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Results for “Organisms, Genetically Modified”

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PubMed2006

A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease.

BACKGROUND & AIMS: The use of living, genetically modified bacteria is an effective approach for topical delivery of immunomodulatory proteins. This strategy circumvents systemic side effects and allows long-term treatment of chronic diseases. However, treatment of patients with a living, genetically modified bacterium raises questions about the safety for human subjects per se and the biologic containment of the transgene. METHODS: We treated Crohn's disease patients with genetically modified Lactococcus lactis (LL-Thy12) in which the thymidylate synthase gene was replaced with a synthetic sequence encoding mature human interleukin-10. Ten patients were included in a placebo-uncontrolled trial. Patients were assessed daily for the presence of potential adverse effects by direct questioning and assessment of disease activity. We evaluated the presence and kinetics of LL-Thy12 release in the stool of patients by conventional culturing and quantitative polymerase chain reaction of LL-Thy12 gene sequences. RESULTS: Treatment with LL-Thy12 was safe because only minor adverse events were present, and a decrease in disease activity was observed. Moreover, fecally recovered LL-Thy12 bacteria were dependent on thymidine for growth and interleukin-10 production, indicating that the containment strategy was effective. CONCLUSIONS: Here we show that the use of genetically modified bacteria for mucosal delivery of proteins is a feasible strategy in human beings. This novel strategy avoids systemic side effects and is biologically contained; therefore it is suitable as maintenance treatment for chronic intestinal disease.

C-Reactive Protein↗

PubMed2005

Can genetically modified Escherichia coli with neutral buoyancy induced by gas vesicles be used as an alternative method to clinorotation for microgravity studies?

Space flight has been shown to affect various bacterial growth parameters. It is proposed that weightlessness allows the cells to remain evenly distributed, consequently altering the chemical makeup of their surrounding fluid, and hence indirectly affecting their physiological behaviour. In support of this argument, ground-based studies using clinostats to partially simulate the quiescent environment attained in microgravity have generally been successful in producing bacterial growth characteristics that mimic responses reported under actual space conditions. A novel approach for evaluating the effects of reduced cell sedimentation is presented here through use of Escherichia coli cultures genetically modified to be neutrally buoyant. Since clinorotation would not (or would only minimally) affect cell distribution of this already near-colloidal cell system, it was hypothesized that the effects on final population density would be eliminated relative to a static control. Gas-vesicle-producing E. coli cultures were grown under clinostat and static conditions and the culture densities at 60 h were compared. As a control, E. coli that do not produce gas vesicles, but were otherwise identical to the experimental strain, were also grown under clinostat and static conditions. As hypothesized, no significant difference was observed in cell populations at 60 h between the clinorotated and static gas-vesicle-producing E. coli cultures, while the cells that did not produce gas vesicles showed a mean increase in population density of 10.5 % (P = 0.001). These results further suggest that the lack of cumulative cell sedimentation is the dominant effect of space flight on non-stirred, in vitro E. coli cultures.

Bioreactors↗

PubMed2005

Quality of transgenic laboratory strains of Cochliomyia hominivorax (Diptera: Calliphoridae).

Genetically modified, mass reared insects present novel possibilities for the future of insect control. One concern about manipulation of insects is a possible loss of strain quality due to the introduction of a foreign gene of any sort into the insect genome. Eight transgenic strains of screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), were compared with the wild-type parental laboratory strain in laboratory culture. Measurements of average fertility, fecundity, larval productivity, and longevity were analyzed. Two transgenic strains had significantly lower larval productivity than controls, one of which was explained by a homozygous lethal insertion of the transgene. Another strain produced significantly fewer eggs than controls. Overall strain characteristics, including measurements from egg, larva, pupa, and adult stages, were compared. Transgenic colonies did not consistently show significantly lower individual or aggregate strain quality characteristics than the control parental colony; hence, the presence of the transgene used to produce the strains tested did not incur a discrete cost to the colonies of laboratory-reared C. hominivorax.

Animals↗

PubMed2001

Microbiological contamination in genetically modified animals and proposals for a microbiological test standard for national universities in Japan.

