Local and Regional Dynamics of Native Maize Seed Lot Use by Small-Scale Producers and Their Impact on Transgene Presence in Three Mexican States.

Mexico harbors over 50% of maize's genetic diversity in the Americas. Native maize varieties are actively managed by small-scale producers within a diverse array of cultivation systems. Seed lot use, exchange and admixture has consequences for the in situ conservation of such varieties. Here we analyze native maize seed management dynamics from 906 small-scale producers surveyed in three Mexican states: Mexico City, Oaxaca and Chiapas. Furthermore, we analyze how their management practices can relate to transgene presence, which was experimentally documented for maize samples associated with the applied surveys. Through a data mining approach, we investigated which practices might be related with a higher probability of transgene presence. The variables found to have a strong spatial association with transgene presence were: for Mexico City, maize producers with larger parcels; for Oaxaca, producer's age (43-46 years) and the sale of seed; for Chiapas, the use of agricultural machinery and younger producers (37-43 years). Additionally, transgene presence and frequency within the socioeconomic regions of Oaxaca and Chiapas was analyzed. In Oaxaca, higher transgene frequencies occurred in regions where transgene presence had been previously reported. In Chiapas, the border regions with Guatemala as well as a region where reproduction of improved seed takes place, the highest proportion of positive samples were found. A detailed mapping of regional seed markets and seed exchange sites together with deployment of national and local biosecurity measures, could help prevent the further spread of transgenes into native maize varieties, as well as improve conservation efforts.

Large scale sampling of Mexican maize landraces for the presence of transgenes.

The presence and levels of transgenic maize in Mexico and the effect this could have on local landraces or closely related species such as teosinte has been the subject of several previous reports, some showing contrasting results. Cultural, social and political factors all affect maize cultivation in Mexico and although since 1998 there has been a moratorium on the commercial cultivation of transgenic maize, Mexico imports maize, mainly from the USA where transgenic cultivars are widely grown. Additionally extensive migration between rural areas in Mexico and the USA and customs of seed exchange between farmers may also play an unintentional role in the establishment of transgenic seed. A comprehensive study of all Mexican maize landraces throughout the country is not feasible, however this report presents data based on analysis of 3204 maize accessions obtained from the central region of Mexico (where permits have never been authorized for cultivation of transgenic maize) and the northern region (where for a short period authorization for experimental plots was granted). The results of the study confirm that transgenes are present in all the geographical areas sampled and were more common in germplasm obtained in the northern region. However, there was no evidence that regions where field trials had been authorized showed higher levels of transgene presence or that the morphology of seed lots harboring transgenic material was significantly modified in favor of expected transgenic phenotypes.

Enhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle Remodeling.

BACKGROUND: Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. METHODS: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. RESULTS: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive ß-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. CONCLUSIONS: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.

Visão geral do desenvolvimento biofarmacêutico de proteinas recombinantes usando o leite de animais transgênicos / Overview of Recombinant Biopharmaceutical Development Using the Milk of Transgenic Livestock

Human plasma for obtaining biotherapeutics has shown considerable limitation in terms of biosafety and scalability. Engineered animal cells in bioreactors provide an alternative source of these complex proteins. However, manufacturing animal cell bioreactor technology can just produce about grams recombinant protein per liter. Besides, animal cell bioreactor has shown a rate limitation of biosynthesis of the post translational modifications (PTMs) in human proteins necessary for physiologic function. PTMs can be provided in nature by the capacity of mammary epithelia of livestock. Transgenic livestock offer a continuous productivity of grams recombinant protein per liter in the milk. Here, we discuss how recombinant proteins made in the milk of transgenic livestock represent a prodigious, selfreplicating bioreactor technology which can bring promising new biotherapeutic treatment paradigms world-wide.

O plasma humano para a obtenção de bioterapêuticos tem mostrado limitaçoes consideráveis em termos de biossegurança e escalabilidade. Células animais cultivadas em biorreatores fornecem uma fonte alternativa para essas proteínas complexas. No entanto, a tecnologia de biorreator de célula animal pode produzir apenas cerca de gramas de proteína recombinante por litro. Além disso, o biorreator de células animais tem mostrado limitacoes na escalabilidade de biossíntese das modificações pós-translacionais (PTMs) nas proteínas humanas necessárias para a função fisiológica. As PTMs podem acontecer naturalmente no epitélio mamário do gado. O gado transgênico oferece uma produtividade contínua de gramas de proteína recombinante por litro no leite. Aqui, discutimos como as proteínas recombinantes feitas no leite de gado transgênico representam uma tecnologia de biorreator auto-replicante prodigiosa que pode trazer novos paradigmas de tratamento bioterapêutico promissores em todo o mundo.

Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering / Empilhamento gênico como estratégia para conferir características de importância agronômica em plantas via engenharia genética

Gene stacking refers to the introduction of two or more transgenes of agronomic interest in the same plant. The main methods for genetically engineering plants with gene stacking involve (i) the simultaneous introduction, by the co-transformation process, and (ii) the sequential introduction of genes using the re-transformation processes or the sexual crossing between separate transgenic events. In general, the choice of the best method varies according to the species of interest and the availability of genetic constructions and preexisting transgenic events. We also present here the use of minichromosome technology as a potential future gene stacking technology. The purpose of this review was to discuss aspects related to the methodology for gene stacking and trait stacking (a gene stacking strategy to combine characteristics of agronomical importance) by genetic engineering. In addition, we presented a list of crops and genes approved commercially that have been used in stacking strategies for combined characteristics and a discussion about the regulatory standards. An increased number of approved and released gene stacking events reached the market in the last decade. Initially, the most common combined characteristics were herbicide tolerance and insect resistance in soybean and maize. Recently, commercially available varieties were released combining these traits with drought tolerance in these commodities. New traits combinations are reaching the farmers fields, including higher quality, disease resistant and nutritional value improved. In other words, gene stacking is growing as a strategy to contribute to food safety and sustainability.(AU)

O empilhamento gênico se refere a introdução de dois ou mais transgenes de interesse agronômico na mesma planta. Os principais métodos de produção de plantas geneticamente modificadas com empilhamento gênico envolvem (i) a introdução simultânea, pelo processo de co-transformação, e (ii) a introdução sequencial de genes, pelos processos de re-transformação ou por cruzamento entre eventos transgênicos. Em geral, a escolha do melhor método varia de acordo com a espécie de interesse e a disponibilidade de construções genéticas e eventos transgênicos preexistentes. Também é apresentado aqui o uso da tecnologia de minicromossomos como tecnologia potencial de empilhamento gênico. O objetivo desta revisão é discutir aspectos relacionados à metodologia para o empilhamento de genes a combinação de características (obtida via empilhamento de genes de interesse agronômico) via engenharia genética. Além de discutir, é apresentado uma lista de culturas e genes aprovados comercialmente que tem sido usado em estratégias de empilhamento e uma discussão sobre normas regulatórias. Um número maior de eventos com empilhamento de genes foi aprovado e liberado no mercado na última década. Inicialmente, a combinação das características de tolerância a herbicidas e resistência a insetos era a mais popular, principalmente em soja e milho. Recentemente, estas características combinadas com tolerância a seca nessas culturas foram liberadas comercialmente. Novas características combinadas estão entrando na lavoura, incluindo aumento da qualidade, resistência a doenças e aumento do valor nutricional. Em outras palavras, o empilhamento gênico está crescendo como tecnologia para contribuir para a segurança alimentar e sustentabilidade.(AU)

Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering / Empilhamento gênico como estratégia para conferir características de importância agronômica em plantas via engenharia genética

ABSTRACT: Gene stacking refers to the introduction of two or more transgenes of agronomic interest in the same plant. The main methods for genetically engineering plants with gene stacking involve (i) the simultaneous introduction, by the co-transformation process, and (ii) the sequential introduction of genes using the re-transformation processes or the sexual crossing between separate transgenic events. In general, the choice of the best method varies according to the species of interest and the availability of genetic constructions and preexisting transgenic events. We also present here the use of minichromosome technology as a potential future gene stacking technology. The purpose of this review was to discuss aspects related to the methodology for gene stacking and trait stacking (a gene stacking strategy to combine characteristics of agronomical importance) by genetic engineering. In addition, we presented a list of crops and genes approved commercially that have been used in stacking strategies for combined characteristics and a discussion about the regulatory standards. An increased number of approved and released gene stacking events reached the market in the last decade. Initially, the most common combined characteristics were herbicide tolerance and insect resistance in soybean and maize. Recently, commercially available varieties were released combining these traits with drought tolerance in these commodities. New traits combinations are reaching the farmer's fields, including higher quality, disease resistant and nutritional value improved. In other words, gene stacking is growing as a strategy to contribute to food safety and sustainability.

