201-positive blood donors by leukapheresis. A commercially available separation system (CliniMACS, Miltenyi Biotec) was used to isolate monocytes by positive selection using CD14-specific immunomagnetic beads. The essentially homogenous starting cell population was cultivated in the presence of granulocyte-macrophage-colony-stimulating factor and interleukin-4 in a closed-bag system in parallel to the standard flask cultivation system. Genetic modification was performed on Day 4. After induction of maturation on Day 5, mature DCs could be harvested and cryopreserved on Day 7. During the cultivation period comparative quality control was performed using flow cytometry, gene expression profiling, and functional assays. Both flasks and bags generated mature genetically modified DCs in similar yields. Surface membrane markers, expression profiles, and functional testing results were comparable. The use of a closed-bag system facilitated clinical applicability of genetically modified DCs. The polyolefin bag-based culture system yields DCs qualitatively and quantitatively comparable to the standard flask preparation. All steps including cryopreservation can be performed in a closed system facilitating standardized, safe, and reproducible preparation of therapeutic cells.

Limited family structure and BRCA gene mutation status in single cases of breast cancer.

PubMed

Weitzel, Jeffrey N; Lagos, Veronica I; Cullinane, Carey A; Gambol, Patricia J; Culver, Julie O; Blazer, Kathleen R; Palomares, Melanie R; Lowstuter, Katrina J; MacDonald, Deborah J

2007-06-20

An autosomal dominant pattern of hereditary breast cancer may be masked by small family size or transmission through males given sex-limited expression. To determine if BRCA gene mutations are more prevalent among single cases of early onset breast cancer in families with limited vs adequate family structure than would be predicted by currently available probability models. A total of 1543 women seen at US high-risk clinics for genetic cancer risk assessment and BRCA gene testing were enrolled in a prospective registry study between April 1997 and February 2007. Three hundred six of these women had breast cancer before age 50 years and no first- or second-degree relatives with breast or ovarian cancers. The main outcome measure was whether family structure, assessed from multigenerational pedigrees, predicts BRCA gene mutation status. Limited family structure was defined as fewer than 2 first- or second-degree female relatives surviving beyond age 45 years in either lineage. Family structure effect and mutation probability by the Couch, Myriad, and BRCAPRO models were assessed with stepwise multiple logistic regression. Model sensitivity and specificity were determined and receiver operating characteristic curves were generated. Family structure was limited in 153 cases (50%). BRCA gene mutations were detected in 13.7% of participants with limited vs 5.2% with adequate family structure. Family structure was a significant predictor of mutation status (odds ratio, 2.8; 95% confidence interval, 1.19-6.73; P = .02). Although none of the models performed well, receiver operating characteristic analysis indicated that modification of BRCAPRO output by a corrective probability index accounting for family structure was the most accurate BRCA gene mutation status predictor (area under the curve, 0.72; 95% confidence interval, 0.63-0.81; P<.001) for single cases of breast cancer. Family structure can affect the accuracy of mutation probability models. Genetic testing guidelines may need to be more inclusive for single cases of breast cancer when the family structure is limited and probability models need to be recreated using limited family history as an actual variable.

Mechanism-based strategies for protein thermostabilization.

PubMed

Mozhaev, V V

1993-03-01

Strategies for stabilizing enzymes can be derived from a two-step model of irreversible inactivation that involves preliminary reversible unfolding, followed by an irreversible step. Reversible unfolding is best prevented by covalent immobilization, whereas methods such as covalent modification of amino acid residues or 'medium engineering' (by the addition of low-molecular-weight compounds) are effective against irreversible 'incorrect' refolding. Genetic modification of the protein sequence is the most effective approach for preventing chemical deterioration.

Benefits and risks associated with genetically modified food products.

PubMed

Kramkowska, Marta; Grzelak, Teresa; Czyżewska, Krystyna

2013-01-01

Scientists employing methods of genetic engineering have developed a new group of living organisms, termed 'modified organisms', which found application in, among others, medicine, the pharmaceutical industry and food distribution. The introduction of transgenic products to the food market resulted in them becoming a controversial topic, with their proponents and contestants. The presented study aims to systematize objective data on the potential benefits and risks resulting from the consumption of transgenic food. Genetic modifications of plants and animals are justified by the potential for improvement of the food situation worldwide, an increase in yield crops, an increase in the nutritional value of food, and the development of pharmaceutical preparations of proven clinical significance. In the opinions of critics, however, transgenic food may unfavourably affect the health of consumers. Therefore, particular attention was devoted to the short- and long-lasting undesirable effects, such as alimentary allergies, synthesis of toxic agents or resistance to antibiotics. Examples arguing for the justified character of genetic modifications and cases proving that their use can be dangerous are innumerable. In view of the presented facts, however, complex studies are indispensable which, in a reliable way, evaluate effects linked to the consumption of food produced with the application of genetic engineering techniques. Whether one backs up or negates transgenic products, the choice between traditional and non-conventional food remains to be decided exclusively by the consumers.

Adding 'epi-' to behaviour genetics: implications for animal domestication.

PubMed

Jensen, Per

2015-01-01

In this review, it is argued that greatly improved understanding of domestication may be gained from extending the field of behaviour genetics to also include epigenetics. Domestication offers an interesting framework of rapid evolutionary changes caused by well-defined selection pressures. Behaviour is an important phenotype in this context, as it represents the primary means of response to environmental challenges. An overview is provided of the evidence for genetic involvement in behavioural control and the presently used methods for finding so-called behaviour genes. This shows that evolutionary changes in behaviour are to a large extent correlated to changes in patterns of gene expression, which brings epigenetics into the focus. This area is concerned with the mechanisms controlling the timing and extent of gene expression, and a lot of focus has been placed on methylation of cytosine in promoter regions, usually associated with genetic downregulation. The review considers the available evidence that environmental input, for example stress, can modify methylation and other epigenetic marks and subsequently affect behaviour. Furthermore, several studies are reviewed, demonstrating that acquired epigenetic modifications can be inherited and cause trans-generational behaviour changes. In conclusion, epigenetics may signify a new paradigm in this respect, as it shows that genomic modifications can be caused by environmental signals, and random mutations in DNA sequence are therefore not the only sources of heritable genetic variation. © 2015. Published by The Company of Biologists Ltd.

Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia.

PubMed

Koehorst-van Putten, H J J; Sudarmonowati, E; Herman, M; Pereira-Bertram, I J; Wolters, A M A; Meima, H; de Vetten, N; Raemakers, C J J M; Visser, R G F

2012-02-01

The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality.

The Future of Weapons of Mass Destruction: Their Nature and Role in 2030

DTIC Science & Technology

2014-06-01

substantial improvements are al- lowed under the rubric of life extension. Other states are not so constrained and may find different ways to develop pure...The foregoing capabilities do not involve genetic manipulation or bioen- gineering; they utilize longstanding biological knowledge and processes. More...sophisticated understanding of biological systems (genomic and proteomic infor- mation) and processes ( genetic modification, bioengineering) for

Invited review: Breeding and ethical perspectives on genetically modified and genome edited cattle.

PubMed

Eriksson, S; Jonas, E; Rydhmer, L; Röcklinsberg, H

2018-01-01

The hot topic of genetic modification and genome editing is sometimes presented as a rapid solution to various problems in the field of animal breeding and genetics. These technologies hold potential for future use in agriculture but we need to be aware of difficulties in large-scale application and integration in breeding schemes. In this review, we discuss applications of both classical genetic modifications (GM) using vectors and genome editing in dairy cattle breeding. We use an interdisciplinary approach considering both ethical and animal breeding perspectives. Decisions on how to make use of these techniques need to be made based not only on what is possible, but on what is reasonable to do. Principles of animal integrity, naturalness, risk perception, and animal welfare issues are examples of ethically relevant factors to consider. These factors also influence public perception and decisions about regulations by authorities. We need to acknowledge that we lack complete understanding of the genetic background of complex traits. It may be difficult, therefore, to predict the full effect of certain modifications in large-scale breeding programs. We present 2 potential applications: genome editing to dispense with dehorning, and insertion of human genes in bovine genomes to improve udder health as an example of classical GM. Both of these cases could be seen as beneficial for animal welfare but they differ in other aspects. In the former case, a genetic variant already present within the species is introduced, whereas in the latter case, transgenic animals are generated-this difference may influence how society regards the applications. We underline that the use of GM, as well as genome editing, of farm animals such as cattle is not independent of the context, and should be considered as part of an entire process, including, for example, the assisted reproduction technology that needs to be used. We propose that breeding organizations and breeding companies should take an active role in ethical discussions about the use of these techniques and thereby signal to society that these questions are being responsibly addressed. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Genome editing of industrial microorganism].

PubMed

Zhu, Linjiang; Li, Qi

2015-03-01

Genome editing is defined as highly-effective and precise modification of cellular genome in a large scale. In recent years, such genome-editing methods have been rapidly developed in the field of industrial strain improvement. The quickly-updating methods thoroughly change the old mode of inefficient genetic modification, which is "one modification, one selection marker, and one target site". Highly-effective modification mode in genome editing have been developed including simultaneous modification of multiplex genes, highly-effective insertion, replacement, and deletion of target genes in the genome scale, cut-paste of a large DNA fragment. These new tools for microbial genome editing will certainly be applied widely, and increase the efficiency of industrial strain improvement, and promote the revolution of traditional fermentation industry and rapid development of novel industrial biotechnology like production of biofuel and biomaterial. The technological principle of these genome-editing methods and their applications were summarized in this review, which can benefit engineering and construction of industrial microorganism.

Germline Modification and Engineering in Avian Species

PubMed Central

Lee, Hong Jo; Lee, Hyung Chul; Han, Jae Yong

2015-01-01

Production of genome-edited animals using germline-competent cells and genetic modification tools has provided opportunities for investigation of biological mechanisms in various organisms. The recently reported programmed genome editing technology that can induce gene modification at a target locus in an efficient and precise manner facilitates establishment of animal models. In this regard, the demand for genome-edited avian species, which are some of the most suitable model animals due to their unique embryonic development, has also increased. Furthermore, germline chimera production through long-term culture of chicken primordial germ cells (PGCs) has facilitated research on production of genome-edited chickens. Thus, use of avian germline modification is promising for development of novel avian models for research of disease control and various biological mechanisms. Here, we discuss recent progress in genome modification technology in avian species and its applications and future strategies. PMID:26333275

Oligo/Polynucleotide-Based Gene Modification: Strategies and Therapeutic Potential

PubMed Central

Sargent, R. Geoffrey; Kim, Soya

2011-01-01

Oligonucleotide- and polynucleotide-based gene modification strategies were developed as an alternative to transgene-based and classical gene targeting-based gene therapy approaches for treatment of genetic disorders. Unlike the transgene-based strategies, oligo/polynucleotide gene targeting approaches maintain gene integrity and the relationship between the protein coding and gene-specific regulatory sequences. Oligo/polynucleotide-based gene modification also has several advantages over classical vector-based homologous recombination approaches. These include essentially complete homology to the target sequence and the potential to rapidly engineer patient-specific oligo/polynucleotide gene modification reagents. Several oligo/polynucleotide-based approaches have been shown to successfully mediate sequence-specific modification of genomic DNA in mammalian cells. The strategies involve the use of polynucleotide small DNA fragments, triplex-forming oligonucleotides, and single-stranded oligodeoxynucleotides to mediate homologous exchange. The primary focus of this review will be on the mechanistic aspects of the small fragment homologous replacement, triplex-forming oligonucleotide-mediated, and single-stranded oligodeoxynucleotide-mediated gene modification strategies as it relates to their therapeutic potential. PMID:21417933

Host-targeted RAD-Seq reveals genetic changes in the coral Oculina patagonica associated with range expansion along the Spanish Mediterranean coast.

PubMed

Leydet, Karine Posbic; Grupstra, Carsten G B; Coma, Rafel; Ribes, Marta; Hellberg, Michael E

2018-06-01

Many organisms are expanding their ranges in response to changing environmental conditions. Understanding the patterns of genetic diversity and adaptation along an expansion front is crucial to assessing a species' long-term success. While next-generation sequencing techniques can reveal these changes in fine detail, ascribing them to a particular species can be difficult for organisms that live in close association with symbionts. Using a novel modified restriction site-associated DNA sequencing (RAD-Seq) protocol to target coral DNA, we collected 595 coral-specific single nucleotide polymorphisms from 189 colonies of the invasive coral Oculina patagonica from the Spanish Mediterranean coast, including established core populations and two expansion fronts. Surprisingly, populations from the recent northern expansion are genetically distinct from the westward expansion and core populations and also harbour greater genetic diversity. We found that temperature may have driven adaptation along the northern expansion, as genome scans for selection found three candidate loci associated with temperature in the north but none in the west. We found no genomic signature of selection associated with artificial substrate, which has been proposed for explaining the rapid spread of O. patagonica. This suggests that this coral is simply an opportunistic colonizer of free space made available by coastal habitat modifications. Our results suggest that unique genetic variation, possibly due to limited dispersal across the Ibiza Channel, an influx of individuals from different depths and/or adaptation to cooler temperatures along the northern expansion front may have facilitated the northward range expansion of O. patagonica in the western Mediterranean. © 2018 John Wiley & Sons Ltd.

Is genetic engineering ever going to take off in forage, turf and bioenergy crop breeding?

PubMed

Wang, Zeng-Yu; Brummer, E Charles

2012-11-01

Genetic engineering offers the opportunity to generate unique genetic variation that is either absent in the sexually compatible gene pool or has very low heritability. The generation of transgenic plants, coupled with breeding, has led to the production of widely used transgenic cultivars in several major cash crops, such as maize, soybean, cotton and canola. The process for regulatory approval of genetically engineered crops is slow and subject to extensive political interference. The situation in forage grasses and legumes is more complicated. Most widely grown forage, turf and bioenergy species (e.g. tall fescue, perennial ryegrass, switchgrass, alfalfa, white clover) are highly self-incompatible and outcrossing. Compared with inbreeding species, they have a high potential to pass their genes to adjacent plants. A major biosafety concern in these species is pollen-mediated transgene flow. Because human consumption is indirect, risk assessment of transgenic forage, turf and bioenergy species has focused on their environmental or ecological impacts. Although significant progress has been made in genetic modification of these species, commercialization of transgenic cultivars is very limited because of the stringent and costly regulatory requirements. To date, the only transgenic forage crop deregulated in the US is 'Roundup Ready' (RR) alfalfa. The approval process for RR alfalfa was complicated, involving several rounds of regulation, deregulation and re-regulation. Nevertheless, commercialization of RR alfalfa is an important step forward in regulatory approval of a perennial outcrossing forage crop. As additional transgenic forage, turf and bioenergy crops are generated and tested, different strategies have been developed to meet regulatory requirements. Recent progress in risk assessment and deregulation of transgenic forage and turf species is summarized and discussed.

Is genetic engineering ever going to take off in forage, turf and bioenergy crop breeding?

PubMed Central

Wang, Zeng-Yu; Brummer, E. Charles

2012-01-01

Background Genetic engineering offers the opportunity to generate unique genetic variation that is either absent in the sexually compatible gene pool or has very low heritability. The generation of transgenic plants, coupled with breeding, has led to the production of widely used transgenic cultivars in several major cash crops, such as maize, soybean, cotton and canola. The process for regulatory approval of genetically engineered crops is slow and subject to extensive political interference. The situation in forage grasses and legumes is more complicated. Scope Most widely grown forage, turf and bioenergy species (e.g. tall fescue, perennial ryegrass, switchgrass, alfalfa, white clover) are highly self-incompatible and outcrossing. Compared with inbreeding species, they have a high potential to pass their genes to adjacent plants. A major biosafety concern in these species is pollen-mediated transgene flow. Because human consumption is indirect, risk assessment of transgenic forage, turf and bioenergy species has focused on their environmental or ecological impacts. Although significant progress has been made in genetic modification of these species, commercialization of transgenic cultivars is very limited because of the stringent and costly regulatory requirements. To date, the only transgenic forage crop deregulated in the US is ‘Roundup Ready’ (RR) alfalfa. The approval process for RR alfalfa was complicated, involving several rounds of regulation, deregulation and re-regulation. Nevertheless, commercialization of RR alfalfa is an important step forward in regulatory approval of a perennial outcrossing forage crop. As additional transgenic forage, turf and bioenergy crops are generated and tested, different strategies have been developed to meet regulatory requirements. Recent progress in risk assessment and deregulation of transgenic forage and turf species is summarized and discussed. PMID:22378838

A formulation of the foundations of genetics and evolution.

