Physico-Chemical Properties and Biodegradability of Genetically Modified Populus trichocarpa and Pinus taeda

NASA Astrophysics Data System (ADS)

Edmunds, Charles Warren

Increasing concerns over greenhouse gas emissions and the finite supply of fossil fuels lead to the goal of utilizing lignocellulosic feedstocks for biofuels, platform chemicals, and biocomposites. Lignin is responsible for the recalcitrance of lignocellulosic biomass and is a major barrier to its deconstruction. Great progress has been made in mapping and modifying the lignin biosynthetic pathway. However, the link between the genetic modification, resulting chemical and physical properties of the wood, and how these properties influence the thermomechanical and recalcitrance to biological and chemical degradation needs further investigation. In this dissertation, the study of modified Populus trichocarpa and Pinus taeda were utilized to accomplish this goal. Thermo-mechanical properties of genetically modified P. trichocarpa with altered lignin content and/or lignin structure were measured with a series of tools including; dynamic mechanical analysis, nuclear magnetic resonance, and wet chemistry techniques. Results demonstrated lignin content and lignin structure likely influence the glass transition temperature (Tg), and that decreased lignin content and the corresponding higher proportion of cell wall carbohydrates may contribute to increased molecular mobility in the wood polymer structure. The effect of lignin biosynthetic pathway modification on biological degradation of these transgenic wood specimens was of interest. However, experimental methods for fungal treatment on small young greenhouse-grown wood specimens are not well established. Therefore, a project was undertaken to develop a method for fungal inoculation and incubation for these unique specimens. Several parameters were tested, and a fungal treatment method was identified with sufficient weight loss after decay and significant reduction in variation of weight loss between replicates compared to previous experiments by direct inoculation of wood with liquid malt extract fungal culture. Utilizing the fungal treatment method which was developed, fungal pretreatment as a potential low-input and environmentally-friendly alternative to conventional pretreatment methods was tested using the white-rot fungus, Ceriporiopsis subvermispora, on wildtype and transgenic P. trichocarpa. In addition to fungal treatment, hot water and dilute acid treatments followed by enzymatic hydrolysis was tested. Results showed no clear relationship between the initial lignin content or syringyl/guaiacyl lignin monomer ratio and weight loss due to fungal treatment. P-hydroxyphenyl lignin monomer degradation of up to 60% during the fungal treatment were observed in cinnamate 3-hydroxylase down-regulated genetic lines. It was demonstrated that fungal treatment in wildtype and several transgenic lines resulted in substantial improvements in sugar yields, up to 2.4-fold increase in glucose yield and 6.7-fold increase in xylose yield after enzymatic hydrolysis. However, some genetic lines showed little benefit from fungal pretreatment, and in general hot water and dilute acid pretreatments showed similar or increased glucose yield compared to fungal treatment. The goal of the last project was to characterize P. taeda which was genetically modified for S lignin production or decreased lignin content. In addition, the amenability to pretreatment and enzymatic hydrolysis were analyzed using hot water and dilute acid pretreatments followed by enzymatic hydrolysis. In the transgenic lines modified for production of syringyl lignin, Maule staining demonstrated the intermittent deposition of syringyl lignin in the secondary xylem, while thioacidolysis showed 13% concentration of S lignin, and solid state NMR demonstrated the occurrence of beta-O-4 linkages in S lignin units. In transgenic lines modified for reduced lignin content, lignin reduction up to 33% was observed, and pretreatment and enzymatic hydrolysis demonstrated increased cellulose conversion in lowlignin samples. These results highlight the potential of softwood to be a viable bioenergy/biochemical feedstock and opens up exciting new avenue of research.

Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research.

PubMed

Siciliano, Giulia; Alano, Pietro

2015-01-01

The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite P. berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria.

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

PubMed

Matsuoka, T; Kuribara, H; Akiyama, H; Miura, H; Goda, Y; Kusakabe, Y; Isshiki, K; Toyoda, M; Hino, A

2001-02-01

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

PubMed

Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

2016-01-01

This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds.

Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

PubMed

Hernández, Marta; Rodríguez-Lázaro, David; Zhang, David; Esteve, Teresa; Pla, Maria; Prat, Salomé

2005-05-04

The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.

A comparative study of human IgE binding to proteins of a genetically modified (GM) soybean and six non-GM soybeans grown in multiple locations.

PubMed

Lu, Mei; Jin, Yuan; Ballmer-Weber, Barbara; Goodman, Richard E

2018-02-01

Prior to commercialization, genetically modified (GM) crops are evaluated to determine the allergenicity of the newly expressed protein. Some regulators require an evaluation of endogenous allergens in commonly allergenic crops including soybean to determine if genetic transformation increased endogenous allergen concentrations, even asking for IgE testing using sera from individual sensitized subjects. Little is known about the variability of the expression of endogenous allergens among non-GM varieties or under different environmental conditions. We tested IgE binding to endogenous allergenic proteins in an experimental non-commercial GM line, a non-GM near-isoline control, and five non-GM commercial soybean lines replicated at three geographically separated locations. One-dimensional (1D) and two-dimensional (2D) immunoblotting and ELISA were performed using serum or plasma from eleven soybean allergic patients. The results of immunoblots and ELISA showed no significant differences in IgE binding between the GM line and its non-GM near-isoline control. However, some distinct differences in IgE binding patterns were observed among the non-GM commercial soybean lines and between different locations, highlighting the inherent variability in endogenous allergenic proteins. Understanding the potential variability in the levels of endogenous allergens is necessary to establish a standard of acceptance for GM soybeans compared to non-GM soybean events and lines. Copyright © 2018. Published by Elsevier Ltd.

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.

Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

PubMed

Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

2013-07-01

Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

Isocost Lines Describe the Cellular Economy of Genetic Circuits

PubMed Central

Gyorgy, Andras; Jiménez, José I.; Yazbek, John; Huang, Hsin-Ho; Chung, Hattie; Weiss, Ron; Del Vecchio, Domitilla

2015-01-01

Genetic circuits in living cells share transcriptional and translational resources that are available in limited amounts. This leads to unexpected couplings among seemingly unconnected modules, which result in poorly predictable circuit behavior. In this study, we determine these interdependencies between products of different genes by characterizing the economy of how transcriptional and translational resources are allocated to the production of proteins in genetic circuits. We discover that, when expressed from the same plasmid, the combinations of attainable protein concentrations are constrained by a linear relationship, which can be interpreted as an isocost line, a concept used in microeconomics. We created a library of circuits with two reporter genes, one constitutive and the other inducible in the same plasmid, without a regulatory path between them. In agreement with the model predictions, experiments reveal that the isocost line rotates when changing the ribosome binding site strength of the inducible gene and shifts when modifying the plasmid copy number. These results demonstrate that isocost lines can be employed to predict how genetic circuits become coupled when sharing resources and provide design guidelines for minimizing the effects of such couplings. PMID:26244745

Genetic variant modifies the effect of N3 PUFAs on DNA methylation of IL6 in the Genetics of Lipid Lowering Drugs and Diet Network study

USDA-ARS?s Scientific Manuscript database

N3 polyunsaturated fatty acids (N3 PUFAs) ameliorate inflammation status with specific regulation on interleukin-6 (IL6) expression. However, the molecular mechanism for this regulation is unclear. Using both cell lines data from Encyclopedia of DNA Elements (ENCODE) consortium and population data f...

On-site detection of stacked genetically modified soybean based on event-specific TM-LAMP and a DNAzyme-lateral flow biosensor.

PubMed

Cheng, Nan; Shang, Ying; Xu, Yuancong; Zhang, Li; Luo, Yunbo; Huang, Kunlun; Xu, Wentao

2017-05-15

Stacked genetically modified organisms (GMO) are becoming popular for their enhanced production efficiency and improved functional properties, and on-site detection of stacked GMO is an urgent challenge to be solved. In this study, we developed a cascade system combining event-specific tag-labeled multiplex LAMP with a DNAzyme-lateral flow biosensor for reliable detection of stacked events (DP305423× GTS 40-3-2). Three primer sets, both event-specific and soybean species-specific, were newly designed for the tag-labeled multiplex LAMP system. A trident-like lateral flow biosensor displayed amplified products simultaneously without cross contamination, and DNAzyme enhancement improved the sensitivity effectively. After optimization, the limit of detection was approximately 0.1% (w/w) for stacked GM soybean, which is sensitive enough to detect genetically modified content up to a threshold value established by several countries for regulatory compliance. The entire detection process could be shortened to 120min without any large-scale instrumentation. This method may be useful for the in-field detection of DP305423× GTS 40-3-2 soybean on a single kernel basis and on-site screening tests of stacked GM soybean lines and individual parent GM soybean lines in highly processed foods. Copyright © 2017 Elsevier B.V. All rights reserved.

Generation of an immortalized mouse embryonic palatal mesenchyme cell line

PubMed Central

Soriano, Philippe

2017-01-01

Palatogenesis is a complex morphogenetic process, disruptions in which result in highly prevalent birth defects in humans. In recent decades, the use of model systems such as genetically-modified mice, mouse palatal organ cultures and primary mouse embryonic palatal mesenchyme (MEPM) cultures has provided significant insight into the molecular and cellular defects underlying cleft palate. However, drawbacks in each of these systems have prevented high-throughput, large-scale studies of palatogenesis in vitro. Here, we report the generation of an immortalized MEPM cell line that maintains the morphology, migration ability, transcript expression and responsiveness to exogenous growth factors of primary MEPM cells, with increased proliferative potential over primary cultures. The immortalization method described in this study will facilitate the generation of palatal mesenchyme cells with an unlimited capacity for expansion from a single genetically-modified mouse embryo and enable mechanistic studies of palatogenesis that have not been possible using primary culture. PMID:28582446

[Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

PubMed

An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

2012-12-01

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

Loss-of-function of a ubiquitin-related modifier promotes the mobilization of the active MITE mPing.

PubMed

Tsukiyama, Takuji; Teramoto, Shota; Yasuda, Kanako; Horibata, Akira; Mori, Nanako; Okumoto, Yutaka; Teraishi, Masayoshi; Saito, Hiroki; Onishi, Akiko; Tamura, Kanako; Tanisaka, Takatoshi

2013-05-01

Miniature inverted-repeat transposable elements (MITEs) are widespread in both prokaryotic and eukaryotic genomes, where their copy numbers can attain several thousands. Little is known, however, about the genetic factor(s) affecting their transpositions. Here, we show that disruption of a gene encoding ubiquitin-like protein markedly enhances the transposition activity of a MITE mPing in intact rice plants without any exogenous stresses. We found that the transposition activity of mPing is far higher in the lines harboring a non-functional allele at the Rurm1 (Rice ubiquitin-related modifier-1) locus than in the wild-type line. Although the alteration of cytosine methylation pattern triggers the activation of transposable elements under exogenous stress conditions, the methylation degrees in the whole genome, the mPing-body region, and the mPing-flanking regions of the non-functional Rurm1 line were unchanged. This study provides experimental evidence for one of the models of genome shock theory that genetic accidents within cells enhance the transposition activities of transposable elements.

Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation

PubMed Central

Farley, Jane S.; Taft, Robert A.

2008-01-01

Background Sperm from C57BL/6 mice are difficult to cryopreserve and recover. Yet, the majority of genetically modified (GM) lines are maintained on this genetic background. Methodology/Principal Findings Reported here is the development of an easily implemented method that consistently yields fertilization rates of 70±5% with this strain. This six-fold increase is achieved by collecting sperm from the vas deferens and epididymis into a cryoprotective medium of 18% raffinose (w/v), 3% skim milk (w/v) and 477 µM monothioglycerol. The sperm suspension is loaded into 0.25 mL French straws and cooled at 37±1°C/min before being plunged and then stored in LN2. Subsequent to storage, the sperm are warmed at 2,232±162°C/min and incubated in in vitro fertilization media for an hour prior to the addition of oocyte cumulus masses from superovulated females. Sperm from 735 GM mouse lines on 12 common genetic backgrounds including C57BL/6J, BALB/cJ, 129S1/SvImJ, FVB/NJ and NOD/ShiLtJ were cryopreserved and recovered. C57BL/6J and BALB/cByJ fertilization rates, using frozen sperm, were slightly reduced compared to rates involving fresh sperm; fertilization rates using fresh or frozen sperm were equivalent in all other lines. Developmental capacity of embryos produced using cryopreserved sperm was equivalent, or superior to, cryopreserved IVF-derived embryos. Conclusions/Significance Combined, these results demonstrate the broad applicability of our approach as an economical and efficient option for archiving and distributing mice. PMID:18665210

Altered Tuber Yield in Genetically Modified High-Amylose and Oil Potato Lines Is Associated With Changed Whole-Plant Nitrogen Economy

PubMed Central

Pourazari, Fereshteh; Andersson, Mariette; Weih, Martin

2018-01-01

Breeding for improved crop quality traits can affect non-target traits related to growth and resource use, and these effects may vary in different cultivation conditions (e. g., greenhouse vs. field). The objectives of this study are to investigate the growth and whole-plant nitrogen (N) economy of two genetically modified (GM) potato lines compared to their non-GM parental varieties and when grown in different cultivation conditions. A high-amylose GM potato line and its parent were grown under field and greenhouse conditions for one growing season in Sweden; and a GM oil potato line and its parent were grown in greenhouse conditions only. Tuber yield, above ground biomass, N uptake efficiency and other plant N economy traits were assessed. In both cultivation conditions, the GM lines produced between 1.5 and two times more tubers as compared with their parents. In the greenhouse, fresh tuber yield and N uptake efficiency were unaffected by the genetic modifications, but the GM-lines produced less tuber biomass per plant-internal N compared to their parents. In the field, the fresh tuber yield was 40% greater in the high-amylose line as compared with its parent; the greater fresh tuber yield in the high-amylose GM line was accomplished by higher water allocation to the harvested tubers, and associated with increased N recovery from soil (+20%), N uptake efficiency (+53%), tuber N content (+20%), and N accumulation (+120%) compared with the non-GM parent. The cultivation conditions influenced the yield and N economy. For example, the final fresh above-ground plant biomass and N pool were considerably higher in the greenhouse conditions, whilst the tuber yield was higher in the field conditions. In conclusion, the genetic modification inducing high accumulation of amylose in potato tubers affected several non-target traits related to plant N economy, and increased the plant N uptake and accumulation efficiency of the field-grown plants. Due to strongly increased plant N accumulation compared to the parental variety, the cultivation of the high-amylose line is expected to require higher N fertilization rates. However, starch productivity per unit land area or soil N still is expected to be higher in the high-amylose line. PMID:29599796

Isocost Lines Describe the Cellular Economy of Genetic Circuits.

PubMed

Gyorgy, Andras; Jiménez, José I; Yazbek, John; Huang, Hsin-Ho; Chung, Hattie; Weiss, Ron; Del Vecchio, Domitilla

2015-08-04

Genetic circuits in living cells share transcriptional and translational resources that are available in limited amounts. This leads to unexpected couplings among seemingly unconnected modules, which result in poorly predictable circuit behavior. In this study, we determine these interdependencies between products of different genes by characterizing the economy of how transcriptional and translational resources are allocated to the production of proteins in genetic circuits. We discover that, when expressed from the same plasmid, the combinations of attainable protein concentrations are constrained by a linear relationship, which can be interpreted as an isocost line, a concept used in microeconomics. We created a library of circuits with two reporter genes, one constitutive and the other inducible in the same plasmid, without a regulatory path between them. In agreement with the model predictions, experiments reveal that the isocost line rotates when changing the ribosome binding site strength of the inducible gene and shifts when modifying the plasmid copy number. These results demonstrate that isocost lines can be employed to predict how genetic circuits become coupled when sharing resources and provide design guidelines for minimizing the effects of such couplings. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

[Genetic improvement of breeding materials in tropical and sub- tropical maize].

PubMed

Sansern, Jampatong; Chaba, Jampatong

2011-12-01

In the present study, 122 maize local cultivars and adapted exotic germplasm from Thailand were used to develop open pollinate varieties (OPVs) using modified ear-to-row scheme, top-cross or test-cross programmes. Ten new maize OPVs with distinct characters were created based on the precise breeding objectives and directional design. The selection of breeding materials was based upon three factors: elite performance, broad adaptability, and genetic diversity. The synthesizing system provided four features: genetic mixing and recombination, equal comparable genetic contribution, mild selection pressure, and maximum intermating for genetic equilibrium (i.e., the female traits were close for the genetic com-positions). Subsequently, Suwan 1 composite and its deritives (Suwan 2, Suwan 3 composite, Suwan 5 and KS24 synthetics), KS6 and KS28 synthetics with the dent type of different origins, and Caripeno DMR composite, KS23, and KS27 synthetics with the dent type of Non-Suwan 1 origin were developed. These OPVs had been improved for 2~13 cycles using S1 recurrent selection method. About 50 inbred lines were developed from these OPVs, and 16 elite single (three-way) crosses were combined and released from these inbred lines. At present, at least one parental inbred line of all the tropical hybrids was derived from Suwan (KS) germplasm in Thailand. Based on the theory of the synthesizing OPVs and developing inbred lines, this paper discussed the genetic moderate diversity, relationship, heterotic group, and patterns for synthesizing OPVs, and inspiration for composed OPVs to heterosis breeding.

Determination of Mycotoxin Production of Fusarium Species in Genetically Modified Maize Varieties by Quantitative Flow Immunocytometry.

PubMed

Bánáti, Hajnalka; Darvas, Béla; Fehér-Tóth, Szilvia; Czéh, Árpád; Székács, András

2017-02-22

Levels of mycotoxins produced by Fusarium species in genetically modified (GM) and near-isogenic maize, were determined using multi-analyte, microbead-based flow immunocytometry with fluorescence detection, for the parallel quantitative determination of fumonisin B1, deoxynivalenol, zearalenone, T-2, ochratoxin A, and aflatoxin B1. Maize varieties included the genetic events MON 810 and DAS-59122-7 , and their isogenic counterparts. Cobs were artificially infested by F. verticillioides and F. proliferatum conidia, and contained F. graminearum and F. sporotrichoides natural infestation. The production of fumonisin B1 and deoxynivalenol was substantially affected in GM maize lines: F. verticillioides , with the addition of F. graminearum and F. sporotrichoides , produced significantly lower levels of fumonisin B1 (~300 mg·kg -1 ) in DAS-59122-7 than in its isogenic line (~580 mg·kg -1 ), while F. proliferatum , in addition to F. graminearum and F. sporotrichoides , produced significantly higher levels of deoxynivalenol (~18 mg·kg -1 ) in MON 810 than in its isogenic line (~5 mg·kg -1 ). Fusarium verticillioides , with F. graminearum and F. sporotrichoides , produced lower amounts of deoxynivalenol and zearalenone than F. proliferatum , with F. graminearum and F. sporotrichoides . T-2 toxin production remained unchanged when considering the maize variety. The results demonstrate the utility of the Fungi-Plex™ quantitative flow immunocytometry method, applied for the high throughput parallel determination of the target mycotoxins.

Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera.

PubMed

Kawahara, Masahiro; Chen, Jianhong; Sogo, Takahiro; Teng, Jinying; Otsu, Makoto; Onodera, Masafumi; Nakauchi, Hiromitsu; Ueda, Hiroshi; Nagamune, Teruyuki

2011-09-01

Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling. Copyright © 2011 Elsevier Ltd. All rights reserved.

Aphid-parasitoid community structure on genetically modified wheat.

PubMed

von Burg, Simone; van Veen, Frank J F; Álvarez-Alfageme, Fernando; Romeis, Jörg

2011-06-23

Since the introduction of genetically modified (GM) plants, one of the main concerns has been their potential effect on non-target insects. Many studies have looked at GM plant effects on single non-target herbivore species or on simple herbivore-natural enemy food chains. Agro-ecosystems, however, are characterized by numerous insect species which are involved in complex interactions, forming food webs. In this study, we looked at transgenic disease-resistant wheat (Triticum aestivum) and its effect on aphid-parasitoid food webs. We hypothesized that the GM of the wheat lines directly or indirectly affect aphids and that these effects cascade up to change the structure of the associated food webs. Over 2 years, we studied different experimental wheat lines under semi-field conditions. We constructed quantitative food webs to compare their properties on GM lines with the properties on corresponding non-transgenic controls. We found significant effects of the different wheat lines on insect community structure up to the fourth trophic level. However, the observed effects were inconsistent between study years and the variation between wheat varieties was as big as between GM plants and their controls. This suggests that the impact of our powdery mildew-resistant GM wheat plants on food web structure may be negligible and potential ecological effects on non-target insects limited.

Visual detection of multiple genetically modified organisms in a capillary array.

PubMed

Shao, Ning; Chen, Jianwei; Hu, Jiaying; Li, Rong; Zhang, Dabing; Guo, Shujuan; Hui, Junhou; Liu, Peng; Yang, Litao; Tao, Sheng-Ce

2017-01-31

There is an urgent need for rapid, low-cost multiplex methodologies for the monitoring of genetically modified organisms (GMOs). Here, we report a C[combining low line]apillary A[combining low line]rray-based L[combining low line]oop-mediated isothermal amplification for M[combining low line]ultiplex visual detection of nucleic acids (CALM) platform for the simple and rapid monitoring of GMOs. In CALM, loop-mediated isothermal amplification (LAMP) primer sets are pre-fixed to the inner surface of capillaries. The surface of the capillary array is hydrophobic while the capillaries are hydrophilic, enabling the simultaneous loading and separation of the LAMP reaction mixtures into each capillary by capillary forces. LAMP reactions in the capillaries are then performed in parallel, and the results are visually detected by illumination with a hand-held UV device. Using CALM, we successfully detected seven frequently used transgenic genes/elements and five plant endogenous reference genes with high specificity and sensitivity. Moreover, we found that measurements of real-world blind samples by CALM are consistent with results obtained by independent real-time PCRs. Thus, with an ability to detect multiple nucleic acids in a single easy-to-operate test, we believe that CALM will become a widely applied technology in GMO monitoring.

Identification of prostate cancer modifier pathways using parental strain expression mapping

PubMed Central

Xu, Qing; Majumder, Pradip K.; Ross, Kenneth; Shim, Yeonju; Golub, Todd R.; Loda, Massimo; Sellers, William R.

2007-01-01

Inherited genetic risk factors play an important role in cancer. However, other than the Mendelian fashion cancer susceptibility genes found in familial cancer syndromes, little is known about risk modifiers that control individual susceptibility. Here we developed a strategy, parental strain expression mapping, that utilizes the homogeneity of inbred mice and genome-wide mRNA expression analyses to directly identify candidate germ-line modifier genes and pathways underlying phenotypic differences among murine strains exposed to transgenic activation of AKT1. We identified multiple candidate modifier pathways and, specifically, the glycolysis pathway as a candidate negative modulator of AKT1-induced proliferation. In keeping with the findings in the murine models, in multiple human prostate expression data set, we found that enrichment of glycolysis pathways in normal tissues was associated with decreased rates of cancer recurrence after prostatectomy. Together, these data suggest that parental strain expression mapping can directly identify germ-line modifier pathways of relevance to human disease. PMID:17978178

Populational survey of arthropods on transgenic common bean expressing the rep gene from Bean golden mosaic virus

PubMed Central

Pinheiro, Patrícia V; Quintela, Eliane D; Junqueira, Ana Maria R; Aragão, Francisco JL; Faria, Josias C

2014-01-01

Genetically modified (GM) crops is considered the fastest adopted crop technology in the history of modern agriculture. However, possible undesirable and unintended effects must be considered during the research steps toward development of a commercial product. In this report we evaluated effects of a common bean virus resistant line on arthropod populations, considered as non-target organisms. This GM bean line (named M1/4) was modified for resistance against Bean golden mosaic virus (BGMV) by expressing a mutated REP protein, which is essential for virus replication. Biosafety studies were performed for a period of three years under field conditions. The abundance of some species was significantly higher in specific treatments in a particular year, but not consistently different in other years. A regular pattern was not observed in the distribution of insects between genetically modified and conventional treatments. Data analyses showed that minor differences observed can be attributed to random variation and were not consistent enough to conclude that the treatments were different. Therefore the present study indicates that the relative abundance of species are similar in transgenic and non-transgenic fields. PMID:24922280

Populational survey of arthropods on transgenic common bean expressing the rep gene from Bean golden mosaic virus.

PubMed

Pinheiro, Patrícia V; Quintela, Eliane D; Junqueira, Ana Maria R; Aragão, Francisco J L; Faria, Josias C

2014-01-01

Genetically modified (GM) crops is considered the fastest adopted crop technology in the history of modern agriculture. However, possible undesirable and unintended effects must be considered during the research steps toward development of a commercial product. In this report we evaluated effects of a common bean virus resistant line on arthropod populations, considered as non-target organisms. This GM bean line (named M1/4) was modified for resistance against Bean golden mosaic virus (BGMV) by expressing a mutated REP protein, which is essential for virus replication. Biosafety studies were performed for a period of three years under field conditions. The abundance of some species was significantly higher in specific treatments in a particular year, but not consistently different in other years. A regular pattern was not observed in the distribution of insects between genetically modified and conventional treatments. Data analyses showed that minor differences observed can be attributed to random variation and were not consistent enough to conclude that the treatments were different. Therefore the present study indicates that the relative abundance of species are similar in transgenic and non-transgenic fields.

Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.

PubMed

Yumlu, Saniye; Stumm, Jürgen; Bashir, Sanum; Dreyer, Anne-Kathrin; Lisowski, Pawel; Danner, Eric; Kühn, Ralf

2017-05-15

Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones. Copyright © 2017 Elsevier Inc. All rights reserved.

Modulation of the Fecal Microbiota in Sprague-Dawley Rats Using Genetically Modified and Isogenic Corn Lines.

PubMed

Li, Penggao; Yang, Chun; Yue, Rong; Zhen, Yaping; Zhuo, Qin; Piao, Jianhua; Yang, Xiaoguang; Xiao, Rong

2018-01-17

This study investigated the composition and proportions of fecal microbiota in Sprague-Dawley rats after consuming two genetically modified (GM) corn lines in comparison with the isogenic corn and the AIN93G standard feed for 10 weeks using bar-coded 16S rRNA gene sequencing. As a result, GM corn did not significantly alter the overall health and alpha-diversity of fecal microbiota. Fecal microbiota structures could be separated into noncorn and corn but not non-GM and GM corn subgroups. Both non-GM and GM corn caused the increase in bacterial populations related to carbohydrates utilization, such as Lactobacillus, Barnesiella, and Bifidobacterium, and the reduction in potentially pathogenic populations, such as Tannerella and Moraxellaceae. In conclusion, similar effects on the fecal microbiota were observed after consuming a GM- and non-GM-corn-based diet for long periods. Further studies are warranted to elucidate the functional relevance of the changes in the proportions of bacterial populations in these diets.

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.

PubMed

Binder, Zev A; Wilson, Kelli M; Salmasi, Vafi; Orr, Brent A; Eberhart, Charles G; Siu, I-Mei; Lim, Michael; Weingart, Jon D; Quinones-Hinojosa, Alfredo; Bettegowda, Chetan; Kassam, Amin B; Olivi, Alessandro; Brem, Henry; Riggins, Gregory J; Gallia, Gary L

2016-01-01

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants. Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.

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

Genome Editing in Human Pluripotent Stem Cells.

PubMed

Carlson-Stevermer, Jared; Saha, Krishanu

2017-01-01

Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.

Selection for Cry3Bb1 resistance in a genetically diverse population of nondiapausing western corn rootworm (Coleoptera: Chrysomelidae).

PubMed

Oswald, Kenneth J; French, B Wade; Nielson, Chad; Bagley, Mark

2011-06-01

Five short-diapause laboratory lines of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), were selected for resistance to MON863, a variety of corn genetically modified with the Bacillus thuringiensis Berliner (Bt) transgene that expresses the Cry3Bb1 delta-endotoxin. Three of the selected lines were developed by incremental increase in the duration of exposure to MON863 over 11 generations (moderate selected lines). Two selected lines were developed from a control group by constant exposure to MON863 for at least 14 d posthatch over seven generations (intense selected lines). At the end of the experiment, survivorship, as measured by adult emergence, was approximately 4 times higher in each of the selected lines reared on MON863 compared with control lines. Estimates of realized heritabilities (h2) were 0.16 and 0.15 for the moderate and intense selected lines, respectively, and are consistent with h2 estimates reported previously from a variety of pest insects. These lines provide data necessary for evaluating the potential for Bt resistance within diabroticite beetles and will be useful for developing improved insect resistance management strategies.

Transgene × Environment Interactions in Genetically Modified Wheat

PubMed Central

Zeller, Simon L.; Kalinina, Olena; Brunner, Susanne; Keller, Beat; Schmid, Bernhard

2010-01-01

Background The introduction of transgenes into plants may cause unintended phenotypic effects which could have an impact on the plant itself and the environment. Little is published in the scientific literature about the interrelation of environmental factors and possible unintended effects in genetically modified (GM) plants. Methods and Findings We studied transgenic bread wheat Triticum aestivum lines expressing the wheat Pm3b gene against the fungus powdery mildew Blumeria graminis f.sp. tritici. Four independent offspring pairs, each consisting of a GM line and its corresponding non-GM control line, were grown under different soil nutrient conditions and with and without fungicide treatment in the glasshouse. Furthermore, we performed a field experiment with a similar design to validate our glasshouse results. The transgene increased the resistance to powdery mildew in all environments. However, GM plants reacted sensitive to fungicide spraying in the glasshouse. Without fungicide treatment, in the glasshouse GM lines had increased vegetative biomass and seed number and a twofold yield compared with control lines. In the field these results were reversed. Fertilization generally increased GM/control differences in the glasshouse but not in the field. Two of four GM lines showed up to 56% yield reduction and a 40-fold increase of infection with ergot disease Claviceps purpurea compared with their control lines in the field experiment; one GM line was very similar to its control. Conclusions Our results demonstrate that, depending on the insertion event, a particular transgene can have large effects on the entire phenotype of a plant and that these effects can sometimes be reversed when plants are moved from the glasshouse to the field. However, it remains unclear which mechanisms underlie these effects and how they may affect concepts in molecular plant breeding and plant evolutionary ecology. PMID:20635001

Transgene x environment interactions in genetically modified wheat.

PubMed

Zeller, Simon L; Kalinina, Olena; Brunner, Susanne; Keller, Beat; Schmid, Bernhard

2010-07-12

The introduction of transgenes into plants may cause unintended phenotypic effects which could have an impact on the plant itself and the environment. Little is published in the scientific literature about the interrelation of environmental factors and possible unintended effects in genetically modified (GM) plants. We studied transgenic bread wheat Triticum aestivum lines expressing the wheat Pm3b gene against the fungus powdery mildew Blumeria graminis f.sp. tritici. Four independent offspring pairs, each consisting of a GM line and its corresponding non-GM control line, were grown under different soil nutrient conditions and with and without fungicide treatment in the glasshouse. Furthermore, we performed a field experiment with a similar design to validate our glasshouse results. The transgene increased the resistance to powdery mildew in all environments. However, GM plants reacted sensitive to fungicide spraying in the glasshouse. Without fungicide treatment, in the glasshouse GM lines had increased vegetative biomass and seed number and a twofold yield compared with control lines. In the field these results were reversed. Fertilization generally increased GM/control differences in the glasshouse but not in the field. Two of four GM lines showed up to 56% yield reduction and a 40-fold increase of infection with ergot disease Claviceps purpurea compared with their control lines in the field experiment; one GM line was very similar to its control. Our results demonstrate that, depending on the insertion event, a particular transgene can have large effects on the entire phenotype of a plant and that these effects can sometimes be reversed when plants are moved from the glasshouse to the field. However, it remains unclear which mechanisms underlie these effects and how they may affect concepts in molecular plant breeding and plant evolutionary ecology.

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.

Identification of Mom12 and Mom13, two novel modifier loci of Apc (Min) -mediated intestinal tumorigenesis.

PubMed

Crist, Richard C; Roth, Jacquelyn J; Lisanti, Michael P; Siracusa, Linda D; Buchberg, Arthur M

2011-04-01

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc (Min/+) mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc (Min/+) mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc (Min) model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc (Min) -mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc (Min/+) mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc (Min/+) controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: 1) Mom12, a dominant modifier linked to the congenic region on chromosome 6, and 2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.

Chronotopes: Forms of Time in Rhetorical Argument

ERIC Educational Resources Information Center

Jack, Jordynn

2006-01-01

The author examines how chronotopes--a term M. M. Bakhtin used to describe space-time relationships in literature--also characterize rhetorical arguments. She uses a case study of a series of debates about genetically modified foods (GMFs) in Canada to illustrate how chronotopes shape arguments along ideological lines. In particular, she suggests…

Real-time PCR array as a universal platform for the detection of genetically modified crops and its application in identifying unapproved genetically modified crops in Japan.

PubMed

Mano, Junichi; Shigemitsu, Natsuki; Futo, Satoshi; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Furui, Satoshi; Kitta, Kazumi

2009-01-14

We developed a novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. Thirty primer-probe sets for the specific detection of GM lines, recombinant DNA (r-DNA) segments, endogenous reference genes, and donor organisms were synthesized, and a 96-well PCR plate was prepared with a different primer-probe in each well as the real-time PCR array. The specificity and sensitivity of the array were evaluated. A comparative analysis with the data and publicly available information on GM crops approved in Japan allowed us to assume the possibility of unapproved GM crop contamination. Furthermore, we designed a Microsoft Excel spreadsheet application, Unapproved GMO Checker version 2.01, which helps process all the data of real-time PCR arrays for the easy assumption of unapproved GM crop contamination. The spreadsheet is available free of charge at http://cse.naro.affrc.go.jp/jmano/index.html .

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

TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.

PubMed

Ramalingam, Sivaprakash; Annaluru, Narayana; Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

2014-01-01

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Exploring the genetics of fertility restoration controlled by Rf1 in common wheat (Triticum aestivum L.) using high-density linkage maps.

PubMed

Geyer, Manuel; Albrecht, Theresa; Hartl, Lorenz; Mohler, Volker

2018-04-01

Hybrid wheat breeding has the potential to significantly increase wheat productivity compared to line breeding. The induction of male sterility by the cytoplasm of Triticum timopheevii Zhuk. is a widely discussed approach to ensure cross-pollination between parental inbred lines in hybrid wheat seed production. As fertility restoration in hybrids with this cytoplasm is often incomplete, understanding the underlying genetics is a prerequisite to apply this technology. A promising component for fertility restoration is the restorer locus Rf1, which was first detected on chromosome 1A of the restorer accession R3. In the present study, we performed quantitative trait locus (QTL) analyses to locate Rf1 and estimate its effect in populations involving the restorer lines R3, R113 and L19. Molecular markers linked to Rf1 in these populations were used to analyse the genomic target region in T. timopheevii accessions and common wheat breeding lines. The QTL analyses revealed that Rf1 interacted with a modifier locus on chromosome 1BS and the restorer locus Rf4 on chromosome 6B. The modifier locus significantly influenced both the penetrance and expressivity of Rf1. Whereas Rf1 exhibited expressivity higher than that of Rf4, the effects of these loci were not additive. Evaluating the marker haplotype for the Rf1 region, we propose that the restoring Rf1 allele may be derived exclusively from T. timopheevii. The present study demonstrates that interactions between restorer and modifier loci play a critical role in fertility restoration of common wheat with the cytoplasm of T. timopheevii.

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.

A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed.

PubMed

Li, Liang; Dong, Mei; An, Na; Liang, Lixia; Wan, Yusong; Jin, Wujun

2015-01-01

Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice.

A Novel Reference Plasmid for the Qualitative Detection of Genetically Modified Rice in Food and Feed

PubMed Central

Dong, Mei; An, Na; Liang, Lixia; Wan, Yusong

2015-01-01

Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice. PMID:26495318

Fast-Flowering Mini-Maize: Seed to Seed in 60 Days

PubMed Central

McCaw, Morgan E.; Wallace, Jason G.; Albert, Patrice S.; Buckler, Edward S.; Birchler, James A.

2016-01-01

Two lines of Zea mays were developed as a short-generation model for maize. The Fast-Flowering Mini-Maize (FFMM) lines A and B are robust inbred lines with a significantly shorter generation time, much smaller stature, and better greenhouse adaptation than traditional maize varieties. Five generations a year are typical. FFMM is the result of a modified double-cross hybrid between four fast-flowering lines: Neuffer’s Early ACR (full color), Alexander’s Early Early Synthetic, Tom Thumb Popcorn, and Gaspe Flint, followed by selection for early flowering and desirable morphology throughout an 11-generation selfing regime. Lines A and B were derived from different progeny of the initial hybrid, and crosses between Mini-Maize A and B exhibit heterosis. The ancestry of each genomic region of Mini-Maize A and B was inferred from the four founder populations using genotyping by sequencing. Other genetic and genomic tools for these lines include karyotypes for both lines A and B, kernel genetic markers y1 (white endosperm) and R1-scm2 (purple endosperm and embryo) introgressed into Mini-Maize A, and ∼24× whole-genome resequencing data for Mini-Maize A. PMID:27440866

Calreticulin and Jak2 as Chaperones for MPL: Insights into MPN Pathogenesis

DTIC Science & Technology

2017-11-01

cell surface; and 2) CRISPR -Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived...line has then been sub-cloned and individual clones were screened for robust Mpl expression using WB before being further modified (Fig. 1). CRISPR ...rescue its function and response to its ligand (Fig. 6). Genetic editing (using CRISPR /Cas9) performed on cells carrying the W272R mutation restored

Laboratory and field studies of guayule modified to overexpress HMGR

USDA-ARS?s Scientific Manuscript database

We report the genetic modification of guayule to overexpress the isoprenoid pathway enzyme HMGR. The rubber content of two-month old in vitro transformed plantlets showed a 65% increase in rubber over the control for one line (HMGR6), and lower resin for another (HMGR2). In field evaluations HMGR6...

Gene flow in genetically modified wheat.

PubMed

Rieben, Silvan; Kalinina, Olena; Schmid, Bernhard; Zeller, Simon L

2011-01-01

Understanding gene flow in genetically modified (GM) crops is critical to answering questions regarding risk-assessment and the coexistence of GM and non-GM crops. In two field experiments, we tested whether rates of cross-pollination differed between GM and non-GM lines of the predominantly self-pollinating wheat Triticum aestivum. In the first experiment, outcrossing was studied within the field by planting "phytometers" of one line into stands of another line. In the second experiment, outcrossing was studied over distances of 0.5-2.5 m from a central patch of pollen donors to adjacent patches of pollen recipients. Cross-pollination and outcrossing was detected when offspring of a pollen recipient without a particular transgene contained this transgene in heterozygous condition. The GM lines had been produced from the varieties Bobwhite or Frisal and contained Pm3b or chitinase/glucanase transgenes, respectively, in homozygous condition. These transgenes increase plant resistance against pathogenic fungi. Although the overall outcrossing rate in the first experiment was only 3.4%, Bobwhite GM lines containing the Pm3b transgene were six times more likely than non-GM control lines to produce outcrossed offspring. There was additional variation in outcrossing rate among the four GM-lines, presumably due to the different transgene insertion events. Among the pollen donors, the Frisal GM line expressing a chitinase transgene caused more outcrossing than the GM line expressing both a chitinase and a glucanase transgene. In the second experiment, outcrossing after cross-pollination declined from 0.7-0.03% over the test distances of 0.5-2.5 m. Our results suggest that pollen-mediated gene flow between GM and non-GM wheat might only be a concern if it occurs within fields, e.g. due to seed contamination. Methodologically our study demonstrates that outcrossing rates between transgenic and other lines within crops can be assessed using a phytometer approach and that gene-flow distances can be efficiently estimated with population-level PCR analyses. © 2011 Rieben et al.

Gene Flow in Genetically Modified Wheat

PubMed Central

Rieben, Silvan; Kalinina, Olena; Schmid, Bernhard; Zeller, Simon L.

2011-01-01

Understanding gene flow in genetically modified (GM) crops is critical to answering questions regarding risk-assessment and the coexistence of GM and non-GM crops. In two field experiments, we tested whether rates of cross-pollination differed between GM and non-GM lines of the predominantly self-pollinating wheat Triticum aestivum. In the first experiment, outcrossing was studied within the field by planting “phytometers” of one line into stands of another line. In the second experiment, outcrossing was studied over distances of 0.5–2.5 m from a central patch of pollen donors to adjacent patches of pollen recipients. Cross-pollination and outcrossing was detected when offspring of a pollen recipient without a particular transgene contained this transgene in heterozygous condition. The GM lines had been produced from the varieties Bobwhite or Frisal and contained Pm3b or chitinase/glucanase transgenes, respectively, in homozygous condition. These transgenes increase plant resistance against pathogenic fungi. Although the overall outcrossing rate in the first experiment was only 3.4%, Bobwhite GM lines containing the Pm3b transgene were six times more likely than non-GM control lines to produce outcrossed offspring. There was additional variation in outcrossing rate among the four GM-lines, presumably due to the different transgene insertion events. Among the pollen donors, the Frisal GM line expressing a chitinase transgene caused more outcrossing than the GM line expressing both a chitinase and a glucanase transgene. In the second experiment, outcrossing after cross-pollination declined from 0.7–0.03% over the test distances of 0.5–2.5 m. Our results suggest that pollen-mediated gene flow between GM and non-GM wheat might only be a concern if it occurs within fields, e.g. due to seed contamination. Methodologically our study demonstrates that outcrossing rates between transgenic and other lines within crops can be assessed using a phytometer approach and that gene-flow distances can be efficiently estimated with population-level PCR analyses. PMID:22216349

Does Wheat Genetically Modified for Disease Resistance Affect Root-Colonizing Pseudomonads and Arbuscular Mycorrhizal Fungi?

PubMed Central

Foetzki, Andrea; Luginbühl, Carolin; Winzeler, Michael; Kneubühler, Yvan; Matasci, Caterina; Mascher-Frutschi, Fabio; Kalinina, Olena; Boller, Thomas; Keel, Christoph; Maurhofer, Monika

2013-01-01

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology. PMID:23372672

Does wheat genetically modified for disease resistance affect root-colonizing pseudomonads and arbuscular mycorrhizal fungi?

PubMed

Meyer, Joana Beatrice; Song-Wilson, Yi; Foetzki, Andrea; Luginbühl, Carolin; Winzeler, Michael; Kneubühler, Yvan; Matasci, Caterina; Mascher-Frutschi, Fabio; Kalinina, Olena; Boller, Thomas; Keel, Christoph; Maurhofer, Monika

2013-01-01

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

The media and genetically modified foods: evidence in support of social amplification of risk.

PubMed

Frewer, Lynn J; Miles, Susan; Marsh, Roy

2002-08-01

Empirical examinations of the "social amplification of risk" framework are rare, partly because of the difficulties in predicting when conditions likely to result in amplification effects will occur. This means that it is difficult to examine changes in risk perception that are contemporaneous with increases and/or decreases in social or media discussion of the risks associated with a particular risk event. However, the collection of attitude data before, during, and after the increased reporting of the risks of genetically modified food in the United Kingdom (spring 1999) has demonstrated that people's risk perceptions do increase and decrease in line with what might be expected upon examination of the amplification and attenuation mechanisms integral to the framework. Perceptions of benefit, however, appeared to be permanently depressed by negative reporting about genetically modified food. Trust in regulatory institutions with responsibility for protecting the public was not affected. It was concluded that the social amplification of risk framework is a useful framework for beginning to explain the potential impact on risk perceptions of a risk event, particularly if that risk event is presented to the public as a new hazard occurring in a crisis context.

Use of Mutant-Assisted Gene Identification and Characterization (MAGIC) to identify novel genetic loci that modify the maize hypersensitive response

USDA-ARS?s Scientific Manuscript database

The partially-dominant, autoactive maize disease resistance gene Rp1-D21 causes hypersensitive response (HR) lesions to form spontaneously on the leaves and stem in the absence of pathogen recognition. The maize nested association mapping (NAM) population consists of 25 200-line subpopulations each...

Assembly line plants take root

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

Comis, D.; Wood, M.

This paper discussed tissue-culture propagation of sugarcane, apple trees, peach trees, citrus, orchids, data palms, and carrots. Tissue-culture propagation is a term used for a variety of techniques used to grow or genetically modify, preserve, or study plant parts in laboratories, from tissue or even a single cell. The author examined the benefits and commercial applications of this propagation process.

Peach (Prunus persica L.).

PubMed

Sabbadini, Silvia; Pandolfini, Tiziana; Girolomini, Luca; Molesini, Barbara; Navacchi, Oriano

2015-01-01

Until now, the application of genetic transformation techniques in peach has been limited by the difficulties in developing efficient regeneration and transformation protocols. Here we describe an efficient regeneration protocol for the commercial micropropagation of GF677 rootstock (Prunus persica × Prunus amygdalus). The method is based on the production, via organogenesis, of meristematic bulk tissues characterized by a high competence for shoot regeneration. This protocol has also been used to obtain GF677 plants genetically engineered with an empty hairpin cassette (hereafter indicated as hp-pBin19), through Agrobacterium tumefaciens-mediated transformation. After 7-8 months of selection on media containing kanamycin, we obtained two genetically modified GF677 lines. PCR and Southern blot analyses were performed to confirm the genetic status.

Influence of simulated weightlessness on the rate of anomalies of the flour beetle Tribolium confusum.

PubMed

Briegleb, W; Neubert, J; Schatz, A; Sinapius, F

1975-01-01

Experiments with Tribolium confusum showed that the morphological characteristics of the beetles are modified by simulated weightlessness (fast running clinostat). Because of possible side effects due to differences in fertility of inbred lines, the first experiments were made with a genetically heterogeneous stock. Thereafter experiments were confirmed with inbred beetles. For both stocks a rise of mainly wing anomalies resulted from rotation of whole cultures of beetles within horizontal tubes. The extent to which these anomalies are teratogenetic or genetic has not yet been analysed in detail.

Guiding plant virus particles to integrin-displaying cells

NASA Astrophysics Data System (ADS)

Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

2012-05-01

Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors. Electronic supplementary information (ESI) available: Synthetic procedures and compound characterization data; assay procedures; additional confocal micrographs at different time points. See DOI: 10.1039/c2nr30571b

Medical hypothesis: bifunctional genetic-hormonal pathways to breast cancer.

PubMed

Davis, D L; Telang, N T; Osborne, M P; Bradlow, H L

1997-04-01

As inherited germ line mutations, such as loss of BRCA1 or AT, account for less than 5% of all breast cancer, most cases involve acquired somatic perturbations. Cumulative lifetime exposure to bioavailable estradiol links most known risk factors (except radiation) for breast cancer. Based on a series of recent experimental and epidemiologic findings, we hypothesize that the multistep process of breast carcinogenesis results from exposure to endogenous or exogenous hormones, including phytoestrogens that directly or indirectly alter estrogen metabolism. Xenohormones are defined as xenobiotic materials that modify hormonal production; they can work bifunctionally, through genetic or hormonal paths, depending on the periods and extent of exposure. As for genetic paths, xenohormones can modify DNA structure or function. As for hormonal paths, two distinct mechanisms can influence the potential for aberrant cell growth: compounds can directly bind with endogenous hormone or growth factor receptors affecting cell proliferation or compounds can modify breast cell proliferation altering the formation of hormone metabolites that influence epithelial-stromal interaction and growth regulation. Beneficial xenohormones, such as indole-3-carbinol, genistein, and other bioflavonoids, may reduce aberrant breast cell proliferation, and influence the rate of DNA repair or apoptosis and thereby influence the genetic or hormonal microenvironments. Upon validation with appropriate in vitro and in vivo studies, biologic markers of the risk for breast cancer, such as hormone metabolites, total bioavailable estradiol, and free radical generators can enhance cancer detection and prevention.

A modified mass selection scheme for creating winter-hardy faba bean (Vicia faba L.) lines with a broad genetic base

USDA-ARS?s Scientific Manuscript database

Winter-hardy faba bean (Vicia faba L.) from northern Europe is represented by a rather narrow gene pool. Limited selection gains for overwintering beyond a maximum of -25°C have restricted the adoption of this crop. Therefore, the faba bean collection maintained by the USDA-ARS National Plant Germpl...

Characterization of Soybean Genetically Modified for Drought Tolerance in Field Conditions

PubMed Central

Fuganti-Pagliarini, Renata; Ferreira, Leonardo C.; Rodrigues, Fabiana A.; Molinari, Hugo B. C.; Marin, Silvana R. R.; Molinari, Mayla D. C.; Marcolino-Gomes, Juliana; Mertz-Henning, Liliane M.; Farias, José R. B.; de Oliveira, Maria C. N.; Neumaier, Norman; Kanamori, Norihito; Fujita, Yasunari; Mizoi, Junya; Nakashima, Kazuo; Yamaguchi-Shinozaki, Kazuko; Nepomuceno, Alexandre L.

2017-01-01

Drought is one of the most stressful environmental factor causing yield and economic losses in many soybean-producing regions. In the last decades, transcription factors (TFs) are being used to develop genetically modified plants more tolerant to abiotic stresses. Dehydration responsive element binding (DREB) and ABA-responsive element-binding (AREB) TFs were introduced in soybean showing improved drought tolerance, under controlled conditions. However, these results may not be representative of the way in which plants behave over the entire season in the real field situation. Thus, the objectives of this study were to analyze agronomical traits and physiological parameters of AtDREB1A (1Ab58), AtDREB2CA (1Bb2193), and AtAREB1 (1Ea2939) GM lines under irrigated (IRR) and non-irrigated (NIRR) conditions in a field experiment, over two crop seasons and quantify transgene and drought-responsive genes expression. Results from season 2013/2014 revealed that line 1Ea2939 showed higher intrinsic water use and leaf area index. Lines 1Ab58 and 1Bb2193 showed a similar behavior to wild-type plants in relation to chlorophyll content. Oil and protein contents were not affected in transgenic lines in NIRR conditions. Lodging, due to plentiful rain, impaired yield from the 1Ea2939 line in IRR conditions. qPCR results confirmed the expression of the inserted TFs and drought-responsive endogenous genes. No differences were identified in the field experiment performed in crop season 2014/2015, probably due to the optimum rainfall volume during the cycle. These field screenings showed promising results for drought tolerance. However, additional studies are needed in further crop seasons and other sites to better characterize how these plants may outperform the WT under field water deficit. PMID:28443101

Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background.

PubMed

Carstea, Ana Claudia; Pirity, Melinda K; Dinnyes, Andras

2009-12-31

In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.

Monitoring of Bt11 and Bt176 genetically modified maize in food sold commercially in Brazil from 2005 to 2007.

PubMed

Dinon, Andréia Z; Bosco, Kenia T; Arisi, Ana Carolina M

2010-07-01

The first genetically modified (GM) maize lines were approved for trading in Brazil after December 2007 and they were T25, MON810, Bt11, NK603 and GA21. The polymerase chain reaction (PCR) method was employed to monitor the presence of Bt11 and nested PCR was used to detect the presence of Bt176 in 81 maize-derived products (maize flour, corn meal, maize flour flakes and polenta) that were sold in Brazilian market from 2005 to 2007, before the release of GM maize in Brazil. The PCR detection limit for Bt11 was 10 g kg(-1) and for nested PCR of Bt176 it was 1 g kg(-1). All Brazilian samples analyzed showed no positive signal for these GM maize events. Bt11 and Bt176 GM maize lines were not detected by specific PCR in 81 maize-derived food samples sold in Brazil from 2005 to 2007, before the commercial release of GM maize in Brazil. These Brazilian food industries were in compliance with the rules stipulated by the current legislation with respect to consumer requirements about GMO labeling.

Detection of Genetically Modified Food: Has Your Food Been Genetically Modified?

ERIC Educational Resources Information Center

Brandner, Diana L.

2002-01-01

Explains the benefits and risks of genetically-modified foods and describes methods for genetically modifying food. Presents a laboratory experiment using a polymerase chain reaction (PCR) test to detect foreign DNA in genetically-modified food. (Contains 18 references.) (YDS)

A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes.

PubMed

Kim, Young-Il; Ryu, Taewoo; Lee, Judong; Heo, Young-Shin; Ahnn, Joohong; Lee, Seung-Jae; Yoo, OokJoon

2010-01-25

Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines). We screened approximately 15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy.

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

PubMed

Dal Bosco, Daniela; Sinski, Iraci; Ritschel, Patrícia S; Camargo, Umberto A; Fajardo, Thor V M; Harakava, Ricardo; Quecini, Vera

2018-06-06

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Neuropeptide S alters anxiety, but not depression-like behaviour in Flinders Sensitive Line rats: a genetic animal model of depression.

PubMed

Wegener, Gregers; Finger, Beate C; Elfving, Betina; Keller, Kirsten; Liebenberg, Nico; Fischer, Christina W; Singewald, Nicolas; Slattery, David A; Neumann, Inga D; Mathé, Aleksander A

2012-04-01

Neuropeptide S (NPS) and its receptor (NPSR) have been implicated in the mediation of anxiolytic-like behaviour in rodents. However, little knowledge is available regarding the NPS system in depression-related behaviours, and whether NPS also exerts anxiolytic effects in an animal model of psychopathology. Therefore, the aim of this work was to characterize the effects of NPS on depression- and anxiety-related parameters, using male and female rats in a well-validated animal model of depression: the Flinders Sensitive Line (FSL), their controls, the Flinders Resistant Line (FRL), and Sprague-Dawley (SD) rats. We found that FSL showed greater immobility in the forced swim test (FST) than FRL, confirming their phenotype. However, NPS did not affect depression-related behaviour in any rat line. No significant differences in baseline anxiety levels between the FSL and FRL strains were observed, but FSL and FRL rats displayed less anxiety-like behaviour compared to SD rats. NPS decreased anxiety-like behaviour on the elevated plus-maze in all strains. The expression of the NPSR in the amygdala, periventricular hypothalamic nucleus, and hippocampus was equal in all male strains, although a trend towards reduced expression within the amygdala was observed in FSL rats compared to SD rats. In conclusion, NPS had a marked anxiolytic effect in FSL, FRL and SD rats, but did not modify the depression-related behaviour in any strain, in spite of the significant differences in innate level between the strains. These findings suggest that NPS specifically modifies anxiety behaviour but cannot overcome/reverse a genetically mediated depression phenotype.

Field trial of insect-resistant and herbicide-tolerant genetically modified cotton (Gossypium hirsutum L.) for environmental risk assessment in Japan.