RESUMO: O empilhamento gênico se refere a introdução de dois ou mais transgenes de interesse agronômico na mesma planta. Os principais métodos de produção de plantas geneticamente modificadas com empilhamento gênico envolvem (i) a introdução simultânea, pelo processo de co-transformação, e (ii) a introdução sequencial de genes, pelos processos de re-transformação ou por cruzamento entre eventos transgênicos. Em geral, a escolha do melhor método varia de acordo com a espécie de interesse e a disponibilidade de construções genéticas e eventos transgênicos preexistentes. Também é apresentado aqui o uso da tecnologia de minicromossomos como tecnologia potencial de empilhamento gênico. O objetivo desta revisão é discutir aspectos relacionados à metodologia para o empilhamento de genes a combinação de características (obtida via empilhamento de genes de interesse agronômico) via engenharia genética. Além de discutir, é apresentado uma lista de culturas e genes aprovados comercialmente que tem sido usado em estratégias de empilhamento e uma discussão sobre normas regulatórias. Um número maior de eventos com empilhamento de genes foi aprovado e liberado no mercado na última década. Inicialmente, a combinação das características de tolerância a herbicidas e resistência a insetos era a mais popular, principalmente em soja e milho. Recentemente, estas características combinadas com tolerância a seca nessas culturas foram liberadas comercialmente. Novas características combinadas estão entrando na lavoura, incluindo aumento da qualidade, resistência a doenças e aumento do valor nutricional. Em outras palavras, o empilhamento gênico está crescendo como tecnologia para contribuir para a segurança alimentar e sustentabilidade.

Agronomic performance of insect-protected and herbicide-tolerant MON 89034 × TC1507 × NK603 × DAS-40278-9 corn is equivalent to that of conventional corn.

Agronomic characteristics of genetically modified (GM) MON 89034 × TC1507 × NK603 × DAS-40278-9 (PowerCore™ Enlist™), MON 89034 × TC1507 × NK603 (PowerCore™), and DAS-40278-9 (Enlist™) corn, a non-GM near-isogenic hybrid, and 2 commercial non-GM hybrids were assessed in a field study to determine if the agronomic performance of the GM corn hybrids is equivalent to that of non-transgenic hybrid corn. The MON 89034 × TC1507 × NK603 × DAS-40278-9 hybrid corn was developed through stacking of 4 individual transgenic events, MON 89034, TC1507, NK603, and DAS-40278-9 by traditional breeding and contains the cry1A.105 and cry2Ab2 (MON 89034), cry1F and pat (TC1507), cp4 epsps (NK603) and aad-1 (DAS-40278-9) transgenes. These transgenes encode the proteins Cry1A.105, Cry2Ab2, and Cry1F, which confer insect resistance, PAT, CP4 EPSPS, and AAD-1, which confer herbicide tolerance. The following agronomic characteristics were assessed in the study: initial and final stand count, seedling vigor, time to silk, time to pollen shed, pollen viability, plant height, ear height, stalk lodging, root lodging, days to maturity, stay green, disease incidence, insect damage, herbicide injury, and yield. The agronomic assessment was conducted in 2 regions of Brazil (Indianopolis-MG; Cravinhos-SP). The agronomic attributes for all GM entries were statistically indistinguishable from the non-GM near-isogenic hybrid. In addition, most of the agronomic assessments fell within the range of the commercial varieties included in the study. Taken together, MON 89034 × TC1507 × NK603 × DAS-40278, MON 89034 × TC1507 × NK603, and DAS-40278-9 were found to be agronomically equivalent to non-GM corn.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Detecting adventitious transgenic events in a maize center of diversity