PubMed

Bahr, Brian Edward

2016-05-01

This paper proposes a formulation of theories of the foundations of genetics and evolution that can be used to mathematically simulate phenotype expression, reproduction, mutation, and natural selection. It will be shown that Mendelian inheritance can be mathematically simulated with expressions involving matrices and that these expressions can also simulate phenomena that are modifications to Mendel's basic principles, like alleles that give rise to quantitative effects and traits that are the expression of multiple alleles and/or multiple genetic loci. Copyright © 2016 Elsevier Inc. All rights reserved.

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.

PubMed

Schwartz, Matthew L; Jorgensen, Erik M

2016-04-01

In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cell-specific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3- to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications. Copyright © 2016 by the Genetics Society of America.

Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery.

PubMed

Volak, Adrienn; LeRoy, Stanley G; Natasan, Jeya Shree; Park, David J; Cheah, Pike See; Maus, Andreas; Fitzpatrick, Zachary; Hudry, Eloise; Pinkham, Kelsey; Gandhi, Sheetal; Hyman, Bradley T; Mu, Dakai; GuhaSarkar, Dwijit; Stemmer-Rachamimov, Anat O; Sena-Esteves, Miguel; Badr, Christian E; Maguire, Casey A

2018-05-16

The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.

76 FR 81894 - Airworthiness Directives; Empresa Brasileira de Aeronautica S.A. (EMBRAER) Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-29

... revising the Airworthiness Limitations Section (ALS) of the Instructions for Continued Airworthiness (ICA... cracks resulted in manufacturer modifications of the ALS of Embraer ERJ 170 Maintenance Review Board... resulted in modifications on the Airworthiness Limitation Section (ALS) of Embraer ERJ 170 Maintenance...

Genetics Home Reference: Walker-Warburg syndrome

MedlinePlus

... also involved in development of this condition. The proteins produced from the genes listed above and others involved in Walker-Warburg syndrome modify a protein called alpha (α)-dystroglycan; this modification, called glycosylation, ...

Conservation and Divergence in the Candida Species Biofilm Matrix Mannan-Glucan Complex Structure, Function, and Genetic Control

PubMed Central

Dominguez, Eddie; Zarnowski, Robert; Sanchez, Hiram; Covelli, Antonio S.; Westler, William M.; Azadi, Parastoo; Nett, Jeniel

2018-01-01

ABSTRACT Candida biofilms resist the effects of available antifungal therapies. Prior studies with Candida albicans biofilms show that an extracellular matrix mannan-glucan complex (MGCx) contributes to antifungal sequestration, leading to drug resistance. Here we implement biochemical, pharmacological, and genetic approaches to explore a similar mechanism of resistance for the three most common clinically encountered non-albicans Candida species (NAC). Our findings reveal that each Candida species biofilm synthesizes a mannan-glucan complex and that the antifungal-protective function of this complex is conserved. Structural similarities extended primarily to the polysaccharide backbone (α-1,6-mannan and β-1,6-glucan). Surprisingly, biochemical analysis uncovered stark differences in the branching side chains of the MGCx among the species. Consistent with the structural analysis, similarities in the genetic control of MGCx production for each Candida species also appeared limited to the synthesis of the polysaccharide backbone. Each species appears to employ a unique subset of modification enzymes for MGCx synthesis, likely accounting for the observed side chain diversity. Our results argue for the conservation of matrix function among Candida spp. While biogenesis is preserved at the level of the mannan-glucan complex backbone, divergence emerges for construction of branching side chains. Thus, the MGCx backbone represents an ideal drug target for effective pan-Candida species biofilm therapy. PMID:29615504

Natural and Genetically Engineered Proteins for Tissue Engineering

PubMed Central

Gomes, Sílvia; Leonor, Isabel B.; Mano, João F.; Reis, Rui L.

2011-01-01

To overcome the limitations of traditionally used autografts, allografts and, to a lesser extent, synthetic materials, there is the need to develop a new generation of scaffolds with adequate mechanical and structural support, control of cell attachment, migration, proliferation and differentiation and with bio-resorbable features. This suite of properties would allow the body to heal itself at the same rate as implant degradation. Genetic engineering offers a route to this level of control of biomaterial systems. The possibility of expressing biological components in nature and to modify or bioengineer them further, offers a path towards multifunctional biomaterial systems. This includes opportunities to generate new protein sequences, new self-assembling peptides or fusions of different bioactive domains or protein motifs. New protein sequences with tunable properties can be generated that can be used as new biomaterials. In this review we address some of the most frequently used proteins for tissue engineering and biomedical applications and describe the techniques most commonly used to functionalize protein-based biomaterials by combining them with bioactive molecules to enhance biological performance. We also highlight the use of genetic engineering, for protein heterologous expression and the synthesis of new protein-based biopolymers, focusing the advantages of these functionalized biopolymers when compared with their counterparts extracted directly from nature and modified by techniques such as physical adsorption or chemical modification. PMID:22058578

Current issues connected with usage of genetically modified crops in production of feed and livestock feeding.

PubMed

Kwiatek, K; Mazur, M; Sieradzki, Z

2008-01-01

Progress, which is brought by new advances in modern molecular biology, allowed interference in the genome of live organisms and gene manipulation. Introducing new genes to the recipient organism enables to give them new features, absent before. Continuous increase in the area of the biotech crops triggers continuous discussion about safety of genetically modified (GM) crops, including food and feed derived from them. Important issue connected with cultivation of genetically modified crops is a horizontal gene transfer and a bacterial antibiotic resistance. Discussion about safety of GM crops concerns also food allergies caused by eating genetically modified food. The problem of genetic modifications of GM crops used for livestock feeding is widely discussed, taking into account Polish feed law.

Improving experimental phases for strong reflections prior to density modification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uervirojnangkoorn, Monarin; University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck; Hilgenfeld, Rolf, E-mail: hilgenfeld@biochem.uni-luebeck.de

A genetic algorithm has been developed to optimize the phases of the strongest reflections in SIR/SAD data. This is shown to facilitate density modification and model building in several test cases. Experimental phasing of diffraction data from macromolecular crystals involves deriving phase probability distributions. These distributions are often bimodal, making their weighted average, the centroid phase, improbable, so that electron-density maps computed using centroid phases are often non-interpretable. Density modification brings in information about the characteristics of electron density in protein crystals. In successful cases, this allows a choice between the modes in the phase probability distributions, and the mapsmore » can cross the borderline between non-interpretable and interpretable. Based on the suggestions by Vekhter [Vekhter (2005 ▶), Acta Cryst. D61, 899–902], the impact of identifying optimized phases for a small number of strong reflections prior to the density-modification process was investigated while using the centroid phase as a starting point for the remaining reflections. A genetic algorithm was developed that optimizes the quality of such phases using the skewness of the density map as a target function. Phases optimized in this way are then used in density modification. In most of the tests, the resulting maps were of higher quality than maps generated from the original centroid phases. In one of the test cases, the new method sufficiently improved a marginal set of experimental SAD phases to enable successful map interpretation. A computer program, SISA, has been developed to apply this method for phase improvement in macromolecular crystallography.« less

Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk

PubMed Central

Cui, Chenchen; Song, Yujie; Liu, Jun; Ge, Hengtao; Li, Qian; Huang, Hui; Hu, Linyong; Zhu, Hongmei; Jin, Yaping; Zhang, Yong

2015-01-01

β-Lactoglobulin (BLG) is a major goat’s milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine. PMID:25994151

Epigenetics and Epigenomics of Plants.

PubMed

Yadav, Chandra Bhan; Pandey, Garima; Muthamilarasan, Mehanathan; Prasad, Manoj

2018-01-23

The genetic material DNA in association with histone proteins forms the complex structure called chromatin, which is prone to undergo modification through certain epigenetic mechanisms including cytosine DNA methylation, histone modifications, and small RNA-mediated methylation. Alterations in chromatin structure lead to inaccessibility of genomic DNA to various regulatory proteins such as transcription factors, which eventually modulates gene expression. Advancements in high-throughput sequencing technologies have provided the opportunity to study the epigenetic mechanisms at genome-wide levels. Epigenomic studies using high-throughput technologies will widen the understanding of mechanisms as well as functions of regulatory pathways in plant genomes, which will further help in manipulating these pathways using genetic and biochemical approaches. This technology could be a potential research tool for displaying the systematic associations of genetic and epigenetic variations, especially in terms of cytosine methylation onto the genomic region in a specific cell or tissue. A comprehensive study of plant populations to correlate genotype to epigenotype and to phenotype, and also the study of methyl quantitative trait loci (QTL) or epiGWAS, is possible by using high-throughput sequencing methods, which will further accelerate molecular breeding programs for crop improvement. Graphical Abstract.

A novel intranuclear RNA vector system for long-term stem cell modification

PubMed Central

Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo

2015-01-01

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk.

PubMed

Cui, Chenchen; Song, Yujie; Liu, Jun; Ge, Hengtao; Li, Qian; Huang, Hui; Hu, Linyong; Zhu, Hongmei; Jin, Yaping; Zhang, Yong

2015-05-21

β-Lactoglobulin (BLG) is a major goat's milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine.

Messenger RNA Delivery for Tissue Engineering and Regenerative Medicine Applications.

PubMed

Patel, Siddharth; Athirasala, Avathamsa; Menezes, Paula P; Ashwanikumar, N; Zou, Ting; Sahay, Gaurav; Bertassoni, Luiz E

2018-06-07

The ability to control cellular processes and precisely direct cellular reprogramming has revolutionized regenerative medicine. Recent advances in in vitro transcribed (IVT) mRNA technology with chemical modifications have led to development of methods that control spatiotemporal gene expression. Additionally, there is a current thrust toward the development of safe, integration-free approaches to gene therapy for translational purposes. In this review, we describe strategies of synthetic IVT mRNA modifications and nonviral technologies for intracellular delivery. We provide insights into the current tissue engineering approaches that use a hydrogel scaffold with genetic material. Furthermore, we discuss the transformative potential of novel mRNA formulations that when embedded in hydrogels can trigger controlled genetic manipulation to regenerate tissues and organs in vitro and in vivo. The role of mRNA delivery in vascularization, cytoprotection, and Cas9-mediated xenotransplantation is additionally highlighted. Harmonizing mRNA delivery vehicle interactions with polymeric scaffolds can be used to present genetic cues that lead to precise command over cellular reprogramming, differentiation, and secretome activity of stem cells-an ultimate goal for tissue engineering.

Creating genetically modified pigs by using nuclear transfer

PubMed Central

Lai, Liangxue; Prather, Randall S

2003-01-01

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture. PMID:14613542

"You Say Tomato, I Say Solanum Lycopersicum Containing Beta-ionone and Phenylacetaldehyde": an Analysis of Connecticut's GMO Labeling Legislation.

PubMed

Nunziato, Travis

2014-01-01

"You Say Tomato, I Say Solanum Lycopersicum Containing Beta-ionone and Phenylacetaldehyde" discusses the importance of requiring labels on products that contain genetically modified organisms, focusing on Connecticut's GMO Labeling statutes, as it is they are the first of their kind in the nation. The article will compare Connecticut's law to the legislation found in Australia, highlighting the positive aspects of Connecticut's bill and identifying its key weaknesses, namely the "trigger clause" found in the statute. Part I will provide an overview of Genetic Modification and provide a brief history of Biotechnology. It will also provide a brief overview of the federal regulatory framework in biotechnology, as well as evaluate the United States Food and Drug Association's role of regulating genetic modification. Part I will conclude by discussing how the American public has shown that labeling GMOs is important, and something that should occur. Part II of this article will explore Connecticut's recent legislation requiring labels on products that contain GMOs. Part III will explore Australia's legislation requiring labels on products containing GMOs, comparing Australia's law to Connecticut's legislation.

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.

PubMed

Vashee, Sanjay; Stockwell, Timothy B; Alperovich, Nina; Denisova, Evgeniya A; Gibson, Daniel G; Cady, Kyle C; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B; Voorhies, Alexander A; Bruening, Eric; Caposio, Patrizia; Früh, Klaus

2017-01-01

Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae . Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae . Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

PubMed Central

Vashee, Sanjay; Stockwell, Timothy B.; Alperovich, Nina; Denisova, Evgeniya A.; Gibson, Daniel G.; Cady, Kyle C.; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B.; Voorhies, Alexander A.; Bruening, Eric; Caposio, Patrizia

2017-01-01

ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes. PMID:28989973

78 FR 69322 - Modification of Regulations Regarding Time Limits for Submission of Information Pertaining to...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-19

... Pertaining to Requests for Sampling in Antidumping Duty Administrative Reviews AGENCY: International Trade... sampling, and comments on sampling in antidumping (AD) administrative reviews. The modifications to the time limits, if adopted, will more clearly prescribe the time for filing requests for sampling in AD...

What Are the Risks and Limitations of Genetic Testing?

MedlinePlus

... does it take to get the results? Will health insurance cover the costs of genetic testing? What are the benefits of genetic testing? What are the risks and limitations of genetic testing? What is genetic ...

Epigenetic regulation of immune checkpoints: another target for cancer immunotherapy?

PubMed

Ali, Mahmoud A; Matboli, Marwa; Tarek, Marwa; Reda, Maged; Kamal, Kamal M; Nouh, Mahmoud; Ashry, Ahmed M; El-Bab, Ahmed Fath; Mesalam, Hend A; Shafei, Ayman El-Sayed; Abdel-Rahman, Omar

2017-01-01

Epigenetic changes in oncogenes and tumor-suppressor genes contribute to carcinogenesis. Understanding the epigenetic and genetic components of tumor immune evasion is crucial. Few cancer genetic mutations have been linked to direct correlations with immune evasion. Studies on the epigenetic modulation of the immune checkpoints have revealed a critical interaction between epigenetic and immune modulation. Epigenetic modifiers can activate many silenced genes. Some of them are immune checkpoints regulators that turn on immune responses and others turn them off resulting in immune evasion. Many forms of epigenetic inheritance mechanisms may play a role in regulation of immune checkpoints including: covalent modifications, noncoding RNA and histone modifications. In this review, we will show how the potential interaction between epigenetic and immune modulation may lead to new approaches for specific epigenome/immunome-targeted therapies for cancer.

Genetic modification of the human germ line: The reasons why this project has no future.

PubMed

Morange, Michel

2015-01-01

Modification of the human germ line has remained a distant but valuable objective for most biologists since the emergence of genetics (and even before). To study the historical transformations of this project, I have selected three periods - the 1930s, at the pinnacle of eugenics, around 1974 when molecular biology triumphed, and today - and have adopted three criteria to estimate the feasibility of this project: the state of scientific knowledge, the existence of suitable tools, and societal demands. Although the long-awaited techniques to modify the germ line are now available, I will show that most of the expectations behind this project have disappeared, or are considered as being reachable by highly different strategies. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Science, governance, and public participation: an analysis of decision making on genetic modification in Aotearoa/New Zealand.

PubMed

Kurian, Priya; Wright, Jeanette

2012-05-01

The acceptance of public participation in science and technology governance in liberal democratic contexts is evident in the institutionalization of a variety of mechanisms for participation in recent decades. Yet questions remain about the extent to which institutions have actually transformed their policy practice to embrace democratic governance of techno-scientific decision making. A critical discourse analysis of the response to public participation by the Environmental Risk ManagementAuthority (ERMA), the key decision-making body on genetic modification in Aotearoa/New Zealand, in a specific case demonstrates that ERMA systematically marginalized concerns raised by the public about risk management, ethics, and ecological, economic, and cultural issues in order to give primacy to a positivist, technological worldview. Such delegitimization of public perspectives pre-empts the possibility of the democratic governance of science.

Fetal alcohol spectrum disorders: gene-environment interactions, predictive biomarkers, and the relationship between structural alterations in the brain and functional outcomes.