PubMed

Asanuma, Yoko; Gondo, Takahiro; Ishigaki, Genki; Inoue, Koichi; Zaita, Norihiro; Muguerza, Melody; Akashi, Ryo

2017-04-03

Japan imports cottonseed mainly from Australia and the USA where more than 96% of all cotton varieties grown are genetically modified (GM). GM crops undergo an environmental risk assessment (ERA) under the Law Concerning the Conservation and Sustainable Use of Biological Diversity before import into Japan. Potential adverse effects on biodiversity are comprehensively assessed based on competitiveness, production of harmful substances and outcrossing ability. Even though imported cottonseed is intended for food and feed uses and not for cultivation, the potential risks from seed spillage during transport must be evaluated. In most cases, the ERA requires data collected from in-country field trials to demonstrate how the GM crop behaves in Japan's environment. Confined field trials in Japan were conducted for the ERA of Lepidoptera-resistant and glufosinate-tolerant GM cotton (Gossypium hirsutum L.) lines GHB119 and T304-40. These lines were compared with conventional varieties for growth habit, morphological characteristics, seed dormancy, and allelopathic activity associated with competitiveness and production of harmful substances. Outcrossing ability was not a concern due to the absence of sexually compatible wild relatives in Japan. Although slight statistical differences were observed between the GM line and its conventional comparator for some morphological characteristics, transgenes or transformation were not considered to be responsible for these differences. The trial demonstrated that competitiveness and production of harmful substances by these GM cotton lines were equivalent to conventional cotton varieties that have a long history of safe use, and no potential adverse effects to biosafety in Japan were observed.

Field trial of insect-resistant and herbicide-tolerant genetically modified cotton (Gossypium hirsutum L.) for environmental risk assessment in Japan

PubMed Central

Asanuma, Yoko; Gondo, Takahiro; Ishigaki, Genki; Inoue, Koichi; Zaita, Norihiro; Muguerza, Melody; Akashi, Ryo

2017-01-01

ABSTRACT Japan imports cottonseed mainly from Australia and the USA where more than 96% of all cotton varieties grown are genetically modified (GM). GM crops undergo an environmental risk assessment (ERA) under the Law Concerning the Conservation and Sustainable Use of Biological Diversity before import into Japan. Potential adverse effects on biodiversity are comprehensively assessed based on competitiveness, production of harmful substances and outcrossing ability. Even though imported cottonseed is intended for food and feed uses and not for cultivation, the potential risks from seed spillage during transport must be evaluated. In most cases, the ERA requires data collected from in-country field trials to demonstrate how the GM crop behaves in Japan's environment. Confined field trials in Japan were conducted for the ERA of Lepidoptera-resistant and glufosinate-tolerant GM cotton (Gossypium hirsutum L.) lines GHB119 and T304-40. These lines were compared with conventional varieties for growth habit, morphological characteristics, seed dormancy, and allelopathic activity associated with competitiveness and production of harmful substances. Outcrossing ability was not a concern due to the absence of sexually compatible wild relatives in Japan. Although slight statistical differences were observed between the GM line and its conventional comparator for some morphological characteristics, transgenes or transformation were not considered to be responsible for these differences. The trial demonstrated that competitiveness and production of harmful substances by these GM cotton lines were equivalent to conventional cotton varieties that have a long history of safe use, and no potential adverse effects to biosafety in Japan were observed. PMID:28510512

Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen.

PubMed

Boase, Murray R; Lewis, David H; Davies, Kevin M; Marshall, Gayle B; Patel, Deepa; Schwinn, Kathy E; Deroles, Simon C

2010-06-13

Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments. A full-length cDNA putatively identified as a F3'5'H (CpF3'5'H) was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the CpF3'5'H encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via Agrobacterium tumefaciens with an antisense CpF3'5'H construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous F3'5'H transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C) in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated. To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results illustrate the complexity involved in modifying a biosynthetic pathway with multiple branch points to different end products and provides important information for future flower colour modification experiments in cyclamen.

Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen

PubMed Central

2010-01-01

Background Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments. Results A full-length cDNA putatively identified as a F3'5'H (CpF3'5'H) was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the CpF3'5'H encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via Agrobacterium tumefaciens with an antisense CpF3'5'H construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous F3'5'H transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C) in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated. Conclusion To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results illustrate the complexity involved in modifying a biosynthetic pathway with multiple branch points to different end products and provides important information for future flower colour modification experiments in cyclamen. PMID:20540805

Growth control of genetically modified cells using an antibody/c-Kit chimera.

PubMed

Kaneko, Etsuji; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

2012-05-01

Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Endpoint visual detection of three genetically modified rice events by loop-mediated isothermal amplification.

PubMed

Chen, Xiaoyun; Wang, Xiaofu; Jin, Nuo; Zhou, Yu; Huang, Sainan; Miao, Qingmei; Zhu, Qing; Xu, Junfeng

2012-11-07

Genetically modified (GM) rice KMD1, TT51-1, and KF6 are three of the most well known transgenic Bt rice lines in China. A rapid and sensitive molecular assay for risk assessment of GM rice is needed. Polymerase chain reaction (PCR), currently the most common method for detecting genetically modified organisms, requires temperature cycling and relatively complex procedures. Here we developed a visual and rapid loop-mediated isothermal amplification (LAMP) method to amplify three GM rice event-specific junction sequences. Target DNA was amplified and visualized by two indicators (SYBR green or hydroxy naphthol blue [HNB]) within 60 min at an isothermal temperature of 63 °C. Different kinds of plants were selected to ensure the specificity of detection and the results of the non-target samples were negative, indicating that the primer sets for the three GM rice varieties had good levels of specificity. The sensitivity of LAMP, with detection limits at low concentration levels (0.01%−0.005% GM), was 10- to 100-fold greater than that of conventional PCR. Additionally, the LAMP assay coupled with an indicator (SYBR green or HNB) facilitated analysis. These findings revealed that the rapid detection method was suitable as a simple field-based test to determine the status of GM crops.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

PubMed

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-08-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. © 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

PubMed Central

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J.; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-01-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%–5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. PMID:24989021

Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen

PubMed Central

Adissu, Hibret A.; Estabel, Jeanne; Sunter, David; Tuck, Elizabeth; Hooks, Yvette; Carragher, Damian M.; Clarke, Kay; Karp, Natasha A.; Project, Sanger Mouse Genetics; Newbigging, Susan; Jones, Nora; Morikawa, Lily; White, Jacqueline K.; McKerlie, Colin

2014-01-01

The Mouse Genetics Project (MGP) at the Wellcome Trust Sanger Institute aims to generate and phenotype over 800 genetically modified mouse lines over the next 5 years to gain a better understanding of mammalian gene function and provide an invaluable resource to the scientific community for follow-up studies. Phenotyping includes the generation of a standardized biobank of paraffin-embedded tissues for each mouse line, but histopathology is not routinely performed. In collaboration with the Pathology Core of the Centre for Modeling Human Disease (CMHD) we report the utility of histopathology in a high-throughput primary phenotyping screen. Histopathology was assessed in an unbiased selection of 50 mouse lines with (n=30) or without (n=20) clinical phenotypes detected by the standard MGP primary phenotyping screen. Our findings revealed that histopathology added correlating morphological data in 19 of 30 lines (63.3%) in which the primary screen detected a phenotype. In addition, seven of the 50 lines (14%) presented significant histopathology findings that were not associated with or predicted by the standard primary screen. Three of these seven lines had no clinical phenotype detected by the standard primary screen. Incidental and strain-associated background lesions were present in all mutant lines with good concordance to wild-type controls. These findings demonstrate the complementary and unique contribution of histopathology to high-throughput primary phenotyping of mutant mice. PMID:24652767

Space Pomology: Dwarf Plums for Fresh Food Production

NASA Technical Reports Server (NTRS)

Spencer, LaShelle; Graham, Thomas; Stutte, Gary; Massa, Gioia; Mickens, Matthew; Wheeler, Raymond

2017-01-01

Recently, USDA ARS researchers genetically modified plums for rapid breeding work and noticed the plants could flower and develop fruit rapidly on relatively small plants. We have tested several of these genetically modified (GM) plums in plant chambers to assess their potential as a space crop. We have been able to clone these genetic lines using cuttings that are rooted using growth regulating compounds. Results showed that the GM plums indeed flower and fruit on small plants in controlled environments similar to what might be used in space, but they require cross-pollination with pollen from a standard plum. Analysis of stomatal conductance and leaf transpiration showed that water use went up in the light period, as expected, and but that GM types typically showed higher conductance than a standard plum. Analysis of tissue showed that fruit could be a good source of potassium and phenolic compounds, which could be beneficial as a bone loss countermeasure (Smith et al., 2014). These findings are all promising for using dwarf GM plums as a supplemental food for space, but further horticultural testing is needed before they are ready.

Real-time polymerase chain reaction detection of cauliflower mosaic virus to complement the 35S screening assay for genetically modified organisms.

PubMed

Cankar, Katarina; Ravnikar, Maja; Zel, Jana; Gruden, Kristina; Toplak, Natasa

2005-01-01

Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.

Aquatic degradation of Cry1Ab protein and decomposition dynamics of transgenic corn leaves under controlled conditions.

PubMed

Böttger, Rita; Schaller, Jörg; Lintow, Sven; Gert Dudel, E

2015-03-01

The increasing cultivation of genetically modified corn plants (Zea mays) during the last decades is suggested as a potential risk to the environment. One of these genetically modified variety expressed the insecticidal Cry1Ab protein originating from Bacillus thuringiensis (Bt), resulting in resistance against Ostrinia nubilalis, the European corn borer. Transgenic litter material is extensively studied regarding the decomposition in soils. However, only a few field studies analyzed the fate of the Cry1Ab protein and the impact of green and senescent leaf litter from corn on the decomposition rate and related ecosystem functions in aquatic environments. Consequently, a microbial litter decomposition experiment was conducted under controlled semi-natural conditions in batch culture using two maize varieties: one variety with Cry1Ab and another one with the appertaining Iso-line as control treatment. The results showed no significant differences between the treatment with Cry1Ab and the Iso-line regarding loss of total mass in dry weight of 43% for Iso-line and 45% for Bt-corn litter, lignin content increased to 137.5% (Iso-line) and 115.7% (Bt-corn), and phenol loss decreased by 53.6% (Iso-line), 62.2% (Bt-corn) during three weeks of the experiment. At the end of the experiment Cry1Ab protein was still detected with 6% of the initial concentration. A slightly but significant lower cellulose content was found for the Cry1Ab treatment compared to the Iso-line litter at the end of the experiment. The significant higher total protein (25%) and nitrogen (25%) content in Bt corn, most likely due to the additionally expression of the transgenic protein, may increase the microbial cellulose degradation and decrease microbial lignin degradation. In conclusion a relevant year by year input of protein and therefore nitrogen rich Bt corn litter into aquatic environments may affect the balanced nutrient turnover in aquatic ecosystems. Copyright © 2014 Elsevier Inc. All rights reserved.

Detection of feral GT73 transgenic oilseed rape (Brassica napus) along railway lines on entry routes to oilseed factories in Switzerland.

PubMed

Hecht, Mirco; Oehen, Bernadette; Schulze, Jürg; Brodmann, Peter; Bagutti, Claudia

2014-01-01

To obtain a reference status prior to cultivation of genetically modified oilseed rape (OSR, Brassica napus L.) in Switzerland, the occurrence of feral OSR was monitored along transportation routes and at processing sites. The focus was set on the detection of (transgenic) OSR along railway lines from the Swiss borders with Italy and France to the respective oilseed processing factories in Southern and Northern Switzerland (Ticino and region of Basel). A monitoring concept was developed to identify sites of largest risk of escape of genetically modified plants into the environment in Switzerland. Transport spillage of OSR seeds from railway goods cars particularly at risk hot spots such as switch yards and (un)loading points but also incidental and continuous spillage were considered. All OSR plants, including their hybridization partners which were collected at the respective monitoring sites were analyzed for the presence of transgenes by real-time PCR. On sampling lengths each of 4.2 and 5.7 km, respectively, 461 and 1,574 plants were sampled in Ticino and the region of Basel. OSR plants were found most frequently along the routes to the oilseed facilities, and in larger amounts on risk hot spots compared to sites of random sampling. At three locations in both monitored regions, transgenic B. napus line GT73 carrying the glyphosate resistance transgenes gox and CP4 epsps were detected (Ticino, 22 plants; in the region of Basel, 159).

Expression of modified tocopherol content and profile in sunflower tissues.

PubMed

Del Moral, Lidia; Fernández-Martínez, José M; Pérez-Vich, Begoña; Velasco, Leonardo

2012-01-30

Alpha-tocopherol is the predominant tocopherol form in sunflower seeds. Sunflower lines that accumulate increased levels of beta-, gamma- and delta-tocopherol in seeds as well as lines with reduced and increased total seed tocopherol content have been developed. The objective of this research was to evaluate whether the modified tocopherol levels are expressed in plant tissues other than seeds. Lines with increased levels of beta-, gamma- and delta-tocopherol in seeds also possessed increased levels of these tocopherols in leaves, roots and pollen. Correlation coefficients for the proportion of individual tocopherols in different plant tissues were significantly positive in all cases, ranging from 0.68 to 0.97. A line with reduced tocopherol content in seeds also showed reduced content in roots and pollen. Genetic modifications producing altered seed tocopherol profiles in sunflower are also expressed in leaves, roots and pollen. Reduced total seed tocopherol content is mainly expressed at the root and pollen level. The expression of tocopherol mutations in other plant tissues will enable further studies on the physiological role of tocopherols and could be of interest for early selection for these traits in breeding programmes. Copyright © 2011 Society of Chemical Industry.

Genetic mouse models of brain ageing and Alzheimer's disease.

PubMed

Bilkei-Gorzo, Andras

2014-05-01

Progression of brain ageing is influenced by a complex interaction of genetic and environmental factors. Analysis of genetically modified animals with uniform genetic backgrounds in a standardised, controlled environment enables the dissection of critical determinants of brain ageing on a molecular level. Human and animal studies suggest that increased load of damaged macromolecules, efficacy of DNA maintenance, mitochondrial activity, and cellular stress defences are critical determinants of brain ageing. Surprisingly, mouse lines with genetic impairment of anti-oxidative capacity generally did not show enhanced cognitive ageing but rather an increased sensitivity to oxidative challenge. Mouse lines with impaired mitochondrial activity had critically short life spans or severe and rapidly progressing neurodegeneration. Strains with impaired clearance in damaged macromolecules or defects in the regulation of cellular stress defences showed alterations in the onset and progression of cognitive decline. Importantly, reduced insulin/insulin-like growth factor signalling generally increased life span but impaired cognitive functions revealing a complex interaction between ageing of the brain and of the body. Brain ageing is accompanied by an increased risk of developing Alzheimer's disease. Transgenic mouse models expressing high levels of mutant human amyloid precursor protein showed a number of symptoms and pathophysiological processes typical for early phase of Alzheimer's disease. Generally, therapeutic strategies effective against Alzheimer's disease in humans were also active in the Tg2576, APP23, APP/PS1 and 5xFAD lines, but a large number of false positive findings were also reported. The 3xtg AD model likely has the highest face and construct validity but further studies are needed. Copyright © 2013 Elsevier Inc. All rights reserved.

Phase I Study of Cellular Immunotherapy for Recurrent/Refractory Malignant Glioma Using Intratumoral Infusions of GRm13Z40-2, An Allogeneic CD8+ Cytolitic T-Cell Line Genetically Modified to Express the IL 13-Zetakine and HyTK and to be Resistant to Glucocorticoids, in Combination With Interleukin-2

ClinicalTrials.gov

2015-06-03

Anaplastic Astrocytoma; Anaplastic Ependymoma; Anaplastic Meningioma; Anaplastic Oligodendroglioma; Brain Stem Glioma; Ependymoblastoma; Giant Cell Glioblastoma; Glioblastoma; Gliosarcoma; Grade III Meningioma; Meningeal Hemangiopericytoma; Mixed Glioma; Pineal Gland Astrocytoma; Brain Tumor

Recent Honey Bee Colony Declines

DTIC Science & Technology

2007-06-20

climate and temperature changes,38 the effects of feed supplements that are produced from transgenic or genetically modified crops, such as high - fructose ... corn syrup ,39 and also the effects of cell phone transmissions and radiation from power lines that may be interfering with a bee’s navigational...podcasts.psu.edu/taxonomy/term/62]. Staple crops such as wheat, corn , and rice do not rely on insect pollination and are mostly wind pollinated

Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines.

PubMed

Košir, Alexandra Bogožalec; Spilsberg, Bjørn; Holst-Jensen, Arne; Žel, Jana; Dobnik, David

2017-08-17

Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.

[Continuously perfused cultivation of genetically-engineered CHO cells producing prothrombin in a modified Super-Spinner].

PubMed

Chen, Z L; Iding, K; Lütkemeyer, D; Lehmann, J

2001-01-01

A Super-Spinner was Modified by mounting a stainless steel filter(pore size 75 microns) to the impeller shaft to retain cells while fresh nutrient is perfused. Using Macroporous microcarrier Cytopore 1, continuously perfused cultivation of a recombinant CHO cell line, CHO2DS producing prothrombin was performed with the perfusion of a protein-free medium DF6S. The cell retention rate was more than 90% during the 24 days continuously perfused cultivation. The viable cell density of CHO2DS and prothrombin concentration reached 4.62 x 10(6)(cells.m/L) and 11.3(mg/L) respectively after 9 days culture.

Attitudes to genetically modified food over time: How trust in organizations and the media cycle predict support.

PubMed

Marques, Mathew D; Critchley, Christine R; Walshe, Jarrod

2015-07-01

This research examined public opinion toward genetically modified plants and animals for food, and how trust in organizations and media coverage explained attitudes toward these organisms. Nationally representative samples (N=8821) over 10 years showed Australians were less positive toward genetically modified animals compared to genetically modified plants for food, especially in years where media coverage was high. Structural equation modeling found that positive attitudes toward different genetically modified organisms for food were significantly associated with higher trust in scientists and regulators (e.g. governments), and with lower trust in watchdogs (e.g. environmental movement). Public trust in scientists and watchdogs was a stronger predictor of attitudes toward the use of genetically modified plants for food than animals, but only when media coverage was low. Results are discussed regarding the moral acceptability of genetically modified organisms for food, the media's role in shaping public opinion, and the role public trust in organizations has on attitudes toward genetically modified organisms. © The Author(s) 2014.

Farmers prevailing perception profiles regarding GM crops: A classification proposal.

PubMed

Almeida, Carla; Massarani, Luisa

2018-04-01

Genetically modified organisms have been at the centre of a major public controversy, involving different interests and actors. While much attention has been devoted to consumer views on genetically modified food, there have been few attempts to understand the perceptions of genetically modified technology among farmers. By investigating perceptions of genetically modified organisms among Brazilian farmers, we intend to contribute towards filling this gap and thereby add the views of this stakeholder group to the genetically modified debate. A comparative analysis of our data and data from other studies indicate there is a complex variety of views on genetically modified organisms among farmers. Despite this diversity, we found variations in such views occur within limited parameters, concerned principally with expectations or concrete experiences regarding the advantages of genetically modified crops, perceptions of risks associated with them, and ethical questions they raise. We then propose a classification of prevailing profiles to represent the spectrum of perceptions of genetically modified organisms among farmers.

Identifying novel genetic determinants of hemostatic balance.

PubMed

Ginsburg, D

2005-08-01

Incomplete penetrance and variable expressivity confound the diagnosis and therapy of most inherited thrombotic and hemorrhagic disorders. For many of these diseases, some or most of this variability is determined by genetic modifiers distinct from the primary disease gene itself. Clues toward identifying such modifier genes may come from studying rare Mendelian disorders of hemostasis. Examples include identification of the cause of combined factor V and VIII deficiency as mutations in the ER Golgi intermediate compartment proteins LMAN1 and MCFD2. These proteins form a cargo receptor that facilitates the transport of factors V and VIII, and presumably other proteins, from the ER to the Golgi. A similar positional cloning approach identified ADAMTS-13 as the gene responsible for familial TTP. Along with the work of many other groups, these findings identified VWF proteolysis by ADAMTS-13 as a key regulatory pathway for hemostasis. Recent advances in mouse genetics also provide powerful tools for the identification of novel genes contributing to hemostatic balance. Genetic studies of inbred mouse lines with unusually high and unusually low plasma VWF levels identified polymorphic variation in the expression of a glycosyltransferase gene, Galgt2, as an important determinant of plasma VWF levels in the mouse. Ongoing studies in mice genetically engineered to carry the factor V Leiden mutation may similarly identify novel genes contributing to thrombosis risk in humans.

Methods for genetic modification of megakaryocytes and platelets.

PubMed

Pendaries, Caroline; Watson, Stephen P; Spalton, Jennifer C

2007-09-01

During recent decades there have been major advances in the fields of thrombosis and haemostasis, in part through development of powerful molecular and genetic technologies. Nevertheless, genetic modification of megakaryocytes and generation of mutant platelets in vitro remains a highly specialized area of research. Developments are hampered by the low frequency of megakaryocytes and their progenitors, a poor efficiency of transfection and a lack of understanding with regard to the mechanism by which megakaryocytes release platelets. Current methods used in the generation of genetically modified megakaryocytes and platelets include mutant mouse models, cell line studies and use of viruses to transform primary megakaryocytes or haematopoietic precursor cells. This review summarizes the advantages, limitations and technical challenges of such methods, with a particular focus on recent successes and advances in this rapidly progressing field including the potential for use in gene therapy for treatment of patients with platelet disorders.

Generation of an immortalized mesenchymal stem cell line producing a secreted biosensor protein for glucose monitoring

PubMed Central

Weisman, Itamar; Romano, Jacob; Ivics, Zoltán; Izsvák, Zsuzsanna; Barkai, Uriel

2017-01-01

Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device. PMID:28949988

Generation of an immortalized mesenchymal stem cell line producing a secreted biosensor protein for glucose monitoring.

PubMed

Siska, Evangelia K; Weisman, Itamar; Romano, Jacob; Ivics, Zoltán; Izsvák, Zsuzsanna; Barkai, Uriel; Petrakis, Spyros; Koliakos, George

2017-01-01

Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.

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.

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

PubMed Central

Ronsyn, Mark W; Daans, Jasmijn; Spaepen, Gie; Chatterjee, Shyama; Vermeulen, Katrien; D'Haese, Patrick; Van Tendeloo, Viggo FI; Van Marck, Eric; Ysebaert, Dirk; Berneman, Zwi N; Jorens, Philippe G; Ponsaerts, Peter

2007-01-01

Background Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. Results First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. Conclusion In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo. PMID:18078525

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord.

PubMed

Ronsyn, Mark W; Daans, Jasmijn; Spaepen, Gie; Chatterjee, Shyama; Vermeulen, Katrien; D'Haese, Patrick; Van Tendeloo, Viggo Fi; Van Marck, Eric; Ysebaert, Dirk; Berneman, Zwi N; Jorens, Philippe G; Ponsaerts, Peter

2007-12-14

Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo.

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (Populus tremula L.).

PubMed

Hoenicka, Hans; Lehnhardt, Denise; Nunna, Suneetha; Reinhardt, Richard; Jeltsch, Albert; Briones, Valentina; Fladung, Matthias

2016-02-01

Differentiation level but not transgene copy number influenced activation of a gene containment system in poplar. Heat treatments promoted CRE gene body methylation. The flower-specific transgene deletion was confirmed. Gene flow between genetic modified trees and their wild relatives is still motive of concern. Therefore, approaches for gene containment are required. In this study, we designed a novel strategy for achieving an inducible and flower-specific transgene removal from poplar trees but still expressing the transgene in the plant body. Hence, pollen carrying transgenes could be used for breeding purposes under controlled conditions in a first phase, and in the second phase genetic modified poplars developing transgene-free pollen grains could be released. This approach is based on the recombination systems CRE/loxP and FLP/frt. Both gene constructs contained a heat-inducible CRE/loxP-based spacer sequence for in vivo assembling of the flower-specific FLP/frt system. This allowed inducible activation of gene containment. The FLP/frt system was under the regulation of a flower-specific promoter, either CGPDHC or PTD. Our results confirmed complete CRE/loxP-based in vivo assembling of the flower-specific transgene excision system after heat treatment in all cells for up to 30 % of regenerants derived from undifferentiated tissue cultures. Degradation of HSP::CRE/loxP spacer after recombination but also persistence as extrachromosomal DNA circles were detected in sub-lines obtained after heat treatments. Furthermore, heat treatment promoted methylation of the CRE gene body. A lower methylation level was detected at CpG sites in transgenic sub-lines showing complete CRE/loxP recombination and persistence of CRE/loxP spacer, compared to sub-lines with incomplete recombination. However, our results suggest that low methylation might be necessary but not sufficient for recombination. The flower-specific FLP/frt-based transgene deletion was confirmed in 6.3 % of flowers.

[Consumer reaction to information on the labels of genetically modified food].

PubMed

Sebastian-Ponce, Miren Itxaso; Sanz-Valero, Javier; Wanden-Berghe, Carmina

2014-02-01

To analyze consumer opinion on genetically modified foods and the information included on the label. A systematic review of the scientific literature on genetically modified food labeling was conducted consulting bibliographic databases (Medline - via PubMed -, EMBASE, ISI-Web of knowledge, Cochrane Library Plus, FSTA, LILACS, CINAHL and AGRICOLA) using the descriptors "organisms, genetically modified" and "food labeling". The search covered the first available date, up to June 2012, selecting relevant articles written in English, Portuguese or Spanish. Forty articles were selected after applying the inclusion and exclusion criteria. All of them should have conducted a population-based intervention focused on consumer awareness of genetically modified foods and their need or not, to include this on the label. The consumers expressed a preference for non-genetically modified products, and added that they were prepared to pay more for this but, ultimately, the product bought was that with the best price, in a market which welcomes new technologies. In 18 of the articles, the population was in favor of obligatory labelling, and in six, in favor of this being voluntary; seven studies showed the consumer knew little about genetically modified food, and in three, the population underestimated the quantity they consumed. Price was an influencing factor in all cases. Label should be homogeneous and clarify the degree of tolerance of genetically modified products in humans, in comparison with those non-genetically modified. Label should also present the content or not of genetically modified products and how these commodities are produced and should be accompanied by the certifying entity and contact information. Consumers express their preference for non-genetically modified products and they even notice that they are willing to pay more for it, but eventually they buy the item with the best price, in a market that welcomes new technologies.

Genetic screens in human cells using the CRISPR-Cas9 system.

PubMed

Wang, Tim; Wei, Jenny J; Sabatini, David M; Lander, Eric S

2014-01-03

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Holst-Jensen, Arne; Žel, Jana

2015-08-18

Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain.

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.

A High-Throughput Arabidopsis Reverse Genetics System

PubMed Central

Sessions, Allen; Burke, Ellen; Presting, Gernot; Aux, George; McElver, John; Patton, David; Dietrich, Bob; Ho, Patrick; Bacwaden, Johana; Ko, Cynthia; Clarke, Joseph D.; Cotton, David; Bullis, David; Snell, Jennifer; Miguel, Trini; Hutchison, Don; Kimmerly, Bill; Mitzel, Theresa; Katagiri, Fumiaki; Glazebrook, Jane; Law, Marc; Goff, Stephen A.

2002-01-01

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org. PMID:12468722

Short-term effects of different genetically modified maize varieties on arthropod food web properties: an experimental field assessment.

PubMed

Szénási, Ágnes; Pálinkás, Zoltán; Zalai, Mihály; Schmitz, Oswald J; Balog, Adalbert

2014-06-17

There is concern that genetically modified (GM) plants may have adverse affects on the arthropod biodiversity comprising agricultural landscapes. The present study report on a two year field experimental test of whether four different genotypic lines, some are novel with no previous field tests, of GM maize hybrids alter the structure of arthropod food webs that they harbour, relative to non-GM maize (control) that is widely used in agriculture. The different GM genotypes produced either Bt toxins, conferred glyphosate tolerance or a combination of the two traits. Quantitative food web analysis, based on short-term assessment assigning a total of 243,896 arthropod individuals collected from the treatments to their positions in food webs, revealed that complex and stable food webs persisted in each maize treatment. Moreover, food web structure remained relatively unchanged by the GM-genotype. The results suggest that at least in short-term period these particular GM maize genotypes will not have adverse effects on arthropod biota of agricultural landscapes.

Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

PubMed

Qi, Jing; Dong, Zhen; Zhang, Yu-Xing

2015-12-01

The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti‑sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti‑sense RNA technology in the genetic improvement of pears and other fruit.

In vitro culture may be the major contributing factor for transgenic versus nontransgenic proteomic plant differences.

PubMed

Fonseca, Cátia; Planchon, Sébastien; Serra, Tânia; Chander, Subhash; Saibo, Nelson J M; Renaut, Jenny; Oliveira, M Margarida; Batista, Rita

2015-01-01

Identification of differences between genetically modified plants and their original counterparts plays a central role in risk assessment strategy. Our main goal was to better understand the relevance of transgene presence, genetic, and epigenetic changes induced by transgene insertion, and in vitro culture in putative unintended differences between a transgenic and its comparator. Thus, we have used multiplex fluorescence 2DE coupled with MS to characterize the proteome of three different rice lines (Oryza sativa L. ssp. japonica cv. Nipponbare): a control conventional line (C), an Agrobacterium-transformed transgenic line (Ta) and a negative segregant (NSb). We observed that Ta and NSb appeared identical (with only one spot differentially abundant--fold difference ≥ 1.5), contrasting with the control (49 spots with fold difference ≥ 1.5, in both Ta and NSb vs. control). Given that in vitro culture was the only event in common between Ta and NSb, we hypothesize that in vitro culture stress was the most relevant condition contributing for the observed proteomic differences. MS protein identification support our hypothesis, indicating that Ta and NSb lines adjusted their metabolic pathways and altered the abundance of several stress related proteins in order to cope with in vitro culture. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetically modified foods and allergy.

PubMed

Lee, T H; Ho, H K; Leung, T F

2017-06-01

2015 marked the 25th anniversary of the commercial use and availability of genetically modified crops. The area of planted biotech crops cultivated globally occupies a cumulative two billion hectares, equivalent to twice the land size of China or the United States. Foods derived from genetically modified plants are widely consumed in many countries and genetically modified soybean protein is extensively used in processed foods throughout the industrialised countries. Genetically modified food technology offers a possible solution to meet current and future challenges in food and medicine. Yet there is a strong undercurrent of anxiety that genetically modified foods are unsafe for human consumption, sometimes fuelled by criticisms based on little or no firm evidence. This has resulted in some countries turning away food destined for famine relief because of the perceived health risks of genetically modified foods. The major concerns include their possible allergenicity and toxicity despite the vigorous testing of genetically modified foods prior to marketing approval. It is imperative that scientists engage the public in a constructive evidence-based dialogue to address these concerns. At the same time, improved validated ways to test the safety of new foods should be developed. A post-launch strategy should be established routinely to allay concerns. Mandatory labelling of genetically modified ingredients should be adopted for the sake of transparency. Such ingredient listing and information facilitate tracing and recall if required.

Genetically engineered microbial biosensors for in situ monitoring of environmental pollution.

PubMed

Shin, Hae Ja

2011-02-01

Microbial biosensors are compact, portable, cost effective, and simple to use, making them seem eminently suitable for the in situ monitoring of environmental pollution. One promising approach for such applications is the fusion of reporter genes with regulatory genes that are dose-dependently responsive to the target chemicals or physiological signals. Their biosensor capabilities, such as target range and sensitivity, could be improved by modification of regulatory genes. Recent uses of such genetically engineered microbial biosensors include the development of portable biosensor kits and high-throughput cell arrays on chips, optic fibers, or other platforms for on-site and on-line monitoring of environmental pollution. This mini-review discusses recent advances in microbial biosensors and their future prospects, with a focus on the development and application of genetically modified microbial biosensors for in situ environmental monitoring.

Detection of Genetically Modified Sugarcane by Using Terahertz Spectroscopy and Chemometrics

NASA Astrophysics Data System (ADS)

Liu, J.; Xie, H.; Zha, B.; Ding, W.; Luo, J.; Hu, C.

2018-03-01

A methodology is proposed to identify genetically modified sugarcane from non-genetically modified sugarcane by using terahertz spectroscopy and chemometrics techniques, including linear discriminant analysis (LDA), support vector machine-discriminant analysis (SVM-DA), and partial least squares-discriminant analysis (PLS-DA). The classification rate of the above mentioned methods is compared, and different types of preprocessing are considered. According to the experimental results, the best option is PLS-DA, with an identification rate of 98%. The results indicated that THz spectroscopy and chemometrics techniques are a powerful tool to identify genetically modified and non-genetically modified sugarcane.

Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Bancelin, Stéphane; Lynch, Barbara; Bonod-Bidaud, Christelle; Ducourthial, Guillaume; Psilodimitrakopoulos, Sotiris; Dokládal, Petr; Allain, Jean-Marc; Schanne-Klein, Marie-Claire; Ruggiero, Florence

2015-12-01

Soft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues.

Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

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

Guseva, Daria; Hannover Medical School, Hannover; Rizvanov, Albert A.

2014-09-05

Highlights: • Gene and cell-based therapies comprise innovative aspects of regenerative medicine. • Genetically modified hUCB-MCs enhanced differentiation of cells in a mouse model of ALS. • Stem cells successfully transformed into micro-glial and endothelial lines in spinal cords. • Over-expressing oct4 and sox2 also induced production of neural marker PGP9.5. • Formation of new nerve cells, secreting trophic factors and neo-vascularisation could improve symptoms in ALS. - Abstract: Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignantmore » transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.« less

Expression of an (Engineered) 4,6-α-Glucanotransferase in Potato Results in Changes in Starch Characteristics

PubMed Central

Xu, Xuan; Dechesne, Annemarie; Visser, Richard G. F.; Trindade, Luisa M.

2016-01-01

Starch structure strongly influences starch physicochemical properties, determining the end uses of starch in various applications. To produce starches with novel structure and exploit the mechanism of starch granule formation, an (engineered) 4, 6-α-glucanotransferase (GTFB) from Lactobacillus reuteri 121 was introduced into two potato genetic backgrounds: amylose-containing line Kardal and amylose-free mutant amf. The resulting starches showed severe changes in granule morphology regardless of genetic backgrounds. Modified starches from amf background exhibited a significant increase in granule size and starch phosphate content relative to the control, while starches from Kardal background displayed a higher digestibility, but did not show changes in granule size and phosphate content. Transcriptome analysis revealed the existence of a mechanism to restore the regular packing of double helices in starch granules, which possibly resulted in the removal of novel glucose chains potentially introduced by the (engineered) GTFB. This amendment mechanics would also explain the difficulties to detect alterations in starch fine structure in the transgenic lines. PMID:27911907

Expression of an (Engineered) 4,6-α-Glucanotransferase in Potato Results in Changes in Starch Characteristics.

PubMed

Xu, Xuan; Dechesne, Annemarie; Visser, Richard G F; Trindade, Luisa M

2016-01-01

Starch structure strongly influences starch physicochemical properties, determining the end uses of starch in various applications. To produce starches with novel structure and exploit the mechanism of starch granule formation, an (engineered) 4, 6-α-glucanotransferase (GTFB) from Lactobacillus reuteri 121 was introduced into two potato genetic backgrounds: amylose-containing line Kardal and amylose-free mutant amf. The resulting starches showed severe changes in granule morphology regardless of genetic backgrounds. Modified starches from amf background exhibited a significant increase in granule size and starch phosphate content relative to the control, while starches from Kardal background displayed a higher digestibility, but did not show changes in granule size and phosphate content. Transcriptome analysis revealed the existence of a mechanism to restore the regular packing of double helices in starch granules, which possibly resulted in the removal of novel glucose chains potentially introduced by the (engineered) GTFB. This amendment mechanics would also explain the difficulties to detect alterations in starch fine structure in the transgenic lines.

Genetic Changes Accompanying the Domestication of Pisum sativum: Is there a Common Genetic Basis to the ‘Domestication Syndrome’ for Legumes?

PubMed Central

Weeden, Norman F.

2007-01-01

Background and Aims The changes that occur during the domestication of crops such as maize and common bean appear to be controlled by relatively few genes. This study investigates the genetic basis of domestication in pea (Pisum sativum) and compares the genes involved with those determined to be important in common bean domestication. Methods Quantitative trait loci and classical genetic analysis are used to investigate and identify the genes modified at three stages of the domestication process. Five recombinant inbred populations involving crosses between different lines representing different stages are examined. Key Results A minimum of 15 known genes, in addition to a relatively few major quantitative trait loci, are identified as being critical to the domestication process. These genes control traits such as pod dehiscence, seed dormancy, seed size and other seed quality characters, stem height, root mass, and harvest index. Several of the genes have pleiotropic effects that in species possessing a more rudimentary genetic characterization might have been interpreted as clusters of genes. Very little evidence for gene clustering was found in pea. When compared with common bean, pea has used a different set of genes to produce the same or similar phenotypic changes. Conclusions Similar to results for common bean, relatively few genes appear to have been modified during the domestication of pea. However, the genes involved are different, and there does not appear to be a common genetic basis to ‘domestication syndrome’ in the Fabaceae. PMID:17660515

Genetic Basis and Genetic Modifiers of β-Thalassemia and Sickle Cell Disease.

PubMed

Thein, Swee Lay

2017-01-01

β-thalassemia and sickle cell disease (SCD) are prototypical Mendelian single gene disorders, both caused by mutations affecting the adult β-globin gene. Despite the apparent genetic simplicity, both disorders display a remarkable spectrum of phenotypic severity and share two major genetic modifiers-α-globin genotype and innate ability to produce fetal hemoglobin (HbF, α 2 γ 2 ).This article provides an overview of the genetic basis for SCD and β-thalassemia, and genetic modifiers identified through phenotype correlation studies. Identification of the genetic variants modifying HbF production in combination with α-globin genotype provide some prediction of disease severity for β-thalassemia and SCD but generation of a personalized genetic risk score to inform prognosis and guide management requires a larger panel of genetic modifiers yet to be discovered.Nonetheless, genetic studies have been successful in characterizing some of the key variants and pathways involved in HbF regulation, providing new therapeutic targets for HbF reactivation.

Evaluation of the safety of a genetically modified DAS-444Ø6-6 soybean meal and hulls in a 90-day dietary toxicity study in rats.

PubMed

Papineni, Sabitha; Murray, Jennifer A; Ricardo, Ekmay; Dunville, Christina M; Sura, Radha Krishna; Thomas, Johnson

2017-11-01

A 90-day sub chronic toxicity study was conducted in rats to evaluate the safety of genetically modified DAS-444Ø6-6 soybeans expressing herbicide tolerant proteins when compared with its conventional comparators (non-transgenic near isoline control soybean and three commercially available non-transgenic line control soybeans). Rats were given diets formulated with either 10% or 20% w/w of soybean meal and 1% or 2% hulls of DAS-444Ø6-6 soybean with an equivalent amount of hulls from an isoline non-transgenic control soybean for at least 90 days. In addition, three separate 20% w/w non-transgenic commercially available soybean varieties were also given to groups of rats to serve as reference controls. Animals were evaluated by cage-side and hand-held detailed clinical observations, ophthalmic examinations, body weights/body weight gains, feed consumption, hematology, prothrombin time, urinalysis, clinical chemistry, selected organ weights, and gross and histopathologic examinations. Under the conditions of this study, the genetically modified DAS-444Ø6-6 diets did not cause any treatment-related effects in rats following 90 days of dietary administration as compared with rats fed diets with soybean of isoline control or commercial reference controls and are considered equivalent to the diets prepared from conventional comparators. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Genetic Contributors to Intergenerational CAG Repeat Instability in Huntington's Disease Knock-In Mice.

PubMed

Neto, João Luís; Lee, Jong-Min; Afridi, Ali; Gillis, Tammy; Guide, Jolene R; Dempsey, Stephani; Lager, Brenda; Alonso, Isabel; Wheeler, Vanessa C; Pinto, Ricardo Mouro

2017-02-01

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in HD families, partly underlying the genetic anticipation seen in this disorder. HD CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest HD knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identification of large repeat size jumps in the knock-in models. Distinct degrees of intergenerational instability were observed between knock-in mice of six background strains, indicating the occurrence of trans-acting genetic modifiers. We also found that lines harboring a neomycin resistance cassette upstream of Htt showed reduced expansion frequency, indicative of a contributing role for sequences in cis, with the expanded repeat as modifiers of intergenerational instability. These results provide a basis for further understanding of the mechanisms underlying intergenerational repeat instability. Copyright © 2017 by the Genetics Society of America.

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.

PubMed

Dehler, Carola E; Boudinot, Pierre; Martin, Samuel A M; Collet, Bertrand

2016-08-01

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.

Transient detection of beta-galactosidase activity in hematopoietic cells, following reinjection of retrovirally marked autologous blood progenitors in patients with breast or ovarian cancer receiving high-dose chemotherapy.

PubMed

Bagnis, Claude; Chabannon, Christian; Gravis, Gwenaelle; Imbert, Anne-Marie; Maroc, Christine; Bardin, Florence; Ladaique, Patrick; Viret, Frédéric; Genre, Dominique; Faucher, Catherine; Stoppa, Anne-Marie; Vey, Norbert; Blaise, Didier; Maraninchi, Dominique; Viens, Patrice; Mannoni, Patrice

2002-02-01

The aim of this report is to demonstrate the feasibility and safety of genetically modifying autologous human blood CD34(+) cells in vitro, with a retroviral vector that encodes a marker gene. The fate of genetically modified cells and their progeny was followed in vivo, after reinfusion in patients treated with high-dose chemotherapy for poor-prognosis breast or ovarian carcinomas. Six patients received genetically modified autologous peripheral blood progenitors, together with unmanipulated aphereses, following high-dose chemotherapy. CD34(+) cells were immunoselected from aphereses, and retrovirally transduced by coculture with the retroviral vector producing cell line, to express a nuclear localized version of E. coli beta-galactosidase, encoded by a defective Moloney-murine leukemia virus-derived retroviral vector. Cells were reinfused to the patients after myeloablation, without prior ex vivo selection. Five out of six patients showed the transient presence of low numbers of beta-galactosidase(+) cells, as detected with an immunocytochemical assay, in the peripheral blood, during the first month following infusion. One patient had beta-galactosidase(+) clonogenic progenitors in her marrow at two months after transplantation, including HPP-CFC; intriguingly, this patient had the lowest percentage of X-gal(+) cells in her graft. Patients experienced side effects that are often observed after high-dose chemotherapy. Feasibility and safety of genetic modification of human hematopoietic stem and progenitor cells are demonstrated by this study. Ex vivo or in vivo selection is not mandatory, even in clinical situations where transduced cells have no survival advantage over wild-type cells; however, significant improvements in gene transfer technology are needed to achieve potentially useful levels of expression in such clinical situations.

Quantitative trait locus analysis of heterosis for plant height and ear height in an elite maize hybrid zhengdan 958 by design III.

PubMed

Li, Hongjian; Yang, Qingsong; Fan, Nannan; Zhang, Ming; Zhai, Huijie; Ni, Zhongfu; Zhang, Yirong

2017-04-17

Plant height (PH) and ear height (EH) are two important agronomic traits in maize selection breeding. F 1 hybrid exhibit significant heterosis for PH and EH as compared to their parental inbred lines. To understand the genetic basis of heterosis controlling PH and EH, we conducted quantitative trait locus (QTL) analysis using a recombinant inbreed line (RIL) based design III population derived from the elite maize hybrid Zhengdan 958 in five environments. A total of 14 environmentally stable QTLs were identified, and the number of QTLs for Z 1 and Z 2 populations was six and eight, respectively. Notably, all the eight environmentally stable QTLs for Z 2 were characterized by overdominance effect (OD), suggesting that overdominant QTLs were the most important contributors to heterosis for PH and EH. Furthermore, 14 environmentally stable QTLs were anchored on six genomic regions, among which four are trait-specific QTLs, suggesting that the genetic basis for PH and EH is partially different. Additionally, qPH.A-1.3, modifying about 10 centimeters of PH, was further validated in backcross populations. The genetic basis for PH and EH is partially different, and overdominant QTLs are important factors for heterosis of PH and EH. A major QTL qPH.A-1.3 may be a desired target for genetic improvement of maize plant height.

The expression of preaxial polydactyly is influenced by modifying genetic elements and is not maintained by chromosomal inversion in an avian biomedical model.

PubMed

Robb, E A; Delany, M E

2012-01-01

Polydactyly (Po) is a common mutation found in many vertebrates. The UCD-Po.003 congenic chicken line was previously characterized for Po inheritance (autosomal dominant) and the mutation was mapped to chromosome 2p. Here, we describe for the first time the range and variability of the phenotype in this congenic line. Further, we studied the hypothesis that a chromosomal inversion was responsible for the maintenance of a large (6.3 Mb) candidate gene region. Fluorescence in situ hybridization employing BACs encompassing a 10.7-Mb region of GGA2p showed that the Po chromosome was normal, i.e. exhibits the wild-type BAC order. Continued fine-mapping along with a change in breeding strategy reduced the size of the causative region to 1.43 Mb. Recent research indicates that the cause of preaxial Po resides within a 794-bp highly conserved zone of polarizing activity regulatory sequence (ZRS) element located in intron 5 of the LMBR1 gene; however, the ZRS polymorphism of interest is found in some but not all breeds of polydactylous chicken. Therefore, we sequenced the ZRS in 101 heterozygous and 30 unaffected (wild-type) individuals to establish the relevance of this region to the Po condition in the UCD-Po.003 congenic line. A single point mutation (C/A at coordinate GGA2p: 8,414,121) within the ZRS segregated with carrier status. The polydactylous UCD-Silkie line also maintains this SNP in addition to a single base deletion. An inheritance analysis of the phenotypic variation in UCD-Po.003 suggests recessive epistasis as the mode of inheritance for the additional modifying genetic elements, residing outside the ZRS, to impact the preaxial polydactyl phenotype. These results contribute to our understanding of the cause of Po in an important vertebrate model. Copyright © 2012 S. Karger AG, Basel.

A natural compromise: a moderate solution to the GMO & "natural" labeling disputes.

PubMed

Amaru, Stephanie

2014-01-01

In the United States, genetically modified (GM) foods are labeled no differently from their natural counterparts, leaving consumers with no mechanism for deciphering genetically modified food content. The Food and Drug Administration (FDA) has not formally defined the term "natural," which is frequently used on food labels despite consumer confusion as to what it means. The FDA should initiate a notice and comment rulemaking addressing the narrow issue of whether use of the word "natural" should be permitted oil GM food labels. Prohibition of the use of"natural" on genetically modified foods would mitigate consumer deception regarding genetically modified food content without significantly disadvantaging genetically modified food producers.

Assessment of the nutritional values of genetically modified wheat, corn, and tomato crops.

PubMed

Venneria, Eugenia; Fanasca, Simone; Monastra, Giovanni; Finotti, Enrico; Ambra, Roberto; Azzini, Elena; Durazzo, Alessandra; Foddai, Maria Stella; Maiani, Giuseppe

2008-10-08

The genetic modification in fruit and vegetables could lead to changes in metabolic pathways and, therefore, to the variation of the molecular pattern, with particular attention to antioxidant compounds not well-described in the literature. The aim of the present study was to compare the quality composition of transgenic wheat ( Triticum durum L.), corn ( Zea mays L.), and tomato ( Lycopersicum esculentum Mill.) to the nontransgenic control with a similar genetic background. In the first experiment, Ofanto wheat cultivar containing the tobacco rab1 gene and nontransgenic Ofanto were used. The second experiment compared two transgenic lines of corn containing Bacillus thuringiensis "Cry toxin" gene (PR33P67 and Pegaso Bt) to their nontransgenic forms. The third experiment was conducted on transgenic tomato ( Lycopersicum esculentum Mill.) containing the Agrobacterium rhizogenes rolD gene and its nontransgenic control (cv. Tondino). Conventional and genetically modified crops were compared in terms of fatty acids content, unsaponifiable fraction of antioxidants, total phenols, polyphenols, carotenoids, vitamin C, total antioxidant activity, and mineral composition. No significant differences were observed for qualitative traits analyzed in wheat and corn samples. In tomato samples, the total antioxidant activity (TAA), measured by FRAP assay, and the naringenin content showed a lower value in genetically modified organism (GMO) samples (0.35 mmol of Fe (2+) 100 g (-1) and 2.82 mg 100 g (-1), respectively), in comparison to its nontransgenic control (0.41 mmol of Fe (2+) 100 g (-1) and 4.17 mg 100 g (-1), respectively). On the basis of the principle of substantial equivalence, as articulated by the World Health Organization, the Organization for Economic Cooperation and Development, and the United Nations Food and Agriculture Organization, these data support the conclusion that GM events are nutritionally similar to conventional varieties of wheat, corn, and tomato on the market today.

Transgenic Mosquitoes - Fact or Fiction?

PubMed

Wilke, André B B; Beier, John C; Benelli, Giovanni

2018-06-01

Technologies for controlling mosquito vectors based on genetic manipulation and the release of genetically modified mosquitoes (GMMs) are gaining ground. However, concrete epidemiological evidence of their effectiveness, sustainability, and impact on the environment and nontarget species is lacking; no reliable ecological evidence on the potential interactions among GMMs, target populations, and other mosquito species populations exists; and no GMM technology has yet been approved by the WHO Vector Control Advisory Group. Our opinion is that, although GMMs may be considered a promising control tool, more studies are needed to assess their true effectiveness, risks, and benefits. Overall, several lines of evidence must be provided before GMM-based control strategies can be used under the integrated vector management framework. Copyright © 2018 Elsevier Ltd. All rights reserved.

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

PubMed Central

Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

2016-01-01

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

Dual targeting of gene delivery by genetic modification of adenovirus serotype 5 fibers and cell-selective transcriptional control.

PubMed

Work, L M; Ritchie, N; Nicklin, S A; Reynolds, P N; Baker, A H

2004-08-01

Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a approximately 1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single- and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression. In rat hepatocytes and endothelial cells, the double modification substantially shifted transduction profiles toward vascular endothelial cells. Furthermore, in intact aortae derived from spontaneously hypertensive rats that display enhanced alphav integrin expression on dysfunctional endothelium, enhanced levels of transduction were observed using the double-modified vector but not in aortae derived from normotensive control rats. Our data indicate that Ad-mediated transduction can be beneficially modified in vitro and in vivo by combining fiber modification and a cell-selective promoter within a single-component vector system.

[Genetically modified plants and food safety. State of the art and discussion in the European Union].

PubMed

Schauzu, M

2004-09-01

Placing genetically modified (GM) plants and derived products on the European Union's (EU) market has been regulated by a Community Directive since 1990. This directive was complemented by a regulation specific for genetically modified and other novel foods in 1997. Specific labelling requirements have been applicable for GM foods since 1998. The law requires a pre-market safety assessment for which criteria have been elaborated and continuously adapted in accordance with the state of the art by national and international bodies and organisations. Consequently, only genetically modified products that have been demonstrated to be as safe as their conventional counterparts can be commercialized. However, the poor acceptance of genetically modified foods has led to a de facto moratorium since 1998. It is based on the lack of a qualified majority of EU member states necessary for authorization to place genetically modified plants and derived foods on the market. New Community Regulations are intended to end this moratorium by providing a harmonized and transparent safety assessment, a centralised authorization procedure, extended labelling provisions and a traceability system for genetically modified organisms (GMO) and derived food and feed.

Consumer reaction to information on the labels of genetically modified food

PubMed Central

Sebastian-Ponce, Miren Itxaso; Sanz-Valero, Javier; Wanden-Berghe, Carmina

2014-01-01

OBJECTIVE To analyze consumer opinion on genetically modified foods and the information included on the label. METHODS A systematic review of the scientific literature on genetically modified food labeling was conducted consulting bibliographic databases (Medline – via PubMed –, EMBASE, ISI-Web of knowledge, Cochrane Library Plus, FSTA, LILACS, CINAHL and AGRICOLA) using the descriptors “organisms, genetically modified” and “food labeling”. The search covered the first available date, up to June 2012, selecting relevant articles written in English, Portuguese or Spanish. RESULTS Forty articles were selected after applying the inclusion and exclusion criteria. All of them should have conducted a population-based intervention focused on consumer awareness of genetically modified foods and their need or not, to include this on the label. The consumers expressed a preference for non-genetically modified products, and added that they were prepared to pay more for this but, ultimately, the product bought was that with the best price, in a market which welcomes new technologies. In 18 of the articles, the population was in favor of obligatory labelling, and in six, in favor of this being voluntary; seven studies showed the consumer knew little about genetically modified food, and in three, the population underestimated the quantity they consumed. Price was an influencing factor in all cases. CONCLUSIONS Label should be homogeneous and clarify the degree of tolerance of genetically modified products in humans, in comparison with those non-genetically modified. Label should also present the content or not of genetically modified products and how these commodities are produced and should be accompanied by the certifying entity and contact information. Consumers express their preference for non-genetically modifiedproducts and they even notice that they are willing to pay more for it, but eventually they buy the item with the best price, in a market that welcomes new technologies. PMID:24789648

Identification of five novel modifier loci of ApcMin harbored in the BXH14 recombinant inbred strain

PubMed Central

Siracusa, Linda D.

2012-01-01

Every year thousands of people in the USA are diagnosed with small intestine and colorectal cancers (CRC). Although environmental factors affect disease etiology, uncovering underlying genetic factors is imperative for risk assessment and developing preventative therapies. Familial adenomatous polyposis is a heritable genetic disorder in which individuals carry germ-line mutations in the adenomatous polyposis coli (APC) gene that predisposes them to CRC. The Apc Min mouse model carries a point mutation in the Apc gene and develops polyps along the intestinal tract. Inbred strain background influences polyp phenotypes in Apc Min mice. Several Modifier of Min (Mom) loci that alter tumor phenotypes associated with the Apc Min mutation have been identified to date. We screened BXH recombinant inbred (RI) strains by crossing BXH RI females with C57BL/6J (B6) Apc Min males and quantitating tumor phenotypes in backcross progeny. We found that the BXH14 RI strain harbors five modifier loci that decrease polyp multiplicity. Furthermore, we show that resistance is determined by varying combinations of these modifier loci. Gene interaction network analysis shows that there are multiple networks with proven gene–gene interactions, which contain genes from all five modifier loci. We discuss the implications of this result for studies that define susceptibility loci, namely that multiple networks may be acting concurrently to alter tumor phenotypes. Thus, the significance of this work resides not only with the modifier loci we identified but also with the combinations of loci needed to get maximal protection against polyposis and the impact of this finding on human disease studies. Abbreviations:APCadenomatous polyposis coliGWASgenome-wide association studiesQTLquantitative trait lociSNPsingle-nucleotide polymorphism. PMID:22637734

Genetic Modifiers and Oligogenic Inheritance

PubMed Central

Kousi, Maria; Katsanis, Nicholas

2015-01-01

Despite remarkable progress in the identification of mutations that drive genetic disorders, progress in understanding the effect of genetic background on the penetrance and expressivity of causal alleles has been modest, in part because of the methodological challenges in identifying genetic modifiers. Nonetheless, the progressive discovery of modifier alleles has improved both our interpretative ability and our analytical tools to dissect such phenomena. In this review, we analyze the genetic properties and behaviors of modifiers as derived from studies in patient populations and model organisms and we highlight conceptual and technological tools used to overcome some of the challenges inherent in modifier mapping and cloning. Finally, we discuss how the identification of these modifiers has facilitated the elucidation of biological pathways and holds the potential to improve the clinical predictive value of primary causal mutations and to develop novel drug targets. PMID:26033081

Not all GMOs are crop plants: non-plant GMO applications in agriculture.