The Biosafety Committee of the Japanese Association of Laboratory Animal Facilities of National Universities (JALAN) investigated recent episodes of microbiological contamination in genetically modified mice (GMM), and the countermeasures taken when the contaminated GMM were introduced into animal facilities, by questionnaires addressed to 53 animal facilities belonging to JALAN and serological tests. Although almost all of the contaminated GMM were accepted with conditions such as rederivation after or before reception and housing in designated rooms, contamination with a spectrum of microorganisms was demonstrated in GMM transferred domestically and from abroad. In serological tests, Mycoplasma pulmonis, mouse parvovirus, and mouse encephalomylitis virus were detected in GMM transferred from domestic facilities and from abroad. The present results of the questionnaires and serological tests suggest that GMM are highly and widely contaminated with microorganisms compared with mice from commercial breeders. Thus, we propose a microbiological requirement, including microbiological status--excellent, common, and minimum--as a guide for the transfer and procurement of mice and rats in Japan.

Animals↗

PubMed2001

Stress-induced evolution and the biosafety of genetically modified microorganisms released into the environment.

This article is focused on the problems of reduction of the risk associated with the deliberate release of genetically modified microorganisms (GMMs) into the environment. Special attention is given to overview the most probable physiological and genetic processes which could be induced in the released GMMs by adverse environmental conditions, namely: (i) activation of quorum sensing and the functions associated with it, (ii) entering into a state of general resistance, (iii) activation of adaptive mutagenesis, adaptive amplifications and transpositions and (iv) stimulation of inter-species gene transfer. To reduce the risks associated with GMMs, the inactivation of their key genes responsible for stress-stimulated increase of viability and evolvability is proposed.

Adaptation, Physiological↗

PubMed2002

A refined method for the detection of baculovirus occlusion bodies in forest terrestrial and aquatic habitats.

A sensitive and efficient method was developed for the detection of genetically modified and wild-type baculovirus occlusion bodies (OB) in forest terrestrial and aquatic habitats. The protocol facilitates the analysis of a large number of samples collected and frozen to maintain viral integrity. Lyophilization was used to standardize the size of both field-collected soil samples and test substrates inoculated with OBs for the determination of minimum detection threshold. To simulate natural conditions, terrestrial test substrates were inoculated at a standardized moisture content determined using a soil pressure plate apparatus. OBs, extracted from lyophilized test substrates by washing, sieving and centrifugation, were subjected to alkaline lysis and viral DNA isolated using a purchased DNA purification kit. PCR amplified DNA was visualized using agarose gel electrophoresis. Minimum detection thresholds in terrestrial substrates were 10(3), 10(2), 10(2) and 10(1) OBs from 0.5 g of lyophilized L, F-H and mineral soil horizons, and 1.0 ml of leachate, respectively. Detection thresholds in aquatic substrates were 10(0) and 10(3) OBs from 1.0 ml of pond water and 1.0 g of bottom sediment, respectively.

Animals↗

PubMed2003

Measuring gene flow from two birdsfoot trefoil (Lotus corniculatus) field trials using transgenes as tracer markers.

Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment. Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants. In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. Plants of Lotus corniculatus L. transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor. Nontransgenic plants belonging to the species L. corniculatus L., L. tenuis Waldst. and Kit. ex Willd, and L. pedunculatus Cav., were utilized as recipients. Two experimental fields were established in two areas of central Italy. Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker. No transgene flow between L. corniculatus transformants and the nontransgenic L. tenuis and L. pedunculatus plants was detected. As regards nontransgenic L. corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot. In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.

Aspartate-Ammonia Ligase↗

PubMed2005

Recombineered Xenopus tropicalis BAC expresses a GFP reporter under the control of Arx transcriptional regulatory elements in transgenic Xenopus laevis embryos.