PubMed

Reynolds, James N; Weinberg, Joanne; Clarren, Sterling; Beaulieu, Christian; Rasmussen, Carmen; Kobor, Michael; Dube, Marie-Pierre; Goldowitz, Daniel

2011-03-01

Prenatal alcohol exposure is a major, preventable cause of behavioral and cognitive deficits in children. Despite extensive research, a unique neurobehavioral profile for children affected by prenatal alcohol exposure remains elusive. A fundamental question that must be addressed is how genetic and environmental factors interact with gestational alcohol exposure to produce neurobehavioral and neurobiological deficits in children. The core objectives of the NeuroDevNet team in fetal alcohol spectrum disorders is to create an integrated research program of basic and clinical investigations that will (1) identify genetic and epigenetic modifications that may be predictive of the neurobehavioral and neurobiological dysfunctions in offspring induced by gestational alcohol exposure and (2) determine the relationship between structural alterations in the brain induced by gestational alcohol exposure and functional outcomes in offspring. The overarching hypothesis to be tested is that neurobehavioral and neurobiological dysfunctions induced by gestational alcohol exposure are correlated with the genetic background of the affected child and/or epigenetic modifications in gene expression. The identification of genetic and/or epigenetic markers that are predictive of the severity of behavioral and cognitive deficits in children affected by gestational alcohol exposure will have a profound impact on our ability to identify children at risk. Copyright © 2011 Elsevier Inc. All rights reserved.

Fetal Alcohol Spectrum Disorders: Gene-Environment Interactions, Predictive Biomarkers, and the Relationship Between Structural Alterations in the Brain and Functional Outcomes

PubMed Central

Reynolds, James N.; Weinberg, Joanne; Clarren, Sterling; Beaulieu, Christian; Rasmussen, Carmen; Kobor, Michael; Dube, Marie-Pierre; Goldowitz, Daniel

2016-01-01

Prenatal alcohol exposure is a major, preventable cause of behavioral and cognitive deficits in children. Despite extensive research, a unique neurobehavioral profile for children affected by prenatal alcohol exposure remains elusive. A fundamental question that must be addressed is how genetic and environmental factors interact with gestational alcohol exposure to produce neurobehavioral and neurobiological deficits in children. The core objectives of the NeuroDevNet team in fetal alcohol spectrum disorders is to create an integrated research program of basic and clinical investigations that will (1) identify genetic and epigenetic modifications that may be predictive of the neurobehavioral and neurobiological dysfunctions in offspring induced by gestational alcohol exposure and (2) determine the relationship between structural alterations in the brain induced by gestational alcohol exposure and functional outcomes in offspring. The overarching hypothesis to be tested is that neurobehavioral and neurobiological dysfunctions induced by gestational alcohol exposure are correlated with the genetic background of the affected child and/or epigenetic modifications in gene expression. The identification of genetic and/or epigenetic markers that are predictive of the severity of behavioral and cognitive deficits in children affected by gestational alcohol exposure will have a profound impact on our ability to identify children at risk. PMID:21575841

Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production.

PubMed

Uchida, Naoya; Demirci, Selami; Haro-Mora, Juan J; Fujita, Atsushi; Raines, Lydia N; Hsieh, Matthew M; Tisdale, John F

2018-06-15

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro . To model therapeutic strategies, we transduced human CD34 + cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34 + cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.

Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture.

PubMed Central

Sánchez-Palomino, S; Rojas, J M; Martínez, M A; Fenyö, E M; Nájera, R; Domingo, E; López-Galíndez, C

1993-01-01

We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes. Images PMID:8474182

Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells.

PubMed

Chen, Xuguang; Nomani, Alireza; Patel, Niket; Nouri, Faranak S; Hatefi, Arash

2018-01-01

Vectors used for stem cell transfection must be non-genotoxic, in addition to possessing high efficiency, because they could potentially transform normal stem cells into cancer-initiating cells. The objective of this research was to bioengineer an efficient vector that can be used for genetic modification of stem cells without any negative somatic or genetic impact. Two types of multifunctional vectors, namely targeted and non-targeted were genetically engineered and purified from E. coli. The targeted vectors were designed to enter stem cells via overexpressed receptors. The non-targeted vectors were equipped with MPG and Pep1 cell penetrating peptides. A series of commercial synthetic non-viral vectors and an adenoviral vector were used as controls. All vectors were evaluated for their efficiency and impact on metabolic activity, cell membrane integrity, chromosomal aberrations (micronuclei formation), gene dysregulation, and differentiation ability of stem cells. The results of this study showed that the bioengineered vector utilizing VEGFR-1 receptors for cellular entry could transfect mesenchymal stem cells with high efficiency without inducing genotoxicity, negative impact on gene function, or ability to differentiate. Overall, the vectors that utilized receptors as ports for cellular entry (viral and non-viral) showed considerably better somato- and genosafety profiles in comparison to those that entered through electrostatic interaction with cellular membrane. The genetically engineered vector in this study demonstrated that it can be safely and efficiently used to genetically modify stem cells with potential applications in tissue engineering and cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

50 CFR 660.409 - Inseason actions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... resource, any adjudicated Indian fishing rights, and the ocean allocation scheme in the fishery management..., the following: (i) Modification of quotas and/or fishing seasons. (ii) Modification of the species... retention regulations. (iii) Modification of recreational bag limits and recreational fishing days per...

50 CFR 660.409 - Inseason actions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... resource, any adjudicated Indian fishing rights, and the ocean allocation scheme in the fishery management..., the following: (i) Modification of quotas and/or fishing seasons. (ii) Modification of the species... retention regulations. (iii) Modification of recreational bag limits and recreational fishing days per...

Utility of CRISPR/Cas9 systems in hematology research.

PubMed

Lucas, Daniel; O'Leary, Heather A; Ebert, Benjamin L; Cowan, Chad A; Tremblay, Cedric S

2017-10-01

Since the end of the 20th century, novel approaches have emerged to manipulate experimental models of hematological disorders so that they more accurately mirror what is observed in the clinical setting. Despite these technological advances, the characterization of crucial genes for benign or malignant hematological disorders remains challenging, given the dynamic nature of the hematopoietic system and the genetic heterogeneity of these disorders. To overcome this limitation, genome-editing technologies have been developed to manipulate the genome specifically via deletion, insertion, or modification of targeted loci. These technologies have progressed swiftly, allowing their common use to investigate genetic function in experimental hematology. Among them, homologous-recombination-mediated targeting technologies have facilitated the manipulation of specific loci by generating knock-out and knock-in models. Despite promoting significant advances in our understanding of the molecular mechanisms involved in hematology, these inefficient, time-consuming, and labor-intensive approaches did not permit the development of cellular or animal models, recapitulating the complexity of hematological disorders. On October 26, 2016, Drs. Ben Ebert and Chad Cowan shared their knowledge of and experience with the utilization of CRISPR for models of myeloid malignancy, disease, and novel therapeutics in an International Society for Experimental Hematology webinar titled "Utility of CRISPR/Cas9 Systems in Hematology Research." Here, we provide an overview of the topics they covered, including their insights into the novel applications of the technique and its strengths and limitations. Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

GM as a route for delivery of sustainable crop protection.

PubMed

Bruce, Toby J A

2012-01-01

Modern agriculture, with its vast monocultures of lush fertilized crops, provides an ideal environment for adapted pests, weeds, and diseases. This vulnerability has implications for food security: when new pesticide-resistant pest biotypes evolve they can devastate crops. Even with existing crop protection measures, approximately one-third yield losses occur globally. Given the projected increase in demand for food (70% by 2050 according to the UN), sustainable ways of preventing these losses are needed. Development of resistant crop cultivars can make an important contribution. However, traditional crop breeding programmes are limited by the time taken to move resistance traits into elite crop genetic backgrounds and the limited gene pools in which to search for novel resistance. Furthermore, resistance based on single genes does not protect against the full spectrum of pests, weeds, and diseases, and is more likely to break down as pests evolve counter-resistance. Although not necessarily a panacea, GM (genetic modification) techniques greatly facilitate transfer of genes and thus provide a route to overcome these constraints. Effective resistance traits can be precisely and conveniently moved into mainstream crop cultivars. Resistance genes can be stacked to make it harder for pests to evolve counter-resistance and to provide multiple resistances to different attackers. GM-based crop protection could substantially reduce the need for farmers to apply pesticides to their crops and would make agricultural production more efficient in terms of resources used (land, energy, water). These benefits merit consideration by environmentalists willing to keep an open mind on the GM debate.

Design of Quiet Rotorcraft Approach Trajectories: Verification Phase

NASA Technical Reports Server (NTRS)

Padula, Sharon L.

2010-01-01

Flight testing that is planned for October 2010 will provide an opportunity to evaluate rotorcraft trajectory optimization techniques. The flight test will involve a fully instrumented MD-902 helicopter, which will be flown over an array of microphones. In this work, the helicopter approach trajectory is optimized via a multiobjective genetic algorithm to improve community noise, passenger comfort, and pilot acceptance. Previously developed optimization strategies are modified to accommodate new helicopter data and to increase pilot acceptance. This paper describes the MD-902 trajectory optimization plus general optimization strategies and modifications that are needed to reduce the uncertainty in noise predictions. The constraints that are imposed by the flight test conditions and characteristics of the MD-902 helicopter limit the testing possibilities. However, the insights that will be gained through this research will prove highly valuable.

Prevention of Age-Related Macular Degeneration.

PubMed

Singh, Niharika; Srinivasan, Sangeetha; Muralidharan, Vinata; Roy, Rupak; V, Jayprakash; Raman, Rajiv

2017-01-01

Age-related macular degeneration (AMD) compromises quality of life. However, the available therapeutic options are limited. This has led to the identification of modifiable risk factors to prevent the development or alter the natural course and prognosis of AMD. The identification and modification of risk factors has the potential for greater public health impact on reducing morbidity from AMD. Likewise, identifying the imaging clues and genetic clues could serve as a guide to recognizing the propensity for progression to severe and end stages of the disease. Several attempts, both successful and unsuccessful, have been made for interventions that could delay the progression of AMD. Of these, pharmacological interventions have shown promising results. The Age-Related Eye Disease Study 1 and 2 have shown the beneficial role of antioxidants in a selected group of patients. Copyright 2017 Asia-Pacific Academy of Ophthalmology.

Characterization and enzymatic hydrolysis of wood from transgenic Pinus taeda engineered with syringyl lignin or reduced lignin content

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edmunds, Charles W.; Peralta, Perry; Kelley, Stephen S.

Softwood is an abundant resource; however, currently its utilization for bioconversion to obtain platform sugars is limited. Pinus taeda trees which were genetically modified to either produce S lignin or to decrease lignin content were characterized with a suite of analytic techniques. Syringyl lignin was visualized in the secondary xylem of one genetic line with Maule staining. Solid-state nuclear magnetic resonance identified the S lignin units were coupled into the lignin through β-O-4 linkages, and thioacidolysis measured approximately 13% S lignin content in the same sample. Reductions of the lignin of as much as 33% were observed in the transgenics.more » To better understand how these modifications affect bioconversion, their amenability to hot water and dilute acid pretreatments and enzymatic hydrolysis was evaluated. Lignin reductions resulted in 1.9-3.2-fold increases in glucose release compared to the control. However, no apparent benefit was observed by S lignin incorporation at the concentrations reported in this study. Finally, these results highlight the potential for softwood cell wall properties to be improved for bioenergy/biochemical applications.« less

Characterization and enzymatic hydrolysis of wood from transgenic Pinus taeda engineered with syringyl lignin or reduced lignin content

DOE PAGES

Edmunds, Charles W.; Peralta, Perry; Kelley, Stephen S.; ...

2017-02-22

Softwood is an abundant resource; however, currently its utilization for bioconversion to obtain platform sugars is limited. Pinus taeda trees which were genetically modified to either produce S lignin or to decrease lignin content were characterized with a suite of analytic techniques. Syringyl lignin was visualized in the secondary xylem of one genetic line with Maule staining. Solid-state nuclear magnetic resonance identified the S lignin units were coupled into the lignin through β-O-4 linkages, and thioacidolysis measured approximately 13% S lignin content in the same sample. Reductions of the lignin of as much as 33% were observed in the transgenics.more » To better understand how these modifications affect bioconversion, their amenability to hot water and dilute acid pretreatments and enzymatic hydrolysis was evaluated. Lignin reductions resulted in 1.9-3.2-fold increases in glucose release compared to the control. However, no apparent benefit was observed by S lignin incorporation at the concentrations reported in this study. Finally, these results highlight the potential for softwood cell wall properties to be improved for bioenergy/biochemical applications.« less

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

PubMed

Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

Epigenetics and obesity: a relationship waiting to be explained.

PubMed

Symonds, Michael E; Budge, Helen; Frazier-Wood, Alexis C

2013-01-01

Obesity can have multifactorial causes that may change with development and are not simply attributable to one's genetic constitution. To date, expensive and laborious genome-wide association studies have only ascribed a small contribution of genetic variants to obesity. The emergence of the field of epigenetics now offers a new paradigm with which to study excess fat mass. Currently, however, there are no compelling epigenetic studies to explain the role of epigenetics in obesity, especially from a developmental perspective. It is clear that until there are advances in the understanding of the main mechanisms by which different fat types, i.e. brown, beige, and white, are established and how these differ between depots and species, population-based studies designed to determine specific aspects of epigenetics will be potentially limited. Obesity is a slowly evolving condition that is not simply explained by changes in the intake of one macronutrient. The latest advances in epigenetics, coupled with the establishment of relevant longitudinal models of obesity, which incorporate functionally relevant end points, may now permit the precise contribution of epigenetic modifications to excess fat mass to be effectively studied. © 2013 S. Karger AG, Basel.

Radiogenomics: a systems biology approach to understanding genetic risk factors for radiotherapy toxicity ?

PubMed Central

Herskind, Carsten; Talbot, Christopher J.; Kerns, Sarah L.; Veldwijk, Marlon R.; Rosenstein, Barry S.; West, Catharine M. L.

2016-01-01

Adverse reactions in normal tissue after radiotherapy (RT) limit the dose that can be given to tumour cells. Since 80% of individual variation in clinical response is estimated to be caused by patient-related factors, identifying these factors might allow prediction of patients with increased risk of developing severe reactions. While inactivation of cell renewal is considered a major cause of toxicity in early-reacting normal tissues, complex interactions involving multiple cell types, cytokines, and hypoxia seem important for late reactions. Here, we review ‘omics’ approaches such as screening of genetic polymorphisms or gene expression analysis, and assess the potential of epigenetic factors, posttranslational modification, signal transduction, and metabolism. Furthermore, functional assays have suggested possible associations with clinical risk of adverse reaction. Pathway analysis incorporating different ‘omics’ approaches may be more efficient in identifying critical pathways than pathway analysis based on single ‘omics’ data sets. Integrating these pathways with functional assays may be powerful in identifying multiple subgroups of RT patients characterized by different mechanisms. Thus ‘omics’ and functional approaches may synergize if they are integrated into radiogenomics ‘systems biology’ to facilitate the goal of individualised radiotherapy. PMID:26944314

Genetics Home Reference: mitochondrial neurogastrointestinal encephalopathy disease

MedlinePlus

... modification) is used as a building block of DNA . Thymidine phosphorylase breaks down thymidine into smaller molecules, ... molecule is damaging to a particular kind of DNA known as mitochondrial DNA or mtDNA. Mitochondria are ...

Five Faces of Cognition: Theoretical Influences on Approaches to Learning Disabilities.

ERIC Educational Resources Information Center

Hresko, Wayne P.; Reid, D. Kim

1981-01-01

The label "cognitive" has been used to designate five substantially different approaches to the study of learning disabilities: information processing, metacognition, genetic epistemology, cognitive behavior modification, and the specific abilities model. (Author)

Plant cell walls to ethanol.

USDA-ARS?s Scientific Manuscript database

Conversion of plant cell walls to ethanol constitutes generation 2 bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation, and separation. Ultimately, it is desired to combine as man...

Laboratory Techniques for the Blind

ERIC Educational Resources Information Center

Tombaugh, Dorothy

1972-01-01

Describes modifications of laboratory procedures for the BSCS Green Version biology, including dissection, microbiology, animal behavior, physiology, biochemistry, and genetics that make the methods suitable for direct experimentation by blind students. Discusses models as substitutes for microscopy. (AL)

Distributed genetic algorithms for the floorplan design problem

NASA Technical Reports Server (NTRS)

Cohoon, James P.; Hegde, Shailesh U.; Martin, Worthy N.; Richards, Dana S.

1991-01-01

Designing a VLSI floorplan calls for arranging a given set of modules in the plane to minimize the weighted sum of area and wire-length measures. A method of solving the floorplan design problem using distributed genetic algorithms is presented. Distributed genetic algorithms, based on the paleontological theory of punctuated equilibria, offer a conceptual modification to the traditional genetic algorithms. Experimental results on several problem instances demonstrate the efficacy of this method and indicate the advantages of this method over other methods, such as simulated annealing. The method has performed better than the simulated annealing approach, both in terms of the average cost of the solutions found and the best-found solution, in almost all the problem instances tried.

