Transgenic Research

Genetic manipulation of carotenoid biosynthesis in higher plants has been the objective of a number of biotechnology programs, e.g. the Golden Rice Program. However, tomato (Solanum lycopersicum L.), which naturally accumulates lycopene in fruits, has attracted the attention of many groups who have manipulated it to increase or diversify carotenoid accumulation. One of the most significant achievements was “HighCaro (HC),” a transgenic tomato plant constitutively expressing the tomato lycopene beta-cyclase (tLcy-b), that produces orange fruits due to the complete conversion of lycopene to β-carotene. In this article we report the results of a field trial conducted in Metaponto (Italy) on HC and on two control genotypes to evaluate the stability of the transgenic trait and their yield performances. Transcriptional regulation of eight genes involved in carotenogenesis was assayed by quantitative real-time PCR (qRT-PCR) analysis on fruits collected at four distinct development stages. Statistical analysis results demonstrated that in field conditions the transgene maintained its ability to induce the conversion of lycopene to β-carotene. Moreover, agronomic performances and fruit quality in the transgenic line were not impaired by this metabolic disturbance. Results of qRT-PCR analysis suggested that transcription of PSY-1, PDS and ZDS genes were developmentally regulated in both genotypes. Unexpectedly, Lcy-b expression in transgenic fruits was also developmentally regulated, despite the fact that the gene was driven by a constitutive promoter. Our data provide evidence that in photosynthetic cells a strict and aspecific mechanism controls the level of transcripts until the onset of chromoplasts differentiation, at which point a gene-specific control on transcription takes place.

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

Wheat seed storage proteins (prolamins) are important for the grain quality because they provide a characteristic texture to wheat flour products. In wheat endosperm cells, prolamins are transported from the Endoplasmic reticulum to Protein storage vacuoles through two distinct pathways—a conventional pathway passing through the Golgi apparatus and an unconventional Golgi-bypassing pathway during which prolamins accumulate in the ER lumen, forming Protein bodies. Unfortunately, transport studies conducted previously achieved limited success because of the seed-specificity of the latter pathway and the multigene architecture of prolamins. To overcome this difficulty, we expressed either of the two families of wheat prolamins, namely α-gliadin or High-molecular-weight subunit of glutenin, in soybean seed, which naturally lacks prolamin-like proteins. SDS-PAGE analysis indicated the successful expression of recombinant wheat prolamins in transgenic soybean seeds. Their accumulation states were quite different—α-gliadin accumulated with partial fragmentation whereas the HMW-glutenin subunit formed disulfide-crosslinked polymers without fragmentation. Immunoelectron microscopy of seed sections revealed that α-gliadin was transported to PSVs whereas HMW-glutenin was deposited in novel ER-derived compartments distinct from PSVs. Observation of a developmental stage of seed cells showed the involvement of post-Golgi Prevacuolar compartments in the transport of α-gliadin. In a similar stage of cells, deposits of HMW-glutenin surrounded by membranes studded with ribosomes were observed confirming the accumulation of this prolamin as ER-derived PBs. Subcellular fractionation analysis supported the electron microscopy observations. Our results should help in better understanding of molecular events during the transport of prolamins in wheat.

In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme (hLZ) in their milk therefore has the potential to positively impact intestinal health. In order to investigate the effect of hLZ-containing milk on the inflammatory response, young pigs were fed pasteurized milk from hLZ or non-transgenic control goats and quantitative real-time PCR was performed to assess local expression of TNF-α, IL-8, and TGF-β1 in the small intestine. Histological changes were also investigated, specifically looking at villi width, length, crypt depth, and lamina propria thickness along with cell counts for intraepithelial lymphocytes and goblet cells. Significantly higher expression of anti-inflammatory cytokine TGF-β1 was seen in the ileum of pigs fed pasteurized milk containing hLZ (P = 0.0478), along with an increase in intraepithelial lymphocytes (P = 0.0255), and decrease in lamina propria thickness in the duodenum (P = 0.0001). Based on these results we conclude that consuming pasteurized milk containing hLZ does not induce an inflammatory response and improves the health of the small intestine in pigs.

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.

Mitochondrial DNA segregation is one of the characteristic modes of mitochondrial inheritance in which the heteroplasmic state of mitochondrial DNA is transmitted to the next generation in variable proportions. To analyze mitochondrial DNA segregation, we produced a heteroplasmic mouse strain with interspecific mitochondrial DNA haplotypes, which contains two types of mitochondrial DNA derived from C57BL/6J and Mus spretus strains. The strain was produced on a C57BL/6J nuclear genomic background by microinjection of donor cytoplasm into fertilized eggs. The PCR-RFLP semi-quantitative analysis method, which was improved to reduce the effect of heteroduplex formation, was used to measure the proportion of heteroplasmic mitochondrial DNA in tissues. Founder mice contained up to approximately 14% of exogenous Mus spretus mitochondrial DNA molecules in their tails, and the detected proportions differed among tissues of the same individual. Heteroplasmic mitochondrial DNA is transmitted to the next generation in varying proportions under the maternal inheritance mode. This mitochondrial heteroplasmic mouse strain and the improved PCR-RFLP measurement system enable analysis of the transmission of heteroplasmic mitochondrial DNA variants between tissues and generations.

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T2–T4 generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.

Multiple genetic modification is necessary for successful xenotransplantation from pigs. However, multiple-genetically modified cells usually suffer from various drug selections and long-term in vitro culture, which have a poor performance for somatic cell nuclear transfer (SCNT) to produce genetically modified pigs. We used to generate GTKO/hCD55/hCD59 triple-gene modified pigs by using drug-selective cell lines for SCNT, but the majority of cloned pigs were transgenic-negative individuals. In this study, to improve the production efficiency of multiple genetically modified pigs, we performed the recloning process by using transgenic porcine fetal fibroblast cells. As a result, two fetuses expressing hCD55 and hCD59 were obtained from 12 live-cloned fetuses, and one carrying high transgene expression was selected as a source of donor cells for recloning. Then we obtained 12 cloned piglets, all GTKO and carrying hCD55 and hCD59. Both hCD55 and hCD59 were expressed in fibroblast cells, but the expression levels of hCD55 and hCD59 were different among these piglets. Furthermore, piglet P5# had the highest expression of hCD55 and hCD59 in fibroblast cells than other piglets. Correspondingly, fibroblast cells of piglet P5# had significantly higher resistance against human serum-mediated cytolysis than those of piglet P11#. In conclusion, our results firstly provide support for improving efficiency of generating multiple genetically modified pig by recloning.

The impact on beneficial insects of proteinase inhibitors expressed in pest-resistant transgenic crops needs to be assessed before the release of these plants into the environment. Three proteinase inhibitors, suitable for incorporation into oilseed rape, were tested on worker bees: the chicken egg white cystatin, oryzacystatin I (OCI) and Bowman-Birk soyabean inhibitor (BBI). Ingestion of low doses of the inhibitors did not cause short-term mortality, and a conditioned proboscis extension assay showed that olfactory learning performances were unchanged when the inhibitors were added to the reward. Long-term ingestion of BBI or OCI did not disrupt total digestive proteolytic activity, but ingestion of BBI induced a new proteinase form, suggesting the existence of a mechanism of control of proteinase synthesis in the honeybee.