PubMed

Hokanson, K E; Dawson, W O; Handler, A M; Schetelig, M F; St Leger, R J

2014-12-01

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.

Genetically Modified Porcine Skin Grafts for Treatment of Severe Burn Injuries

DTIC Science & Technology

2010-07-01

TITLE: Genetically Modified Porcine Skin Grafts for Treatment of Severe Burn Injuries PRINCIPAL INVESTIGATOR: David H. Sachs, M.D...4. TITLE AND SUBTITLE Genetically Modified Porcine Skin Grafts for Treatment of 5a. CONTRACT NUMBER Severe Burn Injuries 5b. GRANT NUMBER...DISTRIBUTION / AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES Burns, skin grafts , genetic

A DWARF MUTATION IN THE RABBIT

PubMed Central

Greene, Harry S. N.

1940-01-01

An hereditary type of dwarfism in the rabbit has been described. In contrast to the dwarfs described in other animals, this type is evident at birth and conforms to the classification, nannosomia primordialis, as used in human pathology. In homozygous form the variation is lethal and produces a miniature individual approximately one-third the size of its normal sibs. Heterozygous animals are approximately two-thirds the size of normal sibs at birth and never attain an equal stature. The expression of the variation is modified by genetic factors carried by a line of cretinoid animals and, rarely, dwarfs derived from crosses with this line survive for 1 to 2 months. The striking changes in such survivors are hypertrophy and hyperplasia of the acidophilic cells of the pituitary and atrophy of the gonads. Such changes are not present in ordinary dwarfs and it is concluded that the acidophilic hyperplasia represents the influence of the modifying factors of the cretinoid line and supplies the growth hormone responsible for survival. The gonadotropic hormone is not supplied by the secretory activity of these cells and as a result the gonads atrophy. The evidence at hand indicates that the primary effect of the dwarfing gene is an inhibition of the secretory functions of the pituitary. In homozygous individuals, the inhibition is complete and the variation is expressed as a lethal dwarf. In heterozygous animals, the function of the organ is altered, producing an undersized individual. The modifying factors of the cretinoid line act either to partially remove the inhibition or to alter the constitution of the animal so that life is possible for a short period without the full complement of pituitary hormones. PMID:19871001

Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J).

PubMed

Lee, Hong Jo; Lee, Kyung Youn; Jung, Kyung Min; Park, Kyung Je; Lee, Ko On; Suh, Jeong-Yong; Yao, Yongxiu; Nair, Venugopal; Han, Jae Yong

2017-12-01

Avian leukosis virus subgroup J (ALV-J), first isolated in the late 1980s, has caused economic losses to the poultry industry in many countries. As all chicken lines studied to date are susceptible to ALV infection, there is enormous interest in developing resistant chicken lines. The ALV-J receptor, chicken Na + /H + exchange 1 (chNHE1) and the critical amino acid sequences involved in viral attachment and entry have already been characterized. However, there are no reported attempts to induce resistance to the virus by targeted genome modification of the receptor sequences. In an attempt to induce resistance to ALV-J infection, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR/Cas9)-based genome editing approaches to modify critical residues of the chNHE1 receptor in chicken cells. The susceptibility of the modified cell lines to ALV-J infection was examined using enhanced green fluorescent protein (EGFP)-expressing marker viruses. We showed that modifying the chNHE1 receptor by artificially generating a premature stop codon induced absolute resistance to viral infection, with mutations of the tryptophan residue at position 38 (Trp38) being very critical. Single-stranded oligodeoxynucleotide (ssODN)-mediated targeted recombination of the Trp38 region revealed that deletions involving the Trp38 residue were most effective in conferring resistance to ALV-J. Moreover, protein structure analysis of the chNHE1 receptor sequence suggested that its intrinsically disordered region undergoes local conformational changes through genetic alteration. Collectively, these results demonstrate that targeted mutations on chNHE1 alter the susceptibility to ALV-J and the technique is expected to contribute to develop disease-resistant chicken lines. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Case of the "Tainted" Taco Shells: A Case Study on Genetically Modified Foods

ERIC Educational Resources Information Center

Taylor, Ann T. S.

2004-01-01

This case study introduces students to the use of genetically modified foods. Students learn how genetically modified plants are made, and then they read primary literature papers to evaluate the environmental, economic, and health issues. (Contains 2 figures.)

Overview of the current status of genetically modified plants in Europe as compared to the USA.

PubMed

Brandt, Peter

2003-07-01

Genetically modified crops have been tested in 1,726 experimental releases in the EU member states and in 7,815 experimental releases in the USA. The global commercial cultivation area of genetically modified crops is likely to reach 50 million hectares in 2001, however, the commercial production of genetically modified crops in the EU amounts to only a few thousand hectares and accounts for only some 0.03% of the world production. A significant gap exists between the more than fifty genetically modified crop species already permitted to be cultivated and to be placed on the market in the USA, Canada and other countries and the five genetically modified crop species permitted for the same use in the EU member states, which are still pending inclusion in the Common Catalogue of agricultural plant species. The further development of the "green gene technology" in the EU will be a matter of public acceptance and administrative legislation.

Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

PubMed

Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

2011-02-01

Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

Evaluation of real-time PCR detection methods for detecting rice products contaminated by rice genetically modified with a CpTI-KDEL-T-nos transgenic construct.

PubMed

Nakamura, Kosuke; Akiyama, Hiroshi; Kawano, Noriaki; Kobayashi, Tomoko; Yoshimatsu, Kayo; Mano, Junichi; Kitta, Kazumi; Ohmori, Kiyomi; Noguchi, Akio; Kondo, Kazunari; Teshima, Reiko

2013-12-01

Genetically modified (GM) rice (Oryza sativa) lines, such as insecticidal Kefeng and Kemingdao, have been developed and found unauthorised in processed rice products in many countries. Therefore, qualitative detection methods for the GM rice are required for the GM food regulation. A transgenic construct for expressing cowpea (Vigna unguiculata) trypsin inhibitor (CpTI) was detected in some imported processed rice products contaminated with Kemingdao. The 3' terminal sequence of the identified transgenic construct for expression of CpTI included an endoplasmic reticulum retention signal coding sequence (KDEL) and nopaline synthase terminator (T-nos). The sequence was identical to that in a report on Kefeng. A novel construct-specific real-time polymerase chain reaction (PCR) detection method for detecting the junction region sequence between the CpTI-KDEL and T-nos was developed. The imported processed rice products were evaluated for the contamination of the GM rice using the developed construct-specific real-time PCR methods, and detection frequency was compared with five event-specific detection methods. The construct-specific detection methods detected the GM rice at higher frequency than the event-specific detection methods. Therefore, we propose that the construct-specific detection method is a beneficial tool for screening the contamination of GM rice lines, such as Kefeng, in processed rice products for the GM food regulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

WONOEP appraisal: new genetic approaches to study epilepsy

PubMed Central

Rossignol, Elsa; Kobow, Katja; Simonato, Michele; Loeb, Jeffrey A.; Grisar, Thierry; Gilby, Krista L.; Vinet, Jonathan; Kadam, Shilpa D.; Becker, Albert J.

2014-01-01

Objective New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming and optogenetic manipulations within epileptic networks are progressively unravelling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiological impacts of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. Methods This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy in Quebec, Canada. Results Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and have revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell-types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knockdown approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. Genetically-encoded cell-type labeling is also providing new means to assess the role of the non-neuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and non-coding RNAs involved in modifying gene expression following seizures. In addition, genetically-based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient iPS cells and genetically-modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Significance Genetics has considerably changed the field of epilepsy research and is paving the way for better diagnosis and therapies for patients with epilepsy. PMID:24965021

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses

PubMed Central

Jean, Christian; Fragnet-Trapp, Laetitia; Rémy, Sylvie; Chabanne-Vautherot, Danièle; Montillet, Guillaume; Fuet, Aurélie; Denesvre, Caroline; Pain, Bertrand

2017-01-01

Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies. PMID:28406989

S-Ketamine Rapidly Reverses Synaptic and Vascular Deficits of Hippocampus in Genetic Animal Model of Depression.

PubMed

Ardalan, Maryam; Wegener, Gregers; Rafati, Ali H; Nyengaard, Jens R

2017-03-01

The neurovascular plasticity of hippocampus is an important theory underlying major depression. Ketamine as a novel glutamatergic antidepressant drug can induce a rapid antidepressant effect within hours. In a mechanistic proof of this concept, we examined whether ketamine leads to an increase in synaptogenesis and vascularization within 24 hours after a single injection in a genetic rat model of depression. Flinders Sensitive Line and Flinders Resistant Line rats were given a single intraperitoneal injection of ketamine (15 mg/kg) or saline. One day later, their behavior was evaluated by a modified forced swim test. Microvessel length was evaluated with global spatial sampling and optical microscopy, whereas the number of asymmetric synapses was quantified through serial section electron microscopy by using physical disector method in the CA1.stratum radiatum area of hippocampus. The immobility time in the forced swim test among Flinders Sensitive Line rats with ketamine treatment was significantly lower compared with Flinders Sensitive Line rats without treatment. The number of nonperforated and perforated synapses was significantly higher in the Flinders Sensitive Line-ketamine vs the Flinders Sensitive Line-vehicle group; however, ketamine did not induce a significant increase in the number of shaft synapses. Additionally, total length of microvessels was significantly increased 1 day after ketamine treatment in Flinders Sensitive Line rats in the hippocampal subregions, including the CA1.stratum radiatum. Our findings indicate that hippocampal vascularization and synaptogenesis is co-regulated rapidly after ketamine, and microvascular elongation may be a supportive factor for synaptic plasticity and neuronal activity. These findings go hand-in-hand with the behavioral observations, where ketamine acts as a potent antidepressant. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Regulating genetically modified food. Policy trajectories, political culture, and risk perceptions in the U.S., Canada, and EU.

PubMed

Wohlers, Anton E

2010-09-01

This paper examines whether national differences in political culture add an explanatory dimension to the formulation of policy in the area of biotechnology, especially with respect to genetically modified food. The analysis links the formulation of protective regulatory policies governing genetically modified food to both country and region-specific differences in uncertainty tolerance levels and risk perceptions in the United States, Canada, and European Union. Based on polling data and document analysis, the findings illustrate that these differences matter. Following a mostly opportunistic risk perception within an environment of high tolerance for uncertainty, policymakers in the United States and Canada modified existing regulatory frameworks that govern genetically modified food in their respective countries. In contrast, the mostly cautious perception of new food technologies and low tolerance for uncertainty among European Union member states has contributed to the creation of elaborate and stringent regulatory policies governing genetically modified food.

Eliminating Glutamatergic Input onto Horizontal Cells Changes the Dynamic Range and Receptive Field Organization of Mouse Retinal Ganglion Cells.

PubMed

Ströh, Sebastian; Puller, Christian; Swirski, Sebastian; Hölzel, Maj-Britt; van der Linde, Lea I S; Segelken, Jasmin; Schultz, Konrad; Block, Christoph; Monyer, Hannah; Willecke, Klaus; Weiler, Reto; Greschner, Martin; Janssen-Bienhold, Ulrike; Dedek, Karin

2018-02-21

In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light responses of transient OFF-α retinal ganglion cells in a newly generated mouse line. In this mouse line, horizontal cell signals were no longer modulated by light. With light response recordings, we show that horizontal cells increase the dynamic range of retinal ganglion cells for contrast and temporal changes and contribute to the center/surround organization of their receptive fields. Copyright © 2018 the authors 0270-6474/18/382015-14$15.00/0.

Biosafety management and commercial use of genetically modified crops in China.

PubMed

Li, Yunhe; Peng, Yufa; Hallerman, Eric M; Wu, Kongming

2014-04-01

As a developing country with relatively limited arable land, China is making great efforts for development and use of genetically modified (GM) crops to boost agricultural productivity. Many GM crop varieties have been developed in China in recent years; in particular, China is playing a leading role in development of insect-resistant GM rice lines. To ensure the safe use of GM crops, biosafety risk assessments are required as an important part of the regulatory oversight of such products. With over 20 years of nationwide promotion of agricultural biotechnology, a relatively well-developed regulatory system for risk assessment and management of GM plants has been developed that establishes a firm basis for safe use of GM crops. So far, a total of seven GM crops involving ten events have been approved for commercial planting, and 5 GM crops with a total of 37 events have been approved for import as processing material in China. However, currently only insect-resistant Bt cotton and disease-resistant papaya have been commercially planted on a large scale. The planting of Bt cotton and disease-resistant papaya have provided efficient protection against cotton bollworms and Papaya ringspot virus (PRSV), respectively. As a consequence, chemical application to these crops has been significantly reduced, enhancing farm income while reducing human and non-target organism exposure to toxic chemicals. This article provides useful information for the colleagues, in particular for them whose mother tongue is not Chinese, to clearly understand the biosafety regulation and commercial use of genetically modified crops in China.

An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

PubMed

Kim, Eunhye; Zheng, Zhong; Jeon, Yubyeol; Jin, Yong-Xun; Hwang, Seon-Ung; Cai, Lian; Lee, Chang-Kyu; Kim, Nam-Hyung; Hyun, Sang-Hwan

2016-01-01

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

Regulated Apoptosis of Genetically-Modified Hematopoietic Stem and Progenitor Cells via an Inducible Caspase-9 Suicide Gene in Rhesus Macaques

PubMed Central

Barese, Cecilia N.; Felizardo, Tania C.; Sellers, Stephanie E.; Keyvanfar, Keyvan; Di Stasi, Antonio; Metzger, Mark E.; Krouse, Allen E.; Donahue, Robert E.; Spencer, David M.; Dunbar, Cynthia E.

2014-01-01

The high risk of insertional oncogenesis reported in clinical trials utilizing integrating retroviral vectors to genetically-modify hematopoietic stem and progenitor cells (HSPC) requires the development of safety strategies to minimize risks associated with novel cell and gene therapies. The ability to ablate genetically modified cells in vivo is desirable, should an abnormal clone emerge. Inclusion of “suicide genes” in vectors to facilitate targeted ablation of vector-containing abnormal clones in vivo is one potential safety approach. We tested whether the inclusion of the “inducible Caspase-9” (iCasp9) suicide gene in a gamma-retroviral vector facilitated efficient elimination of vector-containing HSPCs and their hematopoietic progeny in vivo long-term, in an autologous non-human primate transplantation model. Following stable engraftment of iCasp9 expressing hematopoietic cells in rhesus macaques, administration of AP1903, a chemical inducer of dimerization able to activate iCasp9, specifically eliminated vector-containing cells in all hematopoietic lineages long-term, suggesting activity at the HSPC level. Between 75–94% of vector-containing cells were eliminated by well-tolerated AP1903 dosing, but lack of complete ablation was linked to lower iCasp9 expression in residual cells. Further investigation of resistance mechanisms demonstrated upregulation of Bcl-2 in hematopoietic cell lines transduced with the vector and resistant to AP1903 ablation. These results demonstrate both the potential and the limitations of safety approaches utilizing iCasp9 to HSPC-targeted gene therapy settings, in a model with great relevance to clinical development. PMID:25330775

Genetic modifiers of Duchenne and facioscapulohumeral muscular dystrophies.

PubMed

Hightower, Rylie M; Alexander, Matthew S

2018-01-01

Muscular dystrophy is defined as the progressive wasting of skeletal muscles that is caused by inherited or spontaneous genetic mutations. Next-generation sequencing has greatly improved the accuracy and speed of diagnosis for different types of muscular dystrophy. Advancements in depth of coverage, convenience, and overall reduced cost have led to the identification of genetic modifiers that are responsible for phenotypic variability in affected patients. These genetic modifiers have been postulated to explain key differences in disease phenotypes, including age of loss of ambulation, steroid responsiveness, and the presence or absence of cardiac defects in patients with the same form of muscular dystrophy. This review highlights recent findings on genetic modifiers of Duchenne and facioscapulohumeral muscular dystrophies based on animal and clinical studies. These genetic modifiers hold great promise to be developed into novel therapeutic targets for the treatment of muscular dystrophies. Muscle Nerve 57: 6-15, 2018. © 2017 Wiley Periodicals, Inc.

Genetic Modifiers of Duchenne and Facioscapulohumeral Muscular Dystrophies

PubMed Central

Hightower, Rylie M.; Alexander, Matthew S.

2017-01-01

Muscular dystrophy is defined as the progressive wasting of skeletal muscles that is caused by inherited or spontaneous genetic mutations. Next-generation sequencing (NGS) has greatly improved the accuracy and speed of diagnosis for different types of muscular dystrophy. Advancements in depth of coverage, convenience, and overall reduced cost, have led to the identification of genetic modifiers that are responsible for phenotypic variability in affected patients. These genetic modifiers have been postulated to explain key differences in disease phenotypes including age of loss of ambulation, steroid-responsiveness, and the presence or absence of cardiac defects in patients with the same form of muscular dystrophy. Here we review and highlight recent findings on genetic modifiers of Duchenne and Facioscapulohumeral muscular dystrophies based on animal and clinical studies. These genetic modifiers hold great promise to be developed into novel therapeutic targets for the treatment of muscular dystrophies. PMID:28877560

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

PubMed

Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N

2010-05-01

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

77 FR 7172 - Sequoyah National Wildlife Refuge, Sequoyah, Muskogee, and Haskell Counties, OK; Comprehensive...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-10

.... Scoping for the environmental assessment (EA) on use of specified genetically modified crops in... of genetically modified crops in association with the cooperative farming program was released on... assessment of using specified genetically modified crops into the CCP and determined that an environmental...

HYBRIDIZATION STUDY BETWEEN GENETICALLY MODIFIED BRASSICA NAPUS AND NON-GENETICALLY MODIFIED B. NAPUS AND B. RAPA

EPA Science Inventory

Gene exchange between cultivated crops and wild species has gained significance in recent years because of concerns regarding the potential for gene flow between genetically modified (GM) crops and their domesticated and wild relatives. As part of our ecological effects of gene ...

MATERNAL EFFECTS IN ADVANCED HYBRIDS OF GENETICALLY MODIFIED AND NON-GENETICALLY MODIFIED BRASSICA SPECIES

EPA Science Inventory

Identification of fitness traits potentially impacted by gene flow from genetically modified (GM) crops to compatible relatives is of interest in risk assessments for GM crops. Reciprocal crosses were made between GM canola, Brassica napus cv. RaideRR that expresses CP4 EPSPS fo...

Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer's disease.

PubMed

Yamamoto, Satoshi; Ooshima, Yuki; Nakata, Mitsugu; Yano, Takashi; Matsuoka, Kunio; Watanabe, Sayuri; Maeda, Ryouta; Takahashi, Hideki; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

2013-06-01

Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

Microscope self-calibration based on micro laser line imaging and soft computing algorithms

NASA Astrophysics Data System (ADS)

Apolinar Muñoz Rodríguez, J.

2018-06-01

A technique to perform microscope self-calibration via micro laser line and soft computing algorithms is presented. In this technique, the microscope vision parameters are computed by means of soft computing algorithms based on laser line projection. To implement the self-calibration, a microscope vision system is constructed by means of a CCD camera and a 38 μm laser line. From this arrangement, the microscope vision parameters are represented via Bezier approximation networks, which are accomplished through the laser line position. In this procedure, a genetic algorithm determines the microscope vision parameters by means of laser line imaging. Also, the approximation networks compute the three-dimensional vision by means of the laser line position. Additionally, the soft computing algorithms re-calibrate the vision parameters when the microscope vision system is modified during the vision task. The proposed self-calibration improves accuracy of the traditional microscope calibration, which is accomplished via external references to the microscope system. The capability of the self-calibration based on soft computing algorithms is determined by means of the calibration accuracy and the micro-scale measurement error. This contribution is corroborated by an evaluation based on the accuracy of the traditional microscope calibration.

Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma.

PubMed

Rainusso, N; Brawley, V S; Ghazi, A; Hicks, M J; Gottschalk, S; Rosen, J M; Ahmed, N

2012-03-01

Despite radical surgery and multi-agent chemotherapy, less than one third of patients with recurrent or metastatic osteosarcoma (OS) survive. The limited efficacy of current therapeutic approaches to target tumor-initiating cells (TICs) may explain this dismal outcome. The purpose of this study was to assess the impact of modified T cells expressing a human epidermal growth factor receptor (HER2)-specific chimeric antigen receptor in the OS TIC compartment of human established cell lines. Using the sarcosphere formation assay, we found that OS TICs were resistant to increasing methotrexate concentrations. In contrast, HER2-specific T cells decreased markedly sarcosphere formation capacity and the ability to generate bone tumors in immunodeficient mice after orthotopic transplantation. In vivo, administration of HER2-specific T cells significantly reduced TICs in bulky tumors as judged by decreased sarcosphere forming efficiency in OS cells isolated from explanted tumors. We demonstrate that HER2-specific T cells target drug resistant TICs in established OS cell lines, suggesting that incorporating immunotherapy into current treatment strategies for OS has the potential to improve outcomes.

Is Judgement of Biotechnological Ethical Aspects Related to High School Students' Knowledge?

NASA Astrophysics Data System (ADS)

Črne-Hladnik, Helena; Hladnik, Aleš; Javornik, Branka; Košmelj, Katarina; Peklaj, Cirila

2012-05-01

Quantitative and qualitative studies of various aspects of the perception of biotechnology were conducted among 469 Slovenian high school students of average age 17 years. Our research aimed to explore relationships among students' pre-knowledge of molecular and human genetics, and their attitudes to four specific biotechnological applications. These applications-Bt corn, genetically modified (GM) salmon, somatic and germ line gene therapy (GT)-were investigated from the viewpoints of usefulness, moral acceptance and risk perception. In addition, patterns and quality of moral reasoning related to the biotechnological applications from the aspect of moral acceptability were examined. Clear gender differences were found regarding the relationship between our students' pre-knowledge of genetics and their attitudes to biotechnological applications. While females with a better genetics background expressed a higher risk perception in the case of GM salmon, their similarly well-educated male colleagues emphasized the risk associated with the use of germ line GT. With all four biotechnological applications, patterns of both rationalistic-deontological and teleological-and intuitive moral reasoning were identified. Students with poorer genetics pre-knowledge applied an intuitive pattern of moral reasoning more frequently than their peers with better pre-knowledge. A pattern of emotive reasoning was detected only in the case of GM salmon. A relatively low quality of students' moral reasoning, as demonstrated by their brief and small number of supporting justifications (explanations), show that there is a strong need for practising skills of argumentation about socio-scientific issues in Slovenian high schools on a much larger scale. The implications for future research and classroom applications are discussed.

Zebularine treatment is associated with deletion of FT-B1 leading to an increase in spikelet number in bread wheat.

PubMed

Finnegan, E Jean; Ford, Brett; Wallace, Xiaomei; Pettolino, Filomena; Griffin, Patrick T; Schmitz, Robert J; Zhang, Peng; Barrero, Jose M; Hayden, Matthew J; Boden, Scott A; Cavanagh, Colin A; Swain, Steve M; Trevaskis, Ben

2018-06-01

The number of rachis nodes (spikelets) on a wheat spike is a component of grain yield that correlates with flowering time. The genetic basis regulating flowering in cereals is well understood, but there are reports that flowering time can be modified at a high frequency by selective breeding, suggesting that it may be regulated by both epigenetic and genetic mechanisms. We investigated the role of DNA methylation in regulating spikelet number and flowering time by treating a semi-spring wheat with the demethylating agent, Zebularine. Three lines with a heritable increase in spikelet number were identified. The molecular basis for increased spikelet number was not determined in 2 lines, but the phenotype showed non-Mendelian inheritance, suggesting that it could have an epigenetic basis. In the remaining line, the increased spikelet phenotype behaved as a Mendelian recessive trait and late flowering was associated with a deletion encompassing the floral promoter, FT-B1. Deletion of FT-B1 delayed the transition to reproductive growth, extended the duration of spike development, and increased spikelet number under different temperature regimes and photoperiod. Transiently disrupting DNA methylation can generate novel flowering behaviour in wheat, but these changes may not be sufficiently stable for use in breeding programs. © 2018 John Wiley & Sons Ltd.

Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

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

Fang, Zhen F.; Gai, Hui; Huang, You Z.

2006-11-01

Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ESmore » cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.« less

Brain Tumor Genetic Modification Yields Increased Resistance to Paclitaxel in Physical Confinement

PubMed Central

Bui, Loan; Hendricks, Alissa; Wright, Jamie; Chuong, Cheng-Jen; Davé, Digant; Bachoo, Robert; Kim, Young-tae

2016-01-01

Brain tumor cells remain highly resistant to radiation and chemotherapy, particularly malignant and secondary cancers. In this study, we utilized microchannel devices to examine the effect of a confined environment on the viability and drug resistance of the following brain cancer cell lines: primary cancers (glioblastoma multiforme and neuroblastoma), human brain cancer cell lines (D54 and D54-EGFRvIII), and genetically modified mouse astrocytes (wild type, p53−/−, p53−/− PTEN−/−, p53−/− Braf, and p53−/− PTEN−/− Braf). We found that loss of PTEN combined with Braf activation resulted in higher viability in narrow microchannels. In addition, Braf conferred increased resistance to the microtubule-stabilizing drug Taxol in narrow confinement. Similarly, survival of D54-EGFRvIII cells was unaffected following treatment with Taxol, whereas the viability of D54 cells was reduced by 75% under these conditions. Taken together, our data suggests key targets for anticancer drugs based on cellular genotypes and their specific survival phenotypes during confined migration. PMID:27184621

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

PubMed

Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

2006-09-01

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

[Progress on biosafety assessment of marker genes in genetically modified foods].

PubMed

Yang, Lichen; Yang, Xiaoguang

2003-05-01

Marker genes are useful in facilitating the detection of genetically modified organisms(GMO). These genes play an important role during the early identification stage of GMO development, but they exist in the mature genetically modified crops. So the safety assessment of these genes could not be neglected. In this paper, all the study on the biosafety assessment of marker genes were reviewed, their possible hazards and risks were appraised, and the marker genes proved safe were list too. GMO Labeling the is one important regulations for the development of genetically modified foods in the market. The accurate detecting techniques for GMO are the basis for setting up labeling regulation. In addition, some methods used to remove marker genes in genetically modified foods were introduced in the paper, which can eliminate their biosafety concern thoroughly.

Immunotoxicological evaluation of wheat genetically modified with TaDREB4 gene on BALB/c mice.

PubMed

Liang, Chun Lai; Zhang, Xiao Peng; Song, Yan; Jia, Xu Dong

2013-08-01

To evaluate the immunotoxicological effects of genetically modified wheat with TaDREB4 gene in female BALB/c mice. Female mice weighing 18-22 g were divided into five groups (10 mice/group), which were set as negative control group, common wheat group, parental wheat group, genetically modified wheat group and cyclophosphamide positive control group, respectively. Mice in negative control group and positive control group were fed with AIN93G diet, mice in common wheat group, non-genetically modified parental wheat group and genetically modified wheat group were fed with feedstuffs added corresponding wheat (the proportion is 76%) for 30 days, then body weight, absolute and relative weight of spleen and thymus, white blood cell count, histological examination of immune organ, peripheral blood lymphocytes phenotyping, serum cytokine, serum immunoglobulin, antibody plaque-forming cell, serum half hemolysis value, mitogen-induced splenocyte proliferation, delayed-type hypersensitivity reaction and phagocytic activities of phagocytes were detected. No immunotoxicological effects related to the consumption of the genetically modified wheat were observed in BALB/c mice when compared with parental wheat group, common wheat group and negative control group. From the immunotoxicological point of view, results from this study demonstrate that genetically modified wheat with TaDREB4 gene is as safe as the parental wheat. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

[Genetically modified food--unnecessary controversy?].

PubMed

Tchórz, Michał; Radoniewicz-Chagowska, Anna; Lewandowska-Stanek, Hanna; Szponar, Elzbieta; Szponar, Jarosław

2012-01-01

Fast development of genetic engineering and biotechnology allows use of genetically modified organisms (GMO) more and more in different branches of science and economy. Every year we can see an increase of food amount produced with the use of modification of genetic material. In our supermarkets we can find brand new types of plants, products including genetically modified ingredients or meat from animals fed with food containing GMO. This article presents general information about genetically modified organisms, it also explains the range of genetic manipulation, use of newly developed products and current field area for GMO in the world. Based on scientific data the article presents benefits from development of biotechnology in reference to modified food. It also presents the voice of skeptics who are extremely concerned about the impact of those organisms on human health and natural environment. Problems that appear or can appear as a result of an increase of GMO are very important not only from a toxicologist's or a doctor's point of view but first of all from the point of view of ordinary consumers--all of us.

Implication of Highly Cytotoxic Natural Killer Cells for Esophageal Squamous Cell Carcinoma Treatment.

PubMed

Lim, Kee Siang; Mimura, Kosaku; Kua, Ley-Fang; Shiraishi, Kensuke; Kono, Koji

2018-04-20

Esophageal squamous cell carcinoma (ESCC) is an aggressive upper gastrointestinal cancer and effective treatments are limited. Previous studies reported that natural killer (NK) cells expanded by coculturing with K562-mb15-41BBL feeder cells, a genetically modified K562 leukemia cell line that expresses membrane-bound interleukin (IL)-15 and 41BBL ligand, were highly proliferative and highly cytotoxic. Here, we investigated the potential of expanded NK cells for ESCC treatment. We analyzed both genetic and surface expression levels of NKG2D ligands (NKG2DLs) in ESCC using publicly available microarray data sets and ESCC cell lines. The cytotoxicity of resting and of IL-2-activated NK cells against ESCC cell lines was compared with that of expanded NK cells. We then also investigated the effect of epithelial mesenchymal transition (EMT) inducers, GSK3β inhibitor and epidermal growth factor, on NKG2DLs expressions. As a result, MICA and MICB were significantly overexpressed in ESCC compared with adjacent normal tissues and surface NKG2DLs were expressed in ESCC cell lines. Expanded NK cells were much potent than IL-2-activated and resting NK cells against ESCC cell lines. Blocking of NKG2D with anti-NKG2D monoclonal antibody dampened expanded NK cell cytotoxicity, suggesting that the NKG2DLs-NKG2D interaction is crucial for NK cells to eliminate ESCC cells. EMT inducers concurrently induced EMT and NKG2DLs expression in ESCC cell lines rendering transitioned cells more sensitive to expanded NK cells. In conclusion, expanded NK cells were highly cytotoxic against NKG2DLs-expressing ESCC cells, particularly the EMT phenotype. These results provide a strong rationale for clinical use of these NK cells in ESCC patients.

Multiple organ histopathological changes in broiler chickens fed on genetically modified organism.

PubMed

Cîrnatu, Daniela; Jompan, A; Sin, Anca Ileana; Zugravu, Cornelia Aurelia

2011-01-01

Diet can influence the structural characteristics of internal organs. An experiment involving 130 meat broilers was conducted during 42 days (life term for a meat broiler) to study the effect of feed with protein from genetically modified soy. The 1-day-old birds were randomly allocated to five study groups, fed with soy, sunflower, wheat, fish flour, PC starter. In the diet of each group, an amount of protein from soy was replaced with genetically modified soy (I - 0%, II - 25%, III - 50%, IV - 75%, V - 100% protein from genetically modified soy). The level of protein in soy, either modified, or non-modified, was the same. Organs and carcass weights were measured at about 42 days of age of the birds and histopathology exams were performed during May-June 2009. No statistically significant differences were observed in mortality, growth performance variables or carcass and organ yields between broilers consuming diets produced with genetically modified soybean fractions and those consuming diets produced with near-isoline control soybean fractions. Inflammatory and degenerative liver lesions, muscle hypertrophy, hemorrhagic necrosis of bursa, kidney focal tubular necrosis, necrosis and superficial ulceration of bowel and pancreatic dystrophies were found in tissues from broilers fed on protein from genetically modified soy. Different types of lesions found in our study might be due to other causes (parasites, viral) superimposed but their presence exclusively in groups fed with modified soy raises some serious questions about the consequences of use of this type of feed.

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.

Promoter isolation and characterization of GhAO-like1, a Gossypium hirsutum gene similar to multicopper oxidases that is highly expressed in reproductive organs.

PubMed

Lambret-Frotté, Julia; Artico, Sinara; Muniz Nardeli, Sarah; Fonseca, Fernando; Brilhante Oliveira-Neto, Osmundo; Grossi-de-Sá, Maria Fatima; Alves-Ferreira, Marcio

2016-01-01

Cotton is one of the most economically important cultivated crops. It is the major source of natural fiber for the textile industry and an important target for genetic modification for both biotic stress and herbicide tolerance. Therefore, the characterization of genes and regulatory regions that might be useful for genetic transformation is indispensable. The isolation and characterization of new regulatory regions is of great importance to drive transgene expression in genetically modified crops. One of the major drawbacks in cotton production is pest damage; therefore, the most promising, cost-effective, and sustainable method for pest control is the development of genetically resistant cotton lines. Considering this scenario, our group isolated and characterized the promoter region of a MCO (multicopper oxidase) from Gossypium hirsutum, named GhAO-like1 (ascorbate oxidase-like1). The quantitative expression, together with the in vivo characterization of the promoter region reveals that GhAO-like1 has a flower- and fruit-specific expression pattern. The GUS activity is mainly observed in stamens, as expected considering that the GhAO-like1 regulatory sequence is enriched in cis elements, which have been characterized as a target of reproductive tissue specific transcription factors. Both histological and quantitative analyses in Arabidopsis thaliana have confirmed flower (mainly in stamens) and fruit expression of GhAO-like1. In the present paper, we isolated and characterized both in silico and in vivo the promoter region of the GhAO-like1 gene. The regulatory region of GhAO-like1 might be useful to confer tissue-specific expression in genetically modified plants.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

PubMed Central

Jinadasa, Rasika; Balmus, Gabriel; Gerwitz, Lee; Roden, Jamie; Weiss, Robert; Duhamel, Gerald

2011-01-01

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year. PMID:21490582

Genetic Contributors to Intergenerational CAG Repeat Instability in Huntington’s Disease Knock-In Mice

PubMed Central

Neto, João Luís; Lee, Jong-Min; Afridi, Ali; Gillis, Tammy; Guide, Jolene R.; Dempsey, Stephani; Lager, Brenda; Alonso, Isabel; Wheeler, Vanessa C.; Pinto, Ricardo Mouro

2017-01-01

Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in HD families, partly underlying the genetic anticipation seen in this disorder. HD CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest HD knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identification of large repeat size jumps in the knock-in models. Distinct degrees of intergenerational instability were observed between knock-in mice of six background strains, indicating the occurrence of trans-acting genetic modifiers. We also found that lines harboring a neomycin resistance cassette upstream of Htt showed reduced expansion frequency, indicative of a contributing role for sequences in cis, with the expanded repeat as modifiers of intergenerational instability. These results provide a basis for further understanding of the mechanisms underlying intergenerational repeat instability. PMID:27913616

Biotechnological Advancements for Improving Floral Attributes in Ornamental Plants

PubMed Central

Noman, Ali; Aqeel, Muhammad; Deng, Jianming; Khalid, Noreen; Sanaullah, Tayyaba; Shuilin, He

2017-01-01

Developing new ornamental cultivars with improved floral attributes is a major goal in floriculture. Biotechnological approach together with classical breeding methods has been used to modify floral color, appearance as well as for increasing disease resistance. Transgenic strategies possess immense potential to produce novel flower phenotypes that are not found in nature. Adoption of Genetic engineering has supported the idea of floral trait modification. Ornamental plant attributes like floral color, fragrance, disease resistance, and vase life can be improved by means of genetic manipulation. Therefore, we witness transgenic plant varieties of high aesthetic and commercial value. This review focuses on biotechnological advancements in manipulating key floral traits that contribute in development of diverse ornamental plant lines. Data clearly reveals that regulation of biosynthetic pathways related to characteristics like pigment production, flower morphology and fragrance is both possible and predictable. In spite of their great significance, small number of genetically engineered varieties of ornamental plants has been field tested. Today, novel flower colors production is regarded as chief commercial benefit obtained from transgenic plants. But certain other floral traits are much more important and have high commercial potential. Other than achievements such as novel architecture, modified flower color, etc., very few reports are available regarding successful transformation of other valuable horticultural characteristics. Our review also summarized biotechnological efforts related to enhancement of fragrance and induction of early flowering along with changes in floral anatomy and morphology. PMID:28473834

Biotechnological Advancements for Improving Floral Attributes in Ornamental Plants.

PubMed

Noman, Ali; Aqeel, Muhammad; Deng, Jianming; Khalid, Noreen; Sanaullah, Tayyaba; Shuilin, He

2017-01-01

Developing new ornamental cultivars with improved floral attributes is a major goal in floriculture. Biotechnological approach together with classical breeding methods has been used to modify floral color, appearance as well as for increasing disease resistance. Transgenic strategies possess immense potential to produce novel flower phenotypes that are not found in nature. Adoption of Genetic engineering has supported the idea of floral trait modification. Ornamental plant attributes like floral color, fragrance, disease resistance, and vase life can be improved by means of genetic manipulation. Therefore, we witness transgenic plant varieties of high aesthetic and commercial value. This review focuses on biotechnological advancements in manipulating key floral traits that contribute in development of diverse ornamental plant lines. Data clearly reveals that regulation of biosynthetic pathways related to characteristics like pigment production, flower morphology and fragrance is both possible and predictable. In spite of their great significance, small number of genetically engineered varieties of ornamental plants has been field tested. Today, novel flower colors production is regarded as chief commercial benefit obtained from transgenic plants. But certain other floral traits are much more important and have high commercial potential. Other than achievements such as novel architecture, modified flower color, etc., very few reports are available regarding successful transformation of other valuable horticultural characteristics. Our review also summarized biotechnological efforts related to enhancement of fragrance and induction of early flowering along with changes in floral anatomy and morphology.

First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar.

PubMed

Dubois, A E J; Pagliarani, G; Brouwer, R M; Kollen, B J; Dragsted, L O; Eriksen, F D; Callesen, O; Gilissen, L J W J; Krens, F A; Visser, R G F; Smulders, M J M; Vlieg-Boerstra, B J; Flokstra-de Blok, B J; van de Weg, W E

2015-11-01

Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals. We performed oral food challenges in 21 apple-allergic individuals with Elstar apples which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual analogue scale (VAS). Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges with these downregulated apple lines produced significantly less intense maximal symptoms to the first dose (Vmax1) than with Elstar (Vmax1 Elstar 3.0 mm vs 0.0 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2 mm vs 0.2 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043). Only one subject (5%) remained symptom-free when challenged with the Elstar apple, whereas 43% did so with e-DR1 and 63% with e-DR2. These data show that mRNA silencing of Mal d 1 results in a marked reduction of Mal d 1 gene expression in the fruit and reduction of symptoms when these apples are ingested by allergic subjects. Approximately half of the subjects developed no symptoms whatsoever, and virtually all subjects wished to consume the apple again in the future. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Detection and traceability of genetically modified organisms in the food production chain.

PubMed

Miraglia, M; Berdal, K G; Brera, C; Corbisier, P; Holst-Jensen, A; Kok, E J; Marvin, H J P; Schimmel, H; Rentsch, J; van Rie, J P P F; Zagon, J

2004-07-01

Both labelling and traceability of genetically modified organisms are current issues that are considered in trade and regulation. Currently, labelling of genetically modified foods containing detectable transgenic material is required by EU legislation. A proposed package of legislation would extend this labelling to foods without any traces of transgenics. These new legislations would also impose labelling and a traceability system based on documentation throughout the food and feed manufacture system. The regulatory issues of risk analysis and labelling are currently harmonised by Codex Alimentarius. The implementation and maintenance of the regulations necessitates sampling protocols and analytical methodologies that allow for accurate determination of the content of genetically modified organisms within a food and feed sample. Current methodologies for the analysis of genetically modified organisms are focused on either one of two targets, the transgenic DNA inserted- or the novel protein(s) expressed- in a genetically modified product. For most DNA-based detection methods, the polymerase chain reaction is employed. Items that need consideration in the use of DNA-based detection methods include the specificity, sensitivity, matrix effects, internal reference DNA, availability of external reference materials, hemizygosity versus homozygosity, extrachromosomal DNA, and international harmonisation. For most protein-based methods, enzyme-linked immunosorbent assays with antibodies binding the novel protein are employed. Consideration should be given to the selection of the antigen bound by the antibody, accuracy, validation, and matrix effects. Currently, validation of detection methods for analysis of genetically modified organisms is taking place. In addition, new methodologies are developed, including the use of microarrays, mass spectrometry, and surface plasmon resonance. Challenges for GMO detection include the detection of transgenic material in materials with varying chromosome numbers. The existing and proposed regulatory EU requirements for traceability of genetically modified products fit within a broader tendency towards traceability of foods in general and, commercially, towards products that can be distinguished from each other. Traceability systems document the history of a product and may serve the purpose of both marketing and health protection. In this framework, segregation and identity preservation systems allow for the separation of genetically modified and non-modified products from "farm to fork". Implementation of these systems comes with specific technical requirements for each particular step of the food processing chain. In addition, the feasibility of traceability systems depends on a number of factors, including unique identifiers for each genetically modified product, detection methods, permissible levels of contamination, and financial costs. In conclusion, progress has been achieved in the field of sampling, detection, and traceability of genetically modified products, while some issues remain to be solved. For success, much will depend on the threshold level for adventitious contamination set by legislation. Copryright 2004 Elsevier Ltd.

[Assessment of allergenicity of genetically modified food crops].

PubMed

Schauzu, M; Pöting, A; Rubin, D; Lampen, A

2012-03-01

The placing on the European Union's market of genetically modified crops requires authorization by the European Commission which is based on the proof that the derived foods are as safe as their conventional counterparts. The assessment of potential allergenicity is part of the necessary investigations recommended in the updated Guidance Document of the Scientific Panel on Genetically Modified Organisms (GMO) of the European Food Safety Authority (EFSA), which is based on internationally agreed recommendations. All genetically modified crops which so far have been authorized in the European Union were evaluated by the EFSA GMO Panel which considered it unlikely that their overall allergenicity has been altered.

[Hypothetical link between endometriosis and xenobiotics-associated genetically modified food].

PubMed

Aris, A; Paris, K

2010-12-01

Endometriosis is an oestrogen-dependent inflammatory disease affecting 10 % of reproductive-aged women. Often accompanied by chronic pelvic pain and infertility, endometriosis rigorously interferes with women's quality of life. Although the pathophysiology of endometriosis remains unclear, a growing body of evidence points to the implication of environmental toxicants. Over the last decade, an increase in the incidence of endometriosis has been reported and coincides with the introduction of genetically modified foods in our diet. Even though assessments of genetically modified food risk have not indicated any hazard on human health, xenobiotics-associated genetically modified food, such as pesticides residues and xenoproteins, could be harmful in the long-term. The "low-dose hypothesis", accumulation and biotransformation of pesticides-associated genetically modified food and the multiplied toxicity of pesticides-formulation adjuvants support this hypothesis. This review summarizes toxic effects (in vitro and on animal models) of some xenobiotics-associated genetically modified food, such as glyphosate and Cry1Ab protein, and extrapolates on their potential role in the pathophysiology of endometriosis. Their roles as immune toxicants, pro-oxidants, endocrine disruptors and epigenetic modulators are discussed. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

SeedUSoon: A New Software Program to Improve Seed Stock Management and Plant Line Exchanges between Research Laboratories

PubMed Central

Charavay, Céline; Segard, Stéphane; Pochon, Nathalie; Nussaume, Laurent; Javot, Hélène

2017-01-01

Plant research is supported by an ever-growing collection of mutant or transgenic lines. In the past, a typical basic research laboratory would focus on only a few plant lines that were carefully isolated from collections of lines containing random mutations. The subsequent technological breakthrough in high-throughput sequencing, combined with novel and highly efficient mutagenesis techniques (including site-directed mutagenesis), has led to a recent exponential growth in plant line collections used by individual researchers. Tracking the generation and genetic properties of these genetic resources is thus becoming increasingly challenging for researchers. Another difficulty for researchers is controlling the use of seeds protected by a Material Transfer Agreement, as often only the original recipient of the seeds is aware of the existence of such documents. This situation can thus lead to difficult legal situations. Simultaneously, various institutions and the general public now demand more information about the use of genetically modified organisms (GMOs). In response, researchers are seeking new database solutions to address the triple challenge of research competition, legal constraints, and institutional/public demands. To help plant biology laboratories organize, describe, store, trace, and distribute their seeds, we have developed the new program SeedUSoon, with simplicity in mind. This software contains data management functions that allow the separate tracking of distinct mutations, even in successive crossings or mutagenesis. SeedUSoon reflects the biotechnological diversity of mutations and transgenes contained in any specific line, and the history of their inheritance. It can facilitate GMO certification procedures by distinguishing mutations on the basis of the presence/absence of a transgene, and by recording the technology used for their generation. Its interface can be customized to match the context and rules of any laboratory. In addition, SeedUSoon includes functions to help the laboratory protect intellectual property, export data, and facilitate seed exchange between laboratories. The SeedUSoon program, which is customizable to match individual practices and preferences, provides a powerful toolkit to plant laboratories searching for innovative approaches in laboratory management. PMID:28163712

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.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

PubMed

Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2018-05-01

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Killed whole-HIV vaccine; employing a well established strategy for antiviral vaccines.

PubMed

Kang, C Yong; Gao, Yong

2017-09-12

The development of an efficient prophylactic HIV vaccine has been one of the major challenges in infectious disease research during the last three decades. Here, we present a mini review on strategies employed for the development of HIV vaccines with an emphasis on a well-established vaccine technology, the killed whole-virus vaccine approach. Recently, we reported an evaluation of the safety and the immunogenicity of a genetically modified and killed whole-HIV-1 vaccine designated as SAV001 [1]. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence of the Env signal peptide with that of honeybee melittin to produce an avirulent and replication efficient HIV-1. This genetically modified virus (gmHIV-1 NL4-3 ) was propagated in a human T cell line followed by virus purification and inactivation by aldrithiol-2 and γ-irradiation. We found that SAV001 was well tolerated with no serious adverse events. HIV-1 NL4-3 -specific polymerase chain reaction showed no evidence of vaccine virus replication in participants receiving SAV001 and in human T cells infected in vitro. Furthermore, SAV001 with an adjuvant significantly increased the antibody response to HIV-1 structural proteins. Moreover, antibodies in the plasma from these vaccinations neutralized tier I and tier II of HIV-1 B, A, and D subtypes. These results indicated that the killed whole-HIV vaccine is safe and may trigger appropriate immune responses to prevent HIV infection. Utilization of this killed whole-HIV vaccine strategy may pave the way to develop an effective HIV vaccine.

CoGAPS matrix factorization algorithm identifies transcriptional changes in AP-2alpha target genes in feedback from therapeutic inhibition of the EGFR network

PubMed Central

Thakar, Manjusha; Howard, Jason D.; Kagohara, Luciane T.; Krigsfeld, Gabriel; Ranaweera, Ruchira S.; Hughes, Robert M.; Perez, Jimena; Jones, Siân; Favorov, Alexander V.; Carey, Jacob; Stein-O'Brien, Genevieve; Gaykalova, Daria A.; Ochs, Michael F.; Chung, Christine H.

2016-01-01

Patients with oncogene driven tumors are treated with targeted therapeutics including EGFR inhibitors. Genomic data from The Cancer Genome Atlas (TCGA) demonstrates molecular alterations to EGFR, MAPK, and PI3K pathways in previously untreated tumors. Therefore, this study uses bioinformatics algorithms to delineate interactions resulting from EGFR inhibitor use in cancer cells with these genetic alterations. We modify the HaCaT keratinocyte cell line model to simulate cancer cells with constitutive activation of EGFR, HRAS, and PI3K in a controlled genetic background. We then measure gene expression after treating modified HaCaT cells with gefitinib, afatinib, and cetuximab. The CoGAPS algorithm distinguishes a gene expression signature associated with the anticipated silencing of the EGFR network. It also infers a feedback signature with EGFR gene expression itself increasing in cells that are responsive to EGFR inhibitors. This feedback signature has increased expression of several growth factor receptors regulated by the AP-2 family of transcription factors. The gene expression signatures for AP-2alpha are further correlated with sensitivity to cetuximab treatment in HNSCC cell lines and changes in EGFR expression in HNSCC tumors with low CDKN2A gene expression. In addition, the AP-2alpha gene expression signatures are also associated with inhibition of MEK, PI3K, and mTOR pathways in the Library of Integrated Network-Based Cellular Signatures (LINCS) data. These results suggest that AP-2 transcription factors are activated as feedback from EGFR network inhibition and may mediate EGFR inhibitor resistance. PMID:27650546

DNA extraction methods for detecting genetically modified foods: A comparative study.

PubMed

Elsanhoty, Rafaat M; Ramadan, Mohamed Fawzy; Jany, Klaus Dieter

2011-06-15

The work presented in this manuscript was achieved to compare six different methods for extracting DNA from raw maize and its derived products. The methods that gave higher yield and quality of DNA were chosen to detect the genetic modification in the samples collected from the Egyptian market. The different methods used were evaluated for extracting DNA from maize kernels (without treatment), maize flour (mechanical treatment), canned maize (sweet corn), frozen maize (sweet corn), maize starch, extruded maize, popcorn, corn flacks, maize snacks, and bread made from corn flour (mechanical and thermal treatments). The quality and quantity of the DNA extracted from the standards, containing known percentages of GMO material and from the different food products were evaluated. For qualitative detection of the GMO varieties in foods, the GMOScreen 35S/NOS test kit was used, to screen the genetic modification in the samples. The positive samples for the 35S promoter and/or the NOS terminator were identified by the standard methods adopted by EU. All of the used methods extracted yielded good DNA quality. However, we noted that the purest DNA extract were obtained using the DNA extraction kit (Roche) and this generally was the best method for extracting DNA from most of the maize-derived foods. We have noted that the yield of DNA extracted from maize-derived foods was generally lower in the processed products. The results indicated that 17 samples were positive for the presence of 35S promoter, while 34% from the samples were positive for the genetically modified maize line Bt-176. Copyright © 2010 Elsevier Ltd. All rights reserved.

The beneficial effect of genetically engineered Schwann cells with enhanced motility in peripheral nerve regeneration: review.

PubMed

Gravvanis, A I; Lavdas, A A; Papalois, A; Tsoutsos, D A; Matsas, R

2007-01-01

The importance of Schwann cells in promoting nerve regeneration across a conduit has been extensively reported in the literature, and Schwann cell motility has been acknowledged as a prerequisite for myelination of the peripheral nervous system during regeneration after injury. Review of recent literature and retrospective analysis of our studies with genetically modified Schwann Cells with increased motility in order to identify the underlying mechanism of action and outline the future trends in peripheral nerve repair. Schwann cell transduction with the pREV-retrovirus, for expression of Sialyl-Transferase-X, resulting in conferring Polysialyl-residues (PSA) on NCAM, increases their motility in-vitro and ensures nerve regeneration through silicone tubes after end-to-side neurorraphy in the rat sciatic nerve model, thus significantly promoting fiber maturation and functional outcome. An artificial nerve graft consisting of a type I collagen tube lined with the genetically modified Schwann cells with increased motility, used to bridge a defect in end-to-end fashion in the rat sciatic nerve model, was shown to promote nerve regeneration to a level equal to that of a nerve autograft. The use of genetically engineered Schwann cells with enhanced motility for grafting endoneural tubes promotes axonal regeneration, by virtue of the interaction of the transplanted cells with regenerating axonal growth cones as well as via the recruitment of endogenous Schwann cells. It is envisaged that mixed populations of Schwann cells, expressing PSA and one or more trophic factors, might further enhance the regenerating and remyelinating potential of the lesioned nerves.

Nature and nurture- genes and environment- predict onset and progression of macular degeneration.

PubMed

Sobrin, Lucia; Seddon, Johanna M

2014-05-01

Age-related macular degeneration (AMD) is a common cause of irreversible visual loss and the disease burden is rising world-wide as the population ages. Both environmental and genetic factors contribute to the development of this disease. Among environmental factors, smoking, obesity and dietary factors including antioxidants and dietary fat intake influence onset and progression of AMD. There are also several lines of evidence that link cardiovascular, immune and inflammatory biomarkers to AMD. The genetic etiology of AMD has been and continues to be an intense and fruitful area of investigation. Genome-wide association studies have revealed numerous common variants associated with AMD and sequencing is increasing our knowledge of how rare genetic variants strongly impact disease. Evidence for interactions between environmental, therapeutic and genetic factors is emerging and elucidating the mechanisms of this interplay remains a major challenge in the field. Genotype-phenotype associations are evolving. The knowledge of non-genetic, modifiable risk factors along with information about heritability and genetic risk variants for this disease acquired over the past 25 years have greatly improved patient management and our ability to predict which patients will develop or progress to advanced forms of AMD. Personalized medicine and individualized prevention and treatment strategies may become a reality in the near future. Copyright © 2014. Published by Elsevier Ltd.

Phenotypic plasticity in the range-margin population of the lycaenid butterfly Zizeeria maha

PubMed Central

2010-01-01

Background Many butterfly species have been experiencing the northward range expansion and physiological adaptation, probably due to climate warming. Here, we document an extraordinary field case of a species of lycaenid butterfly, Zizeeria maha, for which plastic phenotypes of wing color-patterns were revealed at the population level in the course of range expansion. Furthermore, we examined whether this outbreak of phenotypic changes was able to be reproduced in a laboratory. Results In the recently expanded northern range margins of this species, more than 10% of the Z. maha population exhibited characteristic color-pattern modifications on the ventral wings for three years. We physiologically reproduced similar phenotypes by an artificial cold-shock treatment of a normal southern population, and furthermore, we genetically reproduced a similar phenotype after selective breeding of a normal population for ten generations, demonstrating that the cold-shock-induced phenotype was heritable and partially assimilated genetically in the breeding line. Similar genetic process might have occurred in the previous and recent range-margin populations as well. Relatively minor modifications expressed in the tenth generation of the breeding line together with other data suggest a role of founder effect in this field case. Conclusions Our results support the notion that the outbreak of the modified phenotypes in the recent range-margin population was primed by the revelation of plastic phenotypes in response to temperature stress and by the subsequent genetic process in the previous range-margin population, followed by migration and temporal establishment of genetically unstable founders in the recent range margins. This case presents not only an evolutionary role of phenotypic plasticity in the field but also a novel evolutionary aspect of range expansion at the species level. PMID:20718993

A design for the control of apoptosis in genetically modified Saccharomyces cerevisiae.

PubMed

Nishida, Nao; Noguchi, Misa; Kuroda, Kouichi; Ueda, Mitsuyoshi

2014-01-01

We have engineered a system that holds potential for use as a safety switch in genetically modified yeasts. Human apoptotic factor BAX (no homolog in yeast), under the control of the FBP1 (gluconeogenesis enzyme) promoter, was conditionally expressed to induce yeast cell apoptosis after glucose depletion. Such systems might prove useful for the safe use of genetically modified organisms.

TEOSINTE BRANCHED1 Regulates Inflorescence Architecture and Development in Bread Wheat (Triticum aestivum)[OPEN

PubMed Central

Greenwood, Julian R.; Bencivenga, Stefano; Cockram, James; Cavanagh, Colin; Swain, Steve M.

2018-01-01

The flowers of major cereals are arranged on reproductive branches known as spikelets, which group together to form an inflorescence. Diversity for inflorescence architecture has been exploited during domestication to increase crop yields, and genetic variation for this trait has potential to further boost grain production. Multiple genes that regulate inflorescence architecture have been identified by studying alleles that modify gene activity or dosage; however, little is known in wheat. Here, we show TEOSINTE BRANCHED1 (TB1) regulates inflorescence architecture in bread wheat (Triticum aestivum) by investigating lines that display a form of inflorescence branching known as “paired spikelets.” We show that TB1 interacts with FLOWERING LOCUS T1 and that increased dosage of TB1 alters inflorescence architecture and growth rate in a process that includes reduced expression of meristem identity genes, with allelic diversity for TB1 found to associate genetically with paired spikelet development in modern cultivars. We propose TB1 coordinates formation of axillary spikelets during the vegetative to floral transition and that alleles known to modify dosage or function of TB1 could help increase wheat yields. PMID:29444813

TEOSINTE BRANCHED1 Regulates Inflorescence Architecture and Development in Bread Wheat (Triticum aestivum).

PubMed

Dixon, Laura E; Greenwood, Julian R; Bencivenga, Stefano; Zhang, Peng; Cockram, James; Mellers, Gregory; Ramm, Kerrie; Cavanagh, Colin; Swain, Steve M; Boden, Scott A

2018-03-01

The flowers of major cereals are arranged on reproductive branches known as spikelets, which group together to form an inflorescence. Diversity for inflorescence architecture has been exploited during domestication to increase crop yields, and genetic variation for this trait has potential to further boost grain production. Multiple genes that regulate inflorescence architecture have been identified by studying alleles that modify gene activity or dosage; however, little is known in wheat. Here, we show TEOSINTE BRANCHED1 ( TB1 ) regulates inflorescence architecture in bread wheat ( Triticum aestivum ) by investigating lines that display a form of inflorescence branching known as "paired spikelets." We show that TB1 interacts with FLOWERING LOCUS T1 and that increased dosage of TB1 alters inflorescence architecture and growth rate in a process that includes reduced expression of meristem identity genes, with allelic diversity for TB1 found to associate genetically with paired spikelet development in modern cultivars. We propose TB1 coordinates formation of axillary spikelets during the vegetative to floral transition and that alleles known to modify dosage or function of TB1 could help increase wheat yields. © 2018 American Society of Plant Biologists. All rights reserved.

Modifications of allergenicity linked to food technologies.

PubMed

Moneret-Vautrin, D A

1998-01-01

The prevalence of food allergies (FA) has increased over the past fifteen years. The reasons suggested are changes in dietary behaviour and the evolution of food technologies. New cases of FA have been described with chayote, rambutan, arguta, pumpkin seeds, custard apple, and with mycoproteins from Fusarium.... Additives using food proteins are at high risk: caseinates, lysozyme, cochineal red, papaïn, alpha-amylase, lactase etc. Heating can reduce allergenicity or create neo-allergens, as well as storage, inducing the synthesis of allergenic stress or PR proteins. Aeroallergens (miles, moulds) contaminate foods and can induce allergic reactions. Involuntary contamination by peanut proteins on production lines is a problem which is not yet solved. Genetically modified plants are at risk of allergenicity, requiring methodological steps of investigations: the comparison of the amino-acid sequence of the transferred protein with the sequence of known allergens, the evaluation of thermo degradability and of the denaturation by pepsin and trypsin are required, as well as the study with sera from patients allergic to the plant producing the gene. The combination of enzymatic hydrolysis, heating, or the development of genetically modified plants may offer new alternatives towards hypoallergenic foods (57 references).

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis.

PubMed

Puttini, Stefania; Ouvrard-Pascaud, Antoine; Palais, Gael; Beggah, Ahmed T; Gascard, Philippe; Cohen-Tannoudji, Michel; Babinet, Charles; Blot-Chabaud, Marcel; Jaisser, Frederic

2005-03-16

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.

Disease-modifying genetic factors in cystic fibrosis.

PubMed

Marson, Fernando A L

2018-05-01

To compile data from the past 10 years regarding the role of modifying genes in cystic fibrosis (CF). CF is a model disease for understanding of the action of modifying genes. Although it is a monogenic (CFTR) autosomal recessive disease, CF presents with wide phenotypic variability. In CF, variability occurs with different intensity among patients by each organ, being organ-specific, resulting from the mutual interaction of environmental and genetic factors, including CFTR mutations and various other genes, most of which are associated with inflammatory processes. In individuals, using precision medicine, gene modification studies have revealed individualized responses to drugs depending on particular CFTR mutations and modifying genes, most of which are alternative ion channels. Studies of modifying genes in CF allow: understanding of clinical variability among patients with the same CFTR genotype; evaluation of precision medicine; understanding of environmental and genetic effects at the organ level; understanding the involvement of genetic variants in inflammatory responses; improvements in genetic counseling; understanding the involvement of genetic variants in inflammatory responses in lung diseases, such as asthma; and understanding the individuality of the person with the disease.

Robust imaging and gene delivery to study human lymphoblastoid cell lines.

PubMed

Jolly, Lachlan A; Sun, Ying; Carroll, Renée; Homan, Claire C; Gecz, Jozef

2018-06-20

Lymphoblastoid cell lines (LCLs) have been by far the most prevalent cell type used to study the genetics underlying normal and disease-relevant human phenotypic variation, across personal to epidemiological scales. In contrast, only few studies have explored the use of LCLs in functional genomics and mechanistic studies. Two major reasons are technical, as (1) interrogating the sub-cellular spatial information of LCLs is challenged by their non-adherent nature, and (2) LCLs are refractory to gene transfection. Methodological details relating to techniques that overcome these limitations are scarce, largely inadequate (without additional knowledge and expertise), and optimisation has never been described. Here we compare, optimise, and convey such methods in-depth. We provide a robust method to adhere LCLs to coverslips, which maintained cellular integrity, morphology, and permitted visualisation of sub-cellular structures and protein localisation. Next, we developed the use of lentiviral-based gene delivery to LCLs. Through empirical and combinatorial testing of multiple transduction conditions, we improved transduction efficiency from 3% up to 48%. Furthermore, we established strategies to purify transduced cells, to achieve sustainable cultures containing >85% transduced cells. Collectively, our methodologies provide a vital resource that enables the use of LCLs in functional cell and molecular biology experiments. Potential applications include the characterisation of genetic variants of unknown significance, the interrogation of cellular disease pathways and mechanisms, and high-throughput discovery of genetic modifiers of disease states among others.

76 FR 78232 - Monsanto Co.; Determination of Nonregulated Status for Soybean Genetically Engineered To Have a...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-16

... peer review of safety tests, and health effects of genetically modified organisms and glyphosate. APHIS...] Monsanto Co.; Determination of Nonregulated Status for Soybean Genetically Engineered To Have a Modified... that there is reason to believe are plant pests. Such genetically engineered organisms and products are...

Childhood chronic inflammatory demyelinating polyneuropathy: an overview of 10 cases in the modern era.

PubMed

Ware, Tyson L; Kornberg, Andrew J; Rodriguez-Casero, M Victoria; Ryan, Monique M

2014-01-01

Chronic inflammatory demyelinating polyneuropathy is a rare condition in children. In this article, we report our experience in the management of 10 cases of childhood chronic inflammatory demyelinating polyneuropathy in a single center, in the era of contrast-enhanced magnetic resonance imaging (MRI), genetic microarray, and chronic inflammatory demyelinating polyneuropathy disease activity status. Robust neurophysiologic abnormalities were present in all cases and both MRI and lumbar puncture were useful adjuncts in diagnosis. Genetic microarray is a simple technique useful in excluding the most common hereditary demyelinating neuropathy. Intravenous immunoglobulin was an effective first-line therapy in most cases, with refractory cases responding to corticosteroids and rituximab. We found the chronic inflammatory demyelinating polyneuropathy disease activity status useful for assessing outcome at final follow-up, whereas the modified Rankin score was better for assessing peak motor disability.

Pyrolysis-field ionization mass spectrometry of rhizodeposits - a new approach to identify potential effects of genetically modified plants on soil organisms.

PubMed

Melnitchouck, Alexei; Leinweber, Peter; Broer, Inge; Eckhardt, Kai-Uwe

2006-01-01

The objectives of the present study were (1) to investigate the qualitative composition of rhizodeposits leached from soils cropped with non-transgenic and genetically modified (GM) potatoes, and disclose if there were GM-specific modifications in potato rhizodeposition, and (2) to compare these results with conventional bulk parameters of microbial activity in soil. We have raised potatoes from a non-transgenic line (Solanum tuberosum L. cv. Désirée) and three GM lines, which expressed a gene for the resistance to kanamycin (DLH 9000) and a gene for T4 lysozyme (DL10 and DL12). A sandy soil placed in 340 cm3-"CombiSart" containers was used, from which the rhizodeposit was leached after a six-week growth period. The freeze-dried leachates were analyzed by pyrolysis-field ionization mass spectrometry (Py-FIMS). The Py-FI mass spectra gave detailed molecular-chemical information about the composition of leachates, indicating that the potato growth generally altered the composition of the soil solution. Moreover, a principal component analysis of the mass spectra showed differences between the leachates from the non-transgenic parent line and the GM potatoes as well as among the latter group. However, these differences in molecular composition could not be assigned to the release of T4-lysozyme into soil. Dehydrogenase activity and substrate-induced soil respiration as more common bulk parameters of soil microbial activity failed to disclose any significant effects of the various potatoes grown. The limitations of the described rhizodeposit leaching and analysis for risk assessment of GM potato cropping under field conditions are discussed critically. However, it could be concluded that the Py-FI mass spectrometric "fingerprint" can be developed as a fast, comprehensive, highly sensitive and reproducible analytical approach to discern any effects GM-crops may exert on soil ecological parameters.

Avoiding genetically modified foods in GMO Ground Zero: A reflective self-narrative.

PubMed

Edwards, Sachi

2015-05-01

I engage in a reflective self-narrative of my experience attempting to maintain a diet free of genetically modified organisms. Social tension over the genetically modified organism industry in Hawai'i, United States, has led to public debates over jobs and social identities. Drawing on local media sources, grassroots organizations, and blog posts, I describe the way this tension has shaped my experience with food, eating, and being with others as a genetically modified organism avoider. I utilize discursive positioning to make sense of my experiences by locating them within the ongoing public conversations that give structure to the daily lives of Hawai'i's residents. © The Author(s) 2015.

How scary! An analysis of visual communication concerning genetically modified organisms in Italy.

PubMed

Ventura, Vera; Frisio, Dario G; Ferrazzi, Giovanni; Siletti, Elena

2017-07-01

Several studies provide evidence of the role of written communication in influencing public perception towards genetically modified organisms, whereas visual communication has been sparsely investigated. This article aims to evaluate the exposure of the Italian population to scary genetically modified organism-related images. A set of 517 images collected through Google are classified considering fearful attributes, and an index that accounts for the scary impact of these images is built. Then, through an ordinary least-squares regression, we estimate the relationship between the Scary Impact Index and a set of variables that describes the context in which the images appear. The results reveal that the first (and most viewed) Google result images contain the most frightful contents. In addition, the agri-food sector in Italy is strongly oriented towards offering a negative representation of genetically modified organisms. Exposure to scary images could be a factor that affects the negative perception of genetically modified organisms in Italy.

Effects of genetically modified T2A-1 rice on the GI health of rats after 90-day supplement

PubMed Central

Yuan, Yanfang; Xu, Wentao; He, Xiaoyun; Liu, Haiyan; Cao, Sishuo; Qi, Xiaozhe; Huang, Kunlun; Luo, Yunbo

2013-01-01

Bacillus thuringiensis insecticidal toxin (Bt) rice will be commercialized as a main food source. Traditional safety assessments on genetically modified products pay little attention on gastrointestinal (GI) health. More data about GI health of Bt rice must be provided to dispel public' doubts about the potential effects on human health. We constructed an improved safety assessment animal model using a basic subchronic toxicity experiment, measuring a range of parameters including microflora composition, intestinal permeability, epithelial structure, fecal enzymes, bacterial activity, and intestinal immunity. Significant differences were found between rice-fed groups and AIN93G-fed control groups in several parameters, whereas no differences were observed between genetically modified and non-genetically modified groups. No adverse effects were found on GI health resulting from genetically modified T2A-1 rice. In conclusion, this study may offer a systematic safety assessment model for GM material with respect to the effects on GI health. PMID:23752350

Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line

PubMed Central

STOCZYNSKA-FIDELUS, EWELINA; PIASKOWSKI, SYLWESTER; PAWLOWSKA, ROZA; SZYBKA, MALGORZATA; PECIAK, JOANNA; HULAS-BIGOSZEWSKA, KRYSTYNA; WINIECKA-KLIMEK, MARTA; RIESKE, PIOTR

2016-01-01

Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research. PMID:26870252

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

PubMed

Yang, Litao; Pan, Aihu; Zhang, Kewei; Guo, Jinchao; Yin, Changsong; Chen, Jianxiu; Huang, Cheng; Zhang, Dabing

2005-08-10

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

Photosynthetic characteristics in genetically modified sense-RbcS silver birch lines.

PubMed

Kontunen-Soppela, Sari; Sillanpää, Maarit; Tuhkanen, Eeva-Maria; Sutinen, Sirkka; Kangasjärvi, Jaakko; Vapaavuori, Elina; Häggman, Hely

2010-07-01

Transgenic silver birch lines carrying extra copies of endogenous small subunit of Rubisco (RbcS)-gene under 35S CaMV promoter were used to study the carbon use efficiency of silver birch (Betula pendula Roth). A five week greenhouse experiment was carried out with four transgenic lines, R3.2, R7.2, E5 and E25, and their corresponding wild types (wt). The first fully developed leaves were used for analyses. Three of the produced lines, R3.2, E5 and E25, differed from the wt lines. Line R3.2 showed an altered growth rhythm; its chlorophyll content, Rubisco amount and activity as well as photosynthetic characteristics were reduced at the beginning of the experiment, which resulted in decreased biomass and growth. In lines E25 and E5, the biomass accumulation was shifted to roots, and in line E25, the total biomass was also reduced. In line E25, the differences were particularly marked in the dry mass, indicating a difference in water use, seen as increased transpiration. Introduction of sense RbcS decreased the Rubisco amount in birch leaves to 80% of wt at times during the tree development, but the lower amount of Rubisco was usually not seen in photosynthesis. The accumulation and distribution of biomass within the plants was altered. 2010 Elsevier GmbH. All rights reserved.

Variable phenotypic expressivity in inbred retinal degeneration mouse lines: A comparative study of C3H/HeOu and FVB/N rd1 mice.

PubMed

van Wyk, Michiel; Schneider, Sabine; Kleinlogel, Sonja

2015-01-01

Recent advances in optogenetics and gene therapy have led to promising new treatment strategies for blindness caused by retinal photoreceptor loss. Preclinical studies often rely on the retinal degeneration 1 (rd1 or Pde6b(rd1)) retinitis pigmentosa (RP) mouse model. The rd1 founder mutation is present in more than 100 actively used mouse lines. Since secondary genetic traits are well-known to modify the phenotypic progression of photoreceptor degeneration in animal models and human patients with RP, negligence of the genetic background in the rd1 mouse model is unwarranted. Moreover, the success of various potential therapies, including optogenetic gene therapy and prosthetic implants, depends on the progress of retinal degeneration, which might differ between rd1 mice. To examine the prospect of phenotypic expressivity in the rd1 mouse model, we compared the progress of retinal degeneration in two common rd1 lines, C3H/HeOu and FVB/N. We followed retinal degeneration over 24 weeks in FVB/N, C3H/HeOu, and congenic Pde6b(+) seeing mouse lines, using a range of experimental techniques including extracellular recordings from retinal ganglion cells, PCR quantification of cone opsin and Pde6b transcripts, in vivo flash electroretinogram (ERG), and behavioral optokinetic reflex (OKR) recordings. We demonstrated a substantial difference in the speed of retinal degeneration and accompanying loss of visual function between the two rd1 lines. Photoreceptor degeneration and loss of vision were faster with an earlier onset in the FVB/N mice compared to C3H/HeOu mice, whereas the performance of the Pde6b(+) mice did not differ significantly in any of the tests. By postnatal week 4, the FVB/N mice expressed significantly less cone opsin and Pde6b mRNA and had neither ERG nor OKR responses. At 12 weeks of age, the retinal ganglion cells of the FVB/N mice had lost all light responses. In contrast, 4-week-old C3H/HeOu mice still had ERG and OKR responses, and we still recorded light responses from C3H/HeOu retinal ganglion cells until the age of 24 weeks. These results show that genetic background plays an important role in the rd1 mouse pathology. Analogous to human RP, the mouse genetic background strongly influences the rd1 phenotype. Thus, different rd1 mouse lines may follow different timelines of retinal degeneration, making exact knowledge of genetic background imperative in all studies that use rd1 models.

Autism-related neuroligin-3 mutation alters social behavior and spatial learning.

PubMed

Jaramillo, Thomas C; Liu, Shunan; Pettersen, Ami; Birnbaum, Shari G; Powell, Craig M

2014-04-01

Multiple candidate genes have been identified for autism spectrum disorders. While some of these genes reach genome-wide significance, others, such as the R451C point mutation in the synaptic cell adhesion molecule neuroligin-3, appear to be rare. Interestingly, two brothers with the same R451C point mutation in neuroligin-3 present clinically on seemingly disparate sides of the autism spectrum. These clinical findings suggest genetic background may play a role in modifying the penetrance of a particular autism-associated mutation. Animal models may contribute additional support for such mutations as functionally relevant and can provide mechanistic insights. Previously, in collaboration with the Südhof laboratory, we reported that mice with an R451C substitution in neuroligin-3 displayed social deficits and enhanced spatial learning. While some of these behavioral abnormalities have since been replicated independently in the Südhof laboratory, observations from the Crawley laboratory failed to replicate these findings in a similar neuroligin-3 mutant mouse model and suggested that genetic background may contribute to variation in observations across laboratories. Therefore, we sought to replicate our findings in the neuroligin-3 R451C point mutant knock-in mouse model (NL3R451C) in a different genetic background. We backcrossed our NL3R451C mouse line onto a 129S2/SvPasCrl genetic background and repeated a subset of our previous behavioral testing. NL3R451C mice on a 129S2/SvPasCrl displayed social deficits, enhanced spatial learning, and increased locomotor activity. These data extend our previous findings that NL3R451C mice exhibit autism-relevant behavioral abnormalities and further suggest that different genetic backgrounds can modify this behavioral phenotype through epistatic genetic interactions. © 2014 International Society for Autism Research, Wiley Periodicals, Inc.

[Methods of identification and assessment of safety of genetically modified microorganisms in manufacture food production].

PubMed

Khovaev, A A; Nesterenko, L N; Naroditskiĭ, B S

2011-01-01

Methods of identification of genetically modified microorganisms (GMM), used in manufacture food on control probes are presented. Results of microbiological and molecular and genetic analyses of food products and their components important in microbiological and genetic expert examination of GMM in foods are considered. Examination of biosafety of GMM are indicated.

The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes.

PubMed

Hirai, Tadayoshi; Kurokawa, Natsuko; Duhita, Narendra; Hiwasa-Tanase, Kyoko; Kato, Kazuhisa; Kato, Ko; Ezura, Hiroshi

2011-09-28

High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.

Strategies and Considerations for Distributing and Recovering Mouse Lines

PubMed Central

Du, Yubin; Xie, Wen; Liu, Chengyu

2012-01-01

As more and more genetically modified mouse lines are being generated, it becomes increasingly common to share animal models among different research institutions. Live mice are routinely transferred between animal facilities. Due to various issues concerning animal welfare, intellectual property rights, colony health status and biohazard, significant paperwork and coordination are required before any animal travel can take place. Shipping fresh or frozen preimplantation embryos, gametes, or reproductive organs can bypass some of the issues associated with live animal transfer, but it requires the receiving facilities to be able to perform delicate and sometimes intricate procedures such as embryo transfer, in vitro fertilization (IVF), or ovary transplantation. Here, we summarize the general requirements for live animal transport and review some of the assisted reproductive technologies (ART) that can be applied to shipping and reviving mouse lines. Intended users of these methods should consult their institution’s responsible official to find out whether each specific method is legal or appropriate in their own animal facilities. PMID:20691859

Selection indices for quality evaluation in wheat breeding.

PubMed

Branlard, G; Pierre, J; Rousset, M

1992-06-01

From multilocation trials involving 125 cultivars of wheat of mainly French and European origin four tests - protein content, Pelshenke, modified Zeleny and the mixograph - were used to establish six selection indices. Three of these indices - IW1, IW2 and IW3 - were calculated in order to evaluate the genetic potentiality of the lines for dough strength as given by the Chopin alveograph. The indices IV1, IV2 and IV3 were established to evaluate loaf volume as measured by the French bread-making standard. A quality index IQ was calculated from the allelic effects of the high-molecular-weight (HMW) subunits of glutenin from 195 cultivars assessed by the Chopin alveograph and the Pelshenke test. Comparison of the relative efficiency of each of the six indices to the individual tests revealed the superiority of the indices over one or several technological parameters. The six selection indices and the quality index were compared using 30 very diverse F4 lines. Their ability to retain the good quality lines is discussed in particular.

Avian Biotechnology.

PubMed

Nakamura, Yoshiaki

2017-01-01

Primordial germ cells (PGCs) generate new individuals through differentiation, maturation and fertilization. This means that the manipulation of PGCs is directly linked to the manipulation of individuals, making PGCs attractive target cells in the animal biotechnology field. A unique biological property of avian PGCs is that they circulate temporarily in the vasculature during early development, and this allows us to access and manipulate avian germ lines. Following the development of a technique for transplantation, PGCs have become central to avian biotechnology, in contrast to the use of embryo manipulation and subsequent transfer to foster mothers, as in mammalian biotechnology. Today, avian PGC transplantation combined with recent advanced manipulation techniques, including cell purification, cryopreservation, depletion, and long-term culture in vitro, have enabled the establishment of genetically modified poultry lines and ex-situ conservation of poultry genetic resources. This chapter introduces the principles, history, and procedures of producing avian germline chimeras by transplantation of PGCs, and the current status of avian germline modification as well as germplasm cryopreservation. Other fundamental avian reproductive technologies are described, including artificial insemination and embryo culture, and perspectives of industrial applications in agriculture and pharmacy are considered, including poultry productivity improvement, egg modification, disease resistance impairment and poultry gene "pharming" as well as gene banking.

Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6)

PubMed Central

Dormady, Shane P.; Zhang, Xin-Min; Basch, Ross S.

2000-01-01

Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively γ-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be γ-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells. PMID:11050245

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.

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

40 CFR 172.45 - Requirement for a notification.

Code of Federal Regulations, 2010 CFR

2010-07-01

... EXPERIMENTAL USE PERMITS Notification for Certain Genetically Modified Microbial Pesticides § 172.45... modified. (2) Nonindigenous microbial pesticides that have not been acted upon by the U.S. Department of... introduction of genetic material that has been deliberately modified. (ii) [Reserved] (2) Testing conducted in...

Coherent spectroscopic methods for monitoring pathogens, genetically modified products and nanostructured materials in colloidal solution

NASA Astrophysics Data System (ADS)

Moguilnaya, T.; Suminov, Y.; Botikov, A.; Ignatov, S.; Kononenko, A.; Agibalov, A.

2017-01-01

We developed the new automatic method that combines the method of forced luminescence and stimulated Brillouin scattering. This method is used for monitoring pathogens, genetically modified products and nanostructured materials in colloidal solution. We carried out the statistical spectral analysis of pathogens, genetically modified soy and nano-particles of silver in water from different regions in order to determine the statistical errors of the method. We studied spectral characteristics of these objects in water to perform the initial identification with 95% probability. These results were used for creation of the model of the device for monitor of pathogenic organisms and working model of the device to determine the genetically modified soy in meat.

Prenatal Air Pollution Exposures, DNA Methyl Transferase Genotypes, and Associations with Newborn LINE1 and Alu Methylation and Childhood Blood Pressure and Carotid Intima-Media Thickness in the Children's Health Study.

PubMed

Breton, Carrie V; Yao, Jin; Millstein, Josh; Gao, Lu; Siegmund, Kimberly D; Mack, Wendy; Whitfield-Maxwell, Lora; Lurmann, Fred; Hodis, Howard; Avol, Ed; Gilliland, Frank D

2016-12-01

Although exposure to ambient air pollutants increases cardiovascular disease risk in adults little is known about the effects of prenatal exposure. Genetic variation and epigenetic alterations are two mechanisms that may influence the effects of early-life exposures on cardiovascular phenotypes. We investigated whether genetic and epigenetic variation modify associations between prenatal air pollution on markers of cardiovascular risk in childhood. We used linear regression analysis to investigate the associations between prenatal pollutants (PM2.5, PM10, NO2, O3), long interspersed nuclear elements (LINE1) and AluYb8 DNA methylation levels measured in newborn blood spot tests, and carotid intima-media thickness (CIMT) and blood pressure (BP) in 459 participants as part of the Children's Health Study. Interaction terms were also included to test for effect modification of these associations by genetic variation in methylation reprogramming genes. Prenatal exposure to NO2 in the third trimester of pregnancy was associated with higher systolic BP in 11-year-old children. Prenatal exposure to multiple air pollutants in the first trimester was associated with lower DNA methylation in LINE1, whereas later exposure to O3 was associated with higher LINE1 methylation levels in newborn blood spots. The magnitude of associations with prenatal air pollution varied according to genotype for 11 SNPs within DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 Beta (DNMT3B), Tet methylcytosine dioxygenase 2 (TET2), and Thymine DNA glycosylase (TDG) genes. Although first-trimester O3 exposure was not associated with CIMT and systolic BP overall, associations within strata of DNMT1 or DNMT3B were observed, and the magnitude and the direction of these associations depended on DNMT1 genotypes. Genetic and epigenetic variation in DNA methylation reprogramming genes and in LINE1 retrotransposons may play important roles in downstream cardiovascular consequences of prenatal air pollution exposure. Citation: Breton CV, Yao J, Millstein J, Gao L, Siegmund KD, Mack W, Whitfield-Maxwell L, Lurmann F, Hodis H, Avol E, Gilliland FD. 2016. Prenatal air pollution exposures, DNA methyl transferase genotypes, and associations with newborn LINE1 and Alu methylation and childhood blood pressure and carotid intima-media thickness in the Children's Health Study. Environ Health Perspect 124:1905-1912; http://dx.doi.org/10.1289/EHP181.

Development of a colloidal gold immunochromatographic strip assay for simple and fast detection of human α-lactalbumin in genetically modified cow milk.

PubMed

Tao, Chenyu; Zhang, Qingde; Feng, Na; Shi, Deshi; Liu, Bang

2016-03-01

The qualitative and quantitative declaration of food ingredients is important to consumers, especially for genetically modified food as it experiences a rapid increase in sales. In this study, we designed an accurate and rapid detection system using colloidal gold immunochromatographic strip assay (GICA) methods to detect genetically modified cow milk. First, we prepared 2 monoclonal antibodies for human α-lactalbumin (α-LA) and measured their antibody titers; the one with the higher titer was used for further experiments. Then, we found the optimal pH value and protein amount of GICA for detection of pure milk samples. The developed strips successfully detected genetically modified cow milk and non-modified cow milk. To determine the sensitivity of GICA, a quantitative ELISA system was used to determine the exact amount of α-LA, and then genetically modified milk was diluted at different rates to test the sensitivity of GICA; the sensitivity was 10 μg/mL. Our results demonstrated that the applied method was effective to detect human α-LA in cow milk. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Emotional attitudes of young people completing secondary schools towards genetic modification of organisms (GMO) and genetically modified foods (GMF).

PubMed

Jurkiewicz, Anna; Zagórski, Jerzy; Bujak, Franciszek; Lachowski, Stanisław; Florek-Łuszczki, Magdalena

2014-01-01

The objective of the study was recognition of the opinions of adolescents completing secondary schools concerning genetically modified organisms and genetically modified food, especially the respondents' emotional attitude towards scientific achievements in the area of live genetically modified organisms. The study covered a group of 500 school adolescents completing secondary school at the level of maturity examination. The study was conducted by the method of a diagnostic survey using a self-designed questionnaire form. Knowledge concerning the possible health effects of consumption of food containing GMO among adolescents competing secondary schools is on a relatively low level; the adolescents examined 'know rather little' or 'very little know' about this problem. In respondents' opinions the results of reliable studies pertaining to the health effects of consumption of GMO 'rather do not exist'. The respondents are against the cultivation of GM plants and breeding of GM animals on own farm in the future. Secondary school adolescents considered that the production of genetically modified food means primarily the enrichment of biotechnological companies, higher income for food producers, and not the elimination of hunger in the world or elimination of many diseases haunting humans.

[Safety assessment of foods derived from genetically modified plants].

PubMed

Pöting, A; Schauzu, M

2010-06-01

The placing of genetically modified plants and derived food on the market falls under Regulation (EC) No. 1829/2003. According to this regulation, applicants need to perform a safety assessment according to the Guidance Document of the Scientific Panel on Genetically Modified Organisms of the European Food Safety Authority (EFSA), which is based on internationally agreed recommendations. This article gives an overview of the underlying legislation as well as the strategy and scientific criteria for the safety assessment, which should generally be based on the concept of substantial equivalence and carried out in relation to an unmodified conventional counterpart. Besides the intended genetic modification, potential unintended changes also have to be assessed with regard to potential adverse effects for the consumer. All genetically modified plants and derived food products, which have been evaluated by EFSA so far, were considered to be as safe as products derived from the respective conventional plants.

[A Study of the Relationship Among Genetic Distances, NIR Spectra Distances, and NIR-Based Identification Model Performance of the Seeds of Maize Iinbred Lines].

PubMed

Liu, Xu; Jia, Shi-qiang; Wang, Chun-ying; Liu, Zhe; Gu, Jian-cheng; Zhai, Wei; Li, Shao-ming; Zhang, Xiao-dong; Zhu, De-hai; Huang, Hua-jun; An, Dong

2015-09-01

This paper explored the relationship among genetic distances, NIR spectra distances and NIR-based identification model performance of the seeds of maize inbred lines. Using 3 groups (total 15 pairs) of maize inbred lines whose genetic distaches are different as experimental materials, we calculates the genetic distance between these seeds with SSR markers and uses Euclidean distance between distributed center points of maize NIR spectrum in the PCA space as the distances of NIR spectrum. BPR method is used to build identification model of inbred lines and the identification accuracy is used as a measure of model identification performance. The results showed that, the correlation of genetic distance and spectra distancesis 0.9868, and it has a correlation of 0.9110 with the identification accuracy, which is highly correlated. This means near-Infrared spectrum of seedscan reflect genetic relationship of maize inbred lines. The smaller the genetic distance, the smaller the distance of spectrum, the poorer ability of model to identify. In practical application, near infrared spectrum analysis technology has the potential to be used to analyze maize inbred genetic relations, contributing much to genetic breeding, identification of species, purity sorting and so on. What's more, when creating a NIR-based identification model, the impact of the maize inbred lines which have closer genetic relationship should be fully considered.

Not all GMOs are crop plants: non-plant GMO applications in agriculture

USDA-ARS?s Scientific Manuscript database

In the time since the tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteri...

Therapeutic potential of intracerebroventricular replacement of modified human β-hexosaminidase B for GM2 gangliosidosis.

PubMed

Matsuoka, Kazuhiko; Tamura, Tomomi; Tsuji, Daisuke; Dohzono, Yukie; Kitakaze, Keisuke; Ohno, Kazuki; Saito, Seiji; Sakuraba, Hitoshi; Itoh, Kohji

2011-06-01

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

Therapeutic Potential of Intracerebroventricular Replacement of Modified Human β-Hexosaminidase B for GM2 Gangliosidosis

PubMed Central

Matsuoka, Kazuhiko; Tamura, Tomomi; Tsuji, Daisuke; Dohzono, Yukie; Kitakaze, Keisuke; Ohno, Kazuki; Saito, Seiji; Sakuraba, Hitoshi; Itoh, Kohji

2011-01-01

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB. PMID:21487393

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

PubMed

Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

2010-01-01

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

Identification of Associations Between Genetic Factors and Asthma that are Modified by Obesity

DTIC Science & Technology

2016-06-01

AFRL-SA-WP-TR-2016-0010 Identification of Associations Between Genetic Factors and Asthma That Are Modified by Obesity Andrew T...Between Genetic Factors and Asthma That Are Modified by Obesity 5a. CONTRACT NUMBER FA8650-13-2-6371 5b. GRANT NUMBER 5c. PROGRAM ELEMENT...among African American women in the Women’s Health Initiative study. 15. SUBJECT TERMS Body mass index, SNP, asthma, obesity , genome, genes 16

Unscented Sampling Techniques For Evolutionary Computation With Applications To Astrodynamic Optimization

DTIC Science & Technology

2016-09-01

to both genetic algorithms and evolution strategies to achieve these goals. The results of this research offer a promising new set of modified ...abs_all.jsp?arnumber=203904 [163] Z. Michalewicz, C. Z. Janikow, and J. B. Krawczyk, “A modified genetic algo- rithm for optimal control problems...Available: http://arc.aiaa.org/doi/abs/10.2514/ 2.7053 375 [166] N. Yokoyama and S. Suzuki, “ Modified genetic algorithm for constrained trajectory

[Impacts of genetically modified soybean leaf residues on Folsomia candida.

PubMed

Zhou, Lin; Wang, Bai Feng; Liu, Xin Ying; Jiang, Ying; Wang, Da Ming; Feng, Shu Dan; Song, Xin Yuan

2016-09-01

When the genetically modified soybean is planted in the field, the expression product of exogenous gene could be exposed in the soil ecosystem and bring potential risk to the soil fauna, with the form of leaves and other debris. A few of genetically modified soybeans developed by China independently were used in our study as materials. They were Phytophthora-resistant soybean harboring hrpZm gene (B4J8049), leaf-feeding insect-resistant soybean harboring Cry1C gene (A2A8001) and Leguminivora glycinivorella-resistant soybean harboring Cry1Iem gene (C802). By feeding Folsomia candida with the three genetically modified soybeans for continuous 60 days, the surviving rate, reproductive rate and changes on the body length of F. candida were studied. The results showed that all the three genetically modified soybeans of B4J8049, A2A8001 and C802 had no significant adverse effects on the growth of F. candida, as an environmental indicator organism. It was initially inferred that they were environmentally safe under short-term exposure, which provided basic data of ecological safety for their wide cultivation.

Genetic basis and detection of unintended effects in genetically modified crop plants

USDA-ARS?s Scientific Manuscript database

In January 2014, an international meeting sponsored by the International Life Sciences Institute/Health and Environmental Sciences Institute and the Canadian Food Inspection Agency titled “Genetic Basis of Unintended Effects in Modified Plants” was held in Ottawa, Canada, bringing together over 75 s...

76 FR 37771 - Monsanto Co.; Availability of Petition, Plant Pest Risk Assessment, and Environmental Assessment...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-28

... Determination of Nonregulated Status for Soybean Genetically Engineered To Have a Modified Fatty Acid Profile... soybean designated as MON 87705, which has been genetically engineered to have a modified fatty acid... our regulations concerning the introduction of certain genetically engineered organisms and products...

Genetic association studies in β-hemoglobinopathies.

PubMed

Thein, Swee Lay

2013-01-01

Characterization of the molecular basis of the β-thalassemias and sickle cell disease (SCD) clearly showed that individuals with the same β-globin genotypes can have extremely diverse clinical severity. Two key modifiers, an innate ability to produce fetal hemoglobin and coinheritance of α-thalassemia, both derived from family and population studies, affect the pathophysiology of both disorders at the primary level. In the past 2 decades, scientific research had applied genetic approaches to identify additional genetic modifiers. The review summarizes recent genetic studies and key genetic modifiers identified and traces the story of fetal hemoglobin genetics, which has led to an emerging network of globin gene regulation. The discoveries have provided insights on new targets for therapeutic intervention and raise possibilities of developing fetal hemoglobin predictive diagnostics for predicting disease severity in the newborn and for integration into prenatal diagnosis to better inform genetic counseling.

K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates

PubMed Central

Straimer, Judith; Gnädig, Nina F.; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D.; Urnov, Fyodor D.; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M.; Ménard, Didier; Fidock, David A.

2015-01-01

The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

Leucine-Rich Repeat Kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinson's disease.

PubMed

Venderova, Katerina; Kabbach, Ghassan; Abdel-Messih, Elizabeth; Zhang, Yi; Parks, Robin J; Imai, Yuzuru; Gehrke, Stephan; Ngsee, Johnny; Lavoie, Matthew J; Slack, Ruth S; Rao, Yong; Zhang, Zhuohua; Lu, Bingwei; Haque, M Emdadul; Park, David S

2009-11-15

Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's disease (PD). However, its physiological and pathological functions are unknown. Therefore, we generated several independent Drosophila lines carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes.

Genetically Modified Foods and Consumer Perspective.

PubMed

Boccia, Flavio; Sarnacchiaro, Pasquale

2015-01-01

Genetically modified food is able to oppose the world's hunger and preserve the environment, even if the patents in this matter are symptomatic of several doubts. And also, transgenic consumption causes problems and skepticism among consumers in several European countries, but above all in Italy, where there is a strong opposition over recent years. So, the present study conducted a research to study the consumption of genetically modified food products by Italian young generation. This research presented the following purposes: firstly, to analyze genetically modified products' consumption among a particular category of consumers; secondly, to implement a quantitative model to understand behaviour about this particular kind of consumption and identify the factors that determine their purchase. The proposed model shows that transgenic consumption is especially linked to knowledge and impact on environment and mankind's health.

Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.

PubMed

Song, Qinxin; Wei, Guijiang; Zhou, Guohua

2014-07-01

A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish.

PubMed

Carroll, Kelli J; North, Trista E

2014-08-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Oceans of Opportunity: Exploring Vertebrate Hematopoiesis in Zebrafish

PubMed Central

Carroll, Kelli J.; North, Trista E.

2015-01-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell (HSPC) biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor and effector cell emergence, expansion and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell-types can be identified and characterized. Further, a myriad of transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of HSPC biology during development, in response to infection or injury, or in the setting of hematological malignancy, continues to deepen, zebrafish will remain essential for exploring the spatio-temporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. PMID:24816275

Dynamic analysis and vibration testing of CFRP drive-line system used in heavy-duty machine tool

NASA Astrophysics Data System (ADS)

Yang, Mo; Gui, Lin; Hu, Yefa; Ding, Guoping; Song, Chunsheng

2018-03-01

Low critical rotary speed and large vibration in the metal drive-line system of heavy-duty machine tool affect the machining precision seriously. Replacing metal drive-line with the CFRP drive-line can effectively solve this problem. Based on the composite laminated theory and the transfer matrix method (TMM), this paper puts forward a modified TMM to analyze dynamic characteristics of CFRP drive-line system. With this modified TMM, the CFRP drive-line of a heavy vertical miller is analyzed. And the finite element modal analysis model of the shafting is established. The results of the modified TMM and finite element analysis (FEA) show that the modified TMM can effectively predict the critical rotary speed of CFRP drive-line. And the critical rotary speed of CFRP drive-line is 20% higher than that of the original metal drive-line. Then, the vibration of the CFRP and the metal drive-line were tested. The test results show that application of the CFRP drive shaft in the drive-line can effectively reduce the vibration of the heavy-duty machine tool.

Safety assessment, detection and traceability, and societal aspects of genetically modified foods. European Network on Safety Assessment of Genetically Modified Food Crops (ENTRANSFOOD). Concluding remarks.

PubMed

Kuiper, H A; König, A; Kleter, G A; Hammes, W P; Knudsen, I

2004-07-01

The most important results from the EU-sponsored ENTRANSFOOD Thematic Network project are reviewed, including the design of a detailed step-wise procedure for the risk assessment of foods derived from genetically modified crops based on the latest scientific developments, evaluation of topical risk assessment issues, and the formulation of proposals for improved risk management and public involvement in the risk analysis process. Copyright 2004 Elsevier Ltd.

Genetic diversity and population structure of the Guinea pig (Cavia porcellus, Rodentia, Caviidae) in Colombia.

PubMed

Burgos-Paz, William; Cerón-Muñoz, Mario; Solarte-Portilla, Carlos

2011-10-01

The aim was to establish the genetic diversity and population structure of three guinea pig lines, from seven production zones located in Nariño, southwest Colombia. A total of 384 individuals were genotyped with six microsatellite markers. The measurement of intrapopulation diversity revealed allelic richness ranging from 3.0 to 6.56, and observed heterozygosity (Ho) from 0.33 to 0.60, with a deficit in heterozygous individuals. Although statistically significant (p < 0.05), genetic differentiation between population pairs was found to be low. Genetic distance, as well as clustering of guinea-pig lines and populations, coincided with the historical and geographical distribution of the populations. Likewise, high genetic identity between improved and native lines was established. An analysis of group probabilistic assignment revealed that each line should not be considered as a genetically homogeneous group. The findings corroborate the absorption of native genetic material into the improved line introduced into Colombia from Peru. It is necessary to establish conservation programs for native-line individuals in Nariño, and control genealogical and production records in order to reduce the inbreeding values in the populations.

Genetic diversity and population structure of the Guinea pig (Cavia porcellus, Rodentia, Caviidae) in Colombia

PubMed Central

Burgos-Paz, William; Cerón-Muñoz, Mario; Solarte-Portilla, Carlos

2011-01-01

The aim was to establish the genetic diversity and population structure of three guinea pig lines, from seven production zones located in Nariño, southwest Colombia. A total of 384 individuals were genotyped with six microsatellite markers. The measurement of intrapopulation diversity revealed allelic richness ranging from 3.0 to 6.56, and observed heterozygosity (Ho) from 0.33 to 0.60, with a deficit in heterozygous individuals. Although statistically significant (p < 0.05), genetic differentiation between population pairs was found to be low. Genetic distance, as well as clustering of guinea-pig lines and populations, coincided with the historical and geographical distribution of the populations. Likewise, high genetic identity between improved and native lines was established. An analysis of group probabilistic assignment revealed that each line should not be considered as a genetically homogeneous group. The findings corroborate the absorption of native genetic material into the improved line introduced into Colombia from Peru. It is necessary to establish conservation programs for native-line individuals in Nariño, and control genealogical and production records in order to reduce the inbreeding values in the populations. PMID:22215979

Genetic Modifiers of Sickle Cell Disease

PubMed Central

Steinberg, Martin H.; Sebastiani, Paola

2015-01-01

Sickle cell anemia is associated with unusual clinical heterogeneity for a Mendelian disorder. Fetal hemoglobin concentration and coincident ∝ thalassemia, both which directly affect the sickle erythrocyte, are the major modulators of the phenotype of disease. Understanding the genetics underlying the heritable subphenotypes of sickle cell anemia would be prognostically useful, could inform personalized therapeutics, and might help the discovery of new “druggable” pathophysiologic targets. Genotype-phenotype association studies have been used to identify novel genetic modifiers. In the future, whole genome sequencing with its promise of discovering hitherto unsuspected variants could add to our understanding of the genetic modifiers of this disease. PMID:22641398

Modifying Knowledge, Emotions, and Attitudes Regarding Genetically Modified Foods

ERIC Educational Resources Information Center

Heddy, Benjamin C.; Danielson, Robert W.; Sinatra, Gale M.; Graham, Jesse

2017-01-01

The purpose of this study was to explore whether conceptual change predicted emotional and attitudinal change while learning about genetically modified foods (GMFs). Participants were 322 college students; half read a refutation text designed to shift conceptual knowledge, emotions, and attitudes, while the other half served as a control group.…

Modifier genes in Mendelian disorders: the example of cystic fibrosis

PubMed Central

Cutting, Garry R.

2011-01-01

In the past three decades, scientists have had immense success in identifying genes and their variants that contribute to an array of diseases. While the identification of such genetic variants has informed our knowledge of the etiologic bases of diseases, there continues to be a substantial gap in our understanding of the factors that modify disease severity. Monogenic diseases provide an opportunity to identify modifiers as they have uniform etiology, detailed phenotyping of affected individuals, and familial clustering. Cystic fibrosis (CF) is among the more common life-shortening recessive disorders that displays wide variability in clinical features and survival. Considerable progress has been made in elucidating the contribution of genetic and nongenetic factors to CF. Allelic variation in CFTR, the gene responsible for CF, correlates with some aspects of the disease. However, lung function, neonatal intestinal obstruction, diabetes, and anthropometry display strong genetic control independent of CFTR, and candidate gene studies have revealed genetic modifiers underlying these traits. The application of genome-wide techniques holds great promise for the identification of novel genetic variants responsible for the heritable features and complications of CF. Since the genetic modifiers are known to alter the course of disease, their protein products become immediate targets for therapeutic intervention. PMID:21175684

ASSESSING POSSIBLE ECOLOGICAL RISKS OF GENETICALLY MODIFIED CROPS: GENE EXPRESSION ASSAYS AND GENETIC MONITORING OF NON-TARGET ORGANISMS

EPA Science Inventory

Widespread planting of genetically modified crops with the Bt transgene pesticide has led to concern over non-target effects of Bt compounds in agroecosystems. While some research suggests that non-target organisms exposed to Bt toxin exhibit reduced fecundity and increased morta...

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.

40 CFR 158.2100 - Microbial pesticides definition and applicability.

Code of Federal Regulations, 2010 CFR

2010-07-01

... to which the organism has been genetically modified. (4) Pest control organisms such as insect... and supported by data required in this subpart. (3) Genetically modified microbial pesticides may be...

National Pork Producers Council Maternal Line National Genetic Evaluation Program: a comparison of sow longevity and trait associations with sow longevity.

PubMed

Serenius, T; Stalder, K J; Baas, T J; Mabry, J W; Goodwin, R N; Johnson, R K; Robison, O W; Tokach, M; Miller, R K

2006-09-01

Data from the National Pork Producers Council Maternal Line National Genetic Evaluation Program were used to compare longevity of sows from 6 commercial genetic lines and to estimate the phenotypic associations of sow longevity with gilt backfat thickness, ADG, age at first farrowing, litter size at first farrowing, litter weight at first farrowing, average feed intake during lactation, and average backfat loss during lactation. The lines evaluated were American Diamond Genetics, Danbred North America, Dekalb-Monsanto DK44, Dekalb-Monsanto GPK347, Newsham Hybrids, and National Swine Registry. The data set contained information from 3,251 gilts, of which 17% had censored longevity records (sows lived longer than 6 parities). The line comparison was carried out by analyzing all lines simultaneously. Because the survival distribution functions differed among genetic lines, later analyses were carried out separately for each genetic line. All analyses were based on the non-parametric proportional hazard (Cox model). Dekalb-Monsanto GPK347 sows had a lower risk of being culled than sows from the other lines. Moreover, the shape of the survival distribution function of the Delkab-Monsanto GPK347 line was different from the other 5 lines. The Dekalb-Monsanto 347 line had lower culling rates because they had lower gilt reproductive failure before the first parity than gilts from the other lines. Within line, sows with lower feed intake and greater backfat loss during lactation had a shorter productive lifetime. Thus, producers should implement management practices having positive effects on sow lactation feed intake. Additionally, the swine genetics industry is challenged to simultaneously improve efficiency of gain of their terminal market pigs and to obtain high feed intake during lactation of their maternal lines for future improvement of sow longevity. Recording sow feed intake and backfat loss during lactation in nucleus and multiplication breeding herds should be considered. Between-line differences in this study indicate that it is possible to select for sow longevity, but more research is needed to determine the most efficient selection methods to improve sow longevity.

Molecular and biochemical identification of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome(s) of bunching onion (A. fistulosum L.).

PubMed

Yaguchi, Shigenori; Hang, Tran Thi Minh; Tsukazaki, Hikaru; Hoa, Vu Quynh; Masuzaki, Shin-ichi; Wako, Tadayuki; Masamura, Noriya; Onodera, Shuichi; Shiomi, Norio; Yamauchi, Naoki; Shigyo, Masayoshi

2009-02-01

To develop the bunching onion (Allium fistulosum L.; genomes, FF) chromosome-specific genetic markers for identifying extra chromosomes, eight shallot (A. cepa L. Aggregatum group; genomes, AA)--A. fistulosum monosomic addition plants (AA+nF) and 62 shallot--A. fistulosum single-alien deletion plants (AAF-nF) were analyzed by 23 different chromosome-specific genetic markers of shallot. The eight monosomic addition plants consisted of one AA+2F, two AA+6F, and five AA+8F. Of the 62 single-alien deletion plants, 60 could be identified as six different single-alien deletion lines (AAF-1F, -3F, -4F, -6F, -7F, and -8F) out of the eight possible types. Several single-alien deletion lines were classified on the basis of leaf and bulb characteristics. AAF-8F had the largest number of expanded leaves of five deletion plants. AAF-7F grew most vigorously, as expressed by its long leaf blade and biggest bulb size. AAF-4F had very small bulbs. AAF-7F and AAF-8F had different bulbs from those of shallot as well as other types of single-alien deletion lines in skin and outer scale color. Regarding the sugar content of the bulb tissues, the single-alien deletion lines showed higher fructan content than shallot. Moreover, shallot could not produce fructan with degree of polymerization (DP) 12 or higher, although the single-alien deletion lines showed DP 20 or higher. The content of S-alk(en)yl-L-cysteine sulfoxide (ACSO) in the single-alien deletion lines was significantly lower than that in shallot. These results indicated that chromosomes from A. fistulosum might carry anonymous factors to increase the highly polymerized fructan production and inhibit the synthesis of ACSO in shallot bulbs. Accordingly, alien chromosomes from A. fistulosum in shallot would contribute to modify the quality of shallot bulbs.

Assessment of endogenous allergenicity of genetically modified plants exemplified by soybean - Where do we stand?

PubMed

Selb, R; Wal, J M; Moreno, F J; Lovik, M; Mills, C; Hoffmann-Sommergruber, K; Fernandez, A

2017-03-01

According to EU regulation, genetically modified (GM) plants considered to be allergenic have to be assessed concerning their endogenous allergens before placement on the EU market, in line with the international standards described in Codex Alimentarius. Under such premises, a quantitative relevant increase in allergens might occur in GM plants as an unintended effect compared with conventionally produced crops, which could pose a risk to consumers. Currently, data showing a connection between dose and allergic sensitisation are scarce since the pathophysiological mechanisms of sensitisation are insufficiently understood. In contrast, data on population dose-distribution relationships acquired by oral food challenge are available showing a connection between quantity of allergenic protein consumed and the population of allergic individuals experiencing reactions. Soybean is currently the only recognised allergenic GM food by law for which EFSA has received applications and was therefore taken as an example for defining an assessment strategy. Identification of potential allergens, methodology for quantification as well as risk assessment considerations, are discussed. A strategy is proposed for the identification, assessment and evaluation of potential hazards/risks concerning endogenous allergenicity in food derived from plants developed by biotechnology. This approach could be expanded to other allergenic foods in the future, whenever required. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomic profiling of liver from Atlantic salmon (Salmo salar) fed genetically modified soy compared to the near-isogenic non-GM line.

PubMed

Sissener, Nini H; Martin, Samuel A M; Cash, Phillip; Hevrøy, Ernst M; Sanden, Monica; Hemre, Gro-Ingunn

2010-06-01

The aim of this study was to investigate potential differences in liver protein expression of Atlantic salmon fed genetically modified (GM) Roundup Ready soy at a high inclusion level (25% inclusion, constituting 21% of crude protein in the diet) for 7 months or a compositionally similar non-GM diet. The liver was selected as the target organ due to its importance in the general metabolism, and 2D gel electrophoresis used as a screening tool. Samples from 12 individual fish from each diet group were evaluated. Of a total of 781 analysed protein spots, only 36 were significantly different by ANOVA (p < 0.05) in abundance between the diet groups. All these spots had low fold differences (1.2-1.6) and high false discovery rate (q = 0.44), indicating minor differences in liver protein synthesis between fish fed GM and non-GM soy. Additionally, low fold differences were observed. Four protein spots were analyzed by liquid chromatography tandem mass spectrometry and identified using a combination of online searches in NCBI and searches in an inhouse database containing salmonid expressed sequence tags and contigs. Follow-up on these proteins by real-time polymerase chain reaction did not identify differences at the transcriptional level.

Evolving phage vectors for cell targeted gene delivery.

PubMed

Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

2002-03-01

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

[Genetically modified food and allergies - an update].

PubMed

Niemann, Birgit; Pöting, Annette; Braeuning, Albert; Lampen, Alfonso

2016-07-01

Approval by the European Commission is mandatory for placing genetically modified plants as food or feed on the market in member states of the European Union (EU). The approval is preceded by a safety assessment based on the guidance of the European Food Safety Authority EFSA. The assessment of allergenicity of genetically modified plants and their newly expressed proteins is an integral part of this assessment process. Guidance documents for the assessment of allergenicity are currently under revision. For this purpose, an expert workshop was conducted in Brussels on June 17, 2015. There, methodological improvements for the assessment of coeliac disease-causing properties of proteins, as well as the use of complex models for in vitro digestion of proteins were discussed. Using such techniques a refinement of the current, proven system of allergenicity assessment of genetically modified plants can be achieved.