76 FR 49368 - Atlantic Highly Migratory Species; Modification of the Retention of Incidentally-Caught Highly...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-10

.... 110112022-1262-02] RIN 0648-BA45 Atlantic Highly Migratory Species; Modification of the Retention of.... SUMMARY: This final rule modifies the permitting requirements and retention limits for Atlantic highly... establishes a retention limit for smoothhound sharks in all Atlantic trawl fisheries. These actions are...

77 FR 50963 - Modification of Regulations Regarding the Definition of Factual Information and Time Limits for...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-23

... DEPARTMENT OF COMMERCE International Trade Administration 19 CFR Part 351 RIN 0625-AA91 Modification of Regulations Regarding the Definition of Factual Information and Time Limits for Submission of... Commerce. ACTION: Proposed rule; extension of comment period. SUMMARY: On July 10, 2012, the Department of...

76 FR 69688 - Modification of the Port Limits of Green Bay, WI

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-09

...)). These ports are listed according to location by districts and are designated as Class A, B, or C. Green... docket as ``Attachment B: Green Bay (Proposed).'' V. Regulatory Requirements A. Signing Authority The.... USCBP-2011-0031] Modification of the Port Limits of Green Bay, WI AGENCY: U.S. Customs and Border...

An HPLC-ICP-MS technique for determination of cadmium-phytochelatins in genetically modified Arabidopsis thaliana.

PubMed

Sadi, Baki B M; Vonderheide, Anne P; Gong, Ji-Ming; Schroeder, Julian I; Shann, Jodi R; Caruso, Joseph A

2008-01-01

A reversed-phase high-performance liquid chromatographic technique was developed to separate cadmium-phytochelatin complexes (Cd-PC2, Cd-PC3, and Cd-PC4) of interest in the plant Arapidopsis thaliana. High-performance liquid chromatography (HPLC) was coupled to an inductively coupled plasma mass spectrometric (ICP-MS) system with some modification to the interface. This was done in order to sustain the plasma with optimum sensitivity for cadmium detection in the presence of the high methanol loads used in the gradient elution of the reversed-phase separation. The detection limits were found to be 91.8 ngl(-1), 77.2 ngl(-1) and 49.2 ngl(-1) for Cd-PC2, Cd-PC3, and Cd-PC4 respectively. The regression coefficients (r2) for Cd-PC2 to Cd-PC4 detection ranged from 0.998 to 0.999. The method was then used to investigate the occurrence and effect of cadmium-phytochelatin complexes in wild-type Arabidopsis and a phytochelatin-deficient mutant cad1-3 that had been genetically modified to ectopically express the wheat TaPCS1 phytochelatin synthase enzyme. The primary complex found in both wild-type and transgenic plants was Cd-PC2. In both lines, higher levels of Cd-PC2 were found in shoots than in roots, showing that phytochelatin synthases contribute to the accumulation of cadmium in shoots, in the Cd-PC2 form. Genetic modification did, however, impact the overall accumulation of Cd. Transgenic plants contained almost two times more cadmium in the form of Cd-PC2 in their roots than did the corresponding wild-type plants. Similarly, the shoot samples of the modified species also contained more (by 1.6 times) cadmium in the form of Cd-PC2 than the wild type. The enhanced role of PC2 in the transgenic Arabidopsis correlates with data showing long-distance transport of Cd in transgenic plants. Targeted transgenic expression of non-native phytochelatin synthases may contribute to improving the efficiency of plants for phytoremediation.

Gene modification therapies: views of parents of people with Down syndrome.

PubMed

Michie, Marsha; Allyse, Megan

2018-06-21

In considering gene modification technologies, the priorities of patient communities must be a central consideration. The purpose of this study is to assess views of families with Down syndrome (DS) regarding potential genome-based interventions. We constructed an anonymous online survey for family members of people with DS. Participants were asked to agree or disagree with scenarios describing hypothetical interventions to silence or significantly alter the physical and cognitive effects of a trisomy 21, and also with scenarios depicting currently available physical interventions. All 532 respondents were parents of people with DS. For each of the five scenarios, over half said they would approve the intervention or would advise their children with DS to do so. Responses to hypothetical prenatal and pediatric cognitive interventions were significantly affected by participants' assessments of the impact of DS on their children's and their families' lives, while physical and adult cognitive scenarios were not. Future interventions to address genetic conditions will impact patient communities and cannot succeed without their input and support. While many parents of people with DS indicated approval for hypothetical genetic therapies, these results indicate a need for continuing dialogue about benefits and drawbacks of gene modification technologies.

Disclosing Pleiotropic Effects during Genetic Risk Assessment for Alzheimer’s Disease: A Randomized, Controlled Trial

PubMed Central

Christensen, Kurt D.; Roberts, J. Scott; Whitehouse, Peter J.; Royal, Charmaine D. M.; Obisesan, Thomas O.; Cupples, L. Adrienne; Vernarelli, Jacqueline A.; Bhatt, Deepak L.; Linnenbringer, Erin; Butson, Melissa B.; Fasaye, Grace-Ann; Uhlmann, Wendy R.; Hiraki, Susan; Wang, Na; Cook-Deegan, Robert; Green, Robert C.

2016-01-01

Background Increasing use of genetic testing raises questions about disclosing secondary findings, including pleiotropic information. Objective To determine the safety and behavioral impact of disclosing modest associations between APOE genotype and coronary artery disease (CAD) risk during APOE-based genetic risk assessments for Alzheimer’s disease (AD). Design Randomized, multicenter equivalence clinical trial Setting Four teaching hospitals Participants 257 asymptomatic adults enrolled, 69% with one AD-affected first degree relative Intervention Disclosing AD and CAD genetic risk information (AD+CAD) versus disclosing only AD genetic risk (AD-only) Measurements Co-primary outcomes were Beck Anxiety Index (BAI) and Center for Epidemiologic Studies Depression Scale (CES-D) scores at 12 months. Secondary outcomes included test-related distress at 12 months, all measures at 6 weeks and 6 months, and health behavior changes at 12 months. Results 12 months after disclosure, mean BAI scores were 3.5 and 3.5 in AD-only and AD+CAD arms (Δ=0.0, 95%CI: −1.0 to 1.0), and mean CES-D scores were 6.4 and 7.1 in AD-only and AD+CAD arms (Δ=0.7, 95%CI: −1.0 to 2.4). Both confidence bounds fell within the equivalence margin of +/−5 points. Among ε4-positive participants, distress was lower in AD+CAD arms than AD-only arms (Δ=−4.8, 95%CI: −8.6 to −1.0) (p=0.031 for disclosure arm x APOE genotype). AD+CAD participants also reported more health behavior changes, regardless of APOE genotype. Limitations Outcomes were self-reported from volunteers without severe anxiety, severe depression, or cognitive problems. Analyses omitted 33 randomized participants. Conclusion Disclosing pleiotropic information did not increase anxiety or depression, and may have decreased distress among those at increased risk for two conditions. Providing risk modification information regarding CAD improved health behaviors. Findings highlight potential benefits of secondary genetic findings disclosure when options exist for decreasing risk. PMID:26810768

Enhanced energy transport in genetically engineered excitonic networks.

PubMed

Park, Heechul; Heldman, Nimrod; Rebentrost, Patrick; Abbondanza, Luigi; Iagatti, Alessandro; Alessi, Andrea; Patrizi, Barbara; Salvalaggio, Mario; Bussotti, Laura; Mohseni, Masoud; Caruso, Filippo; Johnsen, Hannah C; Fusco, Roberto; Foggi, Paolo; Scudo, Petra F; Lloyd, Seth; Belcher, Angela M

2016-02-01

One of the challenges for achieving efficient exciton transport in solar energy conversion systems is precise structural control of the light-harvesting building blocks. Here, we create a tunable material consisting of a connected chromophore network on an ordered biological virus template. Using genetic engineering, we establish a link between the inter-chromophoric distances and emerging transport properties. The combination of spectroscopy measurements and dynamic modelling enables us to elucidate quantum coherent and classical incoherent energy transport at room temperature. Through genetic modifications, we obtain a significant enhancement of exciton diffusion length of about 68% in an intermediate quantum-classical regime.

Possibilities for the evolution of the genetic code from a preceding form

NASA Technical Reports Server (NTRS)

Jukes, T. H.

1973-01-01

Analysis of the interaction between mRNA codons and tRNA anticodons suggests a model for the evolution of the genetic code. Modification of the nucleic acid following the anticodon is at present essential in both eukaryotes and prokaryotes to ensure fidelity of translation of codons starting with A, and the amino acids which could be coded for before the evolution of the modifying enzymes can be deduced.

Heritability of targeted gene modifications induced by plant-optimized CRISPR systems.

PubMed

Mao, Yanfei; Botella, Jose Ramon; Zhu, Jian-Kang

2017-03-01

The Streptococcus-derived CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) system has emerged as a very powerful tool for targeted gene modifications in many living organisms including plants. Since the first application of this system for plant gene modification in 2013, this RNA-guided DNA endonuclease system has been extensively engineered to meet the requirements of functional genomics and crop trait improvement in a number of plant species. Given its short history, the emphasis of many studies has been the optimization of the technology to improve its reliability and efficiency to generate heritable gene modifications in plants. Here we review and analyze the features of customized CRISPR/Cas9 systems developed for plant genetic studies and crop breeding. We focus on two essential aspects: the heritability of gene modifications induced by CRISPR/Cas9 and the factors affecting its efficiency, and we provide strategies for future design of systems with improved activity and heritability in plants.

Epigenetics of reproductive infertility.

PubMed

Das, Laxmidhar; Parbin, Sabnam; Pradhan, Nibedita; Kausar, Chahat; Patra, Samir K

2017-06-01

Infertility is a complex pathophysiological condition. It may caused by specific or multiple physical and physiological factors, including abnormalities in homeostasis, hormonal imbalances and genetic alterations. In recent times various studies implicated that, aberrant epigenetic mechanisms are associated with reproductive infertility. There might be transgenerational effects associated with epigenetic modifications of gametes and studies suggest the importance of alterations in epigenetic modification at early and late stages of gametogenesis. To determine the causes of infertility it is necessary to understand the altered epigenetic modifications of associated gene and mechanisms involved therein. This review is devoted to elucidate the recent mechanistic advances in regulation of genes by epigenetic modification and emphasizes their possible role related to reproductive infertility. It includes environmental, nutritional, hormonal and physiological factors and influence of internal structural architecture of chromatin nucleosomes affecting DNA and histone modifications in both male and female gametes, early embryogenesis and offspring. Finally, we would like to emphasize that research on human infertility by gene knock out of epigenetic modifiers genes must be relied upon animal models.

Pluripotent Stem Cells and Gene Therapy

PubMed Central

Simara, Pavel; Motl, Jason A.; Kaufman, Dan S.

2013-01-01

Human pluripotent stem cells represent an accessible cell source for novel cell-based clinical research and therapies. With the realization of induced pluripotent stem cells (iPSCs), it is possible to produce almost any desired cell type from any patient's cells. Current developments in gene modification methods have opened the possibility for creating genetically corrected human iPSCs for certain genetic diseases that could be used later in autologous transplantation. Promising preclinical studies have demonstrated correction of disease-causing mutations in a number of hematological, neuronal and muscular disorders. This review aims to summarize these recent advances with a focus on iPSC generation techniques, as well as gene modification methods. We will then further discuss some of the main obstacles remaining to be overcome before successful application of human pluripotent stem cell-based therapy arrives in the clinic and what the future of stem cell research may look like. PMID:23353080

Germline Editing: Editors Cautionary.

PubMed

Krishan, K; Kanchan, T; Singh, B; Baryah, N; Puri, S

2018-01-01

This communication is regarding the recent editing of the genome of the human embryo with CRISPR/Cas9 which generated a debate amongst the biological scientists around the world. Editing human germline genes may act as godsend in some serious genetic and other disorders as the genes related to these disorders can be replaced effectively. The scientists are in dilemma whether the human germline gene modification is a boon or bane for the human society. Though editing human germline genes may be an answer to many serious genetic disorders however; it may have unpredictable effects on future generations. The ethical issues regarding the germline editing need further discussion which may have implications on human race and on-going human evolution. Thus, the researchers need to be doubly cautious and some stringent regulations should be framed regarding the various aspects of germ line gene modifications and any potential conflict with nature for future outcome.

Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

PubMed

Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

2015-08-10

The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

Advances in epigenetics and epigenomics for neurodegenerative diseases.

PubMed

Qureshi, Irfan A; Mehler, Mark F

2011-10-01

In the post-genomic era, epigenetic factors-literally those that are "over" or "above" genetic ones and responsible for controlling the expression and function of genes-have emerged as important mediators of development and aging; gene-gene and gene-environmental interactions; and the pathophysiology of complex disease states. Here, we provide a brief overview of the major epigenetic mechanisms (ie, DNA methylation, histone modifications and chromatin remodeling, and non-coding RNA regulation). We highlight the nearly ubiquitous profiles of epigenetic dysregulation that have been found in Alzheimer's and other neurodegenerative diseases. We also review innovative methods and technologies that enable the characterization of individual epigenetic modifications and more widespread epigenomic states at high resolution. We conclude that, together with complementary genetic, genomic, and related approaches, interrogating epigenetic and epigenomic profiles in neurodegenerative diseases represent important and increasingly practical strategies for advancing our understanding of and the diagnosis and treatment of these disorders.

Advances in Epigenetics and Epigenomics for Neurodegenerative Diseases

PubMed Central

Qureshi, Irfan A.

2015-01-01

In the post-genomic era, epigenetic factors—literally those that are “over” or “above” genetic ones and responsible for controlling the expression and function of genes—have emerged as important mediators of development and aging; gene-gene and gene-environmental interactions; and the pathophysiology of complex disease states. Here, we provide a brief overview of the major epigenetic mechanisms (ie, DNA methylation, histone modifications and chromatin remodeling, and non-coding RNA regulation). We highlight the nearly ubiquitous profiles of epigenetic dysregulation that have been found in Alzheimer’s and other neurodegenerative diseases. We also review innovative methods and technologies that enable the characterization of individual epigenetic modifications and more widespread epigenomic states at high resolution. We conclude that, together with complementary genetic, genomic, and related approaches, interrogating epigenetic and epigenomic profiles in neurodegenerative diseases represent important and increasingly practical strategies for advancing our understanding of and the diagnosis and treatment of these disorders. PMID:21671162

Engineering Escherichia coli for improved ethanol production from gluconate.

PubMed

Hildebrand, Amanda; Schlacta, Theresa; Warmack, Rebeccah; Kasuga, Takao; Fan, Zhiliang

2013-10-10

We report on engineering Escherichia coli to produce ethanol at high yield from gluconic acid (gluconate). Knocking out genes encoding for the competing pathways (l-lactate dehydrogenase and pyruvate formate lyase A) in E. coli KO11 eliminated lactate production, lowered the carbon flow toward acetate production, and improved the ethanol yield from 87.5% to 97.5% of the theoretical maximum, while the growth rate of the mutant strain was about 70% of the wild type. The corresponding genetic modifications led to a small improvement of ethanol yield from 101.5% to 106.0% on glucose. Deletion of the pyruvate dehydrogenase gene (pdh) alone improved the ethanol yield from 87.5% to 90.4% when gluconate was a substrate. The growth rate of the mutant strain was identical to that of the wild type. The corresponding genetic modification led to no improvements on ethanol yield on glucose. Copyright © 2013 Elsevier B.V. All rights reserved.

Genetic modification of stem cells for transplantation.

PubMed

Phillips, M Ian; Tang, Yao Liang

2008-01-14

Gene modification of cells prior to their transplantation, especially stem cells, enhances their survival and increases their function in cell therapy. Like the Trojan horse, the gene-modified cell has to gain entrance inside the host's walls and survive and deliver its transgene products. Using cellular, molecular and gene manipulation techniques the transplanted cell can be protected in a hostile environment from immune rejection, inflammation, hypoxia and apoptosis. Genetic engineering to modify cells involves constructing modules of functional gene sequences. They can be simple reporter genes or complex cassettes with gene switches, cell specific promoters and multiple transgenes. We discuss methods to deliver and construct gene cassettes with viral and non-viral delivery, siRNA, and conditional Cre/Lox P. We review the current uses of gene-modified stem cells in cardiovascular disease, diabetes, neurological diseases, (including Parkinson's, Alzheimer's and spinal cord injury repair), bone defects, hemophilia, and cancer.