Background: The genetic diversity of maize in Peru includes several landraces (within race clusters) and modern open pollinated and hybrid cultivars that are grown by farmers across various regions, thereby making this country a secondary center of diversity for this crop. A main topic of controversy in recent years refers to the unintended presence of transgenic events in locally grown cultivars at main centers of crop diversity. Peru does not yet have biosafety regulations to control or permit the growing of genetically modified crops. Hence, the aim of this research was to undertake a survey in the valley of Barranca, where there were recent claims of authorized transgenic maize grown in farmers fields as well as in samples taken from feed storage and grain or seed trade centers. Results: A total of 162 maize samples (134 from fields, 15 from local markets, eight from the collecting centers of poultry companies, from the local trading center and four samples from seed markets) were included for a qualitative detection by the polymerase chain reaction (PCR) of Cauliflower Mosaic Virus (CaMV) 35S promoter (P35S) and nopaline synthase terminator (Tnos) sequences, as well as for six transgenic events, namely BT11, NK603, T25, 176, TC1507 and MON810. The 134 maize samples from farmers fields were negative for Cry1Ab delta-endotoxin insecticidal protein and enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) using lateral flow strips. The PCR analysis did not detect any of the six transgenic events in samples from farmers fields, local markets, seed trading shops and the local collecting center. There were four transgenic events (T25, NK603, MON810 and TC1507) in grain samples from the barns of poultry companies. Conclusions: This research could not detect, at the 95 percent probability level, transgenes in farmers' fields in the valley of Barranca. The four transgenic events in grain samples from barns of poultry companies...

Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector / Transferência de genes heterólogos para células corneanas epiteliais primárias utilizando vetor lentivírus

PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP) reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80 percent confluency) prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS). We evaluated the transduction efficiency (TE) over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells). RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1 percent) and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7 percent. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

OBJETIVO: Avaliar a transferência de genes heterólogos expressando a proteí­na "Green Fluorescent Protein" (GFP) para células corneanas epiteliais primárias ex vivo utilizando vetor lentivírus. MÉTODOS: Tecido corneoescleral de coelhos foi usado para obtenção de suspensão de células corneanas epitelias. As células foram semeadas na densidade de 5×10³ células/cm² e expandidas por 5 dias até uma confluência de 70-80 por cento antes de serem transduzidas. A transferência genética foi monitorada por microscopia fluorescente e por um separador de células ativadas por fluorescência. Foram avaliadas a eficiência de transdução ao longo do tempo e o efeito dose-resposta de diferentes quantidades de partículas virais. Um grupo de células foi analisado pelo separador de células ativadas por fluorescência para avaliar a transdução de células com fenótipo de cé­lulas tronco do epitélio corneano (baseado na exclusão do corante "Hoechst dye"). RESULTADOS: Os vetores lentivírus foram efetivos na transdução de cé­lulas corneanas epiteliais primárias de coelhos ex vivo. Fotodocumentação das células vivas demonstrou células epiteliais de morfologia normal e expressando o gene fluorescente (GFP). A eficiência de transdução ao longo do tempo foi maior no quinto dia após a transdução (14,1 por cento) e demonstrou uma tendência à estabilidade a partir do oitavo dia após a transdução. O número de células transduzidas foi dose-dependente e atingiu 7 por cento com as maiores concentrações de partículas virais. Quando analisadas pelo separador de células ativadas por fluorescência para detecção de células transduzidas e também de células que excluíram o corante "Hoechst dye", foi detectado que células com fenótipo de células tronco do epitélio corneano ("side-population") também foram transduzidas. CONCLUSÕES: Os vetores lentivirais podem transferir genes heterolólogos para células corneanas epiteliais primárias expandidas ex vivo de forma eficiente. Os genes foram expressos de forma estável ao longo do tempo e puderam ser transferidos tanto para células epiteliais maduras como para presumíveis células tronco epiteliais. A eficiência de transdução foi obtida de forma dose-dependente.

Transgenic plants of cassava (Manihot esculenta) with resistance to Basta obtained by Agrobacterium-mediated transformation.

Transgenic plants of cassava (Manihot esculenta) resistant to the herbicide Basta were obtained through Agrobacterium-mediated transformation. The plants also expressed the uidA gene and two were positive for PCR- and/or Southern-based detection of the nptII gene. Somatic-embryo-derived cotyledons were used as source of explants. A non-disarmed Agrobacterium strain (CIAT 1182) was used to transfer the genes of interest into cassava cultivar MPer183. Greenhouse tests of resistance to Basta (Hoechst) showed three plant lines with different levels of tolerance to the herbicide. Based on Southern tests of transgenesis, the transformation efficiency was 1%. The results constitute the first report of the bar gene conferring herbicide resistance to cassava plants.