Genetics, structure, and prevalence of FP967 (CDC Triffid) T-DNA in flax.

PubMed

Young, Lester; Hammerlindl, Joseph; Babic, Vivijan; McLeod, Jamille; Sharpe, Andrew; Matsalla, Chad; Bekkaoui, Faouzi; Marquess, Leigh; Booker, Helen M

2015-01-01

The detection of T-DNA from a genetically modified flaxseed line (FP967, formally CDC Triffid) in a shipment of Canadian flaxseed exported to Europe resulted in a large decrease in the amount of flax planted in Canada. The Canadian flaxseed industry undertook major changes to ensure the removal of FP967 from the supply chain. This study aimed to resolve the genetics and structure of the FP967 transfer DNA (T-DNA). The FP967 T-DNA is thought to be inserted in at single genomic locus. The junction between the T-DNA and genomic DNA consisted of two inverted Right Borders with no Left Border (LB) flanking genomic DNA sequences recovered. This information was used to develop an event-specific quantitative PCR (qPCR) assay. This assay and an existing assay specific to the T-DNA construct were used to determine the genetics and prevalence of the FP967 T-DNA. These data supported the hypothesis that the T-DNA is present at a single location in the genome. The FP967 T-DNA is present at a low level (between 0.01 and 0.1%) in breeder seed lots from 2009 and 2010. None of the 11,000 and 16,000 lines selected for advancement through the Flax Breeding Program in 2010 and 2011, respectively, tested positive for the FP967 T-DNA, however. Most of the FP967 T-DNA sequence was resolved via PCR cloning and next generation sequencing. A 3,720 bp duplication of an internal portion of the T-DNA (including a Right Border) was discovered between the flanking genomic DNA and the LB. An event-specific assay, SAT2-LB, was developed for the junction between this repeat and the LB.

Generating mouse lines for lineage tracing and knockout studies.

PubMed

Kraus, Petra; Sivakamasundari, V; Xing, Xing; Lufkin, Thomas

2014-01-01

In 2007 Capecchi, Evans, and Smithies received the Nobel Prize in recognition for discovering the principles for introducing specific gene modifications in mice via embryonic stem cells, a technology, which has revolutionized the field of biomedical science allowing for the generation of genetically engineered animals. Here we describe detailed protocols based on and developed from these ground-breaking discoveries, allowing for the modification of genes not only to create mutations to study gene function but additionally to modify genes with fluorescent markers, thus permitting the isolation of specific rare wild-type and mutant cell types for further detailed analysis at the biochemical, pathological, and genomic levels.

Genetically Modified Plants: Public and Scientific Perceptions

PubMed Central

2013-01-01

The potential of genetically modified plants to meet the requirements of growing population is not being recognized at present. This is a consequence of concerns raised by the public and the critics about their applications and release into the environment. These include effect on human health and environment, biosafety, world trade monopolies, trustworthiness of public institutions, integrity of regulatory agencies, loss of individual choice, and ethics as well as skepticism about the real potential of the genetically modified plants, and so on. Such concerns are enormous and prevalent even today. However, it should be acknowledged that most of them are not specific for genetically modified plants, and the public should not forget that the conventionally bred plants consumed by them are also associated with similar risks where no information about the gene(s) transfer is available. Moreover, most of the concerns are hypothetical and lack scientific background. Though a few concerns are still to be disproved, it is viewed that, with proper management, these genetically modified plants have immense potential for the betterment of mankind. In the present paper, an overview of the raised concerns and wherever possible reasons assigned to explain their intensity or unsuitability are reviewed. PMID:25937981

Evaluation of genetically-improved (glandless) and genetically-modified low-gossypol cottonseed meal as alternative protein sources in the diet of juvenile southern flounder Paralichthys lethostigma reared in a recirculating

USDA-ARS?s Scientific Manuscript database

Cottonseed meal (CSM) proteins from genetically-improved (glandless) seed (GI-CSM, 52.1% crude protein, CP), genetically-modified low-gossypol seed (GMO-CSM, 56.0% CP) and from an untreated regular (glanded) seed (R-CSM 49.9% CP) were evaluated to replace fish meal (FM) protein (59.5% CP) in juvenil...

A test of the influence of continental axes of orientation on patterns of human gene flow

PubMed Central

Ramachandran, Sohini; Rosenberg, Noah A.

2012-01-01

The geographic distribution of genetic variation reflects trends in past population migrations, and can be used to make inferences about these migrations. It has been proposed that the east-west orientation of the Eurasian landmass facilitated the rapid spread of ancient technological innovations across Eurasia, while the north-south orientation of the Americas led to a slower diffusion of technology there. If the diffusion of technology was accompanied by gene flow, then this hypothesis predicts that genetic differentiation in the Americas along lines of longitude will be greater than that in Eurasia along lines of latitude. We use 678 microsatellite loci from 68 indigenous populations in Eurasia and the Americas to investigate the spatial axes that underlie population-genetic variation. We find that genetic differentiation increases more rapidly along lines of longitude in the Americas than along lines of latitude in Eurasia. Distance along lines of latitude explains a sizeable portion of genetic distance in Eurasia, whereas distance along lines of longitude does not explain a large proportion of Eurasian genetic variation. Genetic differentiation in the Americas occurs along both latitudinal and longitudinal axes and has a greater magnitude than corresponding differentiation in Eurasia, even when adjusting for the lower level of genetic variation in the American populations. These results support the view that continental orientation has influenced migration patterns and has played an important role in determining both the structure of human genetic variation and the distribution and spread of cultural traits. (240 words) PMID:21913175

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…

Genetically Modified Crops and Nuisance: Exploring the Role of Precaution in Private Law

ERIC Educational Resources Information Center

Craik, Neil; Culver, Keith; Siebrasse, Norman

2007-01-01

This article critically considers calls for the precautionary principle to inform judicial decision making in a private law context in light of the Hoffman litigation, where it is alleged that the potential for genetic contamination from genetically modified (GM) crops causes an unreasonable interference with the rights of organic farmers to use…

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…

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 analysis of indefinite division in human cells: Evidence for a cell senescence-related gene(s) on human chromosome 4

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

Yi Ning; Ledbetter, D.H.; Smith, J.R.

1991-07-01

Earlier studies had demonstrated that fusion of normal with immortal human cells yielded hybrids having limited division potential. This indicated that the phenotype of limited proliferation (cellular senescence) is dominant and that immortal cells result from recessive changes in normal growth-regulatory genes. In additional studies, the authors exploited the fact that the immortal phenotype is recessive and, by fusing various immortal human cell lines with each other, identified four complementation groups for indefinite division. Assignment of cell lines to specific groups allowed us to take a focused approach to identify the chromosomes and genes involved in growth regulation that havemore » been modified in immortal cells. They report here that introduction of a normal human chromosome 4 into three immortal cell lines (HeLa, J82, T98G) assigned to complementation group B resulted in loss of proliferation and reversal of the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. This result suggests that a gene(s) involved in cellular senescence and normal growth regulation resides on chromosome 4.« less

Indirect effect of a transgenic wheat on aphids through enhanced powdery mildew resistance.

PubMed

von Burg, Simone; Álvarez-Alfageme, Fernando; Romeis, Jörg

2012-01-01

In agricultural ecosystems, arthropod herbivores and fungal pathogens are likely to colonise the same plant and may therefore affect each other directly or indirectly. The fungus that causes powdery mildew (Blumeria graminis tritici) and cereal aphids are important pests of wheat but interactions between them have seldom been investigated. We studied the effects of powdery mildew of wheat on two cereal aphid species, Metopolophium dirhodum and Rhopalosiphum padi. We hypothesized that aphid number and size will be smaller on powdery mildew-infected plants than on non-infected plants. In a first experiment we used six commercially available wheat varieties whereas in the second experiment we used a genetically modified (GM) mildew-resistant wheat line and its non-transgenic sister line. Because the two lines differed only in the presence of the transgene and in powdery mildew resistance, experiment 2 avoided the confounding effect of variety. In both experiments, the number of M. dirhodum but not of R. padi was reduced by powdery mildew infection. Transgenic mildew-resistant lines therefore harboured bigger aphid populations than the non-transgenic lines. For both aphid species individual size was mostly influenced by aphid number. Our results indicate that plants that are protected from a particular pest (powdery mildew) became more favourable for another pest (aphids).

Indirect Effect of a Transgenic Wheat on Aphids through Enhanced Powdery Mildew Resistance

PubMed Central

von Burg, Simone; Álvarez-Alfageme, Fernando; Romeis, Jörg

2012-01-01

In agricultural ecosystems, arthropod herbivores and fungal pathogens are likely to colonise the same plant and may therefore affect each other directly or indirectly. The fungus that causes powdery mildew (Blumeria graminis tritici) and cereal aphids are important pests of wheat but interactions between them have seldom been investigated. We studied the effects of powdery mildew of wheat on two cereal aphid species, Metopolophium dirhodum and Rhopalosiphum padi. We hypothesized that aphid number and size will be smaller on powdery mildew-infected plants than on non-infected plants. In a first experiment we used six commercially available wheat varieties whereas in the second experiment we used a genetically modified (GM) mildew-resistant wheat line and its non-transgenic sister line. Because the two lines differed only in the presence of the transgene and in powdery mildew resistance, experiment 2 avoided the confounding effect of variety. In both experiments, the number of M. dirhodum but not of R. padi was reduced by powdery mildew infection. Transgenic mildew-resistant lines therefore harboured bigger aphid populations than the non-transgenic lines. For both aphid species individual size was mostly influenced by aphid number. Our results indicate that plants that are protected from a particular pest (powdery mildew) became more favourable for another pest (aphids). PMID:23056284

Between myth and reality: genetically modified maize, an example of a sizeable scientific controversy.

PubMed

Wisniewski, Jean-Pierre; Frangne, Nathalie; Massonneau, Agnès; Dumas, Christian

2002-11-01

Maize is a major crop plant with essential agronomical interests and a model plant for genetic studies. With the development of plant genetic engineering technology, many transgenic strains of this monocotyledonous plant have been produced over the past decade. In particular, field-cultivated insect-resistant Bt-maize hybrids are at the centre of an intense debate between scientists and organizations recalcitrant to genetically modified organisms (GMOs). This debate, which addresses both safety and ethical aspects, has raised questions about the impact of genetically modified (GM) crops on the biodiversity of traditional landraces and on the environment. Here, we review some of the key points of maize genetic history as well as the methods used to stably transform this cereal. We describe the genetically engineered Bt-maizes available for field cultivation and we investigate the controversial reports on their impacts on non-target insects such as the monarch butterfly and on the flow of transgenes into Mexican maize landraces.

Genetic diversity and genetic structure of an endemic Mexican Dusky Rattlesnake (Crotalus triseriatus) in a highly modified agricultural landscape: implications for conservation.

PubMed

Sunny, Armando; Monroy-Vilchis, Octavio; Zarco-González, Martha M; Mendoza-Martínez, Germán David; Martínez-Gómez, Daniel

2015-12-01

It is necessary to determine genetic diversity of fragmented populations in highly modified landscapes to understand how populations respond to land-use change. This information will help guide future conservation and management strategies. We conducted a population genetic study on an endemic Mexican Dusky Rattlesnake (Crotalus triseriatus) in a highly modified landscape near the Toluca metropolitan area, in order to provide crucial information for the conservation of this species. There was medium levels of genetic diversity, with a few alleles and genotypes. We identified three genetically differentiated clusters, likely as a result of different habitat cover type. We also found evidence of an ancestral genetic bottleneck and medium values of effective population size. Inbreeding coefficients were low and there was a moderate gene flow. Our results can be used as a basis for future research and C. triseriatus conservation efforts, particularly considering that the Trans-Mexican Volcanic Belt is heavily impacted by destructive land-use practices.

Biallelic germline and somatic mutations in malignant mesothelioma: multiple mutations in transcription regulators including mSWI/SNF genes.

PubMed

Yoshikawa, Yoshie; Sato, Ayuko; Tsujimura, Tohru; Otsuki, Taiichiro; Fukuoka, Kazuya; Hasegawa, Seiki; Nakano, Takashi; Hashimoto-Tamaoki, Tomoko

2015-02-01

We detected low levels of acetylation for histone H3 tail lysines in malignant mesothelioma (MM) cell lines resistant to histone deacetylase inhibitors. To identify the possible genetic causes related to the low histone acetylation levels, whole-exome sequencing was conducted with MM cell lines established from eight patients. A mono-allelic variant of BRD1 was common to two MM cell lines with very low acetylation levels. We identified 318 homozygous protein-damaging variants/mutations (18-78 variants/mutations per patient); annotation analysis showed enrichment of the molecules associated with mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complexes and co-activators that facilitate initiation of transcription. In seven of the patients, we detected a combination of variants in histone modifiers or transcription factors/co-factors, in addition to variants in mSWI/SNF. Direct sequencing showed that homozygous mutations in SMARCA4, PBRM1 and ARID2 were somatic. In one patient, homozygous germline variants were observed for SMARCC1 and SETD2 in chr3p22.1-3p14.2. These exhibited extended germline homozygosity and were in regions containing somatic mutations, leading to a loss of BAP1 and PBRM1 expression in MM cell line. Most protein-damaging variants were heterozygous in normal tissues. Heterozygous germline variants were often converted into hemizygous variants by mono-allelic deletion, and were rarely homozygous because of acquired uniparental disomy. Our findings imply that MM might develop through the somatic inactivation of mSWI/SNF complex subunits and/or histone modifiers, including BAP1, in subjects that have rare germline variants of these transcription regulators and/or transcription factors/co-factors, and in regions prone to mono-allelic deletion during oncogenesis. © 2014 UICC.

ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors.

PubMed

Cifaldi, Loredana; Romania, Paolo; Falco, Michela; Lorenzi, Silvia; Meazza, Raffaella; Petrini, Stefania; Andreani, Marco; Pende, Daniela; Locatelli, Franco; Fruci, Doriana

2015-03-01

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by MHC class I (MHC-I) molecules. Herein, we demonstrate that genetic or pharmacological inhibition of ERAP1 on human tumor cell lines perturbs their ability to engage several classes of inhibitory receptors by their specific ligands, including killer cell Ig-like receptors (KIR) by classical MHC-I-peptide (pMHC-I) complexes and the lectin-like receptor CD94-NKG2A by nonclassical pMHC-I complexes, in each case leading to natural killer (NK) cell killing. The protective effect of pMHC-I complexes could be restored in ERAP1-deficient settings by the addition of known high-affinity peptides, suggesting that ERAP1 was needed to positively modify the affinity of natural ligands. Notably, ERAP1 inhibition enhanced the ability of NK cells to kill freshly established human lymphoblastoid cell lines from autologous or allogeneic sources, thereby promoting NK cytotoxic activity against target cells that would not be expected because of KIR-KIR ligand matching. Overall, our results identify ERAP1 as a modifier to leverage immune functions that may improve the efficacy of NK cell-based approaches for cancer immunotherapy. ©2015 American Association for Cancer Research.

Recognition of Glioma Stem Cells by Genetically Modified T Cells Targeting EGFRvIII and Development of Adoptive Cell Therapy for Glioma

PubMed Central

Johnson, Laura A.; Davis, Jeremy L.; Zheng, Zhili; Woolard, Kevin D.; Reap, Elizabeth A.; Feldman, Steven A.; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A.; Rosenberg, Steven A.

2012-01-01

Abstract No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application. PMID:22780919

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

PubMed

Morgan, Richard A; Johnson, Laura A; Davis, Jeremy L; Zheng, Zhili; Woolard, Kevin D; Reap, Elizabeth A; Feldman, Steven A; Chinnasamy, Nachimuthu; Kuan, Chien-Tsun; Song, Hua; Zhang, Wei; Fine, Howard A; Rosenberg, Steven A

2012-10-01

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

USEPA Resistance Management Research

EPA Science Inventory

A significant increase in genetically modified corn planting driven by biofuel demand is expected for future planted acreages approaching 80% of total corn plantings in 2009. As demand increases, incidence of farmer non-compliance with mandated non-genetically modified refuge is...

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

Ruegg, Thomas Lawrence; Thelen, Michael P.

The present invention provides for a method of genetically modifying microorganisms to enhance resistance to ionic liquids, host cells genetically modified in accordance with the methods, and methods of using the host cells in a reaction comprising biomass that has been pretreated with ionic liquids.

Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products.

PubMed

Holst-Jensen, Arne; Spilsberg, Bjørn; Arulandhu, Alfred J; Kok, Esther; Shi, Jianxin; Zel, Jana

2016-07-01

The emergence of high-throughput, massive or next-generation sequencing technologies has created a completely new foundation for molecular analyses. Various selective enrichment processes are commonly applied to facilitate detection of predefined (known) targets. Such approaches, however, inevitably introduce a bias and are prone to miss unknown targets. Here we review the application of high-throughput sequencing technologies and the preparation of fit-for-purpose whole genome shotgun sequencing libraries for the detection and characterization of genetically modified and derived products. The potential impact of these new sequencing technologies for the characterization, breeding selection, risk assessment, and traceability of genetically modified organisms and genetically modified products is yet to be fully acknowledged. The published literature is reviewed, and the prospects for future developments and use of the new sequencing technologies for these purposes are discussed.

Development of a qualitative real-time PCR method to detect 19 targets for identification of genetically modified organisms.

PubMed

Peng, Cheng; Wang, Pengfei; Xu, Xiaoli; Wang, Xiaofu; Wei, Wei; Chen, Xiaoyun; Xu, Junfeng

2016-01-01

As the amount of commercially available genetically modified organisms (GMOs) grows recent years, the diversity of target sequences for molecular detection techniques are eagerly needed. Considered as the gold standard for GMO analysis, the real-time PCR technology was optimized to produce a high-throughput GMO screening method. With this method we can detect 19 transgenic targets. The specificity of the assays was demonstrated to be 100 % by the specific amplification of DNA derived from reference material from 20 genetically modified crops and 4 non modified crops. Furthermore, most assays showed a very sensitive detection, reaching the limit of ten copies. The 19 assays are the most frequently used genetic elements present in GM crops and theoretically enable the screening of the known GMO described in Chinese markets. Easy to use, fast and cost efficient, this method approach fits the purpose of GMO testing laboratories.

Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass

PubMed Central

Fu, Chunxiang; Mielenz, Jonathan R.; Xiao, Xirong; Ge, Yaxin; Hamilton, Choo Y.; Rodriguez, Miguel; Chen, Fang; Foston, Marcus; Ragauskas, Arthur; Bouton, Joseph; Dixon, Richard A.; Wang, Zeng-Yu

2011-01-01

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300–400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions. PMID:21321194

Evolutionarily stable disequilibrium: endless dynamics of evolution in a stationary population.

PubMed

Takeuchi, Nobuto; Kaneko, Kunihiko; Hogeweg, Paulien

2016-05-11

Evolution is often conceived as changes in the properties of a population over generations. Does this notion exhaust the possible dynamics of evolution? Life is hierarchically organized, and evolution can operate at multiple levels with conflicting tendencies. Using a minimal model of such conflicting multilevel evolution, we demonstrate the possibility of a novel mode of evolution that challenges the above notion: individuals ceaselessly modify their genetically inherited phenotype and fitness along their lines of descent, without involving apparent changes in the properties of the population. The model assumes a population of primitive cells (protocells, for short), each containing a population of replicating catalytic molecules. Protocells are selected towards maximizing the catalytic activity of internal molecules, whereas molecules tend to evolve towards minimizing it in order to maximize their relative fitness within a protocell. These conflicting evolutionary tendencies at different levels and genetic drift drive the lineages of protocells to oscillate endlessly between high and low intracellular catalytic activity, i.e. high and low fitness, along their lines of descent. This oscillation, however, occurs independently in different lineages, so that the population as a whole appears stationary. Therefore, ongoing evolution can be hidden behind an apparently stationary population owing to conflicting multilevel evolution. © 2016 The Authors.

Effect of hyperbaric oxygen on BDNF-release and neuroprotection: Investigations with human mesenchymal stem cells and genetically modified NIH3T3 fibroblasts as putative cell therapeutics.

PubMed

Schulze, Jennifer; Kaiser, Odett; Paasche, Gerrit; Lamm, Hans; Pich, Andreas; Hoffmann, Andrea; Lenarz, Thomas; Warnecke, Athanasia

2017-01-01

Hyperbaric oxygen therapy (HBOT) is a noninvasive widely applied treatment that increases the oxygen pressure in tissues. In cochlear implant (CI) research, intracochlear application of neurotrophic factors (NTFs) is able to improve survival of spiral ganglion neurons (SGN) after deafness. Cell-based delivery of NTFs such as brain-derived neurotrophic factor (BDNF) may be realized by cell-coating of the surface of the CI electrode. Human mesenchymal stem cells (MSC) secrete a variety of different neurotrophic factors and may be used for the development of a biohybrid electrode in order to release endogenously-derived neuroprotective factors for the protection of residual SGN and for a guided outgrowth of dendrites in the direction of the CI electrode. HBOT could be used to influence cell behaviour after transplantation to the inner ear. The aim of this study was to investigate the effect of HBOT on the proliferation, BDNF-release and secretion of neuroprotective factors. Thus, model cells (an immortalized fibroblast cell line (NIH3T3)-native and genetically modified) and MSCs were repeatedly (3 x - 10 x) exposed to 100% oxygen at different pressures. The effects of HBO on cell proliferation were investigated in relation to normoxic and normobaric conditions (NOR). Moreover, the neuroprotective and neuroregenerative effects of HBO-treated cells were analysed by cultivation of SGN in conditioned medium. Both, the genetically modified NIH3T3/BDNF and native NIH3T3 fibroblasts, showed a highly significant increased proliferation after five days of HBOT in comparison to normoxic controls. By contrast, the number of MSCs was decreased in MSCs treated with 2.0 bar of HBO. Treating SGN cultures with supernatants of fibroblasts and MSCs significantly increased the survival rate of SGN. HBO treatment did not influence (increase / reduce) this effect. Secretome analysis showed that HBO treatment altered the protein expression pattern in MSCs.

Effects of Repeated Social Disruption on the Serotonergic and Dopaminergic Systems in two Genetic Lines of White Leghorn Layers

USDA-ARS?s Scientific Manuscript database

The study was designed to examine whether there are genetic differences in response to repeated social disruption (RSD). Two genetic strains of White Leghorn hens were used in the study; i.e., HGPS (the line selected for high group production and survivability), and DXL (DeKalb XL commercial line). ...

MS-based analytical methodologies to characterize genetically modified crops.

PubMed

García-Cañas, Virginia; Simó, Carolina; León, Carlos; Ibáñez, Elena; Cifuentes, Alejandro

2011-01-01

The development of genetically modified crops has had a great impact on the agriculture and food industries. However, the development of any genetically modified organism (GMO) requires the application of analytical procedures to confirm the equivalence of the GMO compared to its isogenic non-transgenic counterpart. Moreover, the use of GMOs in foods and agriculture faces numerous criticisms from consumers and ecological organizations that have led some countries to regulate their production, growth, and commercialization. These regulations have brought about the need of new and more powerful analytical methods to face the complexity of this topic. In this regard, MS-based technologies are increasingly used for GMOs analysis to provide very useful information on GMO composition (e.g., metabolites, proteins). This review focuses on the MS-based analytical methodologies used to characterize genetically modified crops (also called transgenic crops). First, an overview on genetically modified crops development is provided, together with the main difficulties of their analysis. Next, the different MS-based analytical approaches applied to characterize GM crops are critically discussed, and include "-omics" approaches and target-based approaches. These methodologies allow the study of intended and unintended effects that result from the genetic transformation. This information is considered to be essential to corroborate (or not) the equivalence of the GM crop with its isogenic non-transgenic counterpart. Copyright © 2010 Wiley Periodicals, Inc.

[Plant genetic engineering in Monsanto company: from the first laboratory experiments to worldwide practical use].

PubMed

Konov, A L; Velchev, M; Parcel, D

2005-01-01

The history of modern biotechnology of agricultural plants is briefly considered in the article. Methods of genetic transformation and regeneration of transgenic plants as well as the mechanisms of resistance of genetically modified plants to herbicides and pests are discussed. By the example of genetically modified varieties and hybrids there are shown the ways of solving the problem of weeds and pests. The questions of biosafety legislation in different countries are considered.

Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility.

PubMed

Knudsen, Erik S; Balaji, Uthra; Mannakee, Brian; Vail, Paris; Eslinger, Cody; Moxom, Christopher; Mansour, John; Witkiewicz, Agnieszka K

2018-03-01

Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease with the worst survival rate of common solid tumours. Preclinical models that accurately reflect the genetic and biological diversity of PDAC will be important for delineating features of tumour biology and therapeutic vulnerabilities. 27 primary PDAC tumours were employed for genetic analysis and development of tumour models. Tumour tissue was used for derivation of xenografts and cell lines. Exome sequencing was performed on the originating tumour and developed models. RNA sequencing, histological and functional analyses were employed to determine the relationship of the patient-derived models to clinical presentation of PDAC. The cohort employed captured the genetic diversity of PDAC. From most cases, both cell lines and xenograft models were developed. Exome sequencing confirmed preservation of the primary tumour mutations in developed cell lines, which remained stable with extended passaging. The level of genetic conservation in the cell lines was comparable to that observed with patient-derived xenograft (PDX) models. Unlike historically established PDAC cancer cell lines, patient-derived models recapitulated the histological architecture of the primary tumour and exhibited metastatic spread similar to that observed clinically. Detailed genetic analyses of tumours and derived models revealed features of ex vivo evolution and the clonal architecture of PDAC. Functional analysis was used to elucidate therapeutic vulnerabilities of relevance to treatment of PDAC. These data illustrate that with the appropriate methods it is possible to develop cell lines that maintain genetic features of PDAC. Such models serve as important substrates for analysing the significance of genetic variants and create a unique biorepository of annotated cell lines and xenografts that were established simultaneously from same primary tumour. These models can be used to infer genetic and empirically determined therapeutic sensitivities that would be germane to the patient. 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/.

Construction of the first genetic linkage map of Japanese gentian (Gentianaceae)

PubMed Central

2012-01-01

Background Japanese gentians (Gentiana triflora and Gentiana scabra) are amongst the most popular floricultural plants in Japan. However, genomic resources for Japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. In this study, we developed a genetic linkage map to improve breeding programs of Japanese gentians. Results Enriched simple sequence repeat (SSR) libraries from a G. triflora double haploid line yielded almost 20,000 clones using 454 pyrosequencing technology, 6.7% of which could be used to design SSR markers. To increase the number of molecular markers, we identified three putative long terminal repeat (LTR) sequences using the recently developed inter-primer binding site (iPBS) method. We also developed retrotransposon microsatellite amplified polymorphism (REMAP) markers combining retrotransposon and inter-simple sequence repeat (ISSR) markers. In addition to SSR and REMAP markers, modified amplified fragment length polymorphism (AFLP) and random amplification polymorphic DNA (RAPD) markers were developed. Using 93 BC1 progeny from G. scabra backcrossed with a G. triflora double haploid line, 19 linkage groups were constructed with a total of 263 markers (97 SSR, 97 AFLP, 39 RAPD, and 30 REMAP markers). One phenotypic trait (stem color) and 10 functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility. Conclusions This is the first reported genetic linkage map for Japanese gentians and for any species belonging to the family Gentianaceae. As demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. Our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species. PMID:23186361

The genetic basis of pectoralis major myopathies in modern broiler chicken lines

PubMed Central

Bailey, Richard A.; Watson, Kellie A.; Bilgili, S. F.; Avendano, Santiago

2015-01-01

This is the first report providing estimates of the genetic basis of breast muscle myopathies (BMM) and their relationship with growth and yield in broiler chickens. In addition, this paper addresses the hypothesis that genetic selection for increase breast yield has contributed to the onset of BMM. Data were analyzed from ongoing recording of BMM within the Aviagen breeding program. This study focused on three BMM: deep pectoral myopathy (DPM; binary trait), white striping (WS; 4 categories) and wooden breast (WB; 3 categories). Data from two purebred commercial broiler lines (A and B) were utilized providing greater than 40,000 meat quality records per line. The difference in selection history between these two lines has resulted in contrasting breast yield (BY): 29% for Line A and 21% for Line B. Data were analyzed to estimate genetic parameters using a multivariate animal model including six traits: body weight (BW), processing body weight (PW), BY, DPM, WB, and WS, in addition to the appropriate fixed effects and permanent environmental effect of the dam. Results indicate similar patterns of heritability and genetic correlations for the two lines. Heritabilities (h2) of BW, PW and BY ranged from 0.271–0.418; for DPM and WB h2 <0.1; and for WS h2 ≤0.338. Genetic correlations between the BMM and BW, PW, or BY were ≤0.132 in Line A and ≤0.248 in Line B. This paper demonstrates the polygenic nature of these traits and the low genetic relationships with BW, PW, and BY, which facilitates genetic improvement across all traits in a balanced breeding program. It also highlights the importance of understanding the environmental and/or management factors that contribute greater than 65% of the variance in the incidence of white striping of breast muscle and more than 90% of the variance of the incidence of wooden breast and deep pectoral myopathy in broiler chickens. PMID:26476091

Wild worm embryogenesis harbors ubiquitous polygenic modifier variation.

PubMed

Paaby, Annalise B; White, Amelia G; Riccardi, David D; Gunsalus, Kristin C; Piano, Fabio; Rockman, Matthew V

2015-08-22

Embryogenesis is an essential and stereotypic process that nevertheless evolves among species. Its essentiality may favor the accumulation of cryptic genetic variation (CGV) that has no effect in the wild-type but that enhances or suppresses the effects of rare disruptions to gene function. Here, we adapted a classical modifier screen to interrogate the alleles segregating in natural populations of Caenorhabditis elegans: we induced gene knockdowns and used quantitative genetic methodology to examine how segregating variants modify the penetrance of embryonic lethality. Each perturbation revealed CGV, indicating that wild-type genomes harbor myriad genetic modifiers that may have little effect individually but which in aggregate can dramatically influence penetrance. Phenotypes were mediated by many modifiers, indicating high polygenicity, but the alleles tend to act very specifically, indicating low pleiotropy. Our findings demonstrate the extent of conditional functionality in complex trait architecture.

Resistance Management Research for PIP Crops

EPA Science Inventory

A significant increase in genetically modified corn planting driven by biofuel demand is expected for future planted acreages approaching 80% of total corn plantings in 2009. As demand increases, incidence of farmer non-compliance with mandated non-genetically modified refuge is...

Engineer Novel Anticancer Bioagents

DTIC Science & Technology

2010-10-01

selection (hence to create marker-free genetically modified organism – GMO as required by FDA regulations) have failed. The overall transformation...free genetically modified organism – GMO , as required by FDA regulations). Key Research Status 1. Reconstitution of a complete FK228 biosynthetic

Genetic selection for lifetime reproductive performance.

PubMed

Clutter, A C

2009-01-01

Genetic improvement of sow lifetime reproductive performance has value from both the economic perspectives of pork producers and the pork industry, but also from the perspective of ethical and animal welfare concerns by the general public. Genetic potential for piglets produced from individual litters is a primary determinant of lifetime prolificacy, but females must be able to sustain productivity without injury or death beyond the achievement of positive net present value. Evidence exists for between- and within-line genetic variation in sow lifetime performance, suggesting that improvements may be made by both line choices and genetic selection within lines. However, some of the same barriers to accurate within-line selection that apply to individual litter traits also present challenges to genetic selection for sow lifetime prolificacy: generally low heritabilites, sex-limited expression, expression after the age that animals are typically selected, and unfavorable genetic correlations with other traits in the profit function. In addition, there is an inherent conflict within the genetic nucleus herds where selections take place between the goal of shortened generation interval to accelerate genetic progress and the expression of sow lifetime traits. A proliferation in the industry of commercial multipliers with direct genetic ties and routine record flows to genetic nucleus herds provides a framework for accurate estimates of relevant genetic variances and covariances, and estimation of breeding values for sow lifetime traits that can be used in genetic selection.

Mutational analyses of molecularly cloned satellite tobacco mosaic virus during serial passage in plants: Evidence for hotspots of genetic change

USGS Publications Warehouse

Kurath, G.; Dodds, J.A.

1995-01-01

The high level of genetic diversity and rapid evolution of viral RNA genomes are well documented, but few studies have characterized the rate and nature of ongoing genetic change over time under controlled experimental conditions, especially in plant hosts. The RNA genome of satellite tobacco mosaic virus (STMV) was used as an effective model for such studies because of advantageous features of its genome structure and because the extant genetic heterogeneity of STMV has been characterized previously. In the present study, the process of genetic change over time was studied by monitoring multiple serial passage lines of STMV populations for changes in their consensus sequences. A total of 42 passage lines were initiated by inoculation of tobacco plants with a helper tobamovirus and one of four STMV RNA inocula that were transcribed from full-length infectious STMV clones or extracted from purified STMV type strain virions. Ten serial passages were carried out for each line and the consensus genotypes of progeny STMV populations were assessed for genetic change by RNase protection analyses of the entire 1,059-nt STMV genome. Three different types of genetic change were observed, including the fixation of novel mutations in 9 of 42 lines, mutation at the major heterogeneity site near nt 751 in 5 of the 19 lines inoculated with a single genotype, and selection of a single major genotype in 6 of the 23 lines inoculated with mixed genotypes. Sequence analyses showed that the majority of mutations were single base substitutions. The distribution of mutation sites included three clusters in which mutations occurred at or very near the same site, suggesting hot spots of genetic change in the STMV genome. The diversity of genetic changes in sibling lines is clear evidence for the important role of chance and random sampling events in the process of genetic diversification of STMV virus populations.

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.

Genetic variation in the functional ENG allele inherited from the non-affected parent associates with presence of pulmonary arteriovenous malformation in hereditary hemorrhagic telangiectasia 1 (HHT1) and may influence expression of PTPN14.

PubMed

Letteboer, Tom G W; Benzinou, Michael; Merrick, Christopher B; Quigley, David A; Zhau, Kechen; Kim, Il-Jin; To, Minh D; Jablons, David M; van Amstel, Johannes K P; Westermann, Cornelius J J; Giraud, Sophie; Dupuis-Girod, Sophie; Lesca, Gaetan; Berg, Jonathan H; Balmain, Allan; Akhurst, Rosemary J

2015-01-01

HHT shows clinical variability within and between families. Organ site and prevalence of arteriovenous malformations (AVMs) depend on the HHT causative gene and on environmental and genetic modifiers. We tested whether variation in the functional ENG allele, inherited from the unaffected parent, alters risk for pulmonary AVM in HHT1 mutation carriers who are ENG haploinsufficient. Genetic association was found between rs10987746 of the wild type ENG allele and presence of pulmonary AVM [relative risk = 1.3 (1.0018-1.7424)]. The rs10987746-C at-risk allele associated with lower expression of ENG RNA in a panel of human lymphoblastoid cell lines (P = 0.004). Moreover, in angiogenically active human lung adenocarcinoma tissue, but not in uninvolved quiescent lung, rs10987746-C was correlated with expression of PTPN14 (P = 0.004), another modifier of HHT. Quantitative TAQMAN expression analysis in a panel of normal lung tissues from 69 genetically heterogeneous inter-specific backcross mice, demonstrated strong correlation between expression levels of Eng, Acvrl1, and Ptpn14 (r2 = 0.75-0.9, P < 1 × 10(-12)), further suggesting a direct or indirect interaction between these three genes in lung in vivo. Our data indicate that genetic variation within the single functional ENG gene influences quantitative and/or qualitative differences in ENG expression that contribute to risk of pulmonary AVM in HHT1, and provide correlative support for PTPN14 involvement in endoglin/ALK1 lung biology in vivo. PTPN14 has been shown to be a negative regulator of Yap/Taz signaling, which is implicated in mechanotransduction, providing a possible molecular link between endoglin/ALK1 signaling and mechanical stress. EMILIN2, which showed suggestive genetic association with pulmonary AVM, is also reported to interact with Taz in angiogenesis. Elucidation of the molecular mechanisms regulating these interactions in endothelial cells may ultimately provide more rational choices for HHT therapy.

Genetically Modified Food: Knowledge and Attitude of Teachers and Students

NASA Astrophysics Data System (ADS)

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

2010-10-01

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 expanding technology, genetic engineering, to food production. The results indicated significant difference in understanding of concepts related with genetically engineered food stuffs between teachers and students. The most common ideas about genetically modified food were that cross bred plants and genetically modified plants are not same, GM organisms are produced by inserting a foreign gene into a plant or animal and are high yielding. More teachers thought that genetically engineered food stuffs were unsafe for the environment. Both teachers and students showed number of misconceptions, for example, the pesticidal proteins produced by GM organisms have indirect effects through bioaccumulation, induces production of allergic proteins, genetic engineering is production of new genes, GM plants are leaky sieves and that transgenes are more likely to introgress into wild species than mutated species. In general, more students saw benefits while teachers were cautious about the advantages of genetically engineered food stuffs.

Detection of Genetically Modified Maize in Processed Foods Sold Commercially in Iran by Qualitative PCR

PubMed Central

Rabiei, Maryam; Mehdizadeh, Mehrangiz; Rastegar, Hossein; Vahidi, Hossein; Alebouyeh, Mahmoud

2013-01-01

Detection of genetically modified organisms (GMOs) in food is an important issue for all the subjects involved in food control and customer’s right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt- 11 and MON810 events were detected in some maize samples, while, in none of them Bt- 176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses. PMID:24250568

Detection of genetically modified maize in processed foods sold commercially in iran by qualitative PCR.

PubMed

Rabiei, Maryam; Mehdizadeh, Mehrangiz; Rastegar, Hossein; Vahidi, Hossein; Alebouyeh, Mahmoud

2013-01-01

Detection of genetically modified organisms (GMOs) in food is an important issue for all the subjects involved in food control and customer's right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt- 11 and MON810 events were detected in some maize samples, while, in none of them Bt- 176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses.

Aquaculture: Incorporating risk assessment and risk management into public policies on genetically modified finfish and shellfish

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

Hallerman, E.M.; Kapuscinski, A.R.

Genetically modified finfish and shellfish pose economic benefits to aquaculture, but also pose ecological and genetic risks to ecosystems receiving such organisms. Realization of benefits with minimization of risks posed by a new technology can be addressed through the processes of risk assessment and risk management. Public policies adopted by individual countries will reflect differences in the outocme of risk assessment and risk management processes resulting from differences among the receiving ecosystems and sets of human values at issue. A number of countries and international institutions have begun development of policies for oversight of genetically modified aquatic organisms. In themore » United States, a working group commissioned by the U.S. Department of Agriculture incorporated risk assessment and risk management principles into draft performance standards for safely conducting research with genetically modified finfish and shellfish. The performance standards address research with a broad range of aquatic GMO`s and compliance is intended to be voluntary. In contrast, the Canadian policy mandates adherence to specified guidelines for experiments with transgenic aquatic organisms; establishment as national policy is expended soon.« less

Genetically Engineered Natural Killer Cells as a Means for Adoptive Tumor Immunotherapy.

PubMed

Michen, Susanne; Temme, Achim

2016-01-01

Natural killer (NK) cells are lymphoid cells of the innate immune system; they stand at the first defense line against viruses and transformed cells. NK cells use an array of germline-encoded activating and inhibitory receptors that sense virus-infected cells or malignant cells displaying altered surface expression of activating and inhibitory NK cell ligands. They exert potent cytotoxic responses to cellular targets and thus are candidate effector cells for immunotherapy of cancer. In particular, the genetic engineering of NK cells with chimeric antigen receptors (CARs) against surface-expressed tumor-associated antigens (TAAs) seems promising. In the allogeneic context, gene-modified NK cells compared to T cells may be superior because they are short-lived effector cells and do not cause graft-versus-host disease. Furthermore, their anti-tumoral activity can be augmented by combinatorial use with therapeutic antibodies, chemotherapeutics, and radiation. Today, efforts are being undertaken for large-scale NK-cell expansion and their genetic engineering for adoptive cell transfer. With the recent advances in understanding the complex biological interactions that regulate NK cells, it is expected that the genetic engineering of NK cells and a combinatorial blockade of immune evasion mechanisms are required to exploit the full potential of NK-cell-based immunotherapies.

Resistance Management Monitoring For the US Corn Crop

EPA Science Inventory

Significant increases in genetically modified corn planting are expected for future planted acreages approaching 80% of total corn plantings anticipated by 2009. As demand increases, incidence of farmer non-compliance with mandated non-genetically modified refuge is likely to in...

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury

PubMed Central

Quattrocelli, Mattia; Capote, Joanna; Ohiri, Joyce C.; Warner, James L.; Vo, Andy H.; Hadhazy, Michele; Demonbreun, Alexis R.; Spencer, Melissa J.; McNally, Elizabeth M.

2017-01-01

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor β (TGFβ) pathway, osteopontin encoded by the SPP1 gene and latent TGFβ binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFβ and TGFβ-associated pathways. We identified that increased TGFβ resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFβ and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy. PMID:29065150

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.

PubMed

Quattrocelli, Mattia; Capote, Joanna; Ohiri, Joyce C; Warner, James L; Vo, Andy H; Earley, Judy U; Hadhazy, Michele; Demonbreun, Alexis R; Spencer, Melissa J; McNally, Elizabeth M

2017-10-01

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor β (TGFβ) pathway, osteopontin encoded by the SPP1 gene and latent TGFβ binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFβ and TGFβ-associated pathways. We identified that increased TGFβ resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFβ and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.

Copy number abnormality of acute lymphoblastic leukemia cell lines based on their genetic subtypes.

PubMed

Tomoyasu, Chihiro; Imamura, Toshihiko; Tomii, Toshihiro; Yano, Mio; Asai, Daisuke; Goto, Hiroaki; Shimada, Akira; Sanada, Masashi; Iwamoto, Shotaro; Takita, Junko; Minegishi, Masayoshi; Inukai, Takeshi; Sugita, Kanji; Hosoi, Hajime

2018-05-21

In this study, we performed genetic analysis of 83 B cell precursor acute lymphoblastic leukemia (B-ALL) cell lines. First, we performed multiplex ligation-dependent probe amplification analysis to identify copy number abnormalities (CNAs) in eight genes associated with B-ALL according to genetic subtype. In Ph + B-ALL cell lines, the frequencies of IKZF1, CDKN2A/2B, BTG1, and PAX5 deletion were significantly higher than those in Ph - B-ALL cell lines. The frequency of CDKN2A/2B deletion in KMT2A rearranged cell lines was significantly lower than that in non-KMT2A rearranged cell lines. These findings suggest that CNAs are correlated with genetic subtype in B-ALL cell lines. In addition, we determined that three B-other ALL cell lines had IKZF1 deletions (YCUB-5, KOPN49, and KOPN75); we therefore performed comprehensive genetic analysis of these cell lines. YCUB-5, KOPN49, and KOPN75 had P2RY8-CRLF2, IgH-CRLF2, and PAX5-ETV6 fusions, respectively. Moreover, targeted capture sequencing revealed that YCUB-5 had JAK2 R683I and KRAS G12D, and KOPN49 had JAK2 R683G and KRAS G13D mutations. These data may contribute to progress in the field of leukemia research.

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis.

PubMed

Li, Jingting; Sen, George L

2015-12-14

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

Wild worm embryogenesis harbors ubiquitous polygenic modifier variation

PubMed Central

Paaby, Annalise B; White, Amelia G; Riccardi, David D; Gunsalus, Kristin C; Piano, Fabio; Rockman, Matthew V

2015-01-01

Embryogenesis is an essential and stereotypic process that nevertheless evolves among species. Its essentiality may favor the accumulation of cryptic genetic variation (CGV) that has no effect in the wild-type but that enhances or suppresses the effects of rare disruptions to gene function. Here, we adapted a classical modifier screen to interrogate the alleles segregating in natural populations of Caenorhabditis elegans: we induced gene knockdowns and used quantitative genetic methodology to examine how segregating variants modify the penetrance of embryonic lethality. Each perturbation revealed CGV, indicating that wild-type genomes harbor myriad genetic modifiers that may have little effect individually but which in aggregate can dramatically influence penetrance. Phenotypes were mediated by many modifiers, indicating high polygenicity, but the alleles tend to act very specifically, indicating low pleiotropy. Our findings demonstrate the extent of conditional functionality in complex trait architecture. DOI: http://dx.doi.org/10.7554/eLife.09178.001 PMID:26297805

Investigation of endogenous soybean food allergens by using a 2-dimensional gel electrophoresis approach.

PubMed

Rouquié, David; Capt, Annabelle; Eby, William H; Sekar, Vaithilingam; Hérouet-Guicheney, Corinne

2010-12-01

As part of the safety assessment of genetically modified (GM) soybean, 2-dimensional gel electrophoresis analyses were performed with the isoxaflutole and glyphosate tolerant soybean FG72, its non-GM near-isogenic counterpart (Jack) and three commercial non-GM soybean lines. The objective was to compare the known endogenous human food allergens in seeds in the five different soybean lines in order to evaluate any potential unintended effect(s) of the genetic modification. In total, 37 protein spots representing five well known soybean food allergen groups were quantified in each genotype. Qualitatively, all the allergenic proteins were detected in the different genetic backgrounds. Quantitatively, among 37 protein spots, the levels of accumulation of three allergens were slightly lower in the GM soybean than in the non-GM counterparts. Specifically, while the levels of two of these three allergens fell within the normal range of variation observed in the four non-GM varieties, the level of the third allergen was slightly below the normal range. Overall, there was no significant increase in the level of allergens in FG72 soybean seeds. Therefore, the FG72 soybean can be considered as safe as its non-GM counterpart with regards to endogenous allergenicity. Additional research is needed to evaluate the biological variability in the levels of endogenous soybean allergens and the correlation between level of allergens and allergenic potential in order to improve the interpretation of these data in the safety assessment of GM soybean context. Copyright © 2010 Elsevier Inc. All rights reserved.

Adaptation of the AOAC 2011.25 integrated total dietary fiber assay to determine the dietary fiber and oligosaccharide content of dry edible beans.

PubMed

Kleintop, Adrienne E; Echeverria, Dimas; Brick, Leslie A; Thompson, Henry J; Brick, Mark A

2013-10-09

Dietary fiber (DF) has important health benefits in the human diet. Developing dry edible bean (Phaseolus vulgaris L.) cultivars with improved DF and reduced nondigestible oligosaccharide content is an important goal for dry bean breeders to increase consumer acceptance. To determine if genetic variation exists among dry bean cultivars for DF, two populations of diverse dry bean cultivars/lines that represent two centers of dry bean domestication were evaluated for dietary fiber using the Integrated Total Dietary Fiber Assay (AOAC 2011.25). This assay was adapted to measure water insoluble dietary fiber, water soluble dietary fiber, oligosaccharides raffinose and stachyose, and the calculated total dietary fiber (TDF) content of cooked dry bean seed. The AOAC 2011.25 protocol was modified by using a quick, simple, and sensitive high-performance liquid chromatography method paired with an electrochemical detection method to separate and quantify specific oligosaccharides, and using duplicate samples as replicates to generate statistical information. The TDF of dry bean entries ranged from 20.0 to 27.0% in population I and from 20.6 to 25.7% in population II. Total oligosaccharides ranged from 2.56 to 4.65% in population I and from 2.36 to 3.84% in population II. The results suggest that significant genetic variation exists among dry bean cultivars/lines to allow for genetic selection for improved DF content in dry beans and that the modifications to the AOAC 2011.25 method were suitable for estimating DF in cooked dry edible beans.

Improving the efficiency of feed utilization in poultry by selection. 1. Genetic parameters of anatomy of the gastro-intestinal tract and digestive efficiency.

PubMed

de Verdal, Hugues; Narcy, Agnès; Bastianelli, Denis; Chapuis, Hervé; Même, Nathalie; Urvoix, Séverine; Le Bihan-Duval, Elisabeth; Mignon-Grasteau, Sandrine

2011-07-06

Feed costs represent about 70% of the costs of raising broilers. The main way to decrease these costs is to improve feed efficiency by modification of diet formulation, but one other possibility would be to use genetic selection. Understanding the genetic architecture of the gastro-intestinal tract (GIT) and the impact of the selection criterion on the GIT would be of particular interest. We therefore studied the genetic parameters of AMEn (Apparent metabolisable energy corrected for zero nitrogen balance), feed efficiency, and GIT traits in chickens.Genetic parameters were estimated for 630 broiler chickens of the eighth generation of a divergent selection experiment on AMEn. Birds were reared until 23 d of age and fed a wheat-based diet. The traits measured were body weight (BW), feed conversion ratio (FCR), AMEn, weights of crop, liver, gizzard and proventriculus, and weight, length and density of the duodenum, jejunum and ileum. The heritability estimates of BW, FCR and AMEn were moderate. The heritability estimates were higher for the GIT characteristics except for the weights of the proventriculus and liver. Gizzard weight was negatively correlated with density (weight to length ratio) of duodenum, jejunum and ileum. Proventriculus and gizzard weights were more strongly correlated with AMEn than with FCR, which was not the case for intestine weight and density. GIT traits were largely dependent on genetics and that selecting on AMEn or FCR would modify them. Phenotypic observations carried out in the divergent lines selected on AMEn were consistent with estimated genetic correlations between AMEn and GIT traits.

Relative Contribution of Genetic and Non-genetic Modifiers to Intestinal Obstruction in Cystic Fibrosis

PubMed Central

Blackman, Scott M.; Deering-Brose, Rebecca; McWilliams, Rita; Naughton, Kathleen; Coleman, Barbara; Lai, Teresa; Algire, Marilyn; Beck, Suzanne; Hoover-Fong, Julie; Hamosh, Ada; Fallin, M. Daniele; West, Kristen; Arking, Dan E.; Chakravarti, Aravinda; Cutler, David J.; Cutting, Garry R

2006-01-01

Background & Aims Neonatal intestinal obstruction (meconium ileus or MI) occurs in 15% of patients with cystic fibrosis (CF). Our aim was to determine the relative contribution of genetic and non-genetic modifiers to the development of this major complication of CF. Methods Using clinical data and DNA collected by the CF Twin and Sibling Study, 65 monozygous twin pairs, 23 dizygous twin/triplet sets, and 349 sets of siblings with CF were analyzed for MI status, significant covariates, and genome-wide linkage. Results Specific mutations in CFTR, the gene responsible for CF, correlated with MI indicating a role for CFTR genotype. Monozygous twins showed substantially greater concordance for MI than dizygous twins and siblings (p=1×10−5) demonstrating that modifier genes independent of CFTR contribute substantially to this trait. Regression analysis revealed that MI was correlated with distal intestinal obstruction syndrome (DIOS; p=8×10−4). Unlike MI, concordance analysis indicated that the risk for development of DIOS in CF patients is primarily due to non-genetic factors. Regions of suggestive linkage (logarithm of the odds of linkage >2.0) for modifier genes that cause MI (chromosomes 4q35.1, 8p23.1, and 11q25) or protect from MI (chromosomes 20p11.22 and 21q22.3) were identified by genome-wide analyses. These analyses did not support the existence of a major modifier gene within the CFM1 region on chromosome 19 that had previously been linked to MI. Conclusions The CFTR gene along with two or more modifier genes are the major determinants of intestinal obstruction in newborn CF patients, while intestinal obstruction in older CF patients is primarily due to non-genetic factors. PMID:17030173

Fire Increases Genetic Diversity of Populations of Six-Lined Racerunner.

PubMed

Ragsdale, Alexandria K; Frederick, Bridget M; Dukes, David W; Liebl, Andrea L; Ashton, Kyle G; McCoy, Earl D; Mushinsky, Henry R; Schrey, Aaron W

2016-01-01

Wildfires are highly variable and can disturb habitats, leading to direct and indirect effects on the genetic characteristics of local populations. Florida scrub is a fire-dependent, highly fragmented, and severely threatened habitat. Understanding the effect of fire on genetic characteristics of the species that use this habitat is critically important. We investigated one such lizard, the Six-lined Racerunner (Aspidoscelis sexlineata), which has a strong preference for open areas. We collected Six-lined Racerunners (n = 154) from 11 sites in Highlands County, FL, and defined 2 time-since-last-fire (TSF) categories: recently burned and long unburned. We screened genetic variation at 6 microsatellites to estimate genetic differentiation and compare genetic diversity among sites to determine the relationship with TSF. A clear pattern exists between genetic diversity and TSF in the absence of strong genetic differentiation. Genetic diversity was greater and inbreeding was lower in sites with more recent TSF, and genetic characteristics had significantly larger variance in long unburned sites compared with more recently burned sites. Our results suggest that fire suppression increases variance in genetic characteristics of the Six-lined Racerunner. More generally, fire may benefit genetic characteristics of some species that use fire-dependent habitats and management efforts for such severely fragmented habitat will be challenged by the presence of multiple species with incompatible fire preferences. © The American Genetic Association 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Estimation of genetic parameters and selection of high-yielding, upright common bean lines with slow seed-coat darkening.

PubMed

Alvares, R C; Silva, F C; Melo, L C; Melo, P G S; Pereira, H S

2016-11-21

Slow seed coat darkening is desirable in common bean cultivars and genetic parameters are important to define breeding strategies. The aims of this study were to estimate genetic parameters for plant architecture, grain yield, grain size, and seed-coat darkening in common bean; identify any genetic association among these traits; and select lines that associate desirable phenotypes for these traits. Three experiments were set up in the winter 2012 growing season, in Santo Antônio de Goiás and Brasília, Brazil, including 220 lines obtained from four segregating populations and five parents. A triple lattice 15 x 15 experimental design was used. The traits evaluated were plant architecture, grain yield, grain size, and seed-coat darkening. Analyses of variance were carried out and genetic parameters such as heritability, gain expected from selection, and correlations, were estimated. For selection of superior lines, a "weight-free and parameter-free" index was used. The estimates of genetic variance, heritability, and gain expected from selection were high, indicating good possibility for success in selection of the four traits. The genotype x environment interaction was proportionally more important for yield than for the other traits. There was no strong genetic correlation observed among the four traits, which indicates the possibility of selection of superior lines with many traits. Considering simultaneous selection, it was not possible to join high genetic gains for the four traits. Forty-four lines that combined high yield, more upright plant architecture, slow darkening grains, and commercial grade size were selected.

The evolution of modern agriculture and its future with biotechnology.