Genetic modification of cells for transplantation.

PubMed

Lai, Yi; Drobinskaya, Irina; Kolossov, Eugen; Chen, Chunguang; Linn, Thomas

2008-01-14

Progress in gene therapy has produced promising results that translate experimental research into clinical treatment. Gene modification has been extensively employed in cell transplantation. The main barrier is an effective gene delivery system. Several viral vectors were utilized in end-stage differentiated cells. Recently, successful applications were described with adenovirus-associated vectors. As an alternative, embryonic stem cell- and stem cell-like systems were established for generation of tissue-specified gene-modified cells. Owing to the feasibility for genetic manipulations and the self-renewing potency of these cells they can be used in a way enabling large-scale in vitro production. This approach offers the establishment of in vitro cell culture systems that will deliver sufficient amounts of highly purified, immunoautologous cells suitable for application in regenerative medicine. In this review, the current technology of gene delivery systems to cells is recapitulated and the latest developments for cell transplantation are discussed.

Genetic therapies for RNA mis-splicing diseases.

PubMed

Hammond, Suzan M; Wood, Matthew J A

2011-05-01

RNA mis-splicing diseases account for up to 15% of all inherited diseases, ranging from neurological to myogenic and metabolic disorders. With greatly increased genomic sequencing being performed for individual patients, the number of known mutations affecting splicing has risen to 50-60% of all disease-causing mutations. During the past 10years, genetic therapy directed toward correction of RNA mis-splicing in disease has progressed from theoretical work in cultured cells to promising clinical trials. In this review, we discuss the use of antisense oligonucleotides to modify splicing as well as the principles and latest work in bifunctional RNA, trans-splicing and modification of U1 and U7 snRNA to target splice sites. The success of clinical trials for modifying splicing to treat Duchenne muscular dystrophy opens the door for the use of splicing modification for most of the mis-splicing diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cloning and transgenesis in mammals: implications for xenotransplantation.

PubMed

Piedrahita, Jorge A; Mir, Bashir

2004-01-01

Availability of suitable organs for transplantation remains of major concern and projections indicate that the problem will continue to increase. Therefore, alternatives to the use of human organs for transplantation, continue to be explored including use of stem cells, artificial organs, and organs from other species (xenotransplantation). In xenotransplantation, the species of choice remains the pig due to its physiological similarities to humans, reduced costs, ease of manipulation, and reduced ethical concerns to its use. However, in order to develop pig organs that are suitable for xenotransplantation, complex genetic modification need to be undertaken. These modifications require the introduction of precise genetic changes into the pig that can only be accomplished at this time using somatic cell nuclear transfer. We cover in this review advances in transgenic manipulation and cloning in swine and how the development of these two technologies is critical to the eventual utilization of the pig as a human organ donor.

Alien Genetic Algorithm for Exploration of Search Space

NASA Astrophysics Data System (ADS)

Patel, Narendra; Padhiyar, Nitin

2010-10-01

Genetic Algorithm (GA) is a widely accepted population based stochastic optimization technique used for single and multi objective optimization problems. Various versions of modifications in GA have been proposed in last three decades mainly addressing two issues, namely increasing convergence rate and increasing probability of global minima. While both these. While addressing the first issue, GA tends to converge to a local optima and addressing the second issue corresponds the large computational efforts. Thus, to reduce the contradictory effects of these two aspects, we propose a modification in GA by adding an alien member in the population at every generation. Addition of an Alien member in the current population at every generation increases the probability of obtaining global minima at the same time maintaining higher convergence rate. With two test cases, we have demonstrated the efficacy of the proposed GA by comparing with the conventional GA.

Genetic Modification of Stem Cells for Transplantation

PubMed Central

Phillips, M. Ian; Tang, Yao Liang

2009-01-01

Gene modification of cells for prior to their transplantation, especially stem cells, enhances their survival and increases their function in cell therapy. Like the Trojan horse, the gene modified cell has to gain entrance inside the host’s walls and survive and deliver its transgene products Using cellular, molecular and gene manipulation techniques the transplanted cell can be protected in a hostile environment from immune rejection, inflammation, hypoxia and apoptosis. Genetic engineering to modify cells involves constructing modules of functional gene sequences. They can be simple reporter genes or complex cassettes with gene switches, cell specific promoters and multiple transgenes. We discuss methods to deliver and construct gene cassettes with viral and non viral delivery, siRNA, and conditional Cre/Lox P. We review the current uses of gene modified stem cells in cardiovascular disease, diabetes, neurological diseases,( including Parkinson’s, Alzheimer’s and spinal cord injury repair), bone defects, hemophilia, and cancer. PMID:18031863

A survey of the use of soy in processed Turkish meat products and detection of genetic modification.

PubMed

Ulca, Pelin; Balta, Handan; Senyuva, Hamide Z

2014-01-01

To screen for possible illegal use of soybeans in meat products, the performance characteristics of a commercial polymer chain reaction (PCR) kit for detection of soybean DNA in raw and cooked meat products were established. Minced chicken and beef products containing soybean at levels from 0.1% to 10.0% were analysed by real-time PCR to amplify the soybean lectin gene. The PCR method could reliably detect the addition of soybean at a level of 0.1%. A survey of 38 Turkish processed meat products found only six samples to be negative for the presence of soybean. In 32 (84%) positive samples, 13 (34%) contained levels of soy above 0.1%. Of soybean positive samples, further DNA analysis was conducted by real-time PCR to detect whether genetically modified (GM) soybean had been used. Of 32 meat samples containing soybean, two samples were positive for GM modification.

The genetic incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast.

PubMed

Supekova, Lubica; Zambaldo, Claudio; Choi, Seihyun; Lim, Reyna; Luo, Xiaozhou; Kazane, Stephanie A; Young, Travis S; Schultz, Peter G

2018-05-15

The noncanonical amino acid p-azidomethyl-l-phenylalanine can be genetically incorporated into proteins in bacteria, and has been used both as a spectroscopic probe and for the selective modification of proteins by alkynes using click chemistry. Here we report identification of Escherichia coli tyrosyl tRNA synthetase mutants that allow incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast. When expressed together with the cognate E. coli tRNA CUA Tyr , the new mutant tyrosyl tRNA synthetases directed robust incorporation of p-azidomethyl-l-phenylalanine into a model protein, human superoxide dismutase, in response to the UAG amber nonsense codon. Mass spectrometry analysis of purified superoxide dismutase proteins confirmed the efficient site-specific incorporation of p-azidomethyl-l-phenylalanine. This work provides an additional tool for the selective modification of proteins in eukaryotic cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparative genomic analysis of Helicobacter pylori from Malaysia identifies three distinct lineages suggestive of differential evolution

PubMed Central

Kumar, Narender; Mariappan, Vanitha; Baddam, Ramani; Lankapalli, Aditya K.; Shaik, Sabiha; Goh, Khean-Lee; Loke, Mun Fai; Perkins, Tim; Benghezal, Mohammed; Hasnain, Seyed E.; Vadivelu, Jamuna; Marshall, Barry J.; Ahmed, Niyaz

2015-01-01

The discordant prevalence of Helicobacter pylori and its related diseases, for a long time, fostered certain enigmatic situations observed in the countries of the southern world. Variation in H. pylori infection rates and disease outcomes among different populations in multi-ethnic Malaysia provides a unique opportunity to understand dynamics of host–pathogen interaction and genome evolution. In this study, we extensively analyzed and compared genomes of 27 Malaysian H. pylori isolates and identified three major phylogeographic lineages: hspEastAsia, hpEurope and hpSouthIndia. The analysis of the virulence genes within the core genome, however, revealed a comparable pathogenic potential of the strains. In addition, we identified four genes limited to strains of East-Asian lineage. Our analyses identified a few strain-specific genes encoding restriction modification systems and outlined 311 core genes possibly under differential evolutionary constraints, among the strains representing different ethnic groups. The cagA and vacA genes also showed variations in accordance with the host genetic background of the strains. Moreover, restriction modification genes were found to be significantly enriched in East-Asian strains. An understanding of these variations in the genome content would provide significant insights into various adaptive and host modulation strategies harnessed by H. pylori to effectively persist in a host-specific manner. PMID:25452339

Costs of Limiting Route Optimization to Published Waypoints in the Traffic Aware Planner

NASA Technical Reports Server (NTRS)

Karr, David A.; Vivona, Robert A.; Wing, David J.

2013-01-01

The Traffic Aware Planner (TAP) is an airborne advisory tool that generates optimized, traffic-avoiding routes to support the aircraft crew in making strategic reroute requests to Air Traffic Control (ATC). TAP is derived from a research-prototype self-separation tool, the Autonomous Operations Planner (AOP), in which optimized route modifications that avoid conflicts with traffic and weather, using waypoints at explicit latitudes and longitudes (a technique supported by self-separation concepts), are generated by maneuver patterns applied to the existing route. For use in current-day operations in which trajectory changes must be requested from ATC via voice communication, TAP produces optimized routes described by advisories that use only published waypoints prior to a reconnection waypoint on the existing route. We describe how the relevant algorithms of AOP have been modified to implement this requirement. The modifications include techniques for finding appropriate published waypoints in a maneuver pattern and a method for combining the genetic algorithm of AOP with an exhaustive search of certain types of advisory. We demonstrate methods to investigate the increased computation required by these techniques and to estimate other costs (measured in terms such as time to destination and fuel burned) that may be incurred when only published waypoints are used.

Recent Progress in Stem Cell Modification for Cardiac Regeneration

PubMed Central

Voronina, Natalia; Steinhoff, Gustav

2018-01-01

During the past decades, stem cell-based therapy has acquired a promising role in regenerative medicine. The application of novel cell therapeutics for the treatment of cardiovascular diseases could potentially achieve the ambitious aim of effective cardiac regeneration. Despite the highly positive results from preclinical studies, data from phase I/II clinical trials are inconsistent and the improvement of cardiac remodeling and heart performance was found to be quite limited. The major issues which cardiac stem cell therapy is facing include inefficient cell delivery to the site of injury, accompanied by low cell retention and weak effectiveness of remaining stem cells in tissue regeneration. According to preclinical and clinical studies, various stem cells (adult stem cells, embryonic stem cells, and induced pluripotent stem cells) represent the most promising cell types so far. Beside the selection of the appropriate cell type, researchers have developed several strategies to produce “second-generation” stem cell products with improved regenerative capacity. Genetic and nongenetic modifications, chemical and physical preconditioning, and the application of biomaterials were found to significantly enhance the regenerative capacity of transplanted stem cells. In this review, we will give an overview of the recent developments in stem cell engineering with the goal to facilitate stem cell delivery and to promote their cardiac regenerative activity. PMID:29535769

Cancer cell-selective promoter recognition accompanies antitumor effect by glucocorticoid receptor-targeted gold nanoparticle

NASA Astrophysics Data System (ADS)

Sau, Samaresh; Agarwalla, Pritha; Mukherjee, Sudip; Bag, Indira; Sreedhar, Bojja; Pal-Bhadra, Manika; Patra, Chitta Ranjan; Banerjee, Rajkumar

2014-05-01

Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of `exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of `endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of `endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics.Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of `exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of `endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of `endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr00974f

A Simple Method for Visualization of Locus-Specific H4K20me1 Modifications in Living Caenorhabditis elegans Single Cells.

PubMed

Shinkai, Yoichi; Kuramochi, Masahiro; Doi, Motomichi

2018-05-03

Recently, advances in next-generation sequencing technologies have enabled genome-wide analyses of epigenetic modifications; however, it remains difficult to analyze the states of histone modifications at a single-cell resolution in living multicellular organisms because of the heterogeneity within cellular populations. Here we describe a simple method to visualize histone modifications on the specific sequence of target locus at a single-cell resolution in living Caenorhabditis elegans , by combining the LacO/LacI system and a genetically-encoded H4K20me1-specific probe, "mintbody". We demonstrate that Venus-labeled mintbody and mTurquoise2-labeled LacI can co-localize on an artificial chromosome carrying both the target locus and LacO sequences, where H4K20me1 marks the target locus. We demonstrate that our visualization method can precisely detect H4K20me1 depositions on the her-1 gene sequences on the artificial chromosome, to which the dosage compensation complex binds to regulate sex determination. The degree of H4K20me1 deposition on the her-1 sequences on the artificial chromosome correlated strongly with sex, suggesting that, using the artificial chromosome, this method can reflect context-dependent changes of H4K20me1 on endogenous genomes. Furthermore, we demonstrate live imaging of H4K20me1 depositions on the artificial chromosome. Combined with ChIP assays, this mintbody-LacO/LacI visualization method will enable analysis of developmental and context-dependent alterations of locus-specific histone modifications in specific cells and elucidation of the underlying molecular mechanisms. Copyright © 2018, G3: Genes, Genomes, Genetics.

Examination of arthritis-related work place activity limitations and intermittent disability over four-and-a-half years and its relationship to job modifications and outcomes.

PubMed

Gignac, Monique A M; Cao, Xingshan; Tang, Kenneth; Beaton, Dorcas E

2011-07-01

To examine the type, degree, and episodic nature of arthritis-related work place activity limitations and the consistency of the relationship of activity limitations to job modifications and work place outcomes. Using an interviewer-administered structured questionnaire, individuals with osteoarthritis (OA) or inflammatory arthritis (IA) were interviewed at 4 time points, 18 months apart. At baseline, all participants (n = 490; 381 women, 109 men) were employed. Respondents were recruited using community advertising and from rheumatology and rehabilitation clinics. The Workplace Activity Limitations Scale (WALS) assessed arthritis-related disability with job tasks. Job modifications/accommodations (e.g., scheduling changes), work place outcomes (e.g., absenteeism), demographics, illness, and work context were also measured. Repeated cross-sectional logistic regressions examined levels of WALS disability with job modifications and outcomes at each time point. Similar levels of activity limitations were found comparing OA and IA with fewer difficulties with global aspects of work (e.g., scheduling) than with specific tasks (e.g., working with hands). Three-quarters of the participants had episodic or intermittent WALS difficulty over time. Medium and high levels of work place activity limitations were significantly associated with job modifications, and high WALS difficulty was consistently related to negative work outcomes. Many individuals with arthritis report some difficulty with work place activities. However, these difficulties are often intermittent and may not result in changes to work productivity until they are consistently high. This is important for designing work place interventions and for employers, insurers, and the government to understand to avoid viewing individuals with arthritis as a permanent drain on work place and health resources. Copyright © 2011 by the American College of Rheumatology.

Genome engineering in cattle: recent technological advancements.

PubMed

Wang, Zhongde

2015-02-01

Great strides in technological advancements have been made in the past decade in cattle genome engineering. First, the success of cloning cattle by somatic cell nuclear transfer (SCNT) or chromatin transfer (CT) is a significant advancement that has made obsolete the need for using embryonic stem (ES) cells to conduct cell-mediated genome engineering, whereby site-specific genetic modifications can be conducted in bovine somatic cells via DNA homologous recombination (HR) and whereby genetically engineered cattle can subsequently be produced by animal cloning from the genetically modified cells. With this approach, a chosen bovine genomic locus can be precisely modified in somatic cells, such as to knock out (KO) or knock in (KI) a gene via HR, a gene-targeting strategy that had almost exclusively been used in mouse ES cells. Furthermore, by the creative application of embryonic cloning to rejuvenate somatic cells, cattle genome can be sequentially modified in the same line of somatic cells and complex genetic modifications have been achieved in cattle. Very recently, the development of designer nucleases-such as zinc finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-has enabled highly efficient and more facile genome engineering in cattle. Most notably, by employing such designer nucleases, genomes can be engineered at single-nucleotide precision; this process is now often referred to as genome or gene editing. The above achievements are a drastic departure from the traditional methods of creating genetically modified cattle, where foreign DNAs are randomly integrated into the animal genome, most often along with the integrations of bacterial or viral DNAs. Here, I review the most recent technological developments in cattle genome engineering by highlighting some of the major achievements in creating genetically engineered cattle for agricultural and biomedical applications.

Gene targeting and subsequent site-specific transgenesis at the β-actin (ACTB) locus in common marmoset embryonic stem cells.

PubMed

Shiozawa, Seiji; Kawai, Kenji; Okada, Yohei; Tomioka, Ikuo; Maeda, Takuji; Kanda, Akifumi; Shinohara, Haruka; Suemizu, Hiroshi; James Okano, Hirotaka; Sotomaru, Yusuke; Sasaki, Erika; Okano, Hideyuki

2011-09-01

Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the β-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice.