The aristaless-related homeobox (Arx) gene is expressed in a dynamic pattern in the developing vertebrate forebrain. We identified a bacterial artificial chromosome (BAC) containing the Xenopus tropicalis Arx gene and replaced a portion of the first coding exon with a green fluorescent protein (GFP) expression cassette by homologous recombination in bacteria (recombineering). Transgenic X. laevis embryos obtained by microinjecting the modified BAC expressed GFP in the developing forebrain in a pattern identical to that of the endogenous Arx gene. Thus, this BAC contains transcriptional regulatory elements necessary for regulating proper expression in transgenic frogs. This work demonstrates that transgenesis using recombineered BACs is a viable technique in Xenopus.

Animals↗

PubMed2004

Impact of genetically modified Enterobacter cloacae on indigenous endophytic community of Citrus sinensis seedlings.

Enterobacter cloacae (strain PR2/7), a genetically modified endophyte (GME) in citrus plants, carrying different plasmids (pEC3.0/18, pCelE, pEglA and pGFP), was inoculated into Citrus sinensis seedlings under greenhouse conditions. The impact of this on the indigenous bacterial endophytic community was studied by analyses of 2 different morphologic groups. The germination rates of inoculated seeds were evaluated in greenhouse, and plasmid stability under in vitro conditions. Results demonstrated a great and diverse endophytic community inside plants, and specialization in tissue colonization by some bacterial groups, in different treatments. Shifts in seed germination rate were observed among treatments: in general, the PR2/7 harboring pEglA bacterial clone significantly reduced seed germination, compared to the PR2/7 harboring pEC3.0/18 clone. This suggests that the presence of the pEglA plasmid changes bacteria-seed interactions. The endophytic community of citrus seedlings changed according to treatment. In seedlings treated with the PR2/7 with pEglA clone, the population of group II decreased significantly, within the context of the total endophytic community. These results indicate that the application of GMEs induces shifts in the endophytic bacterial community of citrus seedlings.

Bacteria↗

PubMed2006

Codon modification of T cell receptors allows enhanced functional expression in transgenic human T cells.

Expression of native transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks. Here we show that codon modification of an HPV16E7-specific T cell receptor (TCR), together with omission of mRNA instability motifs and (cryptic) splice sites, leads to a dramatic increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The codon-modified TCRs have been tested in three different configurations in the retroviral vector LZRS: (1) TCRalpha-IRES-GFP in combination with TCRbeta-IRES-NGFR, (2) TCRalpha-IRES-TCRbeta, and (3) TCRalpha-2A-TCRbeta. T cells carrying the codon-modified TCRs are functionally active against target cells loaded with relevant peptide, model tumor cells expressing the specific epitope as well as cervical carcinoma cells. The significant improvements we report here in the functional expression of specific human TCRs will hopefully expedite clinical application of TCR transfer-based immunotherapy.

CD8-Positive T-Lymphocytes↗

PubMed2006

Exclusive overproduction of recombinant versatile peroxidase MnP2 by genetically modified white rot fungus, Pleurotus ostreatus.

By combining a homologous recombinant gene expression system and optimization of the culture conditions, hyper overproduction of Pleurtous ostreatus MnP2 was achieved. Genetically modified P. ostreatus strains with the recombinant mnp2 sequence under the control of sdi1 expression signals, were subjected to agitated culture using media supplemented with wheat bran or its hot-water extract. The best result, whereby 7300 U/l of MnP was produced by a recombinant strain TM2-18, indicated that more than 30-fold overproduction of the recombinant MnP2 compared to the previous result was achieved. On the other hand, no MnP activity was detected for the wild-type strain under the same conditions. Accumulation of the recombinant, but not endogenous, mnp2 transcripts was demonstrated in reverse-transcription PCR experiments. These results indicated that the recombinant MnP2 was exclusively expressed by the recombinant strain. Purified recombinant MnP2 showed almost identical properties to native MnP2 in electrophoresis, spectroscopic and kinetic analyses, including determination of K(m) and V(max) values for Mn(II), H(2)O(2) and veratryl alcohol. Moreover, the recombinant MnP2 directly oxidized a high-molecularweight substrate RNase A in the absence of redox mediators, as does native MnP2. The homologous overproduction system will provide a plat form for exclusive production of mutant or variant peroxidases with a desired property in basidiomycete.

Organisms, Genetically Modified↗

PubMed2006

Manipulation of fish germ cell: visualization, cryopreservation and transplantation.