PubMed

Harlander, Susan K

2002-06-01

Since the dawn of agriculture, humans have been manipulating crops to enhance their quality and yield. Via conventional breeding, seed producers have developed the modern corn hybrids and wheat commonly grown today. Newer techniques, such as radiation breeding, enhanced the seed producers' ability to develop new traits in crops. Then in the 1980's-1990's, scientists began applying genetic engineering techniques to improve crop quality and yield. In contrast to earlier breeding methods, these techniques raised questions about their safety to consumers and the environment. This paper provides an overview of the kinds of genetically modified crops developed and marketed to date and the value they provide farmers and consumers. The safety assessment process required for these crops is contrasted with the lack of a formal process required for traditionally bred crops. While European consumers have expressed concern about foods and animal feeds containing ingredients from genetically modified crops, Americans have largely been unconcerned or unaware of the presence of genetically modified foods on the market. This difference in attitude is reflected in Europe's decision to label foods containing genetically modified ingredients while no such labeling is required in the U.S. In the future, genetic modification will produce a variety of new products with enhanced nutritional or quality attributes.

Genetic architecture underlying convergent evolution of egg-laying behavior in a seed-feeding beetle.

PubMed

Fox, Charles W; Wagner, James D; Cline, Sara; Thomas, Frances Ann; Messina, Frank J

2009-05-01

Independent populations subjected to similar environments often exhibit convergent evolution. An unresolved question is the frequency with which such convergence reflects parallel genetic mechanisms. We examined the convergent evolution of egg-laying behavior in the seed-feeding beetle Callosobruchus maculatus. Females avoid ovipositing on seeds bearing conspecific eggs, but the degree of host discrimination varies among geographic populations. In a previous experiment, replicate lines switched from a small host to a large one evolved reduced discrimination after 40 generations. We used line crosses to determine the genetic architecture underlying this rapid response. The most parsimonious genetic models included dominance and/or epistasis for all crosses. The genetic architecture underlying reduced discrimination in two lines was not significantly different from the architecture underlying differences between geographic populations, but the architecture underlying the divergence of a third line differed from all others. We conclude that convergence of this complex trait may in some cases involve parallel genetic mechanisms.

Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

PubMed

Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

2013-11-01

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

Spatiotemporal patterns of non-genetically modified crops in the era of expansion of genetically modified food

PubMed Central

Sun, Jing; Wu, Wenbin; Tang, Huajun; Liu, Jianguo

2015-01-01

Despite heated debates over the safety of genetically modified (GM) food, GM crops have been expanding rapidly. Much research has focused on the expansion of GM crops. However, the spatiotemporal dynamics of non-genetically modified (non-GM) crops are not clear, although they may have significant environmental and agronomic impacts and important policy implications. To understand the dynamics of non-GM crops and to inform the debates among relevant stakeholders, we conducted spatiotemporal analyses of China’s major non-GM soybean production region, the Heilongjiang Province. Even though the total soybean planting area decreased from 2005 to 2010, surprisingly, there were hotspots of increase. The results also showed hotspots of loss as well as a large decline in the number and continuity of soybean plots. Since China is the largest non-GM soybean producer in the world, the decline of its major production region may signal the continual decline of global non-GM soybeans. PMID:26380899

Dual-reporter surrogate systems for efficient enrichment of genetically modified cells.

PubMed

Ren, Chonghua; Xu, Kun; Liu, Zhongtian; Shen, Juncen; Han, Furong; Chen, Zhilong; Zhang, Zhiying

2015-07-01

Isolation of genetically modified cells generated by designed nucleases are challenging, since they are often phenotypically indistinguishable from their parental cells. To efficiently enrich genetically modified cells, we developed two dual-reporter surrogate systems, namely NHEJ-RPG and SSA-RPG based on NHEJ and SSA repair mechanisms, respectively. Repair and enrichment efficiencies of these two systems were compared using different nucleases. In both CRISPR-Cas9- and ZFNs-induced DSB repair studies, we found that the efficiency and sensitivity of the SSA-RPG reporter with direct repeat length more than 200 bp were much higher than the NHEJ-RPG reporter. By utilizing the SSA-RPG reporter, we achieved the enrichment for indels in several endogenous loci with 6.3- to 34.8-fold of non-selected cells. Thus, the highly sensitive SSA-RPG reporter can be used for activity validation of designed nucleases and efficient enrichment of genetically modified cells. Besides, our systems offer alternative enrichment choices either by puromycin selection or FACS.

Spatiotemporal patterns of non-genetically modified crops in the era of expansion of genetically modified food.

PubMed

Sun, Jing; Wu, Wenbin; Tang, Huajun; Liu, Jianguo

2015-09-18

Despite heated debates over the safety of genetically modified (GM) food, GM crops have been expanding rapidly. Much research has focused on the expansion of GM crops. However, the spatiotemporal dynamics of non-genetically modified (non-GM) crops are not clear, although they may have significant environmental and agronomic impacts and important policy implications. To understand the dynamics of non-GM crops and to inform the debates among relevant stakeholders, we conducted spatiotemporal analyses of China's major non-GM soybean production region, the Heilongjiang Province. Even though the total soybean planting area decreased from 2005 to 2010, surprisingly, there were hotspots of increase. The results also showed hotspots of loss as well as a large decline in the number and continuity of soybean plots. Since China is the largest non-GM soybean producer in the world, the decline of its major production region may signal the continual decline of global non-GM soybeans.

Legal protection of public health through control over genetically modified food.

PubMed

Gutorova, Nataliya; Batyhina, Olena; Trotska, Maryna

2018-01-01

Introduction: Science is constantly being developed which leads to both positive and negative changes in public health and the environment. One of the results of scientific progress is introduction of food based on genetically modified organisms whose effects on human health, to date, remain scantily studied and are ambiguous. The aim: to determine how human health can be influenced by food production based on genetically modified organisms. Materials and methods: international acts, data of international organizations and conclusions of scientists have been examined and used in the study. The article also summarizes information from scientific journals and monographs from a medical and legal point of view with scientific methods. This article is based on dialectical, comparative, analytic, synthetic and comprehensive research methods. Conclusions: Genetically modified organisms are specific human-made organisms being a result of using modern biotechnology techniques. They have both positive and negative effects on human health and the environment. The main disadvantage is not sufficient study of them in various spheres of public life.

Establishing Substantial Equivalence: Proteomics

NASA Astrophysics Data System (ADS)

Lovegrove, Alison; Salt, Louise; Shewry, Peter R.

Wheat is a major crop in world agriculture and is consumed after processing into a range of food products. It is therefore of great importance to determine the consequences (intended and unintended) of transgenesis in wheat and whether genetically modified lines are substantially equivalent to those produced by conventional plant breeding. Proteomic analysis is one of several approaches which can be used to address these questions. Two-dimensional PAGE (2D PAGE) remains the most widely available method for proteomic analysis, but is notoriously difficult to reproduce between laboratories. We therefore describe methods which have been developed as standard operating procedures in our laboratory to ensure the reproducibility of proteomic analyses of wheat using 2D PAGE analysis of grain proteins.

Determination of inorganic elements in blood of mice immunized with Bothrops Snake venom using XRF and NAA

NASA Astrophysics Data System (ADS)

Lopes da Silva, L. F. F.; Zamboni, C. B.; Bahovschi, V.; Metairon, S.; Suzuki, M. F.; Sant'Anna, O. A.; Rizzutto, M. A.

2015-07-01

In this work, mice genetically modified [HIII line] were immunized against different Bothrops snake venoms to produce anti-Bothrops serum (antivenom). The Neutron Activation Analysis (NAA) and Energy Dispersive X-Ray Fluorescence (EDXRF) techniques were used to evaluate Ca and Fe concentrations in blood of these immunized mice in order to establish a potential correlation between both phenotypes: antibody response and blood constituents after Bothrops venom administration. The results were compared with the control group (mice not immunized) and with human being estimative. These data are important for clinical screening of patients submitted to immunological therapy as well as the understanding of the envenoming mechanisms.

Application of a modified selection index for honey bees (Hymenoptera: Apidae).

PubMed

van Engelsdorp, D; Otis, G W

2000-12-01

Nine different genetic families of honey bees (Apis mellifera L.) were compared using summed z-scores (phenotypic values) and a modified selection index (Imod). Imod values incorporated both the phenotypic scores of the different traits and the economic weightings of these traits, as determined by a survey of commercial Ontario beekeepers. Largely because of the high weight all beekeepers place on honey production, a distinct difference between line rankings based on phenotypic scores and Imod scores was apparent, thereby emphasizing the need to properly weight the traits being evaluated to select bee stocks most valuable for beekeepers. Furthermore, when beekeepers who made >10% of their income from queen and nucleus colony sales assigned relative values to the traits used in the Imod calculations, the results differed from those based on weightings assigned by honey producers. Our results underscore the difficulties the North American beekeeping industry must overcome to devise effective methods of evaluating colonies for breeding purposes.

Identification of possible genetic alterations in the breast cancer cell line MCF-7 using high-density SNP genotyping microarray

PubMed Central

Wang, Hui-Yun; Greenawalt, Danielle; Cui, Xiangfeng; Tereshchenko, Irina V; Luo, Minjie; Yang, Qifeng; Azaro, Marco A; Hu, Guohong; Chu, Yi; Li, James Y; Shen, Li; Lin, Yong; Zhang, Lianjun

2009-01-01

Context: Cancer cell lines are used extensively in various research. Knowledge of genetic alterations in these lines is important for understanding mechanisms underlying their biology. However, since paired normal tissues are usually unavailable for comparison, precisely determining genetic alterations in cancer cell lines is difficult. To address this issue, a highly efficient and reliable method is developed. Aims: Establishing a highly efficient and reliable experimental system for genetic profiling of cell lines. Materials and Methods: A widely used breast cancer cell line, MCF-7, was genetically profiled with 4,396 single nucleotide polymorphisms (SNPs) spanning 11 whole chromosomes and two other small regions using a newly developed high-throughput multiplex genotyping approach. Results: The fractions of homozygous SNPs in MCF-7 (13.3%) were significantly lower than those in the control cell line and in 24 normal human individuals (25.1% and 27.4%, respectively). Homozygous SNPs in MCF-7 were found in clusters. The sizes of these clusters were significantly larger than the expected based on random allelic combination. Fourteen such regions were found on chromosomes 1p, 1q, 2q, 6q, 13, 15q, 16q, 17q and 18p in MCF-7 and two in the small regions. Conclusions: These results are generally concordant with those obtained using different approaches but are better in defining their chromosomal positions. The used approach provides a reliable way to detecting possible genetic alterations in cancer cell lines without paired normal tissues. PMID:19439911

Effects of genetic distance on heterosis in a Drosophila melanogaster model system.

PubMed

Jensen, Charlotte; Ørsted, Michael; Kristensen, Torsten Nygaard

2018-05-14

Habitat fragmentation and small population sizes can lead to inbreeding and loss of genetic variation, which can potentially cause inbreeding depression and decrease the ability of populations to adapt to altered environmental conditions. One solution to these genetic problems is the implementation of genetic rescue, which re-establishes gene flow between separated populations. Similar techniques are being used in animal and plant breeding to produce superior production animals and plants. To optimize fitness benefits in genetic rescue programs and to secure high yielding domestic varieties in animal and plant breeding, knowledge on the genetic relatedness of populations being crossed is imperative. In this study, we conducted replicated crosses between isogenic Drosophila melanogaster lines from the Drosophila Genetic Reference Panel. We grouped lines in two genetic distance groups to study the effect of genetic divergence between populations on the expression of heterosis in two fitness components; starvation resistance and reproductive output. We further investigated the transgenerational effects of outcrossing by investigating the fitness consequences in both the F 1 - and the F 3 -generations. High fitness enhancements were observed in hybrid offspring compared to parental lines, especially for reproductive output. However, the level of heterosis declined from the F 1 - to the F 3 -generation. Generally, genetic distance did not have strong impact on the level of heterosis detected, although there were exceptions to this pattern. The best predictor of heterosis was performance of parental lines with poorly performing parental lines showing higher hybrid vigour when crossed, i.e. the potential for heterosis was proportional to the level of inbreeding depression. Overall, our results show that outcrossing can have very strong positive fitness consequences for genetically depauperate populations.

Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes.

PubMed

Espley, Richard V; Bovy, Arnaud; Bava, Christina; Jaeger, Sara R; Tomes, Sumathi; Norling, Cara; Crawford, Jonathan; Rowan, Daryl; McGhie, Tony K; Brendolise, Cyril; Putterill, Jo; Schouten, Henk J; Hellens, Roger P; Allan, Andrew C

2013-05-01

Consumers of whole foods, such as fruits, demand consistent high quality and seek varieties with enhanced health properties, convenience or novel taste. We have raised the polyphenolic content of apple by genetic engineering of the anthocyanin pathway using the apple transcription factor MYB10. These apples have very high concentrations of foliar, flower and fruit anthocyanins, especially in the fruit peel. Independent lines were examined for impacts on tree growth, photosynthesis and fruit characteristics. Fruit were analysed for changes in metabolite and transcript levels. Fruit were also used in taste trials to study the consumer perception of such a novel apple. No negative taste attributes were associated with the elevated anthocyanins. Modification with this one gene provides near isogenic material and allows us to examine the effects on an established cultivar, with a view to enhancing consumer appeal independently of other fruit qualities. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

PubMed

Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A

2015-01-23

The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. Copyright © 2015, American Association for the Advancement of Science.

The genetic basis of pectoralis major myopathies in modern broiler chicken lines.

PubMed

Bailey, Richard A; Watson, Kellie A; Bilgili, S F; Avendano, Santiago

2015-12-01

This is the first report providing estimates of the genetic basis of breast muscle myopathies (BMM) and their relationship with growth and yield in broiler chickens. In addition, this paper addresses the hypothesis that genetic selection for increase breast yield has contributed to the onset of BMM. Data were analyzed from ongoing recording of BMM within the Aviagen breeding program. This study focused on three BMM: deep pectoral myopathy (DPM; binary trait), white striping (WS; 4 categories) and wooden breast (WB; 3 categories). Data from two purebred commercial broiler lines (A and B) were utilized providing greater than 40,000 meat quality records per line. The difference in selection history between these two lines has resulted in contrasting breast yield (BY): 29% for Line A and 21% for Line B. Data were analyzed to estimate genetic parameters using a multivariate animal model including six traits: body weight (BW), processing body weight (PW), BY, DPM, WB, and WS, in addition to the appropriate fixed effects and permanent environmental effect of the dam. Results indicate similar patterns of heritability and genetic correlations for the two lines. Heritabilities (h2) of BW, PW and BY ranged from 0.271-0.418; for DPM and WB h2<0.1; and for WS h2≤0.338. Genetic correlations between the BMM and BW, PW, or BY were ≤0.132 in Line A and ≤0.248 in Line B. This paper demonstrates the polygenic nature of these traits and the low genetic relationships with BW, PW, and BY, which facilitates genetic improvement across all traits in a balanced breeding program. It also highlights the importance of understanding the environmental and/or management factors that contribute greater than 65% of the variance in the incidence of white striping of breast muscle and more than 90% of the variance of the incidence of wooden breast and deep pectoral myopathy in broiler chickens. © The Author 2015. Published by Oxford University Press on behalf of Poultry Science Association.

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs.

PubMed

Bähr, Andrea; Käser, Tobias; Kemter, Elisabeth; Gerner, Wilhelm; Kurome, Mayuko; Baars, Wiebke; Herbach, Nadja; Witter, Kirsti; Wünsch, Annegret; Talker, Stephanie C; Kessler, Barbara; Nagashima, Hiroshi; Saalmüller, Armin; Schwinzer, Reinhard; Wolf, Eckhard; Klymiuk, Nikolai

2016-01-01

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action.

The therapeutic potential of human adipose-derived mesenchymal stem cells producing CXCL10 in a mouse melanoma lung metastasis model.

PubMed

Mirzaei, Hamed; Salehi, Hossein; Oskuee, Reza Kazemi; Mohammadpour, Ali; Mirzaei, Hamid Reza; Sharifi, Mohammad Reza; Salarinia, Reza; Darani, Hossein Yousofi; Mokhtari, Mojgan; Masoudifar, Aria; Sahebkar, Amirhossein; Salehi, Rasoul; Jaafari, Mahmoud Reza

2018-04-10

Interferon γ-induced protein 10 kDa (IP-10) is a potent chemoattractant and has been suggested to enhance antitumor activity and mediate tumor regression through multiple mechanisms of action. Multiple lines of evidence have indicated that genetically-modified adult stem cells represent a potential source for cell-based cancer therapy. In the current study, we assessed therapeutic potential of human adipose derived mesenchymal stem cells (hADSC) genetically-modified to express IP-10 for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A Piggybac vector encoding IP-10 was employed to transfect hADSC ex vivo. Expression and bioactivity of the transgenic protein from hADSCs expressing IP-10 were confirmed prior to in vivo studies. Our results indicated that hADSCs expressing IP-10 could inhibit the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis, TUNEL assay and western blot analysis indicated that hADSCs expressing IP-10 inhibited tumor cell growth, hindered tumor infiltration of Tregs, restricted angiogenesis and significantly prolonged survival. In conclusion, our results demonstrated that targeting metastatic tumor sites by hADSC expressing IP-10 could reduce melanoma tumor growth and lung metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

A genome-wide RNAi screen identifies potential drug targets in a C. elegans model of α1-antitrypsin deficiency.

PubMed

O'Reilly, Linda P; Long, Olivia S; Cobanoglu, Murat C; Benson, Joshua A; Luke, Cliff J; Miedel, Mark T; Hale, Pamela; Perlmutter, David H; Bahar, Ivet; Silverman, Gary A; Pak, Stephen C

2014-10-01

α1-Antitrypsin deficiency (ATD) is a common genetic disorder that can lead to end-stage liver and lung disease. Although liver transplantation remains the only therapy currently available, manipulation of the proteostasis network (PN) by small molecule therapeutics offers great promise. To accelerate the drug-discovery process for this disease, we first developed a semi-automated high-throughput/content-genome-wide RNAi screen to identify PN modifiers affecting the accumulation of the α1-antitrypsin Z mutant (ATZ) in a Caenorhabditis elegans model of ATD. We identified 104 PN modifiers, and these genes were used in a computational strategy to identify human ortholog-ligand pairs. Based on rigorous selection criteria, we identified four FDA-approved drugs directed against four different PN targets that decreased the accumulation of ATZ in C. elegans. We also tested one of the compounds in a mammalian cell line with similar results. This methodology also proved useful in confirming drug targets in vivo, and predicting the success of combination therapy. We propose that small animal models of genetic disorders combined with genome-wide RNAi screening and computational methods can be used to rapidly, economically and strategically prime the preclinical discovery pipeline for rare and neglected diseases with limited therapeutic options. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

PubMed Central

JIN, Li-Fang; LI, Jin-Song

2016-01-01

With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches. PMID:27469251

Endogenous Reference Genes and Their Quantitative Real-Time PCR Assays for Genetically Modified Bread Wheat (Triticum aestivum L.) Detection.

PubMed

Yang, Litao; Quan, Sheng; Zhang, Dabing

2017-01-01

Endogenous reference genes (ERG) and their derivate analytical methods are standard requirements for analysis of genetically modified organisms (GMOs). Development and validation of suitable ERGs is the primary step for establishing assays that monitoring the genetically modified (GM) contents in food/feed samples. Herein, we give a review of the ERGs currently used for GM wheat analysis, such as ACC1, PKABA1, ALMT1, and Waxy-D1, as well as their performances in GM wheat analysis. Also, we discussed one model for developing and validating one ideal RG for one plant species based on our previous research work.

Population and allelic variation of A-to-I RNA editing in human transcriptomes.

PubMed

Park, Eddie; Guo, Jiguang; Shen, Shihao; Demirdjian, Levon; Wu, Ying Nian; Lin, Lan; Xing, Yi

2017-07-28

A-to-I RNA editing is an important step in RNA processing in which specific adenosines in some RNA molecules are post-transcriptionally modified to inosines. RNA editing has emerged as a widespread mechanism for generating transcriptome diversity. However, there remain significant knowledge gaps about the variation and function of RNA editing. In order to determine the influence of genetic variation on A-to-I RNA editing, we integrate genomic and transcriptomic data from 445 human lymphoblastoid cell lines by combining an RNA editing QTL (edQTL) analysis with an allele-specific RNA editing (ASED) analysis. We identify 1054 RNA editing events associated with cis genetic polymorphisms. Additionally, we find that a subset of these polymorphisms is linked to genome-wide association study signals of complex traits or diseases. Finally, compared to random cis polymorphisms, polymorphisms associated with RNA editing variation are located closer spatially to their respective editing sites and have a more pronounced impact on RNA secondary structure. Our study reveals widespread cis variation in RNA editing among genetically distinct individuals and sheds light on possible phenotypic consequences of such variation on complex traits and diseases.

Resistance Management Monitoring for the US Corn Crop to the Illinois Corn Growers Association

EPA Science Inventory

Significant increases in genetically modified corn planting are expected for future planted acreages approaching 80% of total corn plantings anticipated by 2009. As demand increases, incidence of farmer non-compliance with mandated non-genetically modified refuge is likely to in...

TRACKING GENE FLOW FROM A GENETICALLY MODIFIED CREEPING BENTGRASS -- METHODS, MEASURES AND LESSONS LEARNED

EPA Science Inventory

Creeping bentgrass (CBG) expressing an engineered gene for resistance to glyphosate herbicide is one of the first genetically modified (GM) perennial crops to undergo regulatory review for commercial release by the US Department of Agriculture Animal Plant Health and Inspection S...

40 CFR 172.48 - Data requirements for a notification.

Code of Federal Regulations, 2010 CFR

2010-07-01

... PROGRAMS EXPERIMENTAL USE PERMITS Notification for Certain Genetically Modified Microbial Pesticides § 172... methods used to genetically modify the microbial pesticide. (h) The identity and location of the gene... organisms. (d) Information on survival and the ability of the microbial pesticide to increase in numbers...

Electrotransformation and clonal isolation of Rickettsia species

PubMed Central

Riley, Sean P; Macaluso, Kevin R; Martinez, Juan J

2015-01-01

Genetic manipulation of obligate intracellular bacteria of the genus Rickettsia is currently undergoing a rapid period of change. The development of viable genetic tools, including replicative plasmids, transposons, homologous recombination, fluorescent protein-encoding genes, and antibiotic selectable markers has provided the impetus for future research development. This unit is designed to coalesce the basic methods pertaining to creation of genetically modified Rickettsia. The unit describes a series of methods, from inserting exogenous DNA into Rickettsia to the final isolation of genetically modified bacterial clones. Researchers working towards genetic manipulation of Rickettsia or similar obligate intracellular bacteria will find these protocols to be a valuable reference. PMID:26528784

Gone with the Wind: Conceiving of Moral Responsibility in the Case of GMO Contamination.

PubMed

Robaey, Zoë

2016-06-01

Genetically modified organisms are a technology now used with increasing frequency in agriculture. Genetically modified seeds have the special characteristic of being living artefacts that can reproduce and spread; thus it is difficult to control where they end up. In addition, genetically modified seeds may also bring about uncertainties for environmental and human health. Where they will go and what effect they will have is therefore very hard to predict: this creates a puzzle for regulators. In this paper, I use the problem of contamination to complicate my ascription of forward-looking moral responsibility to owners of genetically modified organisms. Indeed, how can owners act responsibly if they cannot know that contamination has occurred? Also, because contamination creates new and unintended ownership, it challenges the ascription of forward-looking moral responsibility based on ownership. From a broader perspective, the question this paper aims to answer is as follows: how can we ascribe forward-looking moral responsibility when the effects of the technologies in question are difficult to know or unknown? To solve this problem, I look at the epistemic conditions for moral responsibility and connect them to the normative notion of the social experiment. Indeed, examining conditions for morally responsible experimentation helps to define a range of actions and to establish the related epistemic virtues that owners should develop in order to act responsibly where genetically modified organisms are concerned.

Environmental biosafety and transgenic potato in a centre of diversity for this crop.

PubMed

Celis, Carolina; Scurrah, Maria; Cowgill, Sue; Chumbiauca, Susana; Green, Jayne; Franco, Javier; Main, Gladys; Kiezebrink, Daan; Visser, Richard G F; Atkinson, Howard J

2004-11-11

The Nuffield Council on Bioethics suggests that introgression of genetic material into related species in centres of crop biodiversity is an insufficient justification to bar the use of genetically modified crops in the developing world. They consider that a precautionary approach to forgo the possible benefits invokes the fallacy of thinking that doing nothing is itself without risk to the poor. Here we report findings relevant to this and other aspects of environmental biosafety for genetically modified potato in its main centre of biodiversity, the central Andes. We studied genetically modified potato clones that provide resistance to nematodes, principal pests of Andean potato crops. We show that there is no harm to many non-target organisms, but gene flow occurs to wild relatives growing near potato crops. If stable introgression were to result, the fitness of these wild species could be altered. We therefore transformed the male sterile cultivar Revolucion to provide a genetically modified nematode-resistant potato to evaluate the benefits that this provides until the possibility of stable introgression to wild relatives is determined. Thus, scientific progress is possible without compromise to the precautionary principle.

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

PubMed

Chen, Hancai; Bodulovic, Greg; Hall, Prudence J; Moore, Andy; Higgins, Thomas J V; Djordjevic, Michael A; Rolfe, Barry G

2009-09-01

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

Combined influence of Bt rice and rice dwarf virus on biological parameters of a non-target herbivore, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae)

PubMed Central

Wang, Qianjin; Han, Naishun; Dang, Cong; Lu, Zengbin; Wang, Fang; Yao, Hongwei; Peng, Yufa; Stanley, David

2017-01-01

The advent of genetically modified (GM) Bt rice creates the possibility of interactions among Bt crops, crop pathogens and non-target herbivores. In particular, information on how pathogen-infected Bt-expressing plants will influence non-target herbivores is necessary to predict the sustainability of GM cropping systems. Laboratory bioassays were conducted to evaluate the potential combined impacts of rice dwarf virus (RDV) and two Bt rice lines, T1C-19 (Cry1C) and T2A-1 (Cry2A), on non-target green rice leafhopper (GRLH), Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae). In the first experiment, GRLHs feeding preference tests on Bt rice lines compared to a parental control rice line, MH63, were conducted. As rice plants were uninfected with RDV, GRLHs generally preferred the control MH63 line over the two Bt lines during the initial 8 h, with no significant preference during the following 64 h. As rice plants were infected with RDV, there were no clear preferences between the Bt rice lines and the control MH63 line. In the second experiment, we assessed the combined influence of RDV-infection status and Bt rice lines on GRLH biological parameters. Egg duration, adult weights, and male adult longevity were significantly affected on RDV-infected Bt rice. Other parameters, egg hatching rate, nymph survival and fecundity were not significantly influenced. We infer that interaction effect among two testing Bt rice lines and RDV will not lead to enlarged pest populations, thus demonstrating that growing these two Bt rice lines will poses negligible risk to GRLH in sustainable rice agroecosystems. Long-term field experiments to monitor the population dynamics of GRLHs at large scale need to be carried out to confirm the current results. PMID:28753622

Combined influence of Bt rice and rice dwarf virus on biological parameters of a non-target herbivore, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae).

PubMed

Wang, Qianjin; Han, Naishun; Dang, Cong; Lu, Zengbin; Wang, Fang; Yao, Hongwei; Peng, Yufa; Stanley, David; Ye, Gongyin

2017-01-01

The advent of genetically modified (GM) Bt rice creates the possibility of interactions among Bt crops, crop pathogens and non-target herbivores. In particular, information on how pathogen-infected Bt-expressing plants will influence non-target herbivores is necessary to predict the sustainability of GM cropping systems. Laboratory bioassays were conducted to evaluate the potential combined impacts of rice dwarf virus (RDV) and two Bt rice lines, T1C-19 (Cry1C) and T2A-1 (Cry2A), on non-target green rice leafhopper (GRLH), Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae). In the first experiment, GRLHs feeding preference tests on Bt rice lines compared to a parental control rice line, MH63, were conducted. As rice plants were uninfected with RDV, GRLHs generally preferred the control MH63 line over the two Bt lines during the initial 8 h, with no significant preference during the following 64 h. As rice plants were infected with RDV, there were no clear preferences between the Bt rice lines and the control MH63 line. In the second experiment, we assessed the combined influence of RDV-infection status and Bt rice lines on GRLH biological parameters. Egg duration, adult weights, and male adult longevity were significantly affected on RDV-infected Bt rice. Other parameters, egg hatching rate, nymph survival and fecundity were not significantly influenced. We infer that interaction effect among two testing Bt rice lines and RDV will not lead to enlarged pest populations, thus demonstrating that growing these two Bt rice lines will poses negligible risk to GRLH in sustainable rice agroecosystems. Long-term field experiments to monitor the population dynamics of GRLHs at large scale need to be carried out to confirm the current results.

A modifier of Huntington's disease onset at the MLH1 locus.

PubMed

Lee, Jong-Min; Chao, Michael J; Harold, Denise; Abu Elneel, Kawther; Gillis, Tammy; Holmans, Peter; Jones, Lesley; Orth, Michael; Myers, Richard H; Kwak, Seung; Wheeler, Vanessa C; MacDonald, Marcy E; Gusella, James F

2017-10-01

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expanded CAG repeat in HTT. Many clinical characteristics of HD such as age at motor onset are determined largely by the size of HTT CAG repeat. However, emerging evidence strongly supports a role for other genetic factors in modifying the disease pathogenesis driven by mutant huntingtin. A recent genome-wide association analysis to discover genetic modifiers of HD onset age provided initial evidence for modifier loci on chromosomes 8 and 15 and suggestive evidence for a locus on chromosome 3. Here, genotyping of candidate single nucleotide polymorphisms in a cohort of 3,314 additional HD subjects yields independent confirmation of the former two loci and moves the third to genome-wide significance at MLH1, a locus whose mouse orthologue modifies CAG length-dependent phenotypes in a Htt-knock-in mouse model of HD. Both quantitative and dichotomous association analyses implicate a functional variant on ∼32% of chromosomes with the beneficial modifier effect that delays HD motor onset by 0.7 years/allele. Genomic DNA capture and sequencing of a modifier haplotype localize the functional variation to a 78 kb region spanning the 3'end of MLH1 and the 5'end of the neighboring LRRFIP2, and marked by an isoleucine-valine missense variant in MLH1. Analysis of expression Quantitative Trait Loci (eQTLs) provides modest support for altered regulation of MLH1 and LRRFIP2, raising the possibility that the modifier affects regulation of both genes. Finally, polygenic modification score and heritability analyses suggest the existence of additional genetic modifiers, supporting expanded, comprehensive genetic analysis of larger HD datasets. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

ASSESSMENT OF ALLERGENIC POTENTIAL OF GENETICALLY MODIFIED FOODS: AN AGENDA FOR FUTURE RESEARCH

EPA Science Inventory

Abstract
Speakers and participants in the Workshop Assessment of the Allergenic Potential of Genetically Modified Foods met in breakout groups to discuss a number of issues including needs for future research. There was agreement that research should move forward quickly in t...

78 FR 25297 - Programmatic Environmental Assessment

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-30

... environmental assessment (PEA) to evaluate the effects of the cultivation and use of genetically modified crops... genetically modified crops (GMCs) on our Refuge System lands. Our PEA will concentrate on the refuges in our... lands are those that have been evaluated and deregulated by the Animal and Plant Health Inspection...

Genetic Variants in the Bone Morphogenic Protein Gene Family Modify the Association between Residential Exposure to Traffic and Peripheral Arterial Disease

EPA Science Inventory

There is a growing literature indicating that genetic variants modify many of the associations between environmental exposures and clinical outcomes, potentially by increasing susceptibility to these exposures. However, genome-scale investigations of these interactions have been ...

Evidence for the establishment and persistence of genetically modified canola populations in the U.S.

EPA Science Inventory

Background/Questions/Methods Concerns surrounding the commercial release of genetically modified crops include the risks of escape from cultivation, naturalization, and the transfer of beneficial traits to native and weedy species. Among the crops commonly grown in the U.S., a l...

Use of spectral imaging for insect resistance monitoring: EPA research on stewardship of Bt crops

EPA Science Inventory

A significant increase in genetically modified corn planting driven by biofuel demand is expected for future growing seasons. As demand increases, incidence of farmer non-compliance with mandated non-genetically modified refuge is likely to increase. As part of the FIFRA regist...

Standing genetic variation as a major contributor to adaptation in the Virginia chicken lines selection experiment.

PubMed

Sheng, Zheya; Pettersson, Mats E; Honaker, Christa F; Siegel, Paul B; Carlborg, Örjan

2015-10-01

Artificial selection provides a powerful approach to study the genetics of adaptation. Using selective-sweep mapping, it is possible to identify genomic regions where allele-frequencies have diverged during selection. To avoid false positive signatures of selection, it is necessary to show that a sweep affects a selected trait before it can be considered adaptive. Here, we confirm candidate, genome-wide distributed selective sweeps originating from the standing genetic variation in a long-term selection experiment on high and low body weight of chickens. Using an intercross between the two divergent chicken lines, 16 adaptive selective sweeps were confirmed based on their association with the body weight at 56 days of age. Although individual additive effects were small, the fixation for alternative alleles across the loci contributed at least 40 % of the phenotypic difference for the selected trait between these lines. The sweeps contributed about half of the additive genetic variance present within and between the lines after 40 generations of selection, corresponding to a considerable portion of the additive genetic variance of the base population. Long-term, single-trait, bi-directional selection in the Virginia chicken lines has resulted in a gradual response to selection for extreme phenotypes without a drastic reduction in the genetic variation. We find that fixation of several standing genetic variants across a highly polygenic genetic architecture made a considerable contribution to long-term selection response. This provides new fundamental insights into the dynamics of standing genetic variation during long-term selection and adaptation.

Fire alters patterns of genetic diversity among 3 lizard species in Florida Scrub habitat.

PubMed

Schrey, Aaron W; Ashton, Kyle G; Heath, Stacy; McCoy, Earl D; Mushinsky, Henry R

2011-01-01

The Florida Sand Skink (Plestiodon reynoldsi), the Florida Scrub Lizard (Sceloporus woodi), and the Six-lined Racerunner (Aspidoscelis sexlineata) occur in the threatened and fire-maintained Florida scrub habitat. Fire may have different consequences to local genetic diversity of these species because they each have different microhabitat preference. We collected tissue samples of each species from 3 sites with different time-since-fire: Florida Sand Skink n = 73, Florida Scrub Lizard n = 70, and Six-lined Racerunner n = 66. We compared the effect of fire on genetic diversity at microsatellite loci for each species. We screened 8 loci for the Florida Sand Skink, 6 loci for the Florida Scrub Lizard, and 6 loci for the Six-lined Racerunner. We also tested 2 potential driving mechanisms for the observed change in genetic diversity, a metapopulation source/sink model and a local demographic model. Genetic diversity varied with fire history, and significant genetic differentiation occurred among sites. The Florida Scrub Lizard had highest genetic variation at more recently burned sites, whereas the Florida Sand Skink and the Six-lined Racerunner had highest genetic variation at less recently burned sites. Habitat preferences of the Florida Sand Skink and the Florida Scrub Lizard may explain their discordant results, and the Six-lined Racerunner may have a more complicated genetic response to fire or is acted on at a different geographic scale than we have investigated. Our results indicate that these species may respond to fire in a more complicated manner than predicted by our metapopulation model or local demographic model. Our results show that the population-level responses in genetic diversity to fire are species-specific mandating conservation management of habitat diversity through a mosaic of burn frequencies.

Production of engineered long-life and male sterile Pelargonium plants

PubMed Central

2012-01-01

Background Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. Results The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3–4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. Conclusion The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers. PMID:22935247

Production of engineered long-life and male sterile Pelargonium plants.

PubMed

García-Sogo, Begoña; Pineda, Benito; Roque, Edelín; Antón, Teresa; Atarés, Alejandro; Borja, Marisé; Beltrán, José Pío; Moreno, Vicente; Cañas, Luis Antonio

2012-08-31

Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3-4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.

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

Impacts of genetic line, gender and season on feeding behavior of grow-finish swine

USDA-ARS?s Scientific Manuscript database

Feeding behavior contains important information that can enable producers to better manage livestock. A study was conducted to quantify these impacts. Data were collected on barrows and gilts (n = 931) from 3 different genetic lines (Landrace x Yorkshire material line with three different sire bree...

Grain quality traits in a sorghum association mapping panel

USDA-ARS?s Scientific Manuscript database

Grain quality traits were analyzed in a diverse sorghum sample set which consisted of 174 sorghum lines (110 non-tannin lines and 64 tannin lines). These samples were previously grouped into five distinct genetic populations which made it possible to compare grain quality traits across the genetic g...

Grain quality traits in sorghum association mapping panel

USDA-ARS?s Scientific Manuscript database

Grain quality traits were analyzed in a diverse sorghum sample set which consisted of 174 sorghum lines (110 non-tannin lines and 64 tannin lines). These samples were previously grouped into five distinct genetic populations which made it possible to compare grain quality traits across the genetic g...

Cell cloning-on-the-spot by using an attachable silicone cylinder.

PubMed

Park, Hong Bum; Son, Wonseok; Chae, Dong Han; Lee, Jisu; Kim, Il-Woung; Yang, Woomi; Sung, Jae Kyu; Lim, Kyu; Lee, Jun Hee; Kim, Kyung-Hee; Park, Jong-Il

2016-06-10

Cell cloning is a laboratory routine to isolate and keep particular properties of cultured cells. Transfected or other genetically modified cells can be selected by the traditional microbiological cloning. In addition, common laboratory cell lines are prone to genotypic drift during their continual culture, so that supplementary cloning steps are often required to maintain correct lineage phenotypes. Here, we designed a silicone-made attachable cloning cylinder, which facilitated an easy and bona fide cloning of interested cells. This silicone cylinder was easy to make, showed competent stickiness to laboratory plastics including culture dishes, and hence enabled secure isolation and culture for days of selected single cells, especially, on the spots of preceding cell-plating dishes under microscopic examination of visible cellular phenotypes. We tested the silicone cylinder in the monoclonal subcloning from a heterogeneous population of a breast cancer cell line, MDA-MB-231, and readily established independent MDA-MB-231 subclones showing different sublineage phenotypes. Copyright © 2016 Elsevier Inc. All rights reserved.

A Cell Line Producing Recombinant Nerve Growth Factor Evokes Growth Responses in Intrinsic and Grafted Central Cholinergic Neurons

NASA Astrophysics Data System (ADS)

Ernfors, Patrik; Ebendal, Ted; Olson, Lars; Mouton, Peter; Stromberg, Ingrid; Persson, Hakan

1989-06-01

The rat β nerve growth factor (NGF) gene was inserted into a mammalian expression vector and cotransfected with a plasmid conferring resistance to neomycin into mouse 3T3 fibroblasts. From this transfection a stable cell line was selected that contains several hundred copies of the rat NGF gene and produces excess levels of recombinant NGF. Such genetically modified cells were implanted into the rat brain as a probe for in vivo effects of NGF on central nervous system neurons. In a model of the cortical cholinergic deficits in Alzheimer disease, we demonstrate a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain cholinergic neurons, as well as stimulation of fiber formation by intact adult intrinsic cholinergic circuits in the cerebral cortex. Adult cholinergic interneurons in intact striatum also sprout vigorously toward implanted fibroblasts. Our results suggest that this model has implications for future treatment of neurodegenerative diseases.

A Cyfip2-Dependent Excitatory Interneuron Pathway Establishes the Innate Startle Threshold.

PubMed

Marsden, Kurt C; Jain, Roshan A; Wolman, Marc A; Echeverry, Fabio A; Nelson, Jessica C; Hayer, Katharina E; Miltenberg, Ben; Pereda, Alberto E; Granato, Michael

2018-04-17

Sensory experiences dynamically modify whether animals respond to a given stimulus, but it is unclear how innate behavioral thresholds are established. Here, we identify molecular and circuit-level mechanisms underlying the innate threshold of the zebrafish startle response. From a forward genetic screen, we isolated five mutant lines with reduced innate startle thresholds. Using whole-genome sequencing, we identify the causative mutation for one line to be in the fragile X mental retardation protein (FMRP)-interacting protein cyfip2. We show that cyfip2 acts independently of FMRP and that reactivation of cyfip2 restores the baseline threshold after phenotype onset. Finally, we show that cyfip2 regulates the innate startle threshold by reducing neural activity in a small group of excitatory hindbrain interneurons. Thus, we identify a selective set of genes critical to establishing an innate behavioral threshold and uncover a circuit-level role for cyfip2 in this process. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Producing primate embryonic stem cells by somatic cell nuclear transfer.

PubMed

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

2007-11-22

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

An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway

PubMed Central

Gonsalves, Foster C.; Klein, Keren; Carson, Brittany B.; Katz, Shauna; Ekas, Laura A.; Evans, Steve; Nagourney, Robert; Cardozo, Timothy; Brown, Anthony M. C.; DasGupta, Ramanuj

2011-01-01

Misregulated β-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of β-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear β-catenin. We show that these inhibitors efficiently block Wnt/β-catenin–induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling. PMID:21393571

Evidence for Absolute Moral Opposition to Genetically Modified Food in the United States.

PubMed

Scott, Sydney E; Inbar, Yoel; Rozin, Paul

2016-05-01

Public opposition to genetic modification (GM) technology in the food domain is widespread (Frewer et al., 2013). In a survey of U.S. residents representative of the population on gender, age, and income, 64% opposed GM, and 71% of GM opponents (45% of the entire sample) were "absolutely" opposed-that is, they agreed that GM should be prohibited no matter the risks and benefits. "Absolutist" opponents were more disgust sensitive in general and more disgusted by the consumption of genetically modified food than were non-absolutist opponents or supporters. Furthermore, disgust predicted support for legal restrictions on genetically modified foods, even after controlling for explicit risk-benefit assessments. This research suggests that many opponents are evidence insensitive and will not be influenced by arguments about risks and benefits. © The Author(s) 2016.

Genetic modifiers of Velo- cardio- facial syndrome/DiGeorge syndrome

PubMed Central

Aggarwal, Vimla S.; Morrow, Bernice E.

2009-01-01

Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), the most common micro-deletion disorder in humans, is characterized by craniofacial, parathyroid and thymic defects as well as cardiac outflow tract malformations. Most patients have a similar hemizygous 3 million base pair deletion on 22q11.2. Studies in mouse have shown that Tbx1, a T- box containing transcription factor present on the deleted region, is likely responsible for the etiology of the syndrome. Furthermore, mutations in TBX1 have been found in rare non-deleted patients. Despite having the same sized deletion, most VCFS/DGS patients exhibit significant clinical variability. Stochastic, environmental and genetic factors likely modify the phenotype of patients with the disorder. Here, we review mouse genetics studies which may help identify genetic modifiers for VCFS/DGS. PMID:18636633

Review: Genetically modified plants for the promotion of human health.

PubMed

Yonekura-Sakakibara, Keiko; Saito, Kazuki

2006-12-01

Plants are attractive biological resources because of their ability to produce a huge variety of chemical compounds, and the familiarity of production in even the most rural settings. Genetic engineering gives plants additional characteristics and value for cultivation and post-harvest. Genetically modified (GM) plants of the "first generation" were conferred with traits beneficial to producers, whereas GM plants in subsequent "generations" are intended to provide beneficial traits for consumers. Golden Rice is a promising example of a GM plant in the second generation, and has overcome a number of obstacles for practical use. Furthermore, consumer-acceptable plants with health-promoting properties that are genetically modified using native genes are being developed. The emerging technology of metabolomics will also support the commercial realization of GM plants by providing comprehensive analyzes of plant biochemical components.

Transdifferentiation of brain-derived neurotrophic factor (BDNF)-secreting mesenchymal stem cells significantly enhance BDNF secretion and Schwann cell marker proteins.

PubMed

Bierlein De la Rosa, Metzere; Sharma, Anup D; Mallapragada, Surya K; Sakaguchi, Donald S

2017-11-01

The use of genetically modified mesenchymal stem cells (MSCs) is a rapidly growing area of research targeting delivery of therapeutic factors for neuro-repair. Cells can be programmed to hypersecrete various growth/trophic factors such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) to promote regenerative neurite outgrowth. In addition to genetic modifications, MSCs can be subjected to transdifferentiation protocols to generate neural cell types to physically and biologically support nerve regeneration. In this study, we have taken a novel approach by combining these two unique strategies and evaluated the impact of transdifferentiating genetically modified MSCs into a Schwann cell-like phenotype. After 8 days in transdifferentiation media, approximately 30-50% of transdifferentiated BDNF-secreting cells immunolabeled for Schwann cell markers such as S100β, S100, and p75 NTR . An enhancement was observed 20 days after inducing transdifferentiation with minimal decreases in expression levels. BDNF production was quantified by ELISA, and its biological activity tested via the PC12-TrkB cell assay. Importantly, the bioactivity of secreted BDNF was verified by the increased neurite outgrowth of PC12-TrkB cells. These findings demonstrate that not only is BDNF actively secreted by the transdifferentiated BDNF-MSCs, but also that it has the capacity to promote neurite sprouting and regeneration. Given the fact that BDNF production remained stable for over 20 days, we believe that these cells have the capacity to produce sustainable, effective, BDNF concentrations over prolonged time periods and should be tested within an in vivo system for future experiments. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFβ signaling in the mammary gland

PubMed Central

2010-01-01

Introduction Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFβ)/Smad pathway as an example. Methods We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse), which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFβ. Results IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFβ. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFβ-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFβ-induced invasion as long as a threshold level of total Smad is exceeded. Conclusions The present work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium. PMID:20942910

Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

PubMed

Cho, Fu-Nan; Chang, Tsung-Hsien; Shu, Chih-Wen; Ko, Ming-Chin; Liao, Shuen-Kuei; Wu, Kang-Hsi; Yu, Ming-Sun; Lin, Shyh-Jer; Hong, Ying-Chung; Chen, Chien-Hsun; Hung, Chien-Hui; Chang, Yu-Hsiang

2014-01-01

Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

Modifier locus mapping of a transgenic F2 mouse population identifies CCDC115 as a novel aggressive prostate cancer modifier gene in humans.

PubMed

Winter, Jean M; Curry, Natasha L; Gildea, Derek M; Williams, Kendra A; Lee, Minnkyong; Hu, Ying; Crawford, Nigel P S

2018-06-11

It is well known that development of prostate cancer (PC) can be attributed to somatic mutations of the genome, acquired within proto-oncogenes or tumor-suppressor genes. What is less well understood is how germline variation contributes to disease aggressiveness in PC patients. To map germline modifiers of aggressive neuroendocrine PC, we generated a genetically diverse F2 intercross population using the transgenic TRAMP mouse model and the wild-derived WSB/EiJ (WSB) strain. The relevance of germline modifiers of aggressive PC identified in these mice was extensively correlated in human PC datasets and functionally validated in cell lines. Aggressive PC traits were quantified in a population of 30 week old (TRAMP x WSB) F2 mice (n = 307). Correlation of germline genotype with aggressive disease phenotype revealed seven modifier loci that were significantly associated with aggressive disease. RNA-seq were analyzed using cis-eQTL and trait correlation analyses to identify candidate genes within each of these loci. Analysis of 92 (TRAMP x WSB) F2 prostates revealed 25 candidate genes that harbored both a significant cis-eQTL and mRNA expression correlations with an aggressive PC trait. We further delineated these candidate genes based on their clinical relevance, by interrogating human PC GWAS and PC tumor gene expression datasets. We identified four genes (CCDC115, DNAJC10, RNF149, and STYXL1), which encompassed all of the following characteristics: 1) one or more germline variants associated with aggressive PC traits; 2) differential mRNA levels associated with aggressive PC traits; and 3) differential mRNA expression between normal and tumor tissue. Functional validation studies of these four genes using the human LNCaP prostate adenocarcinoma cell line revealed ectopic overexpression of CCDC115 can significantly impede cell growth in vitro and tumor growth in vivo. Furthermore, CCDC115 human prostate tumor expression was associated with better survival outcomes. We have demonstrated how modifier locus mapping in mouse models of PC, coupled with in silico analyses of human PC datasets, can reveal novel germline modifier genes of aggressive PC. We have also characterized CCDC115 as being associated with less aggressive PC in humans, placing it as a potential prognostic marker of aggressive PC.

Aptamer modification improves the adenoviral transduction of malignant glioma cells.

PubMed

Chen, Hao; Zheng, Xiaojing; Di, BingYan; Wang, Dongyang; Zhang, Yaling; Xia, Haibin; Mao, Qinwen

2013-12-01

Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Genetic aspects of auto-immune profiles of healthy chickens.

PubMed

Parmentier, Henk K; van der Vaart, Priscilla S; Nieuwland, Mike G B; Savelkoul, Huub F J

2017-09-01

Auto-antibody profiles binding liver antigens differed between chicken lines divergently selected for specific antibody responses to SRBC, and were affected by ageing suggesting both genetic and environmental effects. Presence and levels of IgM and IgG antibodies binding chicken liver cell lysate (CLL) fragments in plasma at 5 weeks of age from 10 individual full sibs and their parents from 5 H srbc and 5 L srbc line families was studied to reveal genetic relations. Non-genetic maternal effects were studied by comparing auto-antibody profiles of 36 weeks old hens from 2 other unrelated lines with the profiles from their chicks at hatch. IgM and IgG antibodies from parents and progeny from both H srbc and L srbc lines bound CLL fragments. Significant line and generation differences and their interactions were found for both isotypes. Higher staining of CLL fragments was usually found for H srbc line birds. Lines were clustered by auto-antibody profiles, but staining by birds of both lines in both generations was very individual for IgG and IgM. The current data with full sibs therefore not supported a genetic basis for auto-antibody profiles. IgG but not IgM auto-antibody profiles of chicks correlated with maternal auto-antibody profiles. The results suggest that the auto-antibody repertoire of healthy chickens is largely stochastically initiated and may be affected by environmental challenges during ageing, but genetic mechanisms may underlie staining intensity of individual bound CLL fragments. The present results suggest that identification of fragments or profiles to be used at early age for genetic selection for health traits is not feasible yet. Secondly, the IgM profile of neonatal chickens seems non-organised independent of the maternal profile, but the neonatal IgG profile is much more related with the maternal profile. Consequences of these findings for disease susceptibility or breeding for optimal health are discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic dissection of a regionally differentiated network for exploratory behavior in Drosophila larvae.

PubMed

Berni, Jimena

2015-05-18

An efficient strategy to explore the environment for available resources involves the execution of random walks where straight line locomotion alternates with changes of direction. This strategy is highly conserved in the animal kingdom, from zooplankton to human hunter-gatherers. Drosophila larvae execute a routine of this kind, performing straight line crawling interrupted at intervals by pause turns that halt crawling and redirect the trajectory of movement. The execution of this routine depends solely on the activity of networks located in the thoracic and abdominal segments of the nervous system, while descending input from the brain serves to modify it in a context-dependent fashion. I used a genetic method to investigate the location and function of the circuitry required for the different elements of exploratory crawling. By using the Slit-Robo axon guidance pathway to target neuronal midline crossing defects selectively to particular regions of the thoracic and abdominal networks, it has been possible to define at least three functions required for the performance of the exploratory routine: (1) symmetrical outputs in thoracic and abdominal segments that generate the crawls; (2) asymmetrical output that is uniquely initiated in the thoracic segments and generates the turns; and (3) an intermittent interruption to crawling that determines the time-dependent transition between crawls and turns. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genetic Dissection of a Regionally Differentiated Network for Exploratory Behavior in Drosophila Larvae

PubMed Central

Berni, Jimena

2015-01-01

Summary An efficient strategy to explore the environment for available resources involves the execution of random walks where straight line locomotion alternates with changes of direction. This strategy is highly conserved in the animal kingdom, from zooplankton to human hunter-gatherers [1–8]. Drosophila larvae execute a routine of this kind, performing straight line crawling interrupted at intervals by pause turns that halt crawling and redirect the trajectory of movement [9–11]. The execution of this routine depends solely on the activity of networks located in the thoracic and abdominal segments of the nervous system, while descending input from the brain serves to modify it in a context-dependent fashion [9]. I used a genetic method to investigate the location and function of the circuitry required for the different elements of exploratory crawling. By using the Slit-Robo axon guidance pathway to target neuronal midline crossing defects selectively to particular regions of the thoracic and abdominal networks, it has been possible to define at least three functions required for the performance of the exploratory routine: (1) symmetrical outputs in thoracic and abdominal segments that generate the crawls; (2) asymmetrical output that is uniquely initiated in the thoracic segments and generates the turns; and (3) an intermittent interruption to crawling that determines the time-dependent transition between crawls and turns. PMID:25959962

Design and Management of Field Trials of Transgenic Cereals

NASA Astrophysics Data System (ADS)

Bedő, Zoltán; Rakszegi, Mariann; Láng, László

The development of gene transformation systems has allowed the introgression of alien genes into plant genomes, thus providing a mechanism for broadening the genetic resources available to plant breeders. The design and the management of field trials vary according to the purpose for which transgenic cereals are developed. Breeders study the phenotypic and genotypic stability of transgenic plants, monitor the increase in homozygosity of transgenic genotypes under field conditions, and develop backcross generations to transfer the introduced genes into secondary transgenic cereal genotypes. For practical purposes, they may also multiply seed of the transgenic lines to produce sufficient amounts of grain for the detailed analysis of trait(s) of interest, to determine the field performance of transgenic lines, and to compare them with the non-transformed parental genotypes. Prior to variety registration, the Distinctness, Uniformity and Stability (DUS) tests and Value for Cultivation and Use (VCU) experiments are carried out in field trials. Field testing includes specific requirements for transgenic cereals to assess potential environmental risks. The capacity of the pollen to survive, establish and disseminate in the field test environment, the potential for gene transfer, the effects of products expressed by the introduced sequences and phenotypic and genotypic instability that might cause deleterious effects must all be specifically monitored, as required by EU Directives 2003/701/EC (1) on the release of genetically modified higher plants in the environment.