Genetic and epigenetic control of plant heat responses

PubMed Central

Liu, Junzhong; Feng, Lili; Li, Jianming; He, Zuhua

2015-01-01

Plants have evolved sophisticated genetic and epigenetic regulatory systems to respond quickly to unfavorable environmental conditions such as heat, cold, drought, and pathogen infections. In particular, heat greatly affects plant growth and development, immunity and circadian rhythm, and poses a serious threat to the global food supply. According to temperatures exposing, heat can be usually classified as warm ambient temperature (about 22–27°C), high temperature (27–30°C) and extremely high temperature (37–42°C, also known as heat stress) for the model plant Arabidopsis thaliana. The genetic mechanisms of plant responses to heat have been well studied, mainly focusing on elevated ambient temperature-mediated morphological acclimation and acceleration of flowering, modulation of circadian clock and plant immunity by high temperatures, and thermotolerance to heat stress. Recently, great progress has been achieved on epigenetic regulation of heat responses, including DNA methylation, histone modifications, histone variants, ATP-dependent chromatin remodeling, histone chaperones, small RNAs, long non-coding RNAs and other undefined epigenetic mechanisms. These epigenetic modifications regulate the expression of heat-responsive genes and function to prevent heat-related damages. This review focuses on recent progresses regarding the genetic and epigenetic control of heat responses in plants, and pays more attention to the role of the major epigenetic mechanisms in plant heat responses. Further research perspectives are also discussed. PMID:25964789

Acaricide resistance mechanisms in Rhipicephalus microplus

USDA-ARS?s Scientific Manuscript database

Acaricide resistance has become widespread in countries where cattle ticks, Rhipicephalus (Boophilus) microplus, are a problem. Resistance arises through genetic changes in a cattle tick population that causes modifications to the target site, increased metabolism or sequestration of the acaricide, ...

Genetic approaches to interfere with malaria transmission by vector mosquitoes

PubMed Central

Wang, Sibao; Jacobs-Lorena, Marcelo

2013-01-01

Malaria remains one of the world’s most devastating diseases, causing over one million deaths every year. The most vulnerable stages of Plasmodium development in the vector mosquito occur in the midgut lumen, making the midgut a prime target for intervention. Mosquito transgenesis and paratransgenesis are two novel strategies that aim at rendering the vector incapable of sustaining Plasmodium development. Mosquito transgenesis involves direct genetic engineering of the mosquito itself for delivery of anti-Plasmodium effector molecules. Conversely, paratransgenesis involves the genetic modification of mosquito symbionts for expression of anti-pathogen effector molecules. Here we consider both genetic manipulation strategies for rendering mosquitoes refractory to Plasmodium infection, and discuss challenges for the translation of laboratory findings to field applications. PMID:23395485

Detection of thermogenesis in rodents in response to anti-obesity drugs and genetic modification

PubMed Central

Arch, Jonathan R. S.; Trayhurn, Paul

2013-01-01

Many compounds and genetic manipulations are claimed to confer resistance to obesity in rodents by raising energy expenditure. Examples taken from recent and older literature, demonstrate that such claims are often based on measurements of energy expenditure after body composition has changed, and depend on comparisons of energy expenditure divided by body weight. This is misleading because white adipose tissue has less influence than lean tissue on energy expenditure. Application of this approach to human data would suggest that human obesity is usually due to a low metabolic rate, which is not an accepted view. Increased energy expenditure per animal is a surer way of demonstrating thermogenesis, but even then it is important to know whether this is due to altered body composition (repartitioning), or increased locomotor activity rather than thermogenesis per se. Regression analysis offers other approaches. The thermogenic response to some compounds has a rapid onset and so cannot be due to altered body composition. These compounds usually mimic or activate the sympathetic nervous system. Thermogenesis occurs in, but may not be confined to, brown adipose tissue. It should not be assumed that weight loss in response to these treatments is due to thermogenesis unless there is a sustained increase in 24-h energy expenditure. Thyroid hormones and fibroblast growth factor 21 also raise energy expenditure before they affect body composition. Some treatments and genetic modifications alter the diurnal rhythm of energy expenditure. It is important to establish whether this is due to altered locomotor activity or efficiency of locomotion. There are no good examples of compounds that do not affect short-term energy expenditure but have a delayed effect. How and under what conditions a genetic modification or compound increases energy expenditure influences the decision on whether to seek drugs for the target or take a candidate drug into clinical studies. PMID:23580228

Modification of COMT-dependent pain sensitivity by psychological stress and sex.

PubMed

Meloto, Carolina B; Bortsov, Andrey V; Bair, Eric; Helgeson, Erika; Ostrom, Cara; Smith, Shad B; Dubner, Ronald; Slade, Gary D; Fillingim, Roger B; Greenspan, Joel D; Ohrbach, Richard; Maixner, William; McLean, Samuel A; Diatchenko, Luda

2016-04-01

Catecholamine-O-methyltransferase (COMT) is a polymorphic gene whose variants affect enzymatic activity and pain sensitivity via adrenergic pathways. Although COMT represents one of the most studied genes in human pain genetics, findings regarding its association with pain phenotypes are not always replicated. Here, we investigated if interactions among functional COMT haplotypes, stress, and sex can modify the effect of COMT genetic variants on pain sensitivity. We tested these interactions in a cross-sectional study, including 2 cohorts, one of 2972 subjects tested for thermal pain sensitivity (Orofacial Pain: Prospective Evaluation and Risk Assessment) and one of 948 subjects with clinical acute pain after motor vehicle collision (post-motor vehicle collision). In both cohorts, the COMT high-pain sensitivity (HPS) haplotype showed robust interaction with stress and number of copies of the HPS haplotype was positively associated with pain sensitivity in nonstressed individuals, but not in stressed individuals. In the post-motor vehicle collision cohort, there was additional modification by sex: the HPS-stress interaction was apparent in males, but not in females. In summary, our findings indicate that stress and sex should be evaluated in association studies aiming to investigate the effect of COMT genetic variants on pain sensitivity.

Monster potential meets potential monster: pros and cons of deploying genetically modified microalgae for biofuels production.

PubMed

Flynn, K J; Mitra, A; Greenwell, H C; Sui, J

2013-02-06

Biofuels production from microalgae attracts much attention but remains an unproven technology. We explore routes to enhance production through modifications to a range of generic microalgal physiological characteristics. Our analysis shows that biofuels production may be enhanced ca fivefold through genetic modification (GM) of factors affecting growth rate, respiration, photoacclimation, photosynthesis efficiency and the minimum cell quotas for nitrogen and phosphorous (N : C and P : C). However, simulations indicate that the ideal GM microalgae for commercial deployment could, on escape to the environment, become a harmful algal bloom species par excellence, with attendant risks to ecosystems and livelihoods. In large measure, this is because an organism able to produce carbohydrate and/or lipid at high rates, providing stock metabolites for biofuels production, will also be able to attain a stoichiometric composition that will be far from optimal as food for the support of zooplankton growth. This composition could suppress or even halt the grazing activity that would otherwise control the microalgal growth in nature. In consequence, we recommend that the genetic manipulation of microalgae, with inherent consequences on a scale comparable to geoengineering, should be considered under strict international regulation.

Pleiotropic analysis of cancer risk loci on esophageal adenocarcinoma risk

PubMed Central

Lee, Eunjung; Stram, Daniel O.; Ek, Weronica E.; Onstad, Lynn E; MacGregor, Stuart; Gharahkhani, Puya; Ye, Weimin; Lagergren, Jesper; Shaheen, Nicholas J.; Murray, Liam J.; Hardie, Laura J; Gammon, Marilie D.; Chow, Wong-Ho; Risch, Harvey A.; Corley, Douglas A.; Levine, David M; Whiteman, David C.; Bernstein, Leslie; Bird, Nigel C.; Vaughan, Thomas L.; Wu, Anna H.

2015-01-01

Background Several cancer-associated loci identified from genome-wide association studies (GWAS) have been associated with risks of multiple cancer sites, suggesting pleiotropic effects. We investigated whether GWAS-identified risk variants for other common cancers are associated with risk of esophageal adenocarcinoma (EA) or its precursor, Barrett's esophagus (BE). Methods We examined the associations between risks of EA and BE and 387 single nucleotide polymorphisms (SNPs) that have been associated with risks of other cancers, by using genotype imputation data on 2,163 control participants and 3,885 (1,501 EA and 2,384 BE) case patients from the Barrett's and Esophageal Adenocarcinoma Genetic Susceptibility Study, and investigated effect modification by smoking history, body mass index (BMI), and reflux/heartburn. Results After correcting for multiple testing, none of the tested 387 SNPs were statistically significantly associated with risk of EA or BE. No evidence of effect modification by smoking, BMI, or reflux/heartburn was observed. Conclusions Genetic risk variants for common cancers identified from GWAS appear not to be associated with risks of EA or BE. Impact To our knowledge, this is the first investigation of pleiotropic genetic associations with risks of EA and BE. PMID:26364162

Monster potential meets potential monster: pros and cons of deploying genetically modified microalgae for biofuels production

PubMed Central

Flynn, K. J.; Mitra, A.; Greenwell, H. C.; Sui, J.

2013-01-01

Biofuels production from microalgae attracts much attention but remains an unproven technology. We explore routes to enhance production through modifications to a range of generic microalgal physiological characteristics. Our analysis shows that biofuels production may be enhanced ca fivefold through genetic modification (GM) of factors affecting growth rate, respiration, photoacclimation, photosynthesis efficiency and the minimum cell quotas for nitrogen and phosphorous (N : C and P : C). However, simulations indicate that the ideal GM microalgae for commercial deployment could, on escape to the environment, become a harmful algal bloom species par excellence, with attendant risks to ecosystems and livelihoods. In large measure, this is because an organism able to produce carbohydrate and/or lipid at high rates, providing stock metabolites for biofuels production, will also be able to attain a stoichiometric composition that will be far from optimal as food for the support of zooplankton growth. This composition could suppress or even halt the grazing activity that would otherwise control the microalgal growth in nature. In consequence, we recommend that the genetic manipulation of microalgae, with inherent consequences on a scale comparable to geoengineering, should be considered under strict international regulation. PMID:24427510

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

PubMed

Johnston, Christopher D; Skeete, Chelsey A; Fomenkov, Alexey; Roberts, Richard J; Rittling, Susan R

2017-01-01

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism's physiology, metabolism, and pathogenesis in human disease.

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia

PubMed Central

Skeete, Chelsey A.; Fomenkov, Alexey; Roberts, Richard J.; Rittling, Susan R.

2017-01-01

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism’s physiology, metabolism, and pathogenesis in human disease. PMID:28934361

Gene therapy: advances, challenges and perspectives

PubMed Central

Gonçalves, Giulliana Augusta Rangel; Paiva, Raquel de Melo Alves

2017-01-01

ABSTRACT The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review. PMID:29091160

Why genetic modification of lignin leads to low-recalcitrance biomass

DOE PAGES

Carmona, Christopher; Langan, Paul; Smith, Jeremy C.; ...

2014-11-11

Genetic modification of plants via down-regulation of cinnamyl alcohol dehydrogenase leads to incorporation of aldehyde groups in the lignin polymer. Moreover, the resulting lignocellulosic biomass has increased bioethanol yield. However, a molecular-scale explanation of this finding is currently lacking. We perform molecular dynamics simulation of the copolymer with hemicellulose of wild type and the genetically modified lignin, in aqueous solution. We find that the non-covalent association with hemicellulose of lignin containing aldehyde groups is reduced compared to the wild-type. This phase separation may increase the cell wall porosity in the mutant plants, thus explaining their easier deconstruction to biofuels. Themore » thermodynamic origin of the reduced lignin-hemicellulose association is found to be a more favorable self-interaction energy and less favorable interaction with hemicellulose for the mutant lignin. Furthermore, reduced hydration water density fluctuations are found for the mutant lignin, implying a more hydrophobic lignin surface. Our results provide a detailed description of how aldehyde incorporation makes lignin more hydrophobic and reduces its association with hemicellulose, thus suggesting that increased lignin hydrophobicity may be an optimal characteristic required for improved biofuel production.« less

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles

PubMed Central

Nandi, Tannistha; Holden, Matthew T.G.; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A.; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J.; Titball, Richard; Chen, Swaine L.; Parkhill, Julian

2015-01-01

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. PMID:25236617

Programming cells by multiplex genome engineering and accelerated evolution.

PubMed

Wang, Harris H; Isaacs, Farren J; Carr, Peter A; Sun, Zachary Z; Xu, George; Forest, Craig R; Church, George M

2009-08-13

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

Towards a systemic paradigm in carcinogenesis: linking epigenetics and genetics.

PubMed

Burgio, Ernesto; Migliore, Lucia

2015-04-01

For at least 30 years cancer has been defined as a genetic disease and explained by the so-called somatic mutation theory (SMT), which has dominated the carcinogenesis field. Criticism of the SMT has recently greatly increased, although still not enough to force all SMT supporters to recognize its limits. Various researchers point out that cancer appears to be a complex process concerning a whole tissue; and that genomic mutations, although variably deleterious and unpredictably important in determining the establishment of the neoplastic phenotype, are not the primary origin for a malignant neoplasia. We attempt to describe the inadequacies of the SMT and demonstrate that epigenetics is a more logical cause of carcinogenesis. Many previous models of carcinogenesis fall into two classes: (i) in which some biological changes inside cells alone lead to malignancy; and (ii) requiring changes in stroma/extracellular matrix. We try to make clear that in the (ii) model genomic instability is induced by persistent signals coming from the microenvironment, provoking epigenetic and genetic modifications in tissue stem cells that can lead to cancer. In this perspective, stochastic mutations of DNA are a critical by-product rather then the primary cause of cancer. Indirect support for such model of carcinogenesis comes from the in vitro and vivo experiments showing apparent 'reversion' of cancer phenotypes obtained via physiological factors of cellular differentiation (cytokines and other signaling molecules) or drugs, even if the key mutations are not 'reversed'.

Germline genome-editing research and its socioethical implications.

PubMed

Ishii, Tetsuya

2015-08-01

Genetically modifying eggs, sperm, and zygotes ('germline' modification) can impact on the entire body of the resulting individual and on subsequent generations. With the advent of genome-editing technology, human germline gene modification is no longer theoretical. Owing to increasing concerns about human germline gene modification, a voluntary moratorium on human genome-editing research and/or the clinical application of human germline genome editing has recently been called for. However, whether such research should be suspended or encouraged warrants careful consideration. The present article reviews recent research on mammalian germline genome editing, discusses the importance of public dialogue on the socioethical implications of human germline genome-editing research, and considers the relevant guidelines and legislation in different countries. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

PubMed

Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A; Villegas, John Paul; Fernandez, Alejandra; Van Pelt, Amelia; Dmitriev, Igor P; Curiel, David T; Moreno, Alberto

2016-01-01

A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective efficacy of adenoviral based malaria vaccines.

Genetic variation in SIRT1 affects susceptibility of lung squamous cell carcinomas in former uranium miners from the Colorado plateau

PubMed Central

Leng, Shuguang; Picchi, Maria A.; Liu, Yushi; Thomas, Cynthia L.; Willis, Derall G.; Bernauer, Amanda M.; Carr, Teara G.; Mabel, Padilla T.; Han, Younghun; Amos, Christopher I.; Lin, Yong; Stidley, Christine A.; Gilliland, Frank D.; Jacobson, Marty R.; Belinsky, Steven A.

