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Gene-chips Prove Transgenes are Clean Genes (GMO Pundit a.k.a David Tribe)


Recent empirical evidence from gene-chips is confirming the considered judgment of geneticists - that genetic engineering is more precise than conventional breeding- is indeed correct.

These gene activity testing devices are providing solid confirmation that transgenic rice, wheat , soybean and thale cress are substantially equivalent to their non-transgenic counterparts.

Gene chips (known also as microarrays) enable the activity of thousands of genes to be measured. Many new studies with these chips listed below convincingly demonstrate that insertion of transgene DNA causes minor perturbation to transcription activity of other genes.

Use of gene-chips to comprehensively survey tens of thousands of genes is revealing that the unintended, unexpected effects of transgenes are small compared to perturbations caused by conventional cross-breeding, which typically perturb an order of magnitude more unrelated genes away from those at the insert target (Baudo 2006, Batista 2008, Cheng 2008, El Ouakfaoui, Miki 2005)

Radiation treatment used to deliberately create mutations in many existing crops also causes many more gene activity perturbation than does transgene insertion (Batista 2008, Dubouzet 2007, Zhang 2006). Note that apart from the intended trait, transgene insertion is generally silent in terms of change to plant phenotype(appearance) (Bouché N, Bouchez D. 2001) .

Yes indeed, transgene inserts are generally clean events as far as unexpected changes to untargeted gene activities. Transgenes are clean genes.


Batista R, Saibo N, Lourenço T, Oliveira MM.
Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion.
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3640-5. Epub 2008 Feb 26.

Instituto Nacional de Saúde Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisbon, Portugal. rita.batista@insa.min-saude.pt

Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding.
We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls:
(i) a gamma-irradiated stable mutant,
(ii) the M1 generation of a 100-Gy gamma-irradiated plant,
(iii) a stable transgenic plant obtained for production of an anticancer antibody,
and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls.

We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering.

Bouché N, Bouchez D.(2001) Curr Opin Plant Biol. 2001 Apr;4(2):111-7.
Arabidopsis gene knockout: phenotypes wanted.

Baudo MM, Lyons R, Powers S, Pastori GM, Edwards KJ, Holdsworth MJ, Shewry PR. (2006) Transgenesis has less impact on the transcriptome of wheat grain than conventional breeding. Plant Biotechnol J. 2006 Jul;4(4):369-80.
Rothamsted Research, Harpenden AL5 2JQ, UK.

Detailed global gene expression profiles have been obtained for a series of transgenic and conventionally bred wheat lines expressing additional genes encoding HMW (high molecular weight) subunits of glutenin, a group of endosperm-specific seed storage proteins known to determine dough strength and therefore bread-making quality. Differences in endosperm and leaf transcriptome profiles between untransformed and derived transgenic lines were consistently extremely small, when analysing plants containing either transgenes only, or also marker genes. Differences observed in gene expression in the endosperm between conventionally bred material were much larger in comparison to differences between transgenic and untransformed lines exhibiting the same complements of gluten subunits. These results suggest that the presence of the transgenes did not significantly alter gene expression and that, at this level of investigation, transgenic plants could be considered substantially equivalent to untransformed parental lines.

Cheng KC, Beaulieu J, Iquira E, Belzile FJ, Fortin MG, Strömvik MV.(2008)
Effect of transgenes on global gene expression in soybean is within the natural range of variation of conventional cultivars.
J Agric Food Chem. 2008 May 14;56(9):3057-67. Epub 2008 Apr 23.
Department of Plant Science, McGill University, 21,111 Lakeshore Road, Sainte Anne de Bellevue, Québec H9X 3V9, Canada.

Current safety assessment for novel crops, including transgenic crops, uses a targeted approach, which relies on compositional analysis. The possibility that transgene expression could lead to unintended effects remains a debated issue. This study used transcriptome profiling as a nontargeted approach to evaluate overall molecular changes in transgenic soybean cultivars. Global gene expression was measured in the first trifoliate leaves of two transgenic and three conventional soybean cultivars using the soybean Affymetrix GeneChip.

It was found that gene expression differs more between the two conventional cultivars than between the transgenics and their closest conventional cultivar investigated and that the magnitudes of differences measured in gene expression and genotype (determined by SSR analysis) do not necessarily correlate. A MySQL database coupled with a CGI Web interface was developed to store and present the results ( http://soyxpress.agrenv.mcgill.ca/). By integrating the microarray data with gene annotations and other soybean data, a comprehensive view of differences in gene expression is explored between cultivars.

Dubouzet JG, Ishihara A, Matsuda F, Miyagawa H, Iwata H, Wakasa K.(2007)
Integrated metabolomic and transcriptomic analyses of high-tryptophan rice expressing a mutant anthranilate synthase alpha subunit.
J Exp Bot. 2007;58(12):3309-21. Epub 2007 Sep 4.

CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan.

Transgenic rice plants overexpressing a mutant rice gene for anthranilate synthase alpha subunit (OASA1D) accumulate large amounts of free tryptophan (Trp) with few adverse effects on the phenotype, except for poor germination and weak seedling growth. Metabolic profiling of 8-d-old seedlings of Nipponbare and two high-Trp lines, HW1 and HW5, by high performance liquid chromatography-photo diode array (HPLC-PDA) confirmed that, relative to Nipponbare, only the peak attributed to Trp was significantly changed in the profiles of the OASA1D lines. More detailed and targeted analysis using HPLC coupled with tandem mass spectrometry revealed that the OASA1D lines had higher levels of anthranilate, tryptamine, and serotonin than Nipponbare, but these metabolites were at much lower levels than free Trp. The levels of phenylalanine (Phe) and tyrosine (Tyr) were not affected by the overproduction of Trp.

Transcriptomic analysis by microarray validated by quantitative Real-Time PCR (qRT-PCR) revealed that at least 12 out of 21 500 genes showed significant differential expression among genotypes. Except for the OASA1D transgene and a putative IAA beta-glucosyltransferase, these were not related to Trp metabolism.
Most importantly, the overexpression of the OASA1D and the consequent accumulation of Trp in these lines had little effect on the overall transcriptome, consistent with the minimal effects on growth and the metabolome. Integrated analysis of the metabolome and transcriptome of these OASA1D transgenic lines indicates that the over-accumulation of free Trp may be partly due to the low activity of Trp decarboxylase or other metabolic genes that directly utilize Trp as a substrate.

El Ouakfaoui S, Miki B.(2005)
The stability of the Arabidopsis transcriptome in transgenic plants expressing the marker genes nptII and uidA.
Plant J. 2005 Mar;41(6):791-800.

Bioproducts and Bioprocesses, Research Branch, Agriculture and Agri-Food Canada, Ottawa, ON K1A 0C6, Canada.

The ATH1 Arabidopsis GeneChip from Affymetrix was used to search for transcriptome changes in Arabidopsis associated with the strong expression of transgenes regulated by constitutive promoters. The insertion and expression of the commonly used marker genes, uidA and nptII, did not induce changes to the expression patterns of the approximately 24 000 genes that were screened under optimal growth conditions and under physiological stress imposed by low temperatures. Approximately 8000 genes (35% of the Arabidopsis genome) underwent changes in gene expression in both wild-type and transgenic plants under abiotic stresses such as salt, dehydration, cold, and heat. This study provides detailed information on the extent of non-targeted or pleiotropic effects of transgenes on plants and shows that the transgenic and non-transgenic plants were equivalent in their global patterns of transcription. This information may help to extend our understanding and interpretation of the principle of substantial equivalence which is used as a first step in the biosafety evaluation of transgenic crops.

Zhang L, Fetch T, Nirmala J, Schmierer D, Brueggeman R, Steffenson B, Kleinhofs A.(2006)
Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley.
Theor Appl Genet. 2006 Sep;113(5):847-55. Epub 2006 Jul 11.

Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA.

Rpg1 is a stem rust resistance gene that has protected barley from severe losses for over 60 years in the US and Canada. It confers resistance to many, but not all, pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron induced deletion mutant, showing susceptibility to stem rust pathotype Pgt-MCC, was identified in barley cv. Morex, which carries Rpg1. Genetic and Rpg1 mRNA and protein expression level analyses showed that the mutation was a suppressor of Rpg1 and was designated Rpr1 (Required for P. graminis resistance).
Genome-wide expression profiling, using the Affymetrix Barley1 GeneChip containing approximately 22,840 probe sets, was conducted with Morex and the rpr1mutant. Of the genes represented on the Barley1 microarray, 20 were up-regulated and 33 were down-regulated by greater than twofold in the mutant, while the Rpg1 mRNA level remained constant. Among the highly down-regulated genes (greater than fourfold), genomic PCR, RT-PCR and Southern analyses identified that three genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at), were deleted in the rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and co-segregated with the rpr1-mediated susceptible phenotype. The loss of resistance was presumed to be due to a mutation in one or more of these genes. However, the possibility exists that there are other genes within the deletions, which are not represented on the Barley1 GeneChip. The Rpr1 gene was not required for Rpg5- and rpg4-mediated stem rust resistance, indicating that it shows specificity to the Rpg1-mediated resistance pathway.