Germ-cell transplantation has many applications in biology and animal husbandry, including investigating the complex processes of germ-cell development and differentiation, producing transgenic animals by genetically modifying germline cells, and creating broodstock systems in which a target species can be produced from a surrogate parent. The germ-cell transplantation technique was initially established in chickens using primordial germ cells (PGCs), and was subsequently extended to mice using spermatogonial stem cells. Recently, we developed the first germ-cell transplantation system in lower vertebrates using fish PGCs and spermatogonia. During mammalian germ-cell transplantation, donor spermatogonial stem cells are introduced into the seminiferous tubules of the recipient testes. By contrast, in the fish germ-cell transplantation system, donor cells are microinjected into the peritoneal cavities of newly hatched embryos; this allows the donor germ cells to migrate towards, and subsequently colonize, the recipient genital ridges. The recipient embryos have immature immune systems, so the donor germ cells can survive and even differentiate into mature gametes in their allogeneic gonads, ultimately leading to the production of normal offspring. In addition, implanted spermatogonia can successfully differentiate into sperm and eggs, respectively, in male and female recipients. The results of transplantation studies in fish are improving our understanding of the development of germ-cell systems during vertebrate evolution.

Animals↗

PubMed2003

Fighting a virus with a virus: a dynamic model for HIV-1 therapy.

A mathematical model examined a potential therapy for controlling viral infections using genetically modified viruses. The control of the infection is an indirect effect of the selective elimination by an engineered virus of infected cells that are the source of the pathogens. Therefore, this engineered virus could greatly compensate for a dysfunctional immune system compromised by AIDS. In vitro studies using engineered viruses have been shown to decrease the HIV-1 load about 1000-fold. However, the efficacy of this potential treatment for reducing the viral load in AIDS patients is unknown. The present model studied the interactions among the HIV-1 virus, its main host cell (activated CD4+ T cells), and a therapeutic engineered virus in an in vivo context; and it examined the conditions for controlling the pathogen. This model predicted a significant drop in the HIV-1 load, but the treatment does not eradicate HIV. A basic estimation using a currently engineered virus indicated an HIV-1 load reduction of 92% and a recovery of host cells to 17% of their normal level. Greater success (98% HIV reduction, 44% host cells recovery) is expected as more competent engineered viruses are designed. These results suggest that therapy using viruses could be an alternative to extend the survival of AIDS patients.

CD4-Positive T-Lymphocytes↗

PubMed2004

Allergy assessment of foods or ingredients derived from biotechnology, gene-modified organisms, or novel foods.

The introduction of novel proteins into foods carries a risk of eliciting allergic reactions in individuals sensitive to the introduced protein and a risk of sensitizing susceptible individuals. No single predictive test exists to perform a hazard assessment in relation to allergenic properties of newly expressed proteins in gene-modified organisms (GMOs). Instead, performance of a weighted risk analysis based on the decision tree approach has been suggested. The individual steps of this analysis comprise sequence homology to known allergens, specific or targeted serum screens for immunoglobulin E (IgE) cross-reactions to known allergens, digestability studies of the proteins in simulated gastric and/or intestinal fluids, and animal studies. These steps are discussed and five examples of risk evaluation of GMOs or novel foods are presented. These include ice-structuring protein derived from fish, microbial transglutaminase, GMO-soybeans, amylase and the Nangai nut.

Allergens↗

PubMed2003

Transgenic swine for biomedicine and agriculture.

Initial technologies for creating transgenic swine only permitted random integration of the construct. However, by combining the technology for homologous recombination in fetal somatic cells with that of nuclear transfer (NT), it is now possible to create specific modifications to the swine genome. The first such example is that of knocking out a gene that is responsible for hyperacute rejection (HAR) when organs from swine are transferred to primates. Because swine are widely used as models of human diseases, there are opportunities for genetic modification to alter these models or to create additional models of human disease. Unfortunately, some of the offspring resulting from NT have abnormal phenotypes. However, it appears that these abnormal phenotypes are a result of epigenetic modifications and, thus, are not transmitted to the offspring of the clones. Although the technique of producing animals with specific genetic modifications by NT has been achieved, improvements to the NT technique as well as improvements in the culture conditions for somatic cells and the techniques for genetic modification are still needed.

Agriculture↗

PubMed2002

Monitoring transfer of recombinant and nonrecombinant plasmids between Lactococcus lactis strains and members of the human gastrointestinal microbiota in vivo--impact of donor cell number and diet.

AIMS: The generation of data of real relevance to the purported risks of DNA transfer from food-borne genetically modified microorganisms (GMMOs) using the human biota associated (HBA) rat model. Plasmid transfer between Lactococcus lactis strains and between donor strains and human gut bacteria was monitored. METHODS AND RESULTS: Transfer of the recombinant plasmid pCK1 and/or the promiscuous nonrecombinant plasmid pAMbeta1 between L. lactis strains was monitored in vivo in HBA rats. No transfer of pCK1 was observed. Transfer of pAMbeta1 was observed to Enterococcus spp. present in the HBA rats. Transconjugants persisted for 30 d and were distributed throughout the gastrointestinal tract. Both HBA rat diet and donor cell numbers impacted on transconjugant numbers. Fewer transconjugants were observed in animals fed a high-fat human type diet, while high levels of plasmid transfer were only observed at doses of donor L. lactis greater than 109 cfu. CONCLUSIONS: The utility of models of the human gut in monitoring DNA transfer events within the gut microbiota was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Such findings give some confidence for the use of GMMOs with recombinant DNA borne on nonconjugative elements in fermented foods. HBA rats are a suitable model for monitoring the fate of food-borne GMMOs.

Animals↗

PubMed2004

Practical moral codes in the transgenic organism debate.

In one study funded by the United States Department of Agriculture, people from North Dakota were interviewed to discover which moral principles they use in evaluating the morality of transgenic organisms and their introduction into markets. It was found that although the moral codes the human subjects employed were very similar, their views on transgenics were vastly different. In this paper, the codes that were used by the respondents are developed, compared to that of the academically composed Belmont Report, and then modified to create the more practical Common Moral Code. At the end, it is shown that the Common Moral Code has inherent inconsistency flaws that might be resolvable, but would require extensive work on the definition of terms and principles. However, the effort is worthwhile, especially if it results in a common moral code that all those involved in the debate are willing to use in negotiating a resolution to their differences.

Advisory Committees↗

PubMed2005

A study in scarlet: enzymes of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis.

The red ketocarotenoid astaxanthin (3,3'-dihydroxy-4,4'-diketo-beta,beta-carotene) is widely used as an additive in feed for the pigmentation of fish and crustaceans and is frequently included in human nutritional supplements as well. There is considerable interest in developing a plant-based biological production process for this valuable carotenoid. Adonis aestivalis (Ranunculaceae) is unusual among plants in synthesizing and accumulating large amounts of astaxanthin and other ketocarotenoids. The formation of astaxanthin requires only the addition of a carbonyl at the number 4 carbon of each beta-ring of zeaxanthin (3,3'-dihydroxy-beta,beta-carotene), a carotenoid typically present in the green tissues of higher plants. We screened an A. aestivalis flower library to identify cDNAs that might encode the enzyme that catalyzes the addition of the carbonyls. Two closely related cDNAs selected in this screen were found to specify polypeptides similar in sequence to plant beta-carotene 3-hydroxylases, enzymes that convert beta-carotene (beta,beta-carotene) into zeaxanthin. The Adonis enzymes, however, exhibited neither 4-ketolase nor 3-hydroxylase activity when presented with beta-carotene as the substrate in Escherichia coli. Instead, the products of the Adonis cDNAs were found to modify beta-rings in two distinctly different ways: desaturation at the 3,4 position and hydroxylation of the number 4 carbon. The 4-hydroxylated carotenoids formed in E. coli were slowly metabolized to yield compounds with ketocarotenoid-like absorption spectra. It is proposed that a 3,4-desaturation subsequent to 4-hydroxylation of the beta-ring leads to the formation of a 4-keto-beta-ring via an indirect and unexpected route: a keto-enol tautomerization.

Adonis↗