2013-01-01

Epidemiological studies of underground miners suggested that occupational exposure to radon causes lung cancer with squamous cell carcinoma (SCC) as the predominant histological type. However, the genetic determinants for susceptibility of radon-induced SCC in miners are unclear. Double-strand breaks induced by radioactive radon daughters are repaired primarily by non-homologous end joining (NHEJ) that is accompanied by the dynamic changes in surrounding chromatin, including nucleosome repositioning and histone modifications. Thus, a molecular epidemiological study was conducted to assess whether genetic variation in 16 genes involved in NHEJ and related histone modification affected susceptibility for SCC in radon-exposed former miners (267 SCC cases and 383 controls) from the Colorado plateau. A global association between genetic variation in the haplotype block where SIRT1 resides and the risk for SCC in miners (P = 0.003) was identified. Haplotype alleles tagged by the A allele of SIRT1 rs7097008 were associated with increased risk for SCC (odds ratio = 1.69, P = 8.2×10−5) and greater survival in SCC cases (hazard ratio = 0.79, P = 0.03) in miners. Functional validation of rs7097008 demonstrated that the A allele was associated with reduced gene expression in bronchial epithelial cells and compromised DNA repair capacity in peripheral lymphocytes. Together, these findings substantiate genetic variation in SIRT1 as a risk modifier for developing SCC in miners and suggest that SIRT1 may also play a tumor suppressor role in radon-induced cancer in miners. PMID:23354305

The Loss and Gain of Functional Amino Acid Residues Is a Common Mechanism Causing Human Inherited Disease

PubMed Central

Lugo-Martinez, Jose; Pejaver, Vikas; Pagel, Kymberleigh A.; Mort, Matthew; Cooper, David N.; Mooney, Sean D.; Radivojac, Predrag

2016-01-01

Elucidating the precise molecular events altered by disease-causing genetic variants represents a major challenge in translational bioinformatics. To this end, many studies have investigated the structural and functional impact of amino acid substitutions. Most of these studies were however limited in scope to either individual molecular functions or were concerned with functional effects (e.g. deleterious vs. neutral) without specifically considering possible molecular alterations. The recent growth of structural, molecular and genetic data presents an opportunity for more comprehensive studies to consider the structural environment of a residue of interest, to hypothesize specific molecular effects of sequence variants and to statistically associate these effects with genetic disease. In this study, we analyzed data sets of disease-causing and putatively neutral human variants mapped to protein 3D structures as part of a systematic study of the loss and gain of various types of functional attribute potentially underlying pathogenic molecular alterations. We first propose a formal model to assess probabilistically function-impacting variants. We then develop an array of structure-based functional residue predictors, evaluate their performance, and use them to quantify the impact of disease-causing amino acid substitutions on catalytic activity, metal binding, macromolecular binding, ligand binding, allosteric regulation and post-translational modifications. We show that our methodology generates actionable biological hypotheses for up to 41% of disease-causing genetic variants mapped to protein structures suggesting that it can be reliably used to guide experimental validation. Our results suggest that a significant fraction of disease-causing human variants mapping to protein structures are function-altering both in the presence and absence of stability disruption. PMID:27564311

The Loss and Gain of Functional Amino Acid Residues Is a Common Mechanism Causing Human Inherited Disease.

PubMed

Lugo-Martinez, Jose; Pejaver, Vikas; Pagel, Kymberleigh A; Jain, Shantanu; Mort, Matthew; Cooper, David N; Mooney, Sean D; Radivojac, Predrag

2016-08-01

Elucidating the precise molecular events altered by disease-causing genetic variants represents a major challenge in translational bioinformatics. To this end, many studies have investigated the structural and functional impact of amino acid substitutions. Most of these studies were however limited in scope to either individual molecular functions or were concerned with functional effects (e.g. deleterious vs. neutral) without specifically considering possible molecular alterations. The recent growth of structural, molecular and genetic data presents an opportunity for more comprehensive studies to consider the structural environment of a residue of interest, to hypothesize specific molecular effects of sequence variants and to statistically associate these effects with genetic disease. In this study, we analyzed data sets of disease-causing and putatively neutral human variants mapped to protein 3D structures as part of a systematic study of the loss and gain of various types of functional attribute potentially underlying pathogenic molecular alterations. We first propose a formal model to assess probabilistically function-impacting variants. We then develop an array of structure-based functional residue predictors, evaluate their performance, and use them to quantify the impact of disease-causing amino acid substitutions on catalytic activity, metal binding, macromolecular binding, ligand binding, allosteric regulation and post-translational modifications. We show that our methodology generates actionable biological hypotheses for up to 41% of disease-causing genetic variants mapped to protein structures suggesting that it can be reliably used to guide experimental validation. Our results suggest that a significant fraction of disease-causing human variants mapping to protein structures are function-altering both in the presence and absence of stability disruption.

A statistical simulation model for field testing of non-target organisms in environmental risk assessment of genetically modified plants.

PubMed

Goedhart, Paul W; van der Voet, Hilko; Baldacchino, Ferdinando; Arpaia, Salvatore

2014-04-01

Genetic modification of plants may result in unintended effects causing potentially adverse effects on the environment. A comparative safety assessment is therefore required by authorities, such as the European Food Safety Authority, in which the genetically modified plant is compared with its conventional counterpart. Part of the environmental risk assessment is a comparative field experiment in which the effect on non-target organisms is compared. Statistical analysis of such trials come in two flavors: difference testing and equivalence testing. It is important to know the statistical properties of these, for example, the power to detect environmental change of a given magnitude, before the start of an experiment. Such prospective power analysis can best be studied by means of a statistical simulation model. This paper describes a general framework for simulating data typically encountered in environmental risk assessment of genetically modified plants. The simulation model, available as Supplementary Material, can be used to generate count data having different statistical distributions possibly with excess-zeros. In addition the model employs completely randomized or randomized block experiments, can be used to simulate single or multiple trials across environments, enables genotype by environment interaction by adding random variety effects, and finally includes repeated measures in time following a constant, linear or quadratic pattern in time possibly with some form of autocorrelation. The model also allows to add a set of reference varieties to the GM plants and its comparator to assess the natural variation which can then be used to set limits of concern for equivalence testing. The different count distributions are described in some detail and some examples of how to use the simulation model to study various aspects, including a prospective power analysis, are provided.

A statistical simulation model for field testing of non-target organisms in environmental risk assessment of genetically modified plants

PubMed Central

Goedhart, Paul W; van der Voet, Hilko; Baldacchino, Ferdinando; Arpaia, Salvatore

2014-01-01

Genetic modification of plants may result in unintended effects causing potentially adverse effects on the environment. A comparative safety assessment is therefore required by authorities, such as the European Food Safety Authority, in which the genetically modified plant is compared with its conventional counterpart. Part of the environmental risk assessment is a comparative field experiment in which the effect on non-target organisms is compared. Statistical analysis of such trials come in two flavors: difference testing and equivalence testing. It is important to know the statistical properties of these, for example, the power to detect environmental change of a given magnitude, before the start of an experiment. Such prospective power analysis can best be studied by means of a statistical simulation model. This paper describes a general framework for simulating data typically encountered in environmental risk assessment of genetically modified plants. The simulation model, available as Supplementary Material, can be used to generate count data having different statistical distributions possibly with excess-zeros. In addition the model employs completely randomized or randomized block experiments, can be used to simulate single or multiple trials across environments, enables genotype by environment interaction by adding random variety effects, and finally includes repeated measures in time following a constant, linear or quadratic pattern in time possibly with some form of autocorrelation. The model also allows to add a set of reference varieties to the GM plants and its comparator to assess the natural variation which can then be used to set limits of concern for equivalence testing. The different count distributions are described in some detail and some examples of how to use the simulation model to study various aspects, including a prospective power analysis, are provided. PMID:24834325

Methods for genetic transformation of filamentous fungi.

PubMed

Li, Dandan; Tang, Yu; Lin, Jun; Cai, Weiwen

2017-10-03

Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of genetic transformation techniques is a precondition that enables scientists to target and modify genes efficiently and may reveal the function of target genes. The method to deliver foreign nucleic acid into cells is the sticking point for fungal genome modification. Up to date, there are some general methods of genetic transformation for fungi, including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation. This article reviews basic protocols and principles of these transformation methods, as well as their advantages and disadvantages.

Commercialising genetically engineered animal biomedical products.

PubMed

Sullivan, Eddie J; Pommer, Jerry; Robl, James M

2008-01-01

Research over the past two decades has increased the quality and quantity of tools available to produce genetically engineered animals. The number of potentially viable biomedical products from genetically engineered animals is increasing. However, moving from cutting-edge research to development and commercialisation of a biomedical product that is useful and wanted by the public has significant challenges. Even early stage development of genetically engineered animal applications requires consideration of many steps, including quality assurance and quality control, risk management, gap analysis, founder animal establishment, cell banking, sourcing of animals and animal-derived material, animal facilities, product collection facilities and processing facilities. These steps are complicated and expensive. Biomedical applications of genetically engineered animals have had some recent successes and many applications are well into development. As researchers consider applications for their findings, having a realistic understanding of the steps involved in the development and commercialisation of a product, produced in genetically engineered animals, is useful in determining the risk of genetic modification to the animal nu. the potential public benefit of the application.

[The genetic control of mouse coat color and its applications in genetics teaching].

PubMed

Xing, Wanjin; Morigen, Morigen

2014-10-01

Mice are the most commonly used mammalian model. The coat colors of mice are typical Mendelian traits, which have various colors such as white, black, yellow and agouti. The inheritance of mouse coat color is usually stated as an example only in teaching the knowledge of recessive lethal alleles. After searched the related literatures and summarized the molecular mechanisms of mouse coat color inheritance, we further expanded the application of this example into the introduction of the basic concepts of alleles and Mendelian laws, demonstration of the gene structure and function, regulation of gene expression, gene interaction, epigenetic modification, quantitative genetics, as well as evolutionary genetics. By running this example through the whole genetics-teaching lectures, we help the student to form a systemic and developmental view of genetic analysis. At the same time, this teaching approach not only highlights the advancement and integrity of genetics, but also results in a good teaching effect on inspiring the students' interest and attracting students' attention.

A cross-sectional study of attitudes about the use of genetic testing for clinical care among patients with an alcohol use disorder.

PubMed

Strobel, Brittany; McManus, Lauren; Leong, Shirley; Blow, Frederic; Slaymaker, Valerie; Berrettini, Wade; Gordon, Adam J; O'Brien, Charles; Oslin, David

2013-01-01

Modification and individualization of medical treatments due to genetic testing has the potential to revolutionize healthcare delivery. As evidence mounts that genetic testing may improve treatment decisions for patients with alcohol use disorder (AUD), we explored patient concerns and attitudes toward genetic testing. Subjects of two USA cross-sectional AUD studies were surveyed regarding their attitudes regarding the use of genetic testing for AUD treatment. Four hundred and fifty-seven participants were surveyed. Overall, subjects showed a high degree of willingness to provide DNA for clinical use and recognized genetics as important to the pathophysiology of a number of disorders including AUD. There were, however, significant concerns expressed related to insurance denial or employment problems. We found that patients enrolled in AUD studies had some concerns about use of genetic testing. The patients in these two samples were, however, willing and knowledgeable about providing DNA samples.

76 FR 22316 - Airworthiness Directives; Pacific Aerospace Limited Model 750XL Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2011-04-21

... pedal assemblies for cracks and incorporating a modification repair scheme if any cracks are found. You... cracks and incorporating a modification repair scheme if any cracks are found. You may obtain further... intervals not to exceed 300 hours time-in-service (TIS) until the modification repair scheme required in...

Comparative analysis of the Corynebacterium glutamicum transcriptome in response to changes in dissolved oxygen levels.

PubMed

Liu, Xiuxia; Yang, Sun; Wang, Fen; Dai, Xiaofeng; Yang, Yankun; Bai, Zhonghu

2017-02-01

The dissolved oxygen (DO) level of a culture of Corynebacterium glutamicum (C. glutamicum) in a bioreactor has a significant impact on the cellular redox potential and the distribution of energy and metabolites. In this study, to gain a deeper understanding of the effects of DO on the metabolism of C. glutamicum, we sought to systematically explore the influence of different DO concentrations on genetic regulation and metabolism through transcriptomic analysis. The results revealed that after 20 h of fermentation, oxygen limitation enhanced the glucose metabolism, pyruvate metabolism and carbon overflow, and restricted NAD + availability. A high oxygen supply enhanced the TCA cycle and reduced glyoxylate metabolism. Several key genes involved in response of C. glutamicum to different oxygen concentrations were examined, which provided suggestions for target site modifications in developing optimized oxygen supply strategies. These data provided new insights into the relationship between oxygen supply and metabolism of C. glutamicum.

Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

PubMed Central

Hagen, Sven; Baumann, Tobias; Wagner, Hanna J.; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M.; Müller, Kristian M.

2014-01-01

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

New Strategies in Engineering T-cell Receptor Gene-Modified T cells to More Effectively Target Malignancies.

PubMed

Schmitt, Thomas M; Stromnes, Ingunn M; Chapuis, Aude G; Greenberg, Philip D

2015-12-01

The immune system, T cells in particular, have the ability to target and destroy malignant cells. However, antitumor immune responses induced from the endogenous T-cell repertoire are often insufficient for the eradication of established tumors, as illustrated by the failure of cancer vaccination strategies or checkpoint blockade for most tumors. Genetic modification of T cells to express a defined T-cell receptor (TCR) can provide the means to rapidly generate large numbers of tumor-reactive T cells capable of targeting tumor cells in vivo. However, cell-intrinsic factors as well as immunosuppressive factors in the tumor microenvironment can limit the function of such gene-modified T cells. New strategies currently being developed are refining and enhancing this approach, resulting in cellular therapies that more effectively target tumors and that are less susceptible to tumor immune evasion. ©2015 American Association for Cancer Research.

Exosomes as nanocarriers for siRNA delivery: paradigms and challenges.

PubMed

Shahabipour, Fahimeh; Banach, Maciej; Sahebkar, Amirhossein

2016-12-01

Exosomes are nano-sized vesicles that facilitate intercellular communications through carrying genetic materials and functional biomolecules. Owing to their unique size and structure, exosomes have emerged as a useful tool to overcome the limitations of siRNA delivery. The use of exosomes as siRNA delivery vehicles lacks certain disadvantages of the existing foreign delivery systems such as viruses, polycationic polymers and liposomes, and introduces several advantages including inherent capacity to pass through biological barriers and escape from phagocytosis by the reticuloendothelial system, as well as being biocompatible, non-toxic, and immunologically inert. Different strategies have been employed to harness exosome-based delivery systems, including surface modification with targeting ligands, and using exosome-display technology, virus-modified exosomes, and exosome-mimetic vesicles. The present review provides a capsule summary of the recent advances and current challenges in the field of exosome-mediated siRNA delivery.

Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation?

PubMed Central

2012-01-01

Mesenchymal stem cells change dramatically during culture expansion. Long-term culture has been suspected to evoke oncogenic transformation: overall, the genome appears to be relatively stable throughout culture but transient clonal aneuploidies have been observed. Oncogenic transformation does not necessarily entail growth advantage in vitro and, therefore, the available methods - such as karyotypic analysis or genomic profiling - cannot exclude this risk. On the other hand, long-term culture is associated with specific senescence-associated DNA methylation (SA-DNAm) changes, particularly in developmental genes. SA-DNAm changes are highly reproducible and can be used to monitor the state of senescence for quality control. Notably, neither telomere attrition nor SA-DNAm changes occur in pluripotent stem cells, which can evade the 'Hayflick limit'. Long-term culture of mesenchymal stem cells seems to involve a tightly regulated epigenetic program. These epigenetic modifications may counteract dominant clones, which are more prone to transformation. PMID:23257053

Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation?

PubMed

Wagner, Wolfgang

2012-12-20

Mesenchymal stem cells change dramatically during culture expansion. Long-term culture has been suspected to evoke oncogenic transformation: overall, the genome appears to be relatively stable throughout culture but transient clonal aneuploidies have been observed. Oncogenic transformation does not necessarily entail growth advantage in vitro and, therefore, the available methods - such as karyotypic analysis or genomic profiling - cannot exclude this risk. On the other hand, long-term culture is associated with specific senescence-associated DNA methylation (SA-DNAm) changes, particularly in developmental genes. SA-DNAm changes are highly reproducible and can be used to monitor the state of senescence for quality control. Notably, neither telomere attrition nor SA-DNAm changes occur in pluripotent stem cells, which can evade the 'Hayflick limit'. Long-term culture of mesenchymal stem cells seems to involve a tightly regulated epigenetic program. These epigenetic modifications may counteract dominant clones, which are more prone to transformation.

Adoptive Cell Therapy for Metastatic Melanoma.

PubMed

Merhavi-Shoham, Efrat; Itzhaki, Orit; Markel, Gal; Schachter, Jacob; Besser, Michal J

Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) is a powerful form of immunotherapy by inducing durable complete responses that significantly extend the survival of melanoma patients. Mutation-derived neoantigens were recently identified as key factors for tumor recognition and rejection by TILs. The isolation of T-cell receptor (TCR) genes directed against neoantigens and their retransduction into peripheral T cells may provide a new form of ACT.Genetic modifications of T cells with chimeric antigen receptors (CARs) have demonstrated remarkable clinical results in hematologic malignancies, but are so far less effective in solid tumors. Only very limited reports exist in melanoma. Progress in CAR T-cell engineering, including neutralization of inhibitory signals or additional safety switches, may open opportunities also in melanoma.We review clinical results and latest developments of adoptive therapies with TILs, T-cell receptor, and CAR-modified T cells and discuss future directions for the treatment of melanoma.