Other "Omics technology papers proving that transgenes are clean;


It is generally accepted that traditional food is safe for the majority of consumers. For the introduction of a new variant or cultivar developed from a traditional crop plant, maximum limits have been set in some cases, e.g., for potato and oilseed rape, to the content of known toxins. The requirements are much more stringent if the crop is developed by using genetic engineering. Why is it so? In a majority of cases seen so far, a new gene, often derived from other plants or microbial species, has been introduced to a non-predetermined location in the plant genome. It is quite feasible to ask the question whether the new gene products are safe or not. Therefore, for all genetically modified crop plants, the safety of the newly introduced proteins needs to be demonstrated before the plants can be released into the market.

Another point of concern is the random integration of the new gene into the plant genome. Both the new gene itself and its site of integration may give rise to unintended adverse effects. For example, transgene integration might interrupt regulatory sequences or open reading frames leading to novel fusion proteins and, thereby, modify plant metabolism.1 These modifications could compromise the safety of the food crops by, for instance, leading to the production of new allergens or toxins. Having the gene and the integration site well characterised should provide a good basis for the safety assessment. However, it is a common practice today to perform a large number of analyses, so-called targeted analyses, to demonstrate that the characteristics of the novel crop are comparable with those of the conventional counterpart, in addition to the intended alterations. Targeted analyses include key macronutrients, micronutrients, antinutrients, and toxins. In certain cases, toxicity studies on experimental animals are advised. And yet, the question about the unintended effects does not seem to be covered in a way that would escape all criticism...continues at GMO Pundit (or alternatively at the original at title link just above)

See also
The current concepts of genetics which can explain much of the detected natural variability in protein and metabolic fingerprints of plants suggested in the previous reports is discussed in Natural GMOs Part 26.

In all publications in which substantial equivalence studies in genetically engineered plants were reported, including ours, the differences in the molecular profile between wt and genetically engineered plant were by orders of magnitude smaller than the differences between varieties under the same environmental conditions or the differences between different environmental conditions in the same variety.


[Fingerprinting and profiling technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). Pundit note]

David A Bender
Department of Biochemistry Molecular Biology, University College London, Gower Street, London WC1E 6BT

"...Of more direct relevance to nutritional sciences, metabolomic techniques will permit identification of biomarkers of health (as opposed to disease), and will therefore permit more precise estimation of appropriate levels of nutrient intake—levels of
Within the European Union, since April 2004, all food and animal feed containing more than 0.9% of ingredients from genetically modified organisms must be so labelled, regardless of whether or not there is any GM material in the final product.14 This means products such as flour, oils and glucose syrups, which were previously considered to be substantially equivalent’ to material from traditional sources, since they contained no modified DNA, will have to be
labelled as GMif they are from a GM source. This will increase public concern about the safety of GM foods.

Christou and Twyman15 reviewed the potential of GM crops to alleviate food insecurity in less developed countries by providing higher yields, resistance to pests and diseases (so increasing the effective yield considerably), ability to grow under adverse conditions (so increasing the land available for crops) and improved nutritional quality. They concluded with the observation that genetic modification of crops is highly politically sensitive. Within the European Union environmental concerns have led to a trade war with the USA, and several southern African countries have refused American food aid that included genetically modified cereals or soy beans.

Metabolic profiling will permit more precise determination of whether a novel or GM product is indeed ‘substantially identical’, or whether there are (possibly significant) differences in hitherto disregarded trace compounds, and should provide valuable evidence to inform consumers about safety.16

Many potentially hazardous compounds occur in plant foods naturally, and sometimes improved varieties that result from traditional plant breeding methods may contain unexpectedly high amounts of toxins. Beier17 cites the case of a new variety of celery that was bred for resistance to pests and wilting (and so had a longer shelf life). Its pest resistance was due to higher than normal concentrations of psoralens, which causes a photosensitive dermatitis through contact with the skin, and its introduction was associated with a significant increase of cases of photodermatitis among grocery employees.Metabolic profiling would, presumably, have been able to detect this abnormal concentration of a toxic compound before the new variety was released into general cultivation..."

Journal of the Science of Food and Agriculture Volume 85, Issue 1 , Pages 7 - 9 Published Online: 3 Nov 2004

14 Anonymous, Regulation no 1829/2003 on Genetically Modified Food and Feed. Off J EU L268:1–23 (2003).

15 Christou P and Twyman RM, The potential of genetically enhanced plants to address food insecurity. Nutr Res Rev 17:23–42 (2004).

16 Cellini F, Chesson A, Colquhoun I, Constable A, Davies HV, Engel KH, Gatehouse AM, Karenlampi S, Kok EJ, Leguay JJ, Lehesranta S, Noteborn HP, Pedersen J and Smith M, Unintended effects and their detection in genetically modified crops. Food Chem Toxicol 42:1089–1125 (2004).

The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment.

This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding.

The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined.