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Transformation Vector (transformation + vector)
Selected AbstractsThe reduction of starch accumulation in transgenic sugarcane cell suspension culture linesBIOTECHNOLOGY JOURNAL, Issue 11 2008Stephanus J. Ferreira Abstract Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and ,-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial ,-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. ,-Amylase activity was increased in the transgenic lines by 1.5,2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production. [source] piggyBac -mediated germline transformation in the beetle Tribolium castaneumINSECT MOLECULAR BIOLOGY, Issue 5 2003M. D. Lorenzen Abstract The lepidopteran transposable element piggyBac can mediate germline insertions in at least four insect orders. It therefore shows promise as a broad-spectrum transformation vector, but applications such as enhancer trapping and transposon-tag mutagenesis are still lacking. We created, cloned, sequenced and genetically mapped a set of piggyBac insertions in the red flour beetle, Tribolium castaneum. Transpositions were precise, and specifically targeted the canonical TTAA recognition sequence. We detected several novel reporter-expression domains, indicating that piggyBac could be used to identify enhancer regions. We also demonstrated that a primary insertion of a non-autonomous element can be efficiently remobilized to non-homologous chromosomes by injection of an immobile helper element into embryos harbouring the primary insertion. These developments suggest potential for more sophisticated methods of piggyBac -mediated genome manipulation. [source] Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vectorINSECT MOLECULAR BIOLOGY, Issue 2 2002N. F. Lobo Mosquito-vectored diseases such as yellow fever and dengue fever continue to have a substantial impact on human populations world-wide. Novel strategies for control of these mosquito vectored diseases can arise through the development of reliable systems for genetic manipulation of the insect vector. A piggyBac vector marked with the Drosophila melanogaster cinnabar (cn) gene was used to transform the white-eyed khw strain of Aedes aegypti. Microinjection of preblastoderm embryos resulted in four families of cinnabar transformed insects. An overall transformation frequency of 4%, with a range of 0% to as high as 13% for individual experiments, was achieved when using a heat-shock induced transposase providing helper plasmid. Southern hybridizations indicated multiple insertion events in three of four transgenic lines, while the presence of duplicated target TTAA sites at either ends of individual insertions confirmed characteristic piggyBac transposition events in these three transgenic lines. The transgenic phenotype has remained stable for more than twenty generations. The transformations effected using the piggyBac element establish the potential of this element as a germ-line transformation vector for Aedine mosquitoes. [source] Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicityPLANT BIOTECHNOLOGY JOURNAL, Issue 2 2008Hortense W. Dodo Summary Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant (P < 0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T0 plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology. [source] Highly similar piggyBac transposase-like sequences from various Bactrocera (Diptera, Tephritidae) speciesINSECT MOLECULAR BIOLOGY, Issue 5 2007M. Bonizzoni Abstract The piggyBac transposable element is currently the vector of choice for transgenesis, enhancer trapping, gene discovery and gene function determination in both insects and mammals. However, the recent discovery of sequences with similarity to piggyBac in a wide diversity of organisms suggests that piggyBac may be horizontally transferred to distantly related species. This has raised concern on the wide-range application of piggyBac -based transformation vectors and their stability. In this paper, the presence of sequences homologous to the piggyBac transposase was investigated in 17 species belonging to six genera within the Tephritidae family, including many pest species for which transformation has already been achieved. piggyBac -like sequences, with a high degree of similarity to the original Trichoplusia ni transposase sequence were identified only in six species of the Bactrocera genus. [source] Bacteriophage WO-B and Wolbachia in natural mosquito hosts: infection incidence, transmission mode and relative densityMOLECULAR ECOLOGY, Issue 9 2006N. CHAUVATCHARIN Abstract Bacteriophages of Wolbachia bacteria have been proposed as a potential transformation tool for genetically modifying mosquito vectors. In this study, we report the presence of the WO-B class of Wolbachia -associated phages among natural populations of several mosquito hosts. Eighty-eight percent (22/25) of Wolbachia -infected mosquito species surveyed were found to contain WO-B phages. WO-B phage orf7 sequence analysis suggested that a single strain of WO-B phage was found in most singly (23/24) or doubly (1/1) Wolbachia -infected mosquitoes. However, the single Wolbachia strain infecting Aedes perplexus was found to harbour at least two different WO-B phages. Phylogenetic analysis suggested that horizontal transmission of WO-B phages has occurred on an evolutionary scale between the Wolbachia residing in mosquitoes. On an ecological scale, a low trend of co-transmission occurred among specific WO-B phages within Wolbachia of each mosquito species. Assessment of the density of WO-B phage by real-time quantitative polymerase chain reaction (RTQ-PCR) revealed an average relative density of 7.76 × 105± 1.61 × 105 orf7 copies per individual mosquito for a single Wolbachia strain infecting mosquitoes, but a threefold higher density in the doubly Wolbachia-infected Aedes albopictus. However, the average combined density of WO-B phage(s) did not correlate with that of their Wolbachia hosts, which varied in different mosquito species. We also confirmed the presence of WO-B-like virus particles in the laboratory colony of Ae. albopictus (KLPP) morphologically, by transmission electron microscopy (TEM). The viral-like particles were detected after purification and filtration of Ae. albopictus ovary extract, suggesting that at least one WO-B-like phage is active (temperate) within the Wolbachia of this mosquito vector. Nevertheless, the idea of utilizing these bacteriophages as transformation vectors still needs more investigation and is likely to be unfeasible. [source] | ||||||||||