MYC association with cancer risk and a new model of MYC-mediated repression.

PubMed

Cole, Michael D

2014-07-01

MYC is one of the most frequently mutated and overexpressed genes in human cancer but the regulation of MYC expression and the ability of MYC protein to repress cellular genes (including itself) have remained mysterious. Recent genome-wide association studies show that many genetic polymorphisms associated with disease risk map to distal regulatory elements that regulate the MYC promoter through large chromatin loops. Cancer risk-associated single-nucleotide polymorphisms (SNPs) contain more potent enhancer activity, promoting higher MYC levels and a greater risk of disease. The MYC promoter is also subject to complex regulatory circuits and limits its own expression by a feedback loop. A model for MYC autoregulation is discussed which involves a signaling pathway between the PTEN (phosphatase and tensin homolog) tumor suppressor and repressive histone modifications laid down by the EZH2 methyltransferase. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Biotechnological production of value-added carotenoids from microalgae: Emerging technology and prospects.

PubMed

Wichuk, Kristine; Brynjólfsson, Sigurður; Fu, Weiqi

2014-01-01

We recently evaluated the relationship between abiotic environmental stresses and lutein biosynthesis in the green microalga Dunaliella salina and suggested a rational design of stress-driven adaptive evolution experiments for carotenoids production in microalgae. Here, we summarize our recent findings regarding the biotechnological production of carotenoids from microalgae and outline emerging technology in this field. Carotenoid metabolic pathways are characterized in several representative algal species as they pave the way for biotechnology development. The adaptive evolution strategy is highlighted in connection with enhanced growth rate and carotenoid metabolism. In addition, available genetic modification tools are described, with emphasis on model species. A brief discussion on the role of lights as limiting factors in carotenoid production in microalgae is also included. Overall, our analysis suggests that light-driven metabolism and the photosynthetic efficiency of microalgae in photobioreactors are the main bottlenecks in enhancing biotechnological potential of carotenoid production from microalgae.

Amino Acids Are an Ineffective Fertilizer for Dunaliella spp. Growth

PubMed Central

Murphree, Colin A.; Dums, Jacob T.; Jain, Siddharth K.; Zhao, Chengsong; Young, Danielle Y.; Khoshnoodi, Nicole; Tikunov, Andrey; Macdonald, Jeffrey; Pilot, Guillaume; Sederoff, Heike

2017-01-01

Autotrophic microalgae are a promising bioproducts platform. However, the fundamental requirements these organisms have for nitrogen fertilizer severely limit the impact and scale of their cultivation. As an alternative to inorganic fertilizers, we investigated the possibility of using amino acids from deconstructed biomass as a nitrogen source in the genus Dunaliella. We found that only four amino acids (glutamine, histidine, cysteine, and tryptophan) rescue Dunaliella spp. growth in nitrogen depleted media, and that supplementation of these amino acids altered the metabolic profile of Dunaliella cells. Our investigations revealed that histidine is transported across the cell membrane, and that glutamine and cysteine are not transported. Rather, glutamine, cysteine, and tryptophan are degraded in solution by a set of oxidative chemical reactions, releasing ammonium that in turn supports growth. Utilization of biomass-derived amino acids is therefore not a suitable option unless additional amino acid nitrogen uptake is enabled through genetic modifications of these algae. PMID:28603530

Brains, genes, and primates.

PubMed

Izpisua Belmonte, Juan Carlos; Callaway, Edward M; Caddick, Sarah J; Churchland, Patricia; Feng, Guoping; Homanics, Gregg E; Lee, Kuo-Fen; Leopold, David A; Miller, Cory T; Mitchell, Jude F; Mitalipov, Shoukhrat; Moutri, Alysson R; Movshon, J Anthony; Okano, Hideyuki; Reynolds, John H; Ringach, Dario; Sejnowski, Terrence J; Silva, Afonso C; Strick, Peter L; Wu, Jun; Zhang, Feng

2015-05-06

One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators, and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive, and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward. Copyright © 2015 Elsevier Inc. All rights reserved.

Brains, Genes and Primates

PubMed Central

Belmonte, Juan Carlos Izpisua; Callaway, Edward M.; Churchland, Patricia; Caddick, Sarah J.; Feng, Guoping; Homanics, Gregg E.; Lee, Kuo-Fen; Leopold, David A.; Miller, Cory T.; Mitchell, Jude F.; Mitalipov, Shoukhrat; Moutri, Alysson R.; Movshon, J. Anthony; Okano, Hideyuki; Reynolds, John H.; Ringach, Dario; Sejnowski, Terrence J.; Silva, Afonso C.; Strick, Peter L.; Wu, Jun; Zhang, Feng

2015-01-01

One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward. PMID:25950631

Anthelmintic metabolism in parasitic helminths: proteomic insights.

PubMed

Brophy, Peter M; MacKintosh, Neil; Morphew, Russell M

2012-08-01

Anthelmintics are the cornerstone of parasitic helminth control. Surprisingly, understanding of the biochemical pathways used by parasitic helminths to detoxify anthelmintics is fragmented, despite the increasing global threat of anthelmintic resistance within the ruminant and equine industries. Reductionist biochemistry has likely over-estimated the enzymatic role of glutathione transferases in anthelmintic metabolism and neglected the potential role of the cytochrome P-450 superfamily (CYPs). Proteomic technologies offers the opportunity to support genomics, reverse genetics and pharmacokinetics, and provide an integrated insight into both the cellular mechanisms underpinning response to anthelmintics and also the identification of biomarker panels for monitoring the development of anthelmintic resistance. To date, there have been limited attempts to include proteomics in anthelmintic metabolism studies. Optimisations of membrane, post-translational modification and interaction proteomic technologies in helminths are needed to especially study Phase I CYPs and Phase III ABC transporter pumps for anthelmintics and their metabolites.

Reliable method for generating double-stranded DNA vectors containing site-specific base modifications.

PubMed

Brégeon, Damien; Doetsch, Paul W

2004-11-01

Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in DNA when it is replicated or transcribed. Lesion bypass by DNA and RNA polymerases has been the subject of numerous investigations. However, knowledge of the in vivo mechanism of transcription lesion bypass is very limited because no robust methodology is available. Here we describe a protocol based on the synthesis of a complementary strand of a circular, single-stranded DNA molecule, which allows for the production of large amounts of double-stranded DNA containing a lesion at a specific position in a transcribed sequence. Such constructs can subsequently be used for lesion bypass studies in vivo by RNA polymerase and to ascertain how these events can be affected by the genetic background of the cells.

Fundamental Design Principles for Transcription-Factor-Based Metabolite Biosensors.

PubMed

Mannan, Ahmad A; Liu, Di; Zhang, Fuzhong; Oyarzún, Diego A

2017-10-20

Metabolite biosensors are central to current efforts toward precision engineering of metabolism. Although most research has focused on building new biosensors, their tunability remains poorly understood and is fundamental for their broad applicability. Here we asked how genetic modifications shape the dose-response curve of biosensors based on metabolite-responsive transcription factors. Using the lac system in Escherichia coli as a model system, we built promoter libraries with variable operator sites that reveal interdependencies between biosensor dynamic range and response threshold. We developed a phenomenological theory to quantify such design constraints in biosensors with various architectures and tunable parameters. Our theory reveals a maximal achievable dynamic range and exposes tunable parameters for orthogonal control of dynamic range and response threshold. Our work sheds light on fundamental limits of synthetic biology designs and provides quantitative guidelines for biosensor design in applications such as dynamic pathway control, strain optimization, and real-time monitoring of metabolism.

Loss of glyphosate efficacy: a changing weed spectrum in Georgia cotton

USDA-ARS?s Scientific Manuscript database

Introduction of glyphosate resistance into crops through genetic modification has revolutionized crop protection. Glyphosate, the proverbial silver bullet, is a broad spectrum herbicide with favorable environmental characteristics and effective broad-spectrum weed control that has greatly improved ...

New pharmacologic treatments for familial hypercholesterolemia.

PubMed

McDonough, Annette; Matura, Lea Ann; Carroll, Diane

2013-10-01

Familial hypercholesterolemias are a group of genetic disorders that cause high levels of low-density lipoprotein (LDL) cholesterol, which can lead to atherosclerosis and premature coronary heart disease. Heart disease is the leading cause of death in U.S. women. A major goal in prevention of cardiovascular disease is identification and modification of risk factors. Lomitapide and mipomersen are two new pharmacologic options for treatment of familial hypercholesterolemia. Both are indicated as an adjunct for the management of homozygous familial hypercholesterolemia, along with lipid-lowering medications and diet modification. © 2013 AWHONN.

Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

PubMed

Nafissi, Nafiseh; Sum, Chi Hong; Wettig, Shawn; Slavcev, Roderick A

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼ 90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics.

Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

PubMed Central

Wettig, Shawn; Slavcev, Roderick A.

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called “Super Sequences” that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

Candidate innate immune system gene expression in the ecological model Daphnia

PubMed Central

Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E.; Little, Tom J.

2011-01-01

The last ten years have witnessed increasing interest in host–pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host–pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia–pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia–Pasteuria system will need to balance a candidate gene approach with more comprehensive approaches to de novo identify immune system genes specific to the Daphnia–Pasteuria interaction. PMID:21550363

Candidate innate immune system gene expression in the ecological model Daphnia.

PubMed

Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E; Little, Tom J

2011-10-01

The last ten years have witnessed increasing interest in host-pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host-pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia-pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia-Pasteuria system will need to balance a candidate gene approach with more comprehensive approaches to de novo identify immune system genes specific to the Daphnia-Pasteuria interaction. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effects of Pharmacological Inhibition and Genetic Deficiency of Plasminogen Activator Inhibitor-1 in Radiation-Induced Intestinal Injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abderrahmani, Rym; Francois, Agnes; Buard, Valerie

2009-07-01

Purpose: To investigate effects of plasminogen activator inhibitor 1 (PAI-1) genetic deficiency and pharmacological PAI-1 inhibition with PAI-039 in a mouse model of radiation-induced enteropathy. Methods and Materials: Wild-type (Wt) and PAI-1{sup -/-} knockout mice received a single dose of 19 Gy to an exteriorized localized intestinal segment. Sham and irradiated Wt mice were treated orally with 1 mg/g of PAI-039. Histological modifications were quantified using a radiation injury score. Moreover, intestinal gene expression was monitored by real-time PCR. Results: At 3 days after irradiation, PAI-039 abolished the radiation-induced increase in the plasma active form of PAI-1 and limited themore » radiation-induced gene expression of transforming growth factor {beta}1 (TGF-{beta}1), CTGF, PAI-1, and COL1A2. Moreover, PAI-039 conferred temporary protection against early lethality. PAI-039 treatment limited the radiation-induced increase of CTGF and PAI-1 at 2 weeks after irradiation but had no effect at 6 weeks. Radiation injuries were less severe in PAI-1{sup -/-} mice than in Wt mice, and despite the beneficial effect, 3 days after irradiation, PAI-039 had no effects on microscopic radiation injuries compared to untreated Wt mice. Conclusions: A genetic deficiency of PAI-1 is associated with amelioration of late radiation enteropathy. Pharmacological inhibition of PAI-1 by PAI-039 positively impacts the early, acute phase increase in plasma PAI-1 and the associated radiation-induced gene expression of inflammatory/extracellular matrix proteins. Since PAI-039 has been shown to inhibit the active form of PAI-1, as opposed to the complete loss of PAI-1 in the knockout animals, these data suggest that a PAI-1 inhibitor could be beneficial in treating radiation-induced tissue injury in acute settings where PAI-1 is elevated.« less

Cell Growth Enhancement

NASA Technical Reports Server (NTRS)

1992-01-01

Exogene Corporation uses advanced technologies to enhance production of bio-processed substances like proteins, antibiotics and amino acids. Among them are genetic modification and a genetic switch. They originated in research for Jet Propulsion Laboratory. Extensive experiments in cell growth through production of hemoglobin to improve oxygen supply to cells were performed. By improving efficiency of oxygen use by cells, major operational expenses can be reduced. Greater product yields result in decreased raw material costs and more efficient use of equipment. A broad range of applications is cited.

Genetic engineering of industrial Saccharomyces cerevisiae strains using a selection/counter-selection approach.

PubMed

Kutyna, Dariusz R; Cordente, Antonio G; Varela, Cristian

2014-01-01

Gene modification of laboratory yeast strains is currently a very straightforward task thanks to the availability of the entire yeast genome sequence and the high frequency with which yeast can incorporate exogenous DNA into its genome. Unfortunately, laboratory strains do not perform well in industrial settings, indicating the need for strategies to modify industrial strains to enable strain development for industrial applications. Here we describe approaches we have used to genetically modify industrial strains used in winemaking.

Topology-changing shape optimization with the genetic algorithm

NASA Astrophysics Data System (ADS)

Lamberson, Steven E., Jr.

The goal is to take a traditional shape optimization problem statement and modify it slightly to allow for prescribed changes in topology. This modification enables greater flexibility in the choice of parameters for the topology optimization problem, while improving the direct physical relevance of the results. This modification involves changing the optimization problem statement from a nonlinear programming problem into a form of mixed-discrete nonlinear programing problem. The present work demonstrates one possible way of using the Genetic Algorithm (GA) to solve such a problem, including the use of "masking bits" and a new modification to the bit-string affinity (BSA) termination criterion specifically designed for problems with "masking bits." A simple ten-bar truss problem proves the utility of the modified BSA for this type of problem. A more complicated two dimensional bracket problem is solved using both the proposed approach and a more traditional topology optimization approach (Solid Isotropic Microstructure with Penalization or SIMP) to enable comparison. The proposed approach is able to solve problems with both local and global constraints, which is something traditional methods cannot do. The proposed approach has a significantly higher computational burden --- on the order of 100 times larger than SIMP, although the proposed approach is able to offset this with parallel computing.

Advances in genetic engineering of the avian genome: "Realising the promise".

PubMed

Doran, Timothy J; Cooper, Caitlin A; Jenkins, Kristie A; Tizard, Mark L V

2016-06-01

This review provides an historic perspective of the key steps from those reported at the 1st Transgenic Animal Research Conference in 1997 through to the very latest developments in avian transgenesis. Eighteen years later, on the occasion of the 10th conference in this series, we have seen breakthrough advances in the use of viral vectors and transposons to transform the germline via the direct manipulation of the chicken embryo, through to the establishment of PGC cultures allowing in vitro modification, expansion into populations to analyse the genetic modifications and then injection of these cells into embryos to create germline chimeras. We have now reached an unprecedented time in the history of chicken transgenic research where we have the technology to introduce precise, targeted modifications into the chicken genome, ranging from; new transgenes that provide improved phenotypes such as increased resilience to economically important diseases; the targeted disruption of immunoglobulin genes and replacement with human sequences to generate transgenic chickens that express "humanised" antibodies for biopharming; and the deletion of specific nucleotides to generate targeted gene knockout chickens for functional genomics. The impact of these advances is set to be realised through applications in chickens, and other bird species as models in scientific research, for novel biotechnology and to protect and improve agricultural productivity.

The Evolution of Sex-Specific Dominance in Response to Sexually Antagonistic Selection.

PubMed

Spencer, Hamish G; Priest, Nicholas K

2016-05-01

Arguments about the evolutionary modification of genetic dominance have a long history in genetics, dating back more than 100 years. Mathematical investigations have shown that modifiers of the level of dominance at the locus of interest can spread at a reasonable rate only if heterozygotes at that locus are common. One hitherto neglected scenario is that of sexually antagonistic selection, which not only is ubiquitous in sexual species but also can generate stable high frequencies of heterozygotes that would appear to facilitate the spread of such modifiers. Here we present a mathematical model that shows that sexually specific dominance modification is a potential outcome of sexually antagonistic selection. Our model predicts that loci with higher levels of sexual conflict should exhibit greater differentiation between males and females in levels of dominance and that the strength of antagonistic selection experienced by one sex should be proportional to the level of dominance modification. We show that evidence from the literature is consistent with these predictions but suggest that empiricists should be alert to the possibility of there being numerous cases of sex-specific dominance. Further, in order to determine the significance of sexual conflict in the evolution of dominance, we need improved measures of sexual conflict and better characterization of loci that modify dominance of genes with sexually antagonistic fitness effects.