A Novel Method to Generate and Expand Clinical-Grade, Genetically Modified, Tumor-Infiltrating Lymphocytes

PubMed Central

Forget, Marie-Andrée; Tavera, René J.; Haymaker, Cara; Ramachandran, Renjith; Malu, Shuti; Zhang, Minying; Wardell, Seth; Fulbright, Orenthial J.; Toth, Chistopher Leroy; Gonzalez, Audrey M.; Thorsen, Shawne T.; Flores, Esteban; Wahl, Arely; Peng, Weiyi; Amaria, Rodabe N.; Hwu, Patrick; Bernatchez, Chantale

2017-01-01

Following the clinical success achieved with the first generation of adoptive cell therapy (ACT) utilizing in vitro expanded tumor-infiltrating lymphocytes (TILs), the second and third generations of TIL ACT are evolving toward the use of genetically modified TIL. TIL therapy generally involves the transfer of a high number of TIL, ranging from 109 to 1011 cells. One of the technical difficulties in genetically modifying TIL, using a retroviral vector, is the ability to achieve large expansion of transduced TIL, while keeping the technique suitable to a Good Manufacturing Practices (GMP) environment. Consequently, we developed and optimized a novel method for the efficient production of large numbers of GMP-grade, gene-modified TIL for the treatment of patients with ACT. The chemokine receptor CXCR2 was used as the gene of interest for methodology development. The optimized procedure is currently used in the production of gene-modified TIL for two clinical trials for the treatment of metastatic melanoma at MD Anderson Cancer Center. PMID:28824634

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

PubMed

Zahradnik, Celine; Kolm, Claudia; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

2014-11-01

In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.

Compositions and methods for increased ethanol titer from biomass

DOEpatents

Jessen, Holly J.; Yi, Jian

2016-11-15

The present application discloses the identification of novel I. orientalis ADH1, ADHa, and ADHb genes, and the production and characterization of genetically modified yeast cells in which these genes were altered. Provided herein are isolated I. orientalis ADH1, ADHa, and ADHb polynucleotides and polypeptides, genetically modified yeast cells that overexpress I. orientalis ADH1 and/or contain deletions or disruptions of ADHa and/or ADHb, and methods of using culturing these modified cells to produce ethanol.

The role of genetic background in susceptibility to chemical warfare nerve agents across rodent and non-human primate models.

PubMed

Matson, Liana M; McCarren, Hilary S; Cadieux, C Linn; Cerasoli, Douglas M; McDonough, John H

2018-01-15

Genetics likely play a role in various responses to nerve agent exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses to environmental stimuli. Mouse strains or selected lines can be used to identify susceptibility based on background genetic features to nerve agent exposure. Additional genetic techniques can then be used to identify mechanisms underlying resistance and sensitivity, with the ultimate goal of developing more effective and targeted therapies. Here, we discuss the available literature on strain and selected line differences in cholinesterase activity levels and response to nerve agent-induced toxicity and seizures. We also discuss the available cholinesterase and toxicity literature across different non-human primate species. The available data suggest that robust genetic differences exist in cholinesterase activity, nerve agent-induced toxicity, and chemical-induced seizures. Available cholinesterase data suggest that acetylcholinesterase activity differs across strains, but are limited by the paucity of carboxylesterase data in strains and selected lines. Toxicity and seizures, two outcomes of nerve agent exposure, have not been fully evaluated for genetic differences, and thus further studies are required to understand baseline strain and selected line differences. Published by Elsevier B.V.

Horizontal gene transfer between bacteria.

PubMed

Heuer, Holger; Smalla, Kornelia

2007-01-01

Horizontal gene transfer (HGT) refers to the acquisition of foreign genes by organisms. The occurrence of HGT among bacteria in the environment is assumed to have implications in the risk assessment of genetically modified bacteria which are released into the environment. First, introduced genetic sequences from a genetically modified bacterium could be transferred to indigenous micro-organisms and alter their genome and subsequently their ecological niche. Second, the genetically modified bacterium released into the environment might capture mobile genetic elements (MGE) from indigenous micro-organisms which could extend its ecological potential. Thus, for a risk assessment it is important to understand the extent of HGT and genome plasticity of bacteria in the environment. This review summarizes the present state of knowledge on HGT between bacteria as a crucial mechanism contributing to bacterial adaptability and diversity. In view of the use of GM crops and microbes in agricultural settings, in this mini-review we focus particularly on the presence and role of MGE in soil and plant-associated bacteria and the factors affecting gene transfer.

Harnessing Genetic Variation in Leaf Angle to Increase Productivity of Sorghum bicolor

PubMed Central

Truong, Sandra K.; McCormick, Ryan F.; Rooney, William L.; Mullet, John E.

2015-01-01

The efficiency with which a plant intercepts solar radiation is determined primarily by its architecture. Understanding the genetic regulation of plant architecture and how changes in architecture affect performance can be used to improve plant productivity. Leaf inclination angle, the angle at which a leaf emerges with respect to the stem, is a feature of plant architecture that influences how a plant canopy intercepts solar radiation. Here we identify extensive genetic variation for leaf inclination angle in the crop plant Sorghum bicolor, a C4 grass species used for the production of grain, forage, and bioenergy. Multiple genetic loci that regulate leaf inclination angle were identified in recombinant inbred line populations of grain and bioenergy sorghum. Alleles of sorghum dwarf-3, a gene encoding a P-glycoprotein involved in polar auxin transport, are shown to change leaf inclination angle by up to 34° (0.59 rad). The impact of heritable variation in leaf inclination angle on light interception in sorghum canopies was assessed using functional-structural plant models and field experiments. Smaller leaf inclination angles caused solar radiation to penetrate deeper into the canopy, and the resulting redistribution of light is predicted to increase the biomass yield potential of bioenergy sorghum by at least 3%. These results show that sorghum leaf angle is a heritable trait regulated by multiple loci and that genetic variation in leaf angle can be used to modify plant architecture to improve sorghum crop performance. PMID:26323882

Genetic Analysis of Recombinant Inbred Lines For Sorghum Bicolor x Perennial S. Propinquum.

USDA-ARS?s Scientific Manuscript database

From an annual S. bicolor x perennial S. propinquum F2 population used in early-generation genetic analysis, we have produced and describe here a recombinant inbred line (RIL) population of 161 F5 genotypes that segregates for rhizomatousness and many other traits. The genetic map of the recombinant...

Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

PubMed

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

PubMed Central

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

KEY ISSUES FOR THE ASSESSMENT OF THE ALLERGENIC POTENTIAL OF GENETICALLY MODIFIED FOODS: BREAKOUT GROUP REPORTS

EPA Science Inventory

Abstract
On the final afternoon of the Workshop, Assessment of the Allergenic Potential of Genetically Modified Foods, speakers and participants met in breakout groups to discuss specific questions in the areas of 1) Use of Human Clinical Data; 2) Animal Models to Assess Food ...

DEVELOPMENT OF A MULTI-TIERED INSECT RESISTANCE MANAGEMENT PROGRAM FOR GENETICALLY MODIFIED CORN HYBRIDS EXPRESSING THE PLANT INCORPORATED PROTECTANT, BACILLUS THURINGIENSIS

EPA Science Inventory

A significant increase in genetically modified corn planting driven by biofuel demand is expected for the 2007 growing season with future planted acreages approaching 80% of total corn plantings anticipated by 2009. As demand increases, incidence of farmer non-compliance with ma...

L-malate production by metabolically engineered escherichia coli

DOEpatents

Zhang, Xueli; Wang, Xuan; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

2015-11-17

A process for the production of malic acid in commercially significant quantities from the carbon compounds by genetically modified bacterial strains (GMBS; also referred to as biocatalysts or genetically modified microorganisms) is disclosed. Microorganisms suitable for the production of malic acid can be cultured in one or two-step processes as disclosed herein.

Pulmonary arterial hypertension associated to systemic erythematous lupus: molecular characterization of 3 cases.

PubMed

Pousada, Guillermo; Lago-Docampo, Mauro; Baloira, Adolfo; Valverde, Diana

2018-03-08

Pulmonary arterial hypertension associated with systemic lupus erythematosus (PAH-SLE) is a rare disease with a low incidence rate. In this study, PAH related genes and genetic modifiers were characterised molecularly in patients with PAH-SLE. Three patients diagnosed with PAH-SLE and 100 control individuals were analysed after signing an informed consent. Two out of the three analysed patients with PAH-SLE were carriers of pathogenic mutations in the genes BMPR2 and ENG. After an in silico analysis, pathogenic mutations were searched for in control individuals and different databases, with negative results, and they were thus functionally analysed. The third patients only showed polymorphisms in the genes BMPR2, ACVRL1 and ENG. Several genetic variants and genetic modifiers were identified in the three analysed patients. These modifiers, along with the pathogenic mutations, could lead to a more severe clinical course in patients with PAH. We present, for the first time, patients with PAH-SLE carrying pathogenic mutations in the main genes related to PAH and alterations in the genetic modifiers. Copyright © 2018 Elsevier España, S.L.U. All rights reserved.

Knowlege of, attitudes toward, and acceptance of genetically modified organisms among prospective teachers of biology, home economics, and grade school in Slovenia.

PubMed

Sorgo, Andrej; Ambrožič-Dolinšek, Jana

2010-05-01

The objective of this study was to investigate knowledge, opinions, and attitudes toward, as well as readiness to accept genetically modified organisms (GMOs) among prospective primary and secondary Slovene teachers. Our findings are that prospective teachers want to take an active role in rejecting or supporting individual GMOs and are aware of the importance of education about genetically modified organism (GMO) items and their potential significance for society. Through cluster analysis, we recognized four clusters of GMOs, separated by degree of genetically modified acceptability. GM plants and microorganisms which are recognized as useful are accepted. They are undecided about organisms used in research or medicine and reject organisms used for food consumption and for fun. There are only weak correlations between knowledge and attitudes and knowledge and acceptance of GMOs, and a strong correlation between attitudes and acceptance. The appropriate strategies and actions for improving university courses in biotechnology are discussed. Copyright © 2010 International Union of Biochemistry and Molecular Biology, Inc.

DNA degradation in genetically modified rice with Cry1Ab by food processing methods: implications for the quantification of genetically modified organisms.

PubMed

Xing, Fuguo; Zhang, Wei; Selvaraj, Jonathan Nimal; Liu, Yang

2015-05-01

Food processing methods contribute to DNA degradation, thereby affecting genetically modified organism detection and quantification. This study evaluated the effect of food processing methods on the relative transgenic content of genetically modified rice with Cry1Ab. In steamed rice and rice noodles, the levels of Cry1Ab were ⩾ 100% and <83%, respectively. Frying and baking in rice crackers contributed to a reduction in Pubi and Cry1Ab, while microwaving caused a decrease in Pubi and an increase in Cry1Ab. The processing methods of sweet rice wine had the most severe degradation effects on Pubi and Cry1Ab. In steamed rice and rice noodles, Cry1Ab was the most stable, followed by SPS and Pubi. However, in rice crackers and sweet rice wine, SPS was the most stable, followed by Cry1Ab and Pubi. Therefore, Cry1Ab is a better representative of transgenic components than is Pubi because the levels of Cry1Ab were less affected compared to Pubi. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genetic and epigenetic status of triple exotic consanguinity cotton introgression lines.

PubMed

He, S P; Sun, J L; Du, X M

2011-10-03

Introgression lines are some of the most important germplasm for breeding applications and other research conducted on cotton crops. The DNA methylation level among 10 introgression lines of cotton (Gossypium hirsutum) and three exotic parental species (G. arboreum, G. thurberi and G. barbadense) were assessed by methylation-sensitive amplified polymorphism (MSAP) technology. The methylation level in the introgression lines ranged from 33.3 to 51.5%. However, the lines PD0111 and PD0113 had the lowest methylation level (34.6 and 33.3%, respectively) due to demethylation of most non-coding sequences. Amplified fragment length polymorphism (AFLP) was used to evaluate the genetic polymorphism in the cotton introgression lines. A high degree of polymorphism was observed in all introgression lines (mean 47.2%) based on AFLP and MSAP analyses. This confirmed the effects of genetic improvement on cotton introgression lines. The low methylation varieties, PD0111 and PD0113 (introgression lines), clustered outside of the introgression lines based on MSAP data, which was incongruent with an AFLP-based dendrogram. This phenomenon could be caused by environmental changes or introgression of exotic DNA fragments.

Infestation of Transgenic Powdery Mildew-Resistant Wheat by Naturally Occurring Insect Herbivores under Different Environmental Conditions

PubMed Central

Álvarez-Alfageme, Fernando; von Burg, Simone; Romeis, Jörg

2011-01-01

A concern associated with the growing of genetically modified (GM) crops is that they could adversely affect non-target organisms. We assessed the impact of several transgenic powdery mildew-resistant spring wheat lines on insect herbivores. The GM lines carried either the Pm3b gene from hexaploid wheat, which confers race-specific resistance to powdery mildew, or the less specific anti-fungal barley seed chitinase and β-1,3-glucanase. In addition to the non-transformed control lines, several conventional spring wheat varieties and barley and triticale were included for comparison. During two consecutive growing seasons, powdery mildew infection and the abundance of and damage by naturally occurring herbivores were estimated under semi-field conditions in a convertible glasshouse and in the field. Mildew was reduced on the Pm3b-transgenic lines but not on the chitinase/glucanase-expressing lines. Abundance of aphids was negatively correlated with powdery mildew in the convertible glasshouse, with Pm3b wheat plants hosting significantly more aphids than their mildew-susceptible controls. In contrast, aphid densities did not differ between GM plants and their non-transformed controls in the field, probably because of low mildew and aphid pressure at this location. Likewise, the GM wheat lines did not affect the abundance of or damage by the herbivores Oulema melanopus (L.) and Chlorops pumilionis Bjerk. Although a previous study has revealed that some of the GM wheat lines show pleiotropic effects under field conditions, their effect on herbivorous insects appears to be low. PMID:21829479

Infestation of transgenic powdery mildew-resistant wheat by naturally occurring insect herbivores under different environmental conditions.

PubMed

Álvarez-Alfageme, Fernando; von Burg, Simone; Romeis, Jörg

2011-01-01

A concern associated with the growing of genetically modified (GM) crops is that they could adversely affect non-target organisms. We assessed the impact of several transgenic powdery mildew-resistant spring wheat lines on insect herbivores. The GM lines carried either the Pm3b gene from hexaploid wheat, which confers race-specific resistance to powdery mildew, or the less specific anti-fungal barley seed chitinase and β-1,3-glucanase. In addition to the non-transformed control lines, several conventional spring wheat varieties and barley and triticale were included for comparison. During two consecutive growing seasons, powdery mildew infection and the abundance of and damage by naturally occurring herbivores were estimated under semi-field conditions in a convertible glasshouse and in the field. Mildew was reduced on the Pm3b-transgenic lines but not on the chitinase/glucanase-expressing lines. Abundance of aphids was negatively correlated with powdery mildew in the convertible glasshouse, with Pm3b wheat plants hosting significantly more aphids than their mildew-susceptible controls. In contrast, aphid densities did not differ between GM plants and their non-transformed controls in the field, probably because of low mildew and aphid pressure at this location. Likewise, the GM wheat lines did not affect the abundance of or damage by the herbivores Oulema melanopus (L.) and Chlorops pumilionis Bjerk. Although a previous study has revealed that some of the GM wheat lines show pleiotropic effects under field conditions, their effect on herbivorous insects appears to be low.

Lifestyle genomics and the metabolic syndrome: A review of genetic variants that influence response to diet and exercise interventions.

PubMed

Fenwick, Peri H; Jeejeebhoy, Khursheed; Dhaliwal, Rupinder; Royall, Dawna; Brauer, Paula; Tremblay, Angelo; Klein, Doug; Mutch, David M

2018-02-05

Metabolic syndrome (MetS) comprises a cluster of risk factors that includes central obesity, dyslipidemia, impaired glucose homeostasis and hypertension. Individuals with MetS have elevated risk of type 2 diabetes and cardiovascular disease; thus placing significant burdens on social and healthcare systems. Lifestyle interventions (comprised of diet, exercise or a combination of both) are routinely recommended as the first line of treatment for MetS. Only a proportion of people respond, and it has been assumed that psychological and social aspects primarily account for these differences. However, the etiology of MetS is multifactorial and stems, in part, on a person's genetic make-up. Numerous single nucleotide polymorphisms (SNPs) are associated with the various components of MetS, and several of these SNPs have been shown to modify a person's response to lifestyle interventions. Consequently, genetic variants can influence the extent to which a person responds to changes in diet and/or exercise. The goal of this review is to highlight SNPs reported to influence the magnitude of change in body weight, dyslipidemia, glucose homeostasis and blood pressure during lifestyle interventions aimed at improving MetS components. Knowledge regarding these genetic variants and their ability to modulate a person's response will provide additional context for improving the effectiveness of personalized lifestyle interventions that aim to reduce the risks associated with MetS.

Genetic variation and population structure of maize inbred lines adapted to the mid-altitude sub-humid maize agro-ecology of Ethiopia using single nucleotide polymorphic (SNP) markers.

PubMed

Ertiro, Berhanu Tadesse; Semagn, Kassa; Das, Biswanath; Olsen, Michael; Labuschagne, Maryke; Worku, Mosisa; Wegary, Dagne; Azmach, Girum; Ogugo, Veronica; Keno, Tolera; Abebe, Beyene; Chibsa, Temesgen; Menkir, Abebe

2017-10-12

Molecular characterization is important for efficient utilization of germplasm and development of improved varieties. In the present study, we investigated the genetic purity, relatedness and population structure of 265 maize inbred lines from the Ethiopian Institute of Agricultural Research (EIAR), the International Maize and Wheat Improvement Centre (CIMMYT) and the International Institute of Tropical Agriculture (IITA) using 220,878 single nucleotide polymorphic (SNP) markers obtained using genotyping by sequencing (GBS). Only 22% of the inbred lines were considered pure with <5% heterogeneity, while the remaining 78% of the inbred lines had a heterogeneity ranging from 5.1 to 31.5%. Pairwise genetic distances among the 265 inbred lines varied from 0.011 to 0.345, with 89% of the pairs falling between 0.301 and 0.345. Only <1% of the pairs had a genetic distance lower than 0.200, which included 14 pairs of sister lines that were nearly identical. Relative kinship analysis showed that the kinship coefficients for 59% of the pairs of lines was close to zero, which agrees with the genetic distance estimates. Principal coordinate analysis, discriminant analysis of principal components (DAPC) and the model-based population structure analysis consistently suggested the presence of three groups, which generally agreed with pedigree information (genetic background). Although not distinct enough, the SNP markers showed some level of separation between the two CIMMYT heterotic groups A and B established based on pedigree and combining ability information. The high level of heterogeneity detected in most of the inbred lines suggested the requirement for purification or further inbreeding except those deliberately maintained at early inbreeding level. The genetic distance and relative kinship analysis clearly indicated the uniqueness of most of the inbred lines in the maize germplasm available for breeders in the mid-altitude maize breeding program of Ethiopia. Results from the present study facilitate the maize breeding work in Ethiopia and germplasm exchange among breeding programs in Africa. We suggest the incorporation of high density molecular marker information in future heterotic group assignments.

Genetic Background Has a Major Impact on Differences in Sleep Resulting from Environmental Influences in Drosophila

PubMed Central

Zimmerman, John E.; Chan, May T.; Jackson, Nicholas; Maislin, Greg; Pack, Allan I.

2012-01-01

Study Objectives: To determine the effect of different genetic backgrounds on demographic and environmental interventions that affect sleep and evaluate variance of these measures; and to evaluate sleep and variance of sleep behaviors in 6 divergent laboratory strains of common origin. Design: Assessment of the effects of age, sex, mating status, food sources, and social experience using video analysis of sleep behavior in 2 different strains of Drosophila, white1118ex (w1118ex) and white Canton-S (wCS10). Sleep was also determined for 6 laboratory strains of Canton-S and 3 inbred lines. The variance of total sleep was determined for all groups and conditions. Measurements and Results: The circadian periods and the effects of age upon sleep were the same between w1118ex and wCS10 strains. However, the w1118ex and wCS10 strains demonstrated genotype-dependent differences in the effects upon sleep of sex, mating status, social experience, and being on different foods. Variance of total sleep was found to differ in a genotype dependent manner for interventions between the w1118ex and wCS10 strains. Six different laboratory Canton-S strains were found to have significantly different circadian periods (P < 0.001) and sleep phenotypes (P < 0.001). Three inbred lines showed reduced variance for sleep measurements. Conclusions: One must control environmental conditions in a rigorously consistent manner to ensure that sleep data may be compared between experiments. Genetic background has a significant impact upon changes in sleep behavior and variance of behavior due to demographic factors and environmental interventions. This represents an opportunity to discover new genes that modify sleep/wake behavior. Citation: Zimmerman JE; Chan MT; Jackson N; Maislin G; Pack AI. Genetic background has a major impact on differences in sleep resulting from environmental influences in Drosophila. SLEEP 2012;35(4):545-557. PMID:22467993

Attitudes towards genetically modified and organic foods.

PubMed

Saher, Marieke; Lindeman, Marjaana; Hursti, Ulla-Kaisa Koivisto

2006-05-01

Finnish students (N=3261) filled out a questionnaire on attitudes towards genetically modified and organic food, plus the rational-experiential inventory, the magical thinking about food and health scale, Schwartz's value survey and the behavioural inhibition scale. In addition, they reported their eating of meat. Structural equation modelling of these measures had greater explanatory power for attitudes towards genetically modified (GM) foods than for attitudes towards organic foods (OF). GM attitudes were best predicted by natural science education and magical food and health beliefs, which mediated the influence of thinking styles. Positive attitudes towards organic food, on the other hand, were more directly related to such individual differences as thinking styles and set of values. The results of the study indicate that OF attitudes are rooted in more fundamental personal attributes than GM attitudes, which are embedded in a more complex but also in a more modifiable network of characteristics.

Mechanisms and Modifiers of Methylmercury-Induced Neurotoxicity

PubMed Central

Fretham, Stephanie JB; Caito, Samuel; Martinez-Finley, Ebany J; Aschner, Michael

2016-01-01

The neurotoxic consequences of methylmercury (MeHg) exposure have long been known, however a complete understanding of the mechanisms underlying this toxicity is elusive. Recent epidemiological and experimental studies have provided many mechanistic insights, particularly into the contribution of genetic and environmental factors that interact with MeHg to modify toxicity. This review will outline cellular processes directly and indirectly affected by MeHg, including oxidative stress, cellular signaling and gene expression, and discuss genetic, environmental and nutritional factors capable of modifying MeHg toxicity. PMID:27795823

Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination.

PubMed

Campos, Samuel K; Barry, Michael A

2004-11-01

There are extensive efforts to develop cell-targeting adenoviral vectors for gene therapy wherein endogenous cell-binding ligands are ablated and exogenous ligands are introduced by genetic means. Although current approaches can genetically manipulate the capsid genes of adenoviral vectors, these approaches can be time-consuming and require multiple steps to produce a modified viral genome. We present here the use of the bacteriophage lambda Red recombination system as a valuable tool for the easy and rapid construction of capsid-modified adenoviral genomes.

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.

The genome architecture of the Collaborative Cross mouse genetic reference population.

PubMed

2012-02-01

The Collaborative Cross Consortium reports here on the development of a unique genetic resource population. The Collaborative Cross (CC) is a multiparental recombinant inbred panel derived from eight laboratory mouse inbred strains. Breeding of the CC lines was initiated at multiple international sites using mice from The Jackson Laboratory. Currently, this innovative project is breeding independent CC lines at the University of North Carolina (UNC), at Tel Aviv University (TAU), and at Geniad in Western Australia (GND). These institutions aim to make publicly available the completed CC lines and their genotypes and sequence information. We genotyped, and report here, results from 458 extant lines from UNC, TAU, and GND using a custom genotyping array with 7500 SNPs designed to be maximally informative in the CC and used a novel algorithm to infer inherited haplotypes directly from hybridization intensity patterns. We identified lines with breeding errors and cousin lines generated by splitting incipient lines into two or more cousin lines at early generations of inbreeding. We then characterized the genome architecture of 350 genetically independent CC lines. Results showed that founder haplotypes are inherited at the expected frequency, although we also consistently observed highly significant transmission ratio distortion at specific loci across all three populations. On chromosome 2, there is significant overrepresentation of WSB/EiJ alleles, and on chromosome X, there is a large deficit of CC lines with CAST/EiJ alleles. Linkage disequilibrium decays as expected and we saw no evidence of gametic disequilibrium in the CC population as a whole or in random subsets of the population. Gametic equilibrium in the CC population is in marked contrast to the gametic disequilibrium present in a large panel of classical inbred strains. Finally, we discuss access to the CC population and to the associated raw data describing the genetic structure of individual lines. Integration of rich phenotypic and genomic data over time and across a wide variety of fields will be vital to delivering on one of the key attributes of the CC, a common genetic reference platform for identifying causative variants and genetic networks determining traits in mammals.

Use of genetically modified bacteria for drug delivery in humans: Revisiting the safety aspect.

PubMed

Wegmann, Udo; Carvalho, Ana Lucia; Stocks, Martin; Carding, Simon R

2017-05-23

The use of live, genetically modified bacteria as delivery vehicles for biologics is of considerable interest scientifically and has attracted significant commercial investment. We have pioneered the use of the commensal gut bacterium Bacteroides ovatus for the oral delivery of therapeutics to the gastrointestinal tract. Here we report on our investigations of the biological safety of engineered B. ovatus bacteria that includes the use of thymineless death as a containment strategy and the potential for the spread of transgenes in vivo in the mammalian gastrointestinal tract. We demonstrate the ability of GM-strains of Bacteroides to survive thymine starvation and overcome it through the exchange of genetic material. We also provide evidence for horizontal gene transfer in the mammalian gastrointestinal tract resulting in transgene-carrying wild type bacteria. These findings sound a strong note of caution on the employment of live genetically modified bacteria for the delivery of biologics.

[Genetically modified organisms in food--production, detection and risks].

PubMed

Zeljezić, Davor

2004-11-01

The first genetically modified plant (GMP) was a tobacco resistant to antibiotics in 1983. In 1996, the first genetically altered crop, a delayed-ripening tomato was commercially released. In the year 2003, the estimated global area of GM crops for was 67.7 million hectares. To produce such a plant a gene of interest has to be isolated from the donor. Together with a promoter, terminator sequence and marker gene it has to be introduced into the plant cell which is then stimulated to generate a whole GMP expressing new characteristics (herbicide/insect resistance, delayed ripening). The last few months have seen a strong public debate over genetically modified organisms which has raised scientific, economic, political, and ethical issues. Some questions concerning the safety of GMPs are still to be answered, and decisions about their future should be based on scientifically validated information.

Genetically engineered T cells to target EGFRvIII expressing glioblastoma.

PubMed

Bullain, Szofia S; Sahin, Ayguen; Szentirmai, Oszkar; Sanchez, Carlos; Lin, Ning; Baratta, Elizabeth; Waterman, Peter; Weissleder, Ralph; Mulligan, Richard C; Carter, Bob S

2009-09-01

Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.

Presynaptic Inputs to Any CNS Projection Neuron Identified by Dual Recombinant Virus Infection

PubMed Central

Bráz, João M.; Wang, Fan; Basbaum, Allan I.

2015-01-01

Although neuroanatomical tracing studies have defined the origin and targets of major projection neurons (PN) of the central nervous system (CNS), there is much less information about the circuits that influence these neurons. Recently, genetic approaches that use Cre recombinase-dependent viral vectors have greatly facilitated such circuit analysis, but these tracing approaches are limited by the availability of Cre-expressing mouse lines and the difficulty in restricting Cre expression to discrete regions of the CNS. Here, we illustrate an alternative approach to drive Cre expression specifically in defined subsets of CNS projection neurons, so as to map both direct and indirect presynaptic inputs to these cells. The method involves a combination of Cre-dependent transneuronal viral tracers that can be used in the adult and that does not require genetically modified mice. To trigger Cre-expression we inject a Cre-expressing adenovirus that is retrogradely transported to the projection neurons of interest. The region containing the retrogradely labeled projection neurons is next injected with Cre-dependent pseudorabies or rabies vectors, which results in labeling of poly- and monosynaptic neuronal inputs, respectively. In proof-of-concept experiments, we used this novel tracing system to study the circuits that engage projection neurons of the superficial dorsal horn of the spinal cord and trigeminal nucleus caudalis, neurons of the parabrachial nucleus of the dorsolateral pons that project to the amygdala and cortically-projecting neurons of the lateral geniculate nucleus. Importantly, because this dual viral tracing method does not require genetically derived Cre-expressing mouse lines, inputs to almost any projection system can be studied and the analysis can be performed in larger animals, such as the rat. PMID:26470056

Genetic modification of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3.

PubMed

Jaleco, A C; Stegmann, A P; Heemskerk, M H; Couwenberg, F; Bakker, A Q; Weijer, K; Spits, H

1999-10-15

Transgenic and gene targeted mice have contributed greatly to our understanding of the mechanisms underlying B-cell development. We describe here a model system that allows us to apply molecular genetic techniques to the analysis of human B-cell development. We constructed a retroviral vector with a multiple cloning site connected to a gene encoding green fluorescent protein by an internal ribosomal entry site. Human CD34(+)CD38(-) fetal liver cells, cultured overnight in a combination of stem cell factor and interleukin-7 (IL-7), could be transduced with 30% efficiency. We ligated the gene encoding the dominant negative helix loop helix (HLH) factor Id3 that inhibits many enhancing basic HLH transcription factors into this vector. CD34(+)CD38(-) FL cells were transduced with Id3-IRES-GFP and cultured with the murine stromal cell line S17. In addition, we cultured the transduced cells in a reaggregate culture system with an SV-transformed human fibroblast cell line (SV19). It was observed that overexpression of Id3 inhibited development of B cells in both culture systems. B-cell development was arrested at a stage before expression of the IL-7Ralpha. The development of CD34(+)CD38(-) cells into CD14(+) myeloid cells in the S17 system was not inhibited by overexpression of Id3. Moreover, Id3(+) cells, although inhibited in their B-cell development, were still able to develop into natural killer (NK) cells when cultured in a combination of Flt-3L, IL-7, and IL-15. These findings confirm the essential role of bHLH factors in B-cell development and demonstrate the feasibility of retrovirus-mediated gene transfer as a tool to genetically modify human B-cell development.

Candidate genetic modifiers for breast and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers

PubMed Central

Peterlongo, Paolo; Chang-Claude, Jenny; Moysich, Kirsten B.; Rudolph, Anja; Schmutzler, Rita K.; Simard, Jacques; Soucy, Penny; Eeles, Rosalind A.; Easton, Douglas F.; Hamann, Ute; Wilkening, Stefan; Chen, Bowang; Rookus, Matti A.; Schmidt, Marjanka K; van der Baan, Frederieke H.; Spurdle, Amanda B.; Walker, Logan C.; Lose, Felicity; Maia, Ana-Teresa; Montagna, Marco; Matricardi, Laura; Lubinski, Jan; Jakubowska, Anna; Gómez Garcia, Encarna B.; Olopade, Olufunmilayo I.; Nussbaum, Robert L.; Nathanson, Katherine L.; Domchek, Susan M.; Rebbeck, Timothy R.; Arun, Banu K.; Karlan, Beth Y.; Orsulic, Sandra; Lester, Jenny; Chung, Wendy K.; Miron, Alex; Southey, Melissa C.; Goldgar, David E.; Buys, Saundra S.; Janavicius, Ramunas; Dorfling, Cecilia M.; van Rensburg, Elizabeth J.; Ding, Yuan Chun; Neuhausen, Susan L.; Hansen, Thomas V. O.; Gerdes, Anne-Marie; Ejlertsen, Bent; Jønson, Lars; Osorio, Ana; Martínez-Bouzas, Cristina; Benitez, Javier; Conway, Edye E.; Blazer, Kathleen R.; Weitzel, Jeffrey N.; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Barile, Monica; Ficarazzi, Filomena; Mariette, Frederique; Fortuzzi, Stefano; Viel, Alessandra; Giannini, Giuseppe; Papi, Laura; Martayan, Aline; Tibiletti, Maria Grazia; Radice, Paolo; Vratimos, Athanassios; Fostira, Florentia; Garber, Judy E.; Donaldson, Alan; Brewer, Carole; Foo, Claire; Evans, D. Gareth R.; Frost, Debra; Eccles, Diana; Brady, Angela; Cook, Jackie; Tischkowitz, Marc; Adlard, Julian; Barwell, Julian; Walker, Lisa; Izatt, Louise; Side, Lucy E.; Kennedy, M. John; Rogers, Mark T.; Porteous, Mary E.; Morrison, Patrick J.; Platte, Radka; Davidson, Rosemarie; Hodgson, Shirley V.; Ellis, Steve; Cole, Trevor; Godwin, Andrew K.; Claes, Kathleen; Van Maerken, Tom; Meindl, Alfons; Gehrig, Andrea; Sutter, Christian; Engel, Christoph; Niederacher, Dieter; Steinemann, Doris; Plendl, Hansjoerg; Kast, Karin; Rhiem, Kerstin; Ditsch, Nina; Arnold, Norbert; Varon-Mateeva, Raymonda; Wappenschmidt, Barbara; Wang-Gohrke, Shan; Bressac-de Paillerets, Brigitte; Buecher, Bruno; Delnatte, Capucine; Houdayer, Claude; Stoppa-Lyonnet, Dominique; Damiola, Francesca; Coupier, Isabelle; Barjhoux, Laure; Venat-Bouvet, Laurence; Golmard, Lisa; Boutry-Kryza, Nadia; Sinilnikova, Olga M.; Caron, Olivier; Pujol, Pascal; Mazoyer, Sylvie; Belotti, Muriel; Piedmonte, Marion; Friedlander, Michael L.; Rodriguez, Gustavo C.; Copeland, Larry J; de la Hoya, Miguel; Segura, Pedro Perez; Nevanlinna, Heli; Aittomäki, Kristiina; van Os, Theo A.M.; Meijers-Heijboer, Hanne E.J.; van der Hout, Annemarie H.; Vreeswijk, Maaike P.G.; Hoogerbrugge, Nicoline; Ausems, Margreet G.E.M.; van Doorn, Helena C.; Collée, J. Margriet; Olah, Edith; Diez, Orland; Blanco, Ignacio; Lazaro, Conxi; Brunet, Joan; Feliubadalo, Lidia; Cybulski, Cezary; Gronwald, Jacek; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Sukiennicki, Grzegorz; Arason, Adalgeir; Chiquette, Jocelyne; Teixeira, Manuel R.; Olswold, Curtis; Couch, Fergus J.; Lindor, Noralane M.; Wang, Xianshu; Szabo, Csilla I.; Offit, Kenneth; Corines, Marina; Jacobs, Lauren; Robson, Mark E.; Zhang, Liying; Joseph, Vijai; Berger, Andreas; Singer, Christian F.; Rappaport, Christine; Kaulich, Daphne Geschwantler; Pfeiler, Georg; Tea, Muy-Kheng M.; Phelan, Catherine M.; Greene, Mark H.; Mai, Phuong L.; Rennert, Gad; Mulligan, Anna Marie; Glendon, Gord; Tchatchou, Sandrine; Andrulis, Irene L.; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Thomassen, Mads; Jensen, Uffe Birk; Laitman, Yael; Rantala, Johanna; von Wachenfeldt, Anna; Ehrencrona, Hans; Askmalm, Marie Stenmark; Borg, Åke; Kuchenbaecker, Karoline B.; McGuffog, Lesley; Barrowdale, Daniel; Healey, Sue; Lee, Andrew; Pharoah, Paul D.P.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Friedman, Eitan

2014-01-01

Background BRCA1 and BRCA2 mutation carriers are at substantially increased risk for developing breast and ovarian cancer. The incomplete penetrance coupled with the variable age at diagnosis in carriers of the same mutation suggests the existence of genetic and non-genetic modifying factors. In this study we evaluated the putative role of variants in many candidate modifier genes. Methods Genotyping data from 15,252 BRCA1 and 8,211 BRCA2 mutation carriers, for known variants (n=3,248) located within or around 445 candidate genes, were available through the iCOGS custom-designed array. Breast and ovarian cancer association analysis was performed within a retrospective cohort approach. Results The observed p-values of association ranged between 0.005-1.000. None of the variants was significantly associated with breast or ovarian cancer risk in either BRCA1 or BRCA2 mutation carriers, after multiple testing adjustments. Conclusion There is little evidence that any of the evaluated candidate variants act as modifiers of breast and/or ovarian cancer risk in BRCA1 or BRCA2 mutation carriers. Impact Genome-wide association studies have been more successful at identifying genetic modifiers of BRCA1/2 penetrance than candidate gene studies. PMID:25336561

Divergent selection on home pen locomotor activity in a chicken model: Selection program, genetic parameters and direct response on activity and body weight

PubMed Central

2017-01-01

General locomotor activity (GLA) in poultry has attracted attention, as it negatively influences production costs (energy expenditure and feed consumption) and welfare parameters (bone strength, litter quality, feather pecking and cannibalism). Laying hen lines diverging in the average level of spontaneous locomotor activity in the home pen were developed by genetic selection using the founder New Hampshire line. Activity was recorded using RFID technology at around five weeks of age during four to five days in the home pen. After initial phenotyping, the least active birds were selected for the low activity line and the most active for the high activity line, with no gene transfer between lines. In each of six generations, approximately ten sires were mated to twenty dams producing 158 to 334 offspring per line per generation. The response to selection was rapid and of a considerable magnitude. In sixth generation, the level of GLA was approximately halved in the low and doubled in the high line compared to the control (7.2, 14.9 and 28.7 recordings/h). Estimated heritability of locomotor activity in the low and high line was 0.38 and 0.33, respectively. Males, in general, were more active than females. High line birds were significantly heavier than low line birds. In fourth, fifth, and sixth generation, low as well as high line birds were lighter than control line birds. This selection experiment demonstrates variation in heritability for GLA and, as a result, genetically diverged lines have been developed. These lines can be used as models for further studies of underlying physiological, neural and molecular genetic mechanisms of spontaneous locomotor activity. PMID:28796792

Divergent selection on home pen locomotor activity in a chicken model: Selection program, genetic parameters and direct response on activity and body weight.

PubMed

Kjaer, Joergen B

2017-01-01

General locomotor activity (GLA) in poultry has attracted attention, as it negatively influences production costs (energy expenditure and feed consumption) and welfare parameters (bone strength, litter quality, feather pecking and cannibalism). Laying hen lines diverging in the average level of spontaneous locomotor activity in the home pen were developed by genetic selection using the founder New Hampshire line. Activity was recorded using RFID technology at around five weeks of age during four to five days in the home pen. After initial phenotyping, the least active birds were selected for the low activity line and the most active for the high activity line, with no gene transfer between lines. In each of six generations, approximately ten sires were mated to twenty dams producing 158 to 334 offspring per line per generation. The response to selection was rapid and of a considerable magnitude. In sixth generation, the level of GLA was approximately halved in the low and doubled in the high line compared to the control (7.2, 14.9 and 28.7 recordings/h). Estimated heritability of locomotor activity in the low and high line was 0.38 and 0.33, respectively. Males, in general, were more active than females. High line birds were significantly heavier than low line birds. In fourth, fifth, and sixth generation, low as well as high line birds were lighter than control line birds. This selection experiment demonstrates variation in heritability for GLA and, as a result, genetically diverged lines have been developed. These lines can be used as models for further studies of underlying physiological, neural and molecular genetic mechanisms of spontaneous locomotor activity.

Seeking perfection: a Kantian look at human genetic engineering.

PubMed

Gunderson, Martin

2007-01-01

It is tempting to argue that Kantian moral philosophy justifies prohibiting both human germ-line genetic engineering and non-therapeutic genetic engineering because they fail to respect human dignity. There are, however, good reasons for resisting this temptation. In fact, Kant's moral philosophy provides reasons that support genetic engineering-even germ-line and non-therapeutic. This is true of Kant's imperfect duties to seek one's own perfection and the happiness of others. It is also true of the categorical imperative. Kant's moral philosophy does, however, provide limits to justifiable genetic engineering.

Competitive Performance of Transgenic Wheat Resistant to Powdery Mildew

PubMed Central

Kalinina, Olena; Zeller, Simon L.; Schmid, Bernhard

2011-01-01

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes. PMID:22132219

Bioreactor expansion of human mesenchymal stem cells according to GMP requirements.

PubMed

Elseberg, Christiane L; Salzig, Denise; Czermak, Peter

2015-01-01

In cell therapy, the use of autologous and allogenic human mesenchymal stem cells is rising. Accordingly, the supply of cells for clinical applications in highest quality is required. As hMSCs are considered as an advanced therapy medicinal products (ATMP), they underlie the requirements of GMP and PAT according to the authorities (FDA and EMA). The production process of these cells must therefore be documented according to GMP, which is usually performed via a GMP protocol based on standard operating procedures. This chapter provides an example of such a GMP protocol for hMSC, here a genetically modified allogenic cell line, based on a production process in a microcarrier-based stirred tank reactor including process monitoring according to PAT and final product quality assurance.

Building lipid barriers: biosynthesis of cutin and suberin.

PubMed

Pollard, Mike; Beisson, Fred; Li, Yonghua; Ohlrogge, John B

2008-05-01

Cutin and suberin are the polymer matrices for lipophilic cell wall barriers. These barriers control the fluxes of gases, water and solutes, and also play roles in protecting plants from biotic and abiotic stresses and in controlling plant morphology. Although they are ubiquitous, cutin and suberin are the least understood of the major plant extracellular polymers. The use of forward and reverse genetic approaches in Arabidopsis has led to the identification of oxidoreductase and acyltransferase genes involved in the biosynthesis of these polymers. However, major questions about the underlying polymer structure, biochemistry, and intracellular versus extracellular assembly remain to be resolved. The analysis of plant lines with modified cutins and suberins has begun to reveal the inter-relationships between the composition and function of these polymers.

Trivial role for NSMCE2 during in vitro proliferation and differentiation of male germline stem cells.

PubMed

Zheng, Yi; Jongejan, Aldo; Mulder, Callista L; Mastenbroek, Sebastiaan; Repping, Sjoerd; Wang, Yinghua; Li, Jinsong; Hamer, Geert

2017-09-01

Spermatogenesis, starting with spermatogonial differentiation, is characterized by ongoing and dramatic alterations in composition and function of chromatin. Failure to maintain proper chromatin dynamics during spermatogenesis may lead to mutations, chromosomal aberrations or aneuploidies. When transmitted to the offspring, these can cause infertility or congenital malformations. The structural maintenance of chromosomes (SMC) 5/6 protein complex has recently been described to function in chromatin modeling and genomic integrity maintenance during spermatogonial differentiation and meiosis. Among the subunits of the SMC5/6 complex, non-SMC element 2 (NSMCE2) is an important small ubiquitin-related modifier (SUMO) ligase. NSMCE2 has been reported to be essential for mouse development, prevention of cancer and aging in adult mice and topological stress relief in human somatic cells. By using in vitro cultured primary mouse spermatogonial stem cells (SSCs), referred to as male germline stem (GS) cells, we investigated the function of NSMCE2 during spermatogonial proliferation and differentiation. We first optimized a protocol to generate genetically modified GS cell lines using CRISPR-Cas9 and generated an Nsmce2 -/- GS cell line. Using this Nsmce2 -/- GS cell line, we found that NSMCE2 was dispensable for proliferation, differentiation and topological stress relief in mouse GS cells. Moreover, RNA sequencing analysis demonstrated that the transcriptome was only minimally affected by the absence of NSMCE2. Only differential expression of Sgsm1 appeared highly significant, but with SGSM1 protein levels being unaffected without NSMCE2. Hence, despite the essential roles of NSMCE2 in somatic cells, chromatin integrity maintenance seems differentially regulated in the germline. © 2017 Society for Reproduction and Fertility.

A proposal regarding best practices for validating the identity of genetic stocks and the effects of genetic variants

USDA-ARS?s Scientific Manuscript database

Colleagues from the medical field have estimated that up to one third of cell lines are contaminated with other cell lines or are misidentified, and in addition, repeated passaging substantially changes cell line properties (reviewed in Hughes et al., 2007). The medical community has therefore begun...

Four chromosome-specific (Gossypium barbadense chromosome 5sh) Upland cotton RILs with improved elongation

USDA-ARS?s Scientific Manuscript database

A chromosome specific recombinant inbred line (CS-B05shRIL) population was created from a cross of TM-1, the genetic standard line of Gossypium hirsutum L. and CS-B05sh, a previously released interspecific chromosome substitution line in which all of the chromosome pairs are genetically similar to T...

Clinical and pathological responses of pigs from two genetically diverse commercial lines to porcine respiratory and reproductive syndrome virus infection

USDA-ARS?s Scientific Manuscript database

The response to infection from porcine reproductive and respiratory syndrome virus (PRRSV) for two genetically diverse commercial pig lines was investigated. Seventy two pigs from each line, aged 6 weeks, were challenged with PRRSV VR-2385, and 66 littermates served as control. The clinical response...

First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses.

PubMed

Choi, Eunsil; Michalski, Chad J; Choo, Seung Ho; Kim, Gyoung Nyoun; Banasikowska, Elizabeth; Lee, Sangkyun; Wu, Kunyu; An, Hwa-Yong; Mills, Anthony; Schneider, Stefan; Bredeek, U Fritz; Coulston, Daniel R; Ding, Shilei; Finzi, Andrés; Tian, Meijuan; Klein, Katja; Arts, Eric J; Mann, Jamie F S; Gao, Yong; Kang, C Yong

2016-11-28

Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1 NL4-3 ) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1 NL4-3 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity. Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1 NL4-3 -specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes. The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.

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.

The Importance of Source: A Mixed Methods Analysis of Undergraduate Students' Attitudes toward Genetically Modified Food

ERIC Educational Resources Information Center

Ruth, Taylor K.; Rumble, Joy N.; Gay, Keegan D.; Rodriguez, Mary T.

2016-01-01

Even though science says genetically modified (GM) foods are safe, many consumers remain skeptical of the technology. Additionally, the scientific community has trouble communicating to the public, causing consumers to make uninformed decisions. The Millennial Generation will have more buying power than any other generation before them, and more…

METHODS FOR DETERMINING EXPOSURE TO AND POTENTIAL ECOLOGICAL EFFECTS OF GENE FLOW FROM GENETICALLY MODIFIED CROPS TO COMPATIBLE RELATIVES

EPA Science Inventory

SCIENCE QUESTIONS:

-Does gene flow occur from genetically modified (GM) crop plants to compatible plants?

-How can it be measured?

-Are there ecological consequences of GM crop gene flow to plant communities?



RESEARCH:

The objectives ...

Assessing Website Quality in Context: Retrieving Information about Genetically Modified Food on the Web

ERIC Educational Resources Information Center

McInerney, Claire R.; Bird, Nora J.

2005-01-01

Introduction: Knowing the credibility of information about genetically modified food on the Internet is critical to the everyday life information seeking of consumers as they form opinions about this nascent agricultural technology. The Website Quality Evaluation Tool (WQET) is a valuable instrument that can be used to determine the credibility of…

Geobacteraceae strains and methods

DOEpatents

Lovley, Derek R.; Nevin, Kelly P.; Yi, Hana

2015-07-07

Embodiments of the present invention provide a method of producing genetically modified strains of electricigenic microbes that are specifically adapted for the production of electrical current in microbial fuel cells, as well as strains produced by such methods and fuel cells using such strains. In preferred embodiments, the present invention provides genetically modified strains of Geobacter sulfurreducens and methods of using such strains.

News Media Use, Informed Issue Evaluation, and South Koreans' Support for Genetically Modified Foods

ERIC Educational Resources Information Center

Kim, Sei-Hill; Kim, Jeong-Nam; Choi, Doo-Hun; Jun, Sangil

2015-01-01

Analyzing survey data on the issue of genetically modified foods in South Korea, this study explores the role of news media in facilitating informed issue evaluation. Respondents who read a newspaper more often were more knowledgeable about the issue. Also, heavy newspaper readers were more able than light readers to hold "consistent"…

Opinion Building on a Socio-Scientific Issue: The Case of Genetically Modified Plants

ERIC Educational Resources Information Center

Ekborg, Margareta

2008-01-01

This paper presents results from a study with the following research questions: (a) are pupils' opinions on genetically modified organisms (GMOs) influenced by biology teaching; and (b) what is important for the opinion pupils hold and how does knowledge work together with other parameters such as values? 64 pupils in an upper secondary school…

Class Teacher Candidates' Opinions on Genetically Modified Organisms (GMO)

ERIC Educational Resources Information Center

Ural Keles, Pinar; Aydin, Suleyman

2017-01-01

This study was conducted to determine the Class teacher candidates' opinions on Genetically Modified Organisms. The study was carried out with 101 teacher candidates who were studying in the 3rd grade of Agri Ibrahim Çeçen University Classroom Teacher Department in 2016-2017 academic year. Of the students who participated in the survey, 56 were…

Learning to Argue as a Biotechnologist: Disprivileging Opposition to Genetically Modified Food

ERIC Educational Resources Information Center

Solli, Anne; Bach, Frank; Åkerman, Björn

2014-01-01

In the public discussion of genetically modified (GM) food the representations of science as a social good, conducted in the public interest to solve major problems are being subjected to intense scrutiny and questioning. Scientists working in these areas have been seen to struggle for the position of science in society. However few in situ…

Minimizing use of fish meal in sunshine bass diets using standard and new varieties of non-genetically modified soybeans

USDA-ARS?s Scientific Manuscript database

Improved plant ingredients are needed to support sustainable culture of carnivorous fish, such as hybrid striped bass (HSB). We are evaluating meals made from new strains of non-genetically-modified soybeans (non-GMO) with high protein and reduced anti-nutritional factors (ANFs) on HSB nutrient dige...

40 CFR 158.2170 - Experimental use permit data requirements-microbial pesticides.

Code of Federal Regulations, 2013 CFR

2013-07-01

... requirements for genetically modified microbial pesticides may include but are not limited to: genetic... genetic stability and exchange; and selected Tier II environmental expression and toxicology tests. ...

40 CFR 158.2170 - Experimental use permit data requirements-microbial pesticides.

Code of Federal Regulations, 2014 CFR

2014-07-01

... requirements for genetically modified microbial pesticides may include but are not limited to: genetic... genetic stability and exchange; and selected Tier II environmental expression and toxicology tests. ...

40 CFR 158.2170 - Experimental use permit data requirements-microbial pesticides.

Code of Federal Regulations, 2012 CFR

2012-07-01

... requirements for genetically modified microbial pesticides may include but are not limited to: genetic... genetic stability and exchange; and selected Tier II environmental expression and toxicology tests. ...

40 CFR 158.2170 - Experimental use permit data requirements-microbial pesticides.

Code of Federal Regulations, 2011 CFR

2011-07-01

... requirements for genetically modified microbial pesticides may include but are not limited to: genetic... genetic stability and exchange; and selected Tier II environmental expression and toxicology tests. ...

[Genetically modified food--great unknown].

PubMed

Cichosz, G; Wiackowski, S K

2012-08-01

Genetically modified food (GMF) creates evident threat to consumers' health. In spite of assurances of biotechnologists, DNA of transgenic plants is instable, so, synthesis of foreign, allergenic proteins is possible. Due to high trypsin inhibitor content the GMF is digested much more slowly what, alike Bt toxin presence, increases probability of alimentary canal diseases. Next threats are bound to the presence of fitoestrogens and residues of Roundup pesticide, that can diminish reproductiveness; and even lead to cancerogenic transformation through disturbance of human hormonal metabolism. In spite of food producers and distributors assurances that food made of GMF raw materials is marked, de facto consumers have no choice. Moreover, along the food law products containing less than 0.9% of GMF protein are not included into genetically modified food.

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

PubMed

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-09-18

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

A Congenic Line of the C57BL/6J Mouse Strain that is Proficient in Melatonin Synthesis.

PubMed

Zhang, Zhijing; Silveyra, Eduardo; Jin, Nange; Ribelayga, Christophe P

2018-05-16

The C57BL/6J (B6) is the most common inbred mouse strain used in biomedical research in the United States. Yet, this strain is notoriously known for being deficient in the biosynthesis of melatonin, an important effector of circadian clocks in the brain and in the retina. Melatonin deficiency in this strain results from non-functional alleles of the genes coding two key enzymes of the melatonin synthesis pathway: arylalkylamine-N-acetyltransferase (Aanat) and N-acetylserotonin-O-methyltransferase (Asmt). By introducing functional alleles of the Aanat and Asmt genes from the melatonin-proficient CBA/CaJ (CBA) mouse strain to B6, we have generated a B6 congenic line that has acquired the capacity of rhythmic melatonin synthesis. In addition, the melatonin-dependent rhythm of dopamine release in the retina is restored in the B6 congenic line. Finally, we have partially characterized the Aanat and Asmt genes of the CBA strain and have identified multiple differences between CBA and B6 alleles, including single nucleotide polymorphism and deletion/insertion of DNA segments of various sizes. As an improved model organism with functional components of the melatonin synthesis pathway and melatonin-dependent circadian regulations, the new line will be useful to researchers studying melatonin physiological functions in a variety of fields including, but not limited to, circadian biology and neuroscience. In particular, the congenic line will be useful to speed up introduction of melatonin production capacity into genetically-modified mouse lines of interest such as knockout lines, many of which are on B6 or mixed B6 backgrounds. The melatonin-proficient B6 congenic line will be widely distributed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

PubMed Central

2011-01-01

Background The recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.). Results A modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation. Conclusions A new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event. PMID:21995716

Monogenic Mouse Models of Autism Spectrum Disorders: Common Mechanisms and Missing Links

PubMed Central

Hulbert, Samuel W.; Jiang, Yong-hui

2016-01-01

Autism Spectrum Disorders (ASDs) present unique challenges in the fields of genetics and neurobiology because of the clinical and molecular heterogeneity underlying these disorders. Genetic mutations found in ASD patients provide opportunities to dissect the molecular and circuit mechanisms underlying autistic behaviors using animal models. Ongoing studies of genetically modified models have offered critical insight into possible common mechanisms arising from different mutations, but links between molecular abnormalities and behavioral phenotypes remain elusive. The challenges encountered in modeling autism in mice demand a new analytic paradigm that integrates behavioral analysis with circuit-level analysis in genetically modified models with strong construct validity. PMID:26733386

Recombination and genetic variance among maize doubled haploids induced from F1 and F2 plants.

PubMed

Sleper, Joshua A; Bernardo, Rex

2016-12-01

Inducing maize doubled haploids from F 2 plants (DHF2) instead of F 1 plants (DHF1) led to more recombination events. However, the best DHF2 lines did not outperform the best DHF1 lines. Maize (Zea mays L.) breeders rely on doubled haploid (DH) technology for fast and efficient production of inbreds. Breeders can induce DH lines most quickly from F 1 plants (DHF1), or induce DH lines from F 2 plants (DHF2) to allow selection prior to DH induction and have more recombinations. Our objective was to determine if the additional recombinations in maize DHF2 lines lead to a larger genetic variance and a superior mean of the best lines. A total of 311 DHF1 and 241 DHF2 lines, derived from the same biparental cross, were crossed to two testers and evaluated in multilocation trials in Europe and the US. The mean number of recombinations per genome was 14.48 among the DHF1 lines and 21.38 among the DHF1 lines. The means of the DHF1 and DHF2 lines did not differ for yield, moisture, and plant height. The genetic variance was higher among DHF2 lines than among DHF1 lines for moisture, but not for yield and plant height. The ratio of repulsion to coupling linkages, which was estimated from genomewide marker effects, was higher among DHF1 lines than among DHF2 lines for moisture, but not for yield and plant height. The higher genetic variance for moisture among DHF2 lines did not lead to lower moisture of the best 10 % of the lines. Our results indicated that the decision of inducing DH lines from F 1 or F 2 plants needs to be made from considerations other than the performance of the resulting DHF1 or DHF2 lines.

[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).

Genetic load makes cancer cells more sensitive to common drugs: evidence from Cancer Cell Line Encyclopedia.

PubMed

Pavel, Ana B; Korolev, Kirill S

2017-05-16

Genetic alterations initiate tumors and enable the evolution of drug resistance. The pro-cancer view of mutations is however incomplete, and several studies show that mutational load can reduce tumor fitness. Given its negative effect, genetic load should make tumors more sensitive to anticancer drugs. Here, we test this hypothesis across all major types of cancer from the Cancer Cell Line Encyclopedia, which provides genetic and expression data of 496 cell lines together with their response to 24 common anticancer drugs. We found that the efficacy of 9 out of 24 drugs showed significant association with genetic load in a pan-cancer analysis. The associations for some tissue-drug combinations were remarkably strong, with genetic load explaining up to 83% of the variance in the drug response. Overall, the role of genetic load depended on both the drug and the tissue type with 10 tissues being particularly vulnerable to genetic load. We also identified changes in gene expression associated with increased genetic load, which included cell-cycle checkpoints, DNA damage and apoptosis. Our results show that genetic load is an important component of tumor fitness and can predict drug sensitivity. Beyond being a biomarker, genetic load might be a new, unexplored vulnerability of cancer.

The Influence of LINE-1 and SINE Retrotransposons on Mammalian Genomes.

PubMed

Richardson, Sandra R; Doucet, Aurélien J; Kopera, Huira C; Moldovan, John B; Garcia-Perez, José Luis; Moran, John V

2015-04-01

Transposable elements have had a profound impact on the structure and function of mammalian genomes. The retrotransposon Long INterspersed Element-1 (LINE-1 or L1), by virtue of its replicative mobilization mechanism, comprises ∼17% of the human genome. Although the vast majority of human LINE-1 sequences are inactive molecular fossils, an estimated 80-100 copies per individual retain the ability to mobilize by a process termed retrotransposition. Indeed, LINE-1 is the only active, autonomous retrotransposon in humans and its retrotransposition continues to generate both intra-individual and inter-individual genetic diversity. Here, we briefly review the types of transposable elements that reside in mammalian genomes. We will focus our discussion on LINE-1 retrotransposons and the non-autonomous Short INterspersed Elements (SINEs) that rely on the proteins encoded by LINE-1 for their mobilization. We review cases where LINE-1-mediated retrotransposition events have resulted in genetic disease and discuss how the characterization of these mutagenic insertions led to the identification of retrotransposition-competent LINE-1s in the human and mouse genomes. We then discuss how the integration of molecular genetic, biochemical, and modern genomic technologies have yielded insight into the mechanism of LINE-1 retrotransposition, the impact of LINE-1-mediated retrotransposition events on mammalian genomes, and the host cellular mechanisms that protect the genome from unabated LINE-1-mediated retrotransposition events. Throughout this review, we highlight unanswered questions in LINE-1 biology that provide exciting opportunities for future research. Clearly, much has been learned about LINE-1 and SINE biology since the publication of Mobile DNA II thirteen years ago. Future studies should continue to yield exciting discoveries about how these retrotransposons contribute to genetic diversity in mammalian genomes.

Possibility of modifying the growth trajectory in Raeini Cashmere goat.

PubMed

Ghiasi, Heydar; Mokhtari, M S

2018-03-27

The objective of this study was to investigate the possibility of modifying the growth trajectory in Raeini Cashmere goat breed. In total, 13,193 records on live body weight collected from 4788 Raeini Cashmere goats were used. According to Akanke's information criterion (AIC), the sing-trait random regression model included fourth-order Legendre polynomial for direct and maternal genetic effect; maternal and individual permanent environmental effect was the best model for estimating (co)variance components. The matrices of eigenvectors for (co)variances between random regression coefficients of direct additive genetic were used to calculate eigenfunctions, and different eigenvector indices were also constructed. The obtained results showed that the first eigenvalue explained 79.90% of total genetic variance. Therefore, changing the body weights applying the first eigenfunction will be obtained rapidly. Selection based on the first eigenvector will cause favorable positive genetic gains for all body weight considered from birth to 12 months of age. For modifying the growth trajectory in Raeini Cashmere goat, the selection should be based on the second eigenfunction. The second eigenvalue accounted for 14.41% of total genetic variance for body weights that is low in comparison with genetic variance explained by the first eigenvalue. The complex patterns of genetic change in growth trajectory observed under the third and fourth eigenfunction and low amount of genetic variance explained by the third and fourth eigenvalues.

Quantitative detection method for Roundup Ready soybean in food using duplex real-time PCR MGB chemistry.

PubMed

Samson, Maria Cristina; Gullì, Mariolina; Marmiroli, Nelson

2010-07-01

Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non-authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost-efficient solution. Construct-specific primers and probe were developed for quantitative analysis of Roundup Ready soybean (RRS) event glyphosate-tolerant soybean (GTS) 40-3-2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS- and Le1-specific quantitative real-time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. In this study, a duplex qRTPCR using TaqMan minor groove binder-non-fluorescent quencher (MGB-NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40-3-2 that can be used for practical monitoring in processed food products.

A 90-day subchronic feeding study of genetically modified rice expressing Cry1Ab protein in Sprague-Dawley rats.

PubMed

Song, Huan; He, Xiaoyun; Zou, Shiying; Zhang, Teng; Luo, Yunbo; Huang, Kunlun; Zhu, Zhen; Xu, Wentao

2015-04-01

Bacillus thuringiensis (Bt) transgenic rice line (mfb-MH86) expressing a synthetic cry1Ab gene can be protected against feeding damage from Lepidopteran insects, including Sesamia inferens, Chilo suppressalis, Tryporyza incertulas and Cnaphalocrocis medinalis. Rice flour from mfb-MH86 and its near-isogenic control MH86 was separately formulated into rodent diets at concentrations of 17.5, 35 and 70 % (w/w) for a 90-day feeding test with rats, and all of the diets were nutritionally balanced. In this study, the responses of rats fed diets containing mfb-MH86 were compared to those of rats fed flour from MH86. Overall health, body weight and food consumption were comparable between groups fed diets containing mfb-MH86 and MH86. Blood samples were collected prior to sacrifice and a few significant differences (p < 0.05) were observed in haematological and biochemical parameters between rats fed genetically modified (GM) and non-GM diets. However, the values of these parameters were within the normal ranges of values for rats of this age and sex, thus not considered treatment related. In addition, upon sacrifice a large number of organs were weighed, macroscopic and histopathological examinations were performed with only minor changes to report. In conclusion, these results demonstrated that no toxic effect was observed in the conditions of the experiment, based on the different parameters assessed. GM rice mfb-MH86 is as safe and nutritious as non-GM rice.

Conservation priorities for the different lines of Dutch Red and White Friesian cattle change when relationships with other breeds are taken into account.

PubMed

Hulsegge, B; Calus, M P L; Oldenbroek, J K; Windig, J J

2017-02-01

From a genetic point of view, the selection of breeds and animals within breeds for conservation in a national gene pool can be based on a maximum diversity strategy. This implies that priority is given to conservation of breeds and animals that diverge most and overlap of conserved diversity is minimized. This study investigated the genetic diversity in the Dutch Red and White Friesian (DFR) cattle breed and its contribution to the total genetic diversity in the pool of the Dutch dairy breeds. All Dutch cattle breeds are clearly distinct, except for Dutch Friesian breed (DF) and DFR and have their own specific genetic identity. DFR has a small but unique contribution to the total genetic diversity of Dutch cattle breeds and is closely related to the Dutch Friesian breed. Seven different lines are distinguished within the DFR breed and all contribute to the diversity of the DFR breed. Two lines show the largest contributions to the genetic diversity in DFR. One of these lines comprises unique diversity both within the breed and across all cattle breeds. The other line comprises unique diversity for the DFR but overlaps with the Holstein Friesian breed. There seems to be no necessity to conserve the other five lines separately, because their level of differentiation is very low. This study illustrates that, when taking conservation decisions for a breed, it is worthwhile to take into account the population structure of the breed itself and the relationships with other breeds. © 2016 Blackwell Verlag GmbH.

Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: consistent loss of chromosome 13 and amplification of chromosome 20.

PubMed

Jin, Yuesheng; Zhang, Hao; Tsao, Sai Wah; Jin, Charlotte; Lv, Mei; Strömbeck, Bodil; Wiegant, Joop; Wan, Thomas Shek Kong; Yuen, Po Wing; Kwong, Yok-Lam

2004-01-01

This study aimed at identifying the genetic events involved in immortalization of ovarian epithelial cells, which might be important steps in ovarian carcinogenesis. The genetic profiles of five human ovarian surface epithelial (HOSE) cell lines immortalized by retroviral transfection of the human papillomavirus (HPV) E6/E7 genes were thoroughly characterized by chromosome banding and fluorescence in situ hybridization (FISH), at various passages pre- and post-crisis. In pre-crisis, most cells had simple, non-clonal karyotypic changes. Telomere association was the commonest aberration, suggesting that tolermase dysfunction might be an important genetic event leading to cellular crisis. After immortalization post-crisis, however, the karyotypic patterns were non-random. Loss of genetic materials was a characteristic feature. The commonest numerical aberrations were -13, -14, -16, -17, -18, and +5. Among them, loss of chromosome 13 was common change observed in all lines. The only recurrent structural aberration was homogeneously staining regions (hsr) observed in three lines. FISH and combined binary ratio labeling (COBRA)-FISH showed in two cases that the hsrs were derived from chromosome 20. Clonal evolution was observed in four of the lines. In one line, hsr was the only change shared by all subclones, suggesting that it might be a primary event in cell immortalization. The results of the present study suggested that loss of chromosome 13 and the amplification of chromosome 20 might be early genetic events involved in ovarian cell immortalization, and might be useful targets for the study of genomic aberrations in ovarian carcinogenesis.

A fast hidden line algorithm with contour option. M.S. Thesis

NASA Technical Reports Server (NTRS)

Thue, R. E.

1984-01-01

The JonesD algorithm was modified to allow the processing of N-sided elements and implemented in conjunction with a 3-D contour generation algorithm. The total hidden line and contour subsystem is implemented in the MOVIE.BYU Display package, and is compared to the subsystems already existing in the MOVIE.BYU package. The comparison reveals that the modified JonesD hidden line and contour subsystem yields substantial processing time savings, when processing moderate sized models comprised of 1000 elements or less. There are, however, some limitations to the modified JonesD subsystem.

Comparative assessment of genetic and epigenetic variation among regenerants of potato (Solanum tuberosum) derived from long-term nodal tissue-culture and cell selection.

PubMed

Dann, Alison L; Wilson, Calum R

2011-04-01

Three long-term nodal tissued cultured Russet Burbank potato clones and nine thaxtomin A-treated regenerant lines, derived from the nodal lines, were assessed for genetic and epigenetic (in the form of DNA methylation) differences by AFLP and MSAP. The treated regenerant lines were originally selected for superior resistance to common scab disease and acceptable tuber yield in pot and field trials. The long-term, tissue culture clone lines exhibited genetic (8.75-15.63% polymorphisms) and epigenetic (12.56-26.13% polymorphisms) differences between them and may represent a stress response induced by normal plant growth disruption. The thaxtomin A-treated regenerant lines exhibited much higher significant (p < 0.05) genetic (2-29.38%) and epigenetic (45.22-51.76%) polymorphisms than the nodal cultured parent clones. Methylation-sensitive mutations accumulated within the regenerant lines are significantly correlated (p < 0.05) to disease resistance. However, linking phenotypic differences that could be of benefit to potato growers, to single gene sequence polymorphisms in a tetraploid plant such as the potato would be extremely difficult since it is assumed many desirable traits are under polygenic control.

Immunological characteristics and response to lipopolysaccharide of mouse lines selectively bred with natural and acquired immunities.

PubMed

Narahara, Hiroki; Sakai, Eri; Katayama, Masafumi; Ohtomo, Yukiko; Yamamoto, Kanako; Takemoto, Miki; Aso, Hisashi; Ohwada, Shyuichi; Mohri, Yasuaki; Nishimori, Katsuhiko; Isogai, Emiko; Yamaguchi, Takahiro; Fukuda, Tomokazu

2012-05-01

Genetic improvement of resistance to infectious diseases is a challenging goal in animal breeding. Infection resistance involves multiple immunological characteristics, including natural and acquired immunity. In the present study, we developed an experimental model based on genetic selection, to improve immunological phenotypes. We selectively established three mouse lines based on phagocytic activity, antibody production and the combination of these two phenotypes. We analyzed the immunological characteristics of these lines using a lipopolysaccharide (LPS), which is one of the main components of Gram-negative bacteria. An intense immunological reaction was induced in each of the three mouse lines. Severe loss of body weight and liver damage were observed, and a high level of cytokine messenger RNA was detected in the liver tissue. The mouse line established using a combination of the two selection standards showed unique characteristics relative to the mouse lines selected on the basis of a single phenotype. Our results indicate that genetic selection and breeding is effective, even for immunological phenotypes with a relatively low heritability. Thus, it may be possible to improve resistance to infectious diseases by means of genetic selection. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate.

PubMed

Chhapekar, Sushil; Raghavendrarao, Sanagala; Pavan, Gadamchetty; Ramakrishna, Chopperla; Singh, Vivek Kumar; Phanindra, Mullapudi Lakshmi Venkata; Dhandapani, Gurusamy; Sreevathsa, Rohini; Ananda Kumar, Polumetla

2015-05-01

Highly tolerant herbicide-resistant transgenic rice was developed by expressing codon-modified synthetic CP4--EPSPS. The transformants could tolerate up to 1% commercial glyphosate and has the potential to be used for DSR (direct-seeded rice). Weed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T0, T1 and T2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1% of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.

Unraveling Genetic Modifiers in the Gria4 Mouse Model of Absence Epilepsy

PubMed Central

Frankel, Wayne N.; Mahaffey, Connie L.; McGarr, Tracy C.; Beyer, Barbara J.; Letts, Verity A.

2014-01-01

Absence epilepsy (AE) is a common type of genetic generalized epilepsy (GGE), particularly in children. AE and GGE are complex genetic diseases with few causal variants identified to date. Gria4 deficient mice provide a model of AE, one for which the common laboratory inbred strain C3H/HeJ (HeJ) harbors a natural IAP retrotransposon insertion in Gria4 that reduces its expression 8-fold. Between C3H and non-seizing strains such as C57BL/6, genetic modifiers alter disease severity. Even C3H substrains have surprising variation in the duration and incidence of spike-wave discharges (SWD), the characteristic electroencephalographic feature of absence seizures. Here we discovered extensive IAP retrotransposition in the C3H substrain, and identified a HeJ-private IAP in the Pcnxl2 gene, which encodes a putative multi-transmembrane protein of unknown function, resulting in decreased expression. By creating new Pcnxl2 frameshift alleles using TALEN mutagenesis, we show that Pcnxl2 deficiency is responsible for mitigating the seizure phenotype – making Pcnxl2 the first known modifier gene for absence seizures in any species. This finding gave us a handle on genetic complexity between strains, directing us to use another C3H substrain to map additional modifiers including validation of a Chr 15 locus that profoundly affects the severity of SWD episodes. Together these new findings expand our knowledge of how natural variation modulates seizures, and highlights the feasibility of characterizing and validating modifiers in mouse strains and substrains in the post-genome sequence era. PMID:25010494

Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids

PubMed Central

2013-01-01

Background Genetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood. Results We investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations. Conclusions Our results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of somaclonal variation in rice, which is concomitant with epigenetic alterations. Perturbed expression by tissue culture of a set of 41 genes encoding for enzymes involved in DNA repair and DNA methylation is associated with both genetic and epigenetic alterations. There exist fundamental differences among distinct genotypes, pure-lines, hybrids and tetraploids, in propensities of generating both genetic and epigenetic alterations under the tissue culture condition. Parent-of-origin has a conspicuous effect on the alteration frequencies. PMID:23642214

Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids.

PubMed

Wang, Xiaoran; Wu, Rui; Lin, Xiuyun; Bai, Yan; Song, Congdi; Yu, Xiaoming; Xu, Chunming; Zhao, Na; Dong, Yuzhu; Liu, Bao

2013-05-05

Genetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood. We investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations. Our results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of somaclonal variation in rice, which is concomitant with epigenetic alterations. Perturbed expression by tissue culture of a set of 41 genes encoding for enzymes involved in DNA repair and DNA methylation is associated with both genetic and epigenetic alterations. There exist fundamental differences among distinct genotypes, pure-lines, hybrids and tetraploids, in propensities of generating both genetic and epigenetic alterations under the tissue culture condition. Parent-of-origin has a conspicuous effect on the alteration frequencies.

Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

PubMed Central

Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

2016-01-01

Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

Genetically Modified Food in Perspective: An Inquiry-Based Curriculum to Help Middle School Students Make Sense of Tradeoffs. Research Report

ERIC Educational Resources Information Center

Seethaler, Sherry; Linn, Marcia

2004-01-01

To understand how students learn about science controversy, this study examines students' reasoning about tradeoffs in the context of a technology-enhanced curriculum about genetically modified food. The curriculum was designed and refined based on the Scaffolded Knowledge Integration Framework to help students sort and integrate their initial…

Awareness and support of release of genetically modified "sterile" mosquitoes, Key West, Florida, USA.

PubMed

Ernst, Kacey C; Haenchen, Steven; Dickinson, Katherine; Doyle, Michael S; Walker, Kathleen; Monaghan, Andrew J; Hayden, Mary H

2015-02-01

After a dengue outbreak in Key West, Florida, during 2009-2010, authorities, considered conducting the first US release of male Aedes aegypti mosquitoes genetically modified to prevent reproduction. Despite outreach and media attention, only half of the community was aware of the proposal; half of those were supportive. Novel public health strategies require community engagement.

Efficacy of genetically modified Bt toxins alone and in combinations against pink bollworm resistant to Cry1Ac and Cry2Ab

USDA-ARS?s Scientific Manuscript database

Evolution of resistance in pests threatens the long-term success of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and ...

The Development and Validation of the GMOAS, an Instrument Measuring Secondary School Students' Attitudes towards Genetically Modified Organisms

ERIC Educational Resources Information Center

Herodotou, Christothea; Kyza, Eleni A.; Nicolaidou, Iolie; Hadjichambis, Andreas; Kafouris, Dimitris; Terzian, Freda

2012-01-01

Genetically modified organisms (GMOs) is a rapidly evolving area of scientific innovation and an issue receiving global attention. Attempts to devise usable instruments that assess people's attitudes towards this innovation have been rare and non-systematic. The aim of this paper is to present the development and validation of the genetically…

Three-generation reproduction toxicity study of genetically modified rice with insect resistant genes.

PubMed

Hu, Yichun; Zhuo, Qin; Gong, Zhaolong; Piao, Jianhua; Yang, Xiaoguang

2017-01-01

In the present work, we evaluated the three generation reproductive toxicity of the genetically modified rice with insectresistant cry1Ac and sck genes. 120 Sprague-Dawley (SD) rats were divided into three groups which were fed with genetically modified rice diet (GM group), parental control rice diet (PR group) and AIN-93 control diet (both used as negative control) respectively. Bodyweight, food consumption, reproductive data, hematological parameters, serum chemistry, relative organ weights and histopathology for each generation were examined respectively. All the hematology and serum chemistry parameters, organ/body weight indicators were within the normal range or no change to the adverse direction was observed, although several differences in hematology and serum chemistry parameters (WBC, BUN, LDH of male rat, PLT, PCT, MPV of female rats), reproductive data (rate of morphologically abnormal sperm) were observed between GM rice group and two control groups. No macroscopic or histological adverse effects were found or considered as treatment-related, either. Overall, the three generation study of genetically modified rice with cry1Ac and sck genes at a high level showed no unintended adverse effects on rats's reproductive system. Copyright © 2016. Published by Elsevier Ltd.

Preliminary assessment of framework conditions for release of genetically modified mosquitoes in Burkina Faso.

PubMed

De Freece, Chenoa; Paré Toé, Léa; Esposito, Fulvio; Diabaté, Abdoulaye; Favia, Guido

2014-09-01

Genetically modified mosquitoes (GMMs) are emerging as a measure to control mosquito-borne diseases, but before any genetically modified organisms (GMOs) are released into the environment, it is imperative to establish regulatory standards incorporating public engagement. A previous project in Burkina Faso introduced a type of genetically modified cotton [Bacillus thuringiensis (Bt)] cotton) that produces insecticide, and incorporated policies on public engagement. We explored the perspectives of Burkinabè (citizens of Burkina Faso) on bio-agricultural exposure to GMOs and their receptiveness to the use of GMOs. Interviews were conducted in a village (Bondoukuy) and with representatives from stakeholder organizations. The population may be very receptive to the use of GMMs against malaria, but may voice unfounded concerns that GMMs can transmit other diseases. It is important to constantly supply the population with correct and factual information. Investigating the application of Burkina Faso's biotechnology policies with regard to Bt cotton has shown that it may be conceivable in the future to have open discussions about the merits of GMM release. © The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Electrochemical sensor for multiplex screening of genetically modified DNA: identification of biotech crops by logic-based biomolecular analysis.

PubMed

Liao, Wei-Ching; Chuang, Min-Chieh; Ho, Ja-An Annie

2013-12-15

Genetically modified (GM) technique, one of the modern biomolecular engineering technologies, has been deemed as profitable strategy to fight against global starvation. Yet rapid and reliable analytical method is deficient to evaluate the quality and potential risk of such resulting GM products. We herein present a biomolecular analytical system constructed with distinct biochemical activities to expedite the computational detection of genetically modified organisms (GMOs). The computational mechanism provides an alternative to the complex procedures commonly involved in the screening of GMOs. Given that the bioanalytical system is capable of processing promoter, coding and species genes, affirmative interpretations succeed to identify specified GM event in terms of both electrochemical and optical fashions. The biomolecular computational assay exhibits detection capability of genetically modified DNA below sub-nanomolar level and is found interference-free by abundant coexistence of non-GM DNA. This bioanalytical system, furthermore, sophisticates in array fashion operating multiplex screening against variable GM events. Such a biomolecular computational assay and biosensor holds great promise for rapid, cost-effective, and high-fidelity screening of GMO. Copyright © 2013 Elsevier B.V. All rights reserved.

Exploring Middle School Students' Understanding of Three Conceptual Models in Genetics

ERIC Educational Resources Information Center

Freidenreich, Hava Bresler; Duncan, Ravit Golan; Shea, Nicole

2011-01-01

Genetics is the cornerstone of modern biology and a critical aspect of scientific literacy. Research has shown, however, that many high school graduates lack fundamental understandings in genetics necessary to make informed decisions about issues and emerging technologies in this domain, such as genetic screening, genetically modified foods, etc.…

Metabolomics of Genetically Modified Crops

PubMed Central

Simó, Carolina; Ibáñez, Clara; Valdés, Alberto; Cifuentes, Alejandro; García-Cañas, Virginia

2014-01-01

Metabolomic-based approaches are increasingly applied to analyse genetically modified organisms (GMOs) making it possible to obtain broader and deeper information on the composition of GMOs compared to that obtained from traditional analytical approaches. The combination in metabolomics of advanced analytical methods and bioinformatics tools provides wide chemical compositional data that contributes to corroborate (or not) the substantial equivalence and occurrence of unintended changes resulting from genetic transformation. This review provides insight into recent progress in metabolomics studies on transgenic crops focusing mainly in papers published in the last decade. PMID:25334064

Background controlled QTL mapping in pure-line genetic populations derived from four-way crosses

PubMed Central

Zhang, S; Meng, L; Wang, J; Zhang, L

2017-01-01

Pure lines derived from multiple parents are becoming more important because of the increased genetic diversity, the possibility to conduct replicated phenotyping trials in multiple environments and potentially high mapping resolution of quantitative trait loci (QTL). In this study, we proposed a new mapping method for QTL detection in pure-line populations derived from four-way crosses, which is able to control the background genetic variation through a two-stage mapping strategy. First, orthogonal variables were created for each marker and used in an inclusive linear model, so as to completely absorb the genetic variation in the mapping population. Second, inclusive composite interval mapping approach was implemented for one-dimensional scanning, during which the inclusive linear model was employed to control the background variation. Simulation studies using different genetic models demonstrated that the new method is efficient when considering high detection power, low false discovery rate and high accuracy in estimating quantitative trait loci locations and effects. For illustration, the proposed method was applied in a reported wheat four-way recombinant inbred line population. PMID:28722705

Background controlled QTL mapping in pure-line genetic populations derived from four-way crosses.

PubMed

Zhang, S; Meng, L; Wang, J; Zhang, L

2017-10-01

Pure lines derived from multiple parents are becoming more important because of the increased genetic diversity, the possibility to conduct replicated phenotyping trials in multiple environments and potentially high mapping resolution of quantitative trait loci (QTL). In this study, we proposed a new mapping method for QTL detection in pure-line populations derived from four-way crosses, which is able to control the background genetic variation through a two-stage mapping strategy. First, orthogonal variables were created for each marker and used in an inclusive linear model, so as to completely absorb the genetic variation in the mapping population. Second, inclusive composite interval mapping approach was implemented for one-dimensional scanning, during which the inclusive linear model was employed to control the background variation. Simulation studies using different genetic models demonstrated that the new method is efficient when considering high detection power, low false discovery rate and high accuracy in estimating quantitative trait loci locations and effects. For illustration, the proposed method was applied in a reported wheat four-way recombinant inbred line population.

Cisgenic Rvi6 scab-resistant apple lines show no differences in Rvi6 transcription when compared with conventionally bred cultivars.

PubMed

Chizzali, Cornelia; Gusberti, Michele; Schouten, Henk J; Gessler, Cesare; Broggini, Giovanni A L

2016-03-01

The expression of the apple scab resistance gene Rvi6 in different apple cultivars and lines is not modulated by biotic or abiotic factors. All commercially important apple cultivars are susceptible to Venturia inaequalis, the causal organism of apple scab. A limited number of apple cultivars were bred to express the resistance gene Vf from the wild apple genotype Malus floribunda 821. Positional cloning of the Vf locus allowed the identification of the Rvi6 (formerly HcrVf2) scab resistance gene that was subsequently used to generate cisgenic apple lines. It is important to understand and compare how this resistance gene is transcribed and modulated during infection in conventionally bred cultivars and in cisgenic lines. The aim of this work was to study the transcription pattern of Rvi6 in three classically bred apple cultivars and six lines of 'Gala' genetically modified to express Rvi6. Rvi6 transcription was analyzed at two time points using quantitative real-time PCR (RT-qPCR) following inoculation with V. inaequalis conidia or water. Rvi6 transcription was assessed in relation to five reference genes. β-Actin, RNAPol, and UBC were the most suited to performing RT-qPCR experiments on Malus × domestica. Inoculation with V. inaequalis conidia under conditions conducive to scab infection failed to produce any significant changes to the transcription level of Rvi6. Rvi6 expression levels were inconsistent in response to external treatments in the different apple cultivars, and transgenic, intragenic or cisgenic lines.

Population dynamics of Sesamia inferens on transgenic rice expressing Cry1Ac and CpTI in southern China.

PubMed

Han, Lanzhi; Liu, Peilei; Wu, Kongming; Peng, Yufa; Wang, Feng

2008-10-01

Genetically modified insect-resistant rice lines containing the cry1Ac gene from Bacillus thuringiensis (Bt) or the CpTI (cowpea trypsin inhibitor) gene developed for the management of lepidopterous pests are highly resistant to the major target pests, Chilo suppressalis (Walker), Cnaphalocrocis medinalis (Guenée), and Scirpophaga incertulas (Walker), in the main rice-growing areas of China. However, the effects of these transgenic lines on Sesamia inferens (Walker), an important lepidopterous rice pest, are currently unknown. Because different insect species have varying susceptibility to Bt insecticidal proteins that may affect population dynamics, research into the effects of these transgenic rice lines on the population dynamics of S. inferens was conducted in Fuzhou, southern China, in 2005 and 2006. The results of laboratory, field cage, and field plot experiments show that S. inferens has comparatively high susceptibility to the transgenic line during the early growing season, with significant differences observed in larval density and infestation levels between transgenic and control lines. Because of a decrease in Cry1Ac levels in the plant as it ages, the transgenic line provided only a low potential for population suppression late in the growing season. There is a correlation between the changing expression of Cry1Ac and the impact of transgenic rice on the population dynamics of S. inferens during the season. These results indicate that S. inferens may become a major pest in fields of prospective commercially released transgenic rice, and more attention should be paid to developing an effective alternative management strategy.

High-Throughput Cryopreservation of Plant Cell Cultures for Functional Genomics

PubMed Central

Ogawa, Yoichi; Sakurai, Nozomu; Oikawa, Akira; Kai, Kosuke; Morishita, Yoshihiko; Mori, Kumiko; Moriya, Kanami; Fujii, Fumiko; Aoki, Koh; Suzuki, Hideyuki; Ohta, Daisaku; Saito, Kazuki; Shibata, Daisuke

2012-01-01

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell–LSP solution mixtures was kept at −30°C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics. PMID:22437846

A cell line resource derived from honey bee (Apis mellifera) embryonic tissues.

PubMed

Goblirsch, Michael J; Spivak, Marla S; Kurtti, Timothy J

2013-01-01

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

Beyond the Mouse Monopoly: Studying the Male Germ Line in Domestic Animal Models

PubMed Central

González, Raquel; Dobrinski, Ina

2015-01-01

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals. PMID:25991701

Pressurized Pepsin Digestion in Proteomics

PubMed Central

López-Ferrer, Daniel; Petritis, Konstantinos; Robinson, Errol W.; Hixson, Kim K.; Tian, Zhixin; Lee, Jung Hwa; Lee, Sang-Won; Tolić, Nikola; Weitz, Karl K.; Belov, Mikhail E.; Smith, Richard D.; Paša-Tolić, Ljiljana

2011-01-01

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome. PMID:20627868

BIOTECHNOLOGY/ECORISK

EPA Science Inventory

This research effort is designed to provide the risk assessment community with modern genetic tools for evaluating long-term risks of genetically modified (GM) crops. Molecular population genetic data can potentially reveal information about long-term trends in both pest populat...

Whole-body transcriptome of selectively bred, resistant-, control-, and susceptible-line rainbow trout following experimental challenge with Flavobacterium psychrophilum

PubMed Central

Marancik, David; Gao, Guangtu; Paneru, Bam; Ma, Hao; Hernandez, Alvaro G.; Salem, Mohamed; Yao, Jianbo; Palti, Yniv; Wiens, Gregory D.

2014-01-01

Genetic improvement for enhanced disease resistance in fish is an increasingly utilized approach to mitigate endemic infectious disease in aquaculture. In domesticated salmonid populations, large phenotypic variation in disease resistance has been identified but the genetic basis for altered responsiveness remains unclear. We previously reported three generations of selection and phenotypic validation of a bacterial cold water disease (BCWD) resistant line of rainbow trout, designated ARS-Fp-R. This line has higher survival after infection by either standardized laboratory challenge or natural challenge as compared to two reference lines, designated ARS-Fp-C (control) and ARS-Fp-S (susceptible). In this study, we utilized 1.1 g fry from the three genetic lines and performed RNA-seq to measure transcript abundance from the whole body of naive and Flavobacterium psychrophilum infected fish at day 1 (early time-point) and at day 5 post-challenge (onset of mortality). Sequences from 24 libraries were mapped onto the rainbow trout genome reference transcriptome of 46,585 predicted protein coding mRNAs that included 2633 putative immune-relevant gene transcripts. A total of 1884 genes (4.0% genome) exhibited differential transcript abundance between infected and mock-challenged fish (FDR < 0.05) that included chemokines, complement components, tnf receptor superfamily members, interleukins, nod-like receptor family members, and genes involved in metabolism and wound healing. The largest number of differentially expressed genes occurred on day 5 post-infection between naive and challenged ARS-Fp-S line fish correlating with high bacterial load. After excluding the effect of infection, we identified 21 differentially expressed genes between the three genetic lines. In summary, these data indicate global transcriptome differences between genetic lines of naive animals as well as differentially regulated transcriptional responses to infection. PMID:25620978

Genetic variation in heat tolerance-related traits in a population of wheat multiple synthetic derivatives

PubMed Central

Elbashir, Awad A. E.; Gorafi, Yasir S. A.; Tahir, Izzat S. A.; Elhashimi, Ashraf. M. A.; Abdalla, Modather G. A.; Tsujimoto, Hisashi

2017-01-01

In wheat (Triticum aestivum L.) high temperature (≥30°C) during grain filling leads to considerable reduction in grain yield. We studied 400 multiple synthetic derivatives (MSD) lines to examine the genetic variability of heat stress–adaptive traits and to identify new sources of heat tolerance to be used in wheat breeding programs. The experiment was arranged in an augmented randomized complete block design in four environments in Sudan. A wide range of genetic variability was found in most of the traits in all environments. For all traits examined, we found MSD lines that showed better performance than their parent ‘Norin 61’ and two adapted Sudanese cultivars. Using the heat tolerance efficiency, we identified 13 highly heat-tolerant lines and several lines with intermediate heat tolerance and good yield potential. We also identified lines with alleles that can be used to increase wheat yield potential. Our study revealed that the use of the MSD population is an efficient way to explore the genetic variation in Ae. tauschii for wheat breeding and improvement. PMID:29398942

Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Štebih, Dejan; Morisset, Dany; Holst-Jensen, Arne; Žel, Jana

2018-01-01

The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency. Here we describe two protocols for multiplex quantification of GM maize events: (1) nondiscriminating, with multiplex quantification of targets as a group (12 GM maize lines) and (2) discriminating, with multiplex quantification of individual targets (events). The first enables the quantification of twelve European Union authorized GM maize events as a group with only two assays, but does not permit determination of the individual events present. The second protocol enables the quantification of four individual targets (three GM events and one endogene) in a single reaction. Both protocols can be modified for quantification of any other DNA target.

Proteomics insight into the biological safety of transgenic modification of rice as compared with conventional genetic breeding and spontaneous genotypic variation.

PubMed

Gong, Chun Yan; Li, Qi; Yu, Hua Tao; Wang, Zizhang; Wang, Tai

2012-05-04

The potential of unintended effects caused by transgenic events is a key issue in the commercialization of genetically modified (GM) crops. To investigate whether transgenic events cause unintended effects, we used comparative proteomics approaches to evaluate proteome differences in seeds from 2 sets of GM indica rice, herbicide-resistant Bar68-1 carrying bar and insect-resistant 2036-1a carrying cry1Ac/sck, and their respective controls D68 and MH86, as well as indica variety MH63, a parental line for breeding MH86, and japonica variety ZH10. This experimental design allowed for comparing proteome difference caused by transgenes, conventional genetic breeding, and natural genetic variation. Proteomics analysis revealed the maximum numbers of differentially expressed proteins between indica and japonica cultivars, second among indica varieties with relative small difference between MH86 and MH63, and the minimum between GM rice and respective control, thus indicating GM events do not substantially alter proteome profiles as compared with conventional genetic breeding and natural genetic variation. Mass spectrometry analysis revealed 234 proteins differentially expressed in the 6 materials, and these proteins were involved in different cellular and metabolic processes with a prominent skew toward metabolism (31.2%), protein synthesis and destination (25.2%), and defense response (22.4%). In these seed proteomes, proteins implicated in the 3 prominent biological processes showed significantly different composite expression patterns and were major factors differentiating japonica and indica cultivars, as well as indica varieties. Thus, metabolism, protein synthesis and destination, and defense response in seeds are important in differentiating rice cultivars and varieties.

Genetic background influences age-related decline in visual and nonvisual retinal responses, circadian rhythms, and sleep☆

PubMed Central

Banks, Gareth; Heise, Ines; Starbuck, Becky; Osborne, Tamzin; Wisby, Laura; Potter, Paul; Jackson, Ian J.; Foster, Russell G.; Peirson, Stuart N.; Nolan, Patrick M.

2015-01-01

The circadian system is entrained to the environmental light/dark cycle via retinal photoreceptors and regulates numerous aspects of physiology and behavior, including sleep. These processes are all key factors in healthy aging showing a gradual decline with age. Despite their importance, the exact mechanisms underlying this decline are yet to be fully understood. One of the most effective tools we have to understand the genetic factors underlying these processes are genetically inbred mouse strains. The most commonly used reference mouse strain is C57BL/6J, but recently, resources such as the International Knockout Mouse Consortium have started producing large numbers of mouse mutant lines on a pure genetic background, C57BL/6N. Considering the substantial genetic diversity between mouse strains we expect there to be phenotypic differences, including differential effects of aging, in these and other strains. Such differences need to be characterized not only to establish how different mouse strains may model the aging process but also to understand how genetic background might modify age-related phenotypes. To ascertain the effects of aging on sleep/wake behavior, circadian rhythms, and light input and whether these effects are mouse strain-dependent, we have screened C57BL/6J, C57BL/6N, C3H-HeH, and C3H-Pde6b+ mouse strains at 5 ages throughout their life span. Our data show that sleep, circadian, and light input parameters are all disrupted by the aging process. Moreover, we have cataloged a number of strain-specific aging effects, including the rate of cataract development, decline in the pupillary light response, and changes in sleep fragmentation and the proportion of time spent asleep. PMID:25179226

Genetic background influences age-related decline in visual and nonvisual retinal responses, circadian rhythms, and sleep.

PubMed

Banks, Gareth; Heise, Ines; Starbuck, Becky; Osborne, Tamzin; Wisby, Laura; Potter, Paul; Jackson, Ian J; Foster, Russell G; Peirson, Stuart N; Nolan, Patrick M

2015-01-01

The circadian system is entrained to the environmental light/dark cycle via retinal photoreceptors and regulates numerous aspects of physiology and behavior, including sleep. These processes are all key factors in healthy aging showing a gradual decline with age. Despite their importance, the exact mechanisms underlying this decline are yet to be fully understood. One of the most effective tools we have to understand the genetic factors underlying these processes are genetically inbred mouse strains. The most commonly used reference mouse strain is C57BL/6J, but recently, resources such as the International Knockout Mouse Consortium have started producing large numbers of mouse mutant lines on a pure genetic background, C57BL/6N. Considering the substantial genetic diversity between mouse strains we expect there to be phenotypic differences, including differential effects of aging, in these and other strains. Such differences need to be characterized not only to establish how different mouse strains may model the aging process but also to understand how genetic background might modify age-related phenotypes. To ascertain the effects of aging on sleep/wake behavior, circadian rhythms, and light input and whether these effects are mouse strain-dependent, we have screened C57BL/6J, C57BL/6N, C3H-HeH, and C3H-Pde6b+ mouse strains at 5 ages throughout their life span. Our data show that sleep, circadian, and light input parameters are all disrupted by the aging process. Moreover, we have cataloged a number of strain-specific aging effects, including the rate of cataract development, decline in the pupillary light response, and changes in sleep fragmentation and the proportion of time spent asleep. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Analytical criteria for performance characteristics of IgE binding methods for evaluating safety of biotech food products.

PubMed

Holzhauser, Thomas; Ree, Ronald van; Poulsen, Lars K; Bannon, Gary A

2008-10-01

There is detailed guidance on how to perform bioinformatic analyses and enzymatic degradation studies for genetically modified crops under consideration for approval by regulatory agencies; however, there is no consensus in the scientific community on the details of how to perform IgE serum studies. IgE serum studies are an important safety component to acceptance of genetically modified crops when the introduced protein is novel, the introduced protein is similar to known allergens, or the crop is allergenic. In this manuscript, we describe the characteristics of the reagents, validation of assay performance, and data analysis necessary to optimize the information obtained from serum testing of novel proteins and genetically modified (GM) crops and to make results more accurate and comparable between different investigations.

Standards for gene therapy clinical trials based on pro-active risk assessment in a London NHS Teaching Hospital Trust.

PubMed

Bamford, K B; Wood, S; Shaw, R J

2005-02-01

Conducting gene therapy clinical trials with genetically modified organisms as the vectors presents unique safety and infection control issues. The area is governed by a range of legislation and guidelines, some unique to this field, as well as those pertinent to any area of clinical work. The relevant regulations covering gene therapy using genetically modified vectors are reviewed and illustrated with the approach taken by a large teaching hospital NHS Trust. Key elements were Trust-wide communication and involvement of staff in a pro-active approach to risk management, with specific emphasis on staff training and engagement, waste management, audit and record keeping. This process has led to the development of proposed standards for clinical trials involving genetically modified micro-organisms.

Use of Traditional and Genetically Modified Probiotics in Human Health: What Does the Future Hold?

PubMed

Bermúdez-Humarán, Luis G; Langella, Philippe

2017-09-01

Probiotics are live, nonpathogenic microorganisms that confer benefits to human health when administered in adequate amounts. Among the frequent proposed health benefits attributed to probiotics, their ability to interact with the host immune system is now well demonstrated. Although history has revealed that probiotics were part of fermented foods in the past, clinicians have started to use them therapeutically in regular diets. Moreover, the use of genetically modified probiotics to deliver molecules of therapeutic interest is gaining importance as an extension of the probiotic concept. This chapter summarizes some of the recent findings and perspectives on the use of both traditional and genetically modified probiotics to treat human diseases as well as what the future may hold concerning the use of these probiotics in humans.

[Labeling of food containing genetically modified organisms: international policies and Brazilian legislation].

PubMed

Costa, Thadeu Estevam Moreira Maramaldo; Marin, Victor Augustus

2011-08-01

The increase in surface area planted with genetically modified crops, with the subsequent transfer of such crops into the general environment for commercial trade, has raised questions about the safety of these products. The introduction of the Cartagena Protocol on Biosafety has led to the need to produce information and ensure training in this area for the implementation of policies on biosafety and for decision-making on the part of governments at the national, regional and international level. This article presents two main standpoints regarding the labeling of GM products (one adopted by the United States and the other by the European Union), as well as the position adopted by Brazil and its current legislation on labeling and commercial release of genetically modified (GM) products.

Genetic characterization of Russian honey bee stock selected for improved resistance to Varroa destructor.

PubMed

Bourgeois, A Lelania; Rinderer, Thomas E

2009-06-01

Maintenance of genetic diversity among breeding lines is important in selective breeding and stock management. The Russian Honey Bee Breeding Program has strived to maintain high levels of heterozygosity among its breeding lines since its inception in 1997. After numerous rounds of selection for resistance to tracheal and varroa mites and improved honey production, 18 lines were selected as the core of the program. These lines were grouped into three breeding blocks that were crossbred to improve overall heterozygosity levels of the population. Microsatellite DNA data demonstrated that the program has been successful. Heterozygosity and allelic richness values are high and there are no indications of inbreeding among the three blocks. There were significant levels of genetic structure measured among the three blocks. Block C was genetically distinct from both blocks A and B (F(ST) = 0.0238), whereas blocks A and B did not differ from each other (F(ST) = 0.0074). The same pattern was seen for genic (based on numbers of alleles) differentiation. Genetic distance, as measured by chord distance, indicates that all of the 18 lines are equally distant, with minimal clustering. The data indicate that the overall design of the breeding program has been successful in maintaining high levels of diversity and avoiding problems associated with inbreeding.

Bone Cell Bioenergetics and Skeletal Energy Homeostasis

PubMed Central

Riddle, Ryan C.; Clemens, Thomas L.

2017-01-01

The rising incidence of metabolic diseases worldwide has prompted renewed interest in the study of intermediary metabolism and cellular bioenergetics. The application of modern biochemical methods for quantitating fuel substrate metabolism with advanced mouse genetic approaches has greatly increased understanding of the mechanisms that integrate energy metabolism in the whole organism. Examination of the intermediary metabolism of skeletal cells has been sparked by a series of unanticipated observations in genetically modified mice that suggest the existence of novel endocrine pathways through which bone cells communicate their energy status to other centers of metabolic control. The recognition of this expanded role of the skeleton has in turn led to new lines of inquiry directed at defining the fuel requirements and bioenergetic properties of bone cells. This article provides a comprehensive review of historical and contemporary studies on the metabolic properties of bone cells and the mechanisms that control energy substrate utilization and bioenergetics. Special attention is devoted to identifying gaps in our current understanding of this new area of skeletal biology that will require additional research to better define the physiological significance of skeletal cell bioenergetics in human health and disease. PMID:28202599

Novel Phenotype Issues Raised in Cross-National Epidemiological Research on Drug Dependence

PubMed Central

Anthony, James C.

2010-01-01

Stage-transition models based on the American Diagnostic and Statistical Manual (DSM) generally are applied in epidemiology and genetics research on drug dependence syndromes associated with cannabis, cocaine, and other internationally regulated drugs (IRD). Difficulties with DSM stage-transition models have surfaced during cross-national research intended to provide a truly global perspective, such as the work of the World Mental Health Surveys (WMHS) Consortium. Alternative simpler dependence-related phenotypes are possible, including population-level count process models for steps early and before coalescence of clinical features into a coherent syndrome (e.g., zero-inflated Poisson regression). Selected findings are reviewed, based on ZIP modeling of alcohol, tobacco, and IRD count processes, with an illustration that may stimulate new research on genetic susceptibility traits. The annual National Surveys on Drug Use and Health can be readily modified for this purpose, along the lines of a truly anonymous research approach that can help make NSDUH-type cross-national epidemiological surveys more useful in the context of subsequent genome wide association (GWAS) research and post-GWAS investigations with a truly global health perspective. PMID:20201862

CRISPR-Cas9: from Genome Editing to Cancer Research

PubMed Central

Chen, Si; Sun, Heng; Miao, Kai; Deng, Chu-Xia

2016-01-01

Cancer development is a multistep process triggered by innate and acquired mutations, which cause the functional abnormality and determine the initiation and progression of tumorigenesis. Gene editing is a widely used engineering tool for generating mutations that enhance tumorigenesis. The recent developed clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) system renews the genome editing approach into a more convenient and efficient way. By rapidly introducing genetic modifications in cell lines, organs and animals, CRISPR-Cas9 system extends the gene editing into whole genome screening, both in loss-of-function and gain-of-function manners. Meanwhile, the system accelerates the establishment of animal cancer models, promoting in vivo studies for cancer research. Furthermore, CRISPR-Cas9 system is modified into diverse innovative tools for observing the dynamic bioprocesses in cancer studies, such as image tracing for targeted DNA, regulation of transcription activation or repression. Here, we view recent technical advances in the application of CRISPR-Cas9 system in cancer genetics, large-scale cancer driver gene hunting, animal cancer modeling and functional studies. PMID:27994508

Fall armyworm migration across the Lesser Antilles and the potential for genetic exchanges between North and South American populations

PubMed Central

Nagoshi, Rodney N.; Hay-Roe, Mirian; Khan, Ayub; Murúa, M. Gabriela; Silvie, Pierre; Vergara, Clorinda; Westbrook, John

2017-01-01

The fall armyworm, Spodoptera frugiperda (J. E. Smith)(Lepidoptera: Noctuidae), is an important agricultural pest of the Western Hemisphere noted for its broad host range, long distance flight capabilities, and a propensity to develop resistance to pesticides that includes a subset of those used in genetically modified corn varieties. These characteristics exacerbate the threat fall armyworm poses to agriculture, with the potential that a resistance trait arising in one geographical location could rapidly disseminate throughout the hemisphere. A region of particular concern is the Caribbean, where a line of islands that extends from Florida to Venezuela provides a potential migratory pathway between populations from North and South America that could allow for consistent and substantial genetic interactions. In this study, surveys of populations from Peru, Bolivia, Paraguay, and Trinidad & Tobago expand on previous work in South America that indicates a generally homogeneous population with respect to haplotype markers. This population differs from that found in most of the Lesser Antilles where a combination of genetic and meteorological observations is described that indicate fall armyworm migration from Puerto Rico to as far south as Barbados, but does not support significant incursion into Trinidad & Tobago and South America. Air transport projections demonstrate that the wind patterns in the Caribbean region are not conducive to consistent flight along the north-south orientation of the Lesser Antilles, supporting the conclusion that such migration is minor and sporadic, providing few opportunities for genetic exchanges. The implications of these findings on the dissemination of deleterious traits between the two Western Hemisphere continents are discussed. PMID:28166292

Population structure and genetic diversity in a commercial maize breeding program assessed with SSR and SNP markers.

PubMed

Van Inghelandt, Delphine; Melchinger, Albrecht E; Lebreton, Claude; Stich, Benjamin

2010-05-01

Information about the genetic diversity and population structure in elite breeding material is of fundamental importance for the improvement of crops. The objectives of our study were to (a) examine the population structure and the genetic diversity in elite maize germplasm based on simple sequence repeat (SSR) markers, (b) compare these results with those obtained from single nucleotide polymorphism (SNP) markers, and (c) compare the coancestry coefficient calculated from pedigree records with genetic distance estimates calculated from SSR and SNP markers. Our study was based on 1,537 elite maize inbred lines genotyped with 359 SSR and 8,244 SNP markers. The average number of alleles per locus, of group specific alleles, and the gene diversity (D) were higher for SSRs than for SNPs. Modified Roger's distance (MRD) estimates and membership probabilities of the STRUCTURE matrices were higher for SSR than for SNP markers but the germplasm organization in four heterotic pools was consistent with STRUCTURE results based on SSRs and SNPs. MRD estimates calculated for the two marker systems were highly correlated (0.87). Our results suggested that the same conclusions regarding the structure and the diversity of heterotic pools could be drawn from both markers types. Furthermore, although our results suggested that the ratio of the number of SSRs and SNPs required to obtain MRD or D estimates with similar precision is not constant across the various precision levels, we propose that between 7 and 11 times more SNPs than SSRs should be used for analyzing population structure and genetic diversity.

How much does transgenesis affect wheat allergenicity?: Assessment in two GM lines over-expressing endogenous genes.

PubMed

Lupi, R; Denery-Papini, S; Rogniaux, H; Lafiandra, D; Rizzi, C; De Carli, M; Moneret-Vautrin, D A; Masci, S; Larré, C

2013-03-27

Wheat kernel albumins/globulins (A/G) and gluten proteins are responsible for baker's asthma and food allergy in atopic subjects. Although no commercial genetically modified wheats are currently being grown, they are under study and the allergenicity of GM products is a major concern. In order to establish the expected and unexpected effects of genetic transformation on allergenicity and also to carry out a safety assessment of genetic transformation, two GM wheat lines (bread and pasta wheat) transformed with endogenous genes were compared to their untransformed counterparts (wt), first by an allergenomic approach, and second, using ELISA with sera from patients suffering from food allergy to wheat and baker's asthma. The 2D immunoblots performed on sera from patients suffering from food allergy and baker's asthma on the A/G fraction of the four lines (two GM and two wt) revealed comparable IgE-binding profiles. A total of 109 IgE-binding spots were analyzed by mass spectrometry, and most of the proteins identified had already been described as allergens or potential allergens. Only two IgE-binding proteins were specific to one GM line. The concentration of specific IgE against the A/G fractions of GM wheat lines and their wt genotypes differed for some sera. The originality of our paper is to relate the transformation of wheat lines with their potential allergenicity using patient sera, such focus has never been done before in wheat and should be of interest to the researches working in this field. Another interesting point of this paper is the study of two types of allergies (respiratory and food) on two wheat genotypes and their GM which reveals that some allergens already known in respiratory allergy could be involved in children suffering from wheat food allergy. In this paper we used a classical 2D proteomic analysis and the protein identifications were performed by mass spectrometry after spot picking and in gel trypsin hydrolysis. Concerning the LC-MS/MS analyses classical software and parameters were used as described in Material and methods. We worked on wheat which is actually not fully sequenced that was a difficulty; we therefore searched against two databanks (proteins and ESTs) in order to compare the results. Moreover all proteins reported in our paper were identified with at least three unique peptides. The identified proteins were checked for their potential allergenicity. In order to have a best interpretation of protein identified in terms of potential allergens, BLAST alignments were performed by using an allergen databank (SDAP). This allows the determination of the cross-reactivity of these identified proteins with known allergens of other species and also the prediction of a potential allergenicity. Copyright © 2013 Elsevier B.V. All rights reserved.

Application of synthetic biology in cyanobacteria and algae

PubMed Central

Wang, Bo; Wang, Jiangxin; Zhang, Weiwen; Meldrum, Deirdre R.

2012-01-01

Cyanobacteria and algae are becoming increasingly attractive cell factories for producing renewable biofuels and chemicals due to their ability to capture solar energy and CO2 and their relatively simple genetic background for genetic manipulation. Increasing research efforts from the synthetic biology approach have been made in recent years to modify cyanobacteria and algae for various biotechnological applications. In this article, we critically review recent progresses in developing genetic tools for characterizing or manipulating cyanobacteria and algae, the applications of genetically modified strains for synthesizing renewable products such as biofuels and chemicals. In addition, the emergent challenges in the development and application of synthetic biology for cyanobacteria and algae are also discussed. PMID:23049529

Application of laws, policies, and guidance from the United States and Canada to the regulation of food and feed derived from genetically modified crops: interpretation of composition data.

PubMed

Price, William D; Underhill, Lynne

2013-09-04

With the development of recombinant DNA techniques for genetically modifying plants to exhibit beneficial traits, laws and regulations were adopted to ensure the safety of food and feed derived from such plants. This paper focuses on the regulation of genetically modified (GM) plants in Canada and the United States, with emphasis on the results of the compositional analysis routinely utilized as an indicator of possible unintended effects resulting from genetic modification. This work discusses the mandate of Health Canada and the Canadian Food Inspection Agency as well as the U.S. Food and Drug Administration's approach to regulating food and feed derived from GM plants. This work also addresses how publications by the Organisation for Economic Co-operation and Development and Codex Alimentarius fit, particularly with defining the importance and purpose of compositional analysis. The importance of study design, selection of comparators, use of literature, and commercial variety reference values is also discussed.