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Transgenic Protein (transgenic + protein)
Selected AbstractsComparison of conventional FASTA identity searches with the 80 amino acid sliding window FASTA search for the elucidation of potential identities to known allergensMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 8 2007Gregory S. Ladics Abstract Food and Agriculture Organization/World Health Organization (FAO/WHO) recommended that IgE cross-reactivity between a transgenic protein and allergen be considered when there is ,F 35% identity over a sliding "window" of 80 amino acids. Our objective was to evaluate the false positive and negative rates observed using the FAO/WHO versus conventional FASTA analyses. Data used as queries against allergen databases and analyzed to assess false positive rates included: 1102 hypothetical corn ORFs; 907 randomly selected proteins; 89 randomly selected corn proteins; and 97 corn seed proteins. To evaluate false negative rates of both methods: Bet v 1a along with several crossreacting fruit/vegetable allergens and a bean ,-amylase inhibitor were used as queries. Both methods were also evaluated for their ability to detect a putative nonallergenic test protein containing a sequence derived from Ara h 1. FASTA versions 3.3t0 and 3.4t25 were utilized. Data indicate a conventional FASTA analysis produced fewer false positives and equivalent false negative rates. Conventional FASTA versus sliding window derived E scores were generally more significant. Results suggest a conventional FASTA search provides more relevant identity to the query protein and better reflects the functional similarities between proteins. It is recommended that the conventional FASTA analysis be conducted to compare identities of proteins to allergens. [source] Comparative proteomic and transcriptional profiling of a bread wheat cultivar and its derived transgenic line overexpressing a low molecular weight glutenin subunit gene in the endospermPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2008Federico Scossa Abstract We carried out a parallel transcriptional and proteomic comparison of seeds from a transformed bread wheat line that overexpresses a transgenic low molecular weight glutenin subunit gene relative to the corresponding nontransformed genotype. Proteomic analyses showed that, during seed development, several classes of endosperm proteins were differentially accumulated in the transformed endosperm. As a result of the strong increase in the amount of the transgenic protein, the endogenous glutenin subunit, all subclasses of gliadins, and metabolic as well as chloroform/methanol soluble proteins were diminished in the transgenic genotype. The differential accumulation detected by proteomic analyses, both in mature and developing seeds, was paralleled by the corresponding changes in transcript levels detected by microarray experiments. Our results suggest that the most evident effect of the strong overexpression of the transgenic glutenin gene consists in a global compensatory response involving a significant decrease in the amounts of polypeptides belonging to the prolamin superfamily. It is likely that such compensation is a consequence of the diversion of amino acid reserves and translation machinery to the synthesis of the transgenic glutenin subunit. [source] Sustained delivery of therapeutic concentrations of human clotting factor IX , a comparison of adenoviral and AAV vectors administered in uteroTHE JOURNAL OF GENE MEDICINE, Issue 1 2002Holm Schneider Abstract Background Prenatal somatic gene therapy has been considered for genetic disorders presenting with morbidity at birth. Haemophilia is associated with an increased risk of catastrophic perinatal bleeding complications such as intracranial haemorrhage, which could be prevented by gene transfer in utero. Prenatal gene therapy may be more promising than postnatal treatment, as the fetus may be more amenable to uptake and integration of therapeutic DNA and the immaturity of its immune system may permit life-long immune tolerance of the transgenic protein, thus avoiding the dominant problem in haemophilia treatment, the formation of inhibitory antibodies. Methods Adenovirus serotype 5-derived or AAV serotype 2-derived vectors carrying human clotting factor IX (hfIX) cDNA or a reporter gene were administered intramuscularly, intraperitoneally or intravascularly to late-gestation mouse fetuses. Both vector types were evaluated with respect to the kinetics of hfIX delivery to the systemic circulation and possible immune responses against the vector or the transgene product. Results Mice treated in utero by intramuscular injection of an adenoviral vector carrying hfIX cDNA exhibited high-level gene expression at birth and therapeutic , albeit continuously decreasing , plasma concentrations of hfIX over the entire 6 months of the study. Adenoviral vector spread to multiple organs was detected by polymerase chain reaction (PCR). Intramuscular, intraperitoneal or intravascular application of AAV vectors carrying hfIX cDNA led to much lower plasma concentrations of hfIX shortly after birth, which appeared to decline during the first month of life but stabilized in some of the mice at detectable levels. No signs of immune responses were found, either against the different viral vectors or against hfIX. Conclusion This study demonstrates for the first time that sustained systemic delivery of a therapeutic protein can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy in utero and provides a basis for considering this concept as a preventive therapeutic strategy for haemophilia and perhaps also for other plasma protein deficiencies. Copyright © 2002 John Wiley & Sons, Ltd. [source] Gene therapy for diseases of the cornea , a reviewCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 2 2010Keryn A Williams PhD Abstract The cornea is particularly suited to gene therapy. The cornea is readily accessible, normally transparent, and is somewhat sequestrated from the general circulation and the systemic immune system. The principle of genetic therapy for the cornea is to use an appropriate vector system to transfer a gene to the cornea itself, or to the ocular environs, or systemically, so that a transgenic protein will be expressed that will modulate congenital or acquired disease. The protein may be structural such as a collagen, or functionally active such as an enzyme, cytokine or growth factor that may modulate a pathological process. Alternatively, gene expression may be silenced by the use of modalities such as antisense oligonucleotides. Interestingly, despite a very considerable amount of work in animal models, clinical translation directed to gene therapy of the human cornea has been minimal. This is in contrast to gene therapy for monogenic inherited diseases of the retina, where promising early results of clinical trials for Leber's congenital amaurosis have already been published and a number of other trials are ongoing. [source] A new inducible adenoviral expression system that responds to inflammatory stimuli in vivoTHE JOURNAL OF GENE MEDICINE, Issue 12 2006Gang Cai Abstract Background Gene transfer using inducible promoters, which control expression of transgenic proteins in response to physiological conditions, may have significant advantages. In this study, we tried to achieve an inducible adenoviral expression system for physiologically responsive gene therapy of autoimmune or inflammatory diseases. Methods A luciferase reporter vector with a hybrid promoter containing the human IL-1, enhancer region (,3690 to , 2720) and the human CIITA promoter IV (,399 to + 2) was constructed. A replication-deficient adenovirus was engineered with luciferase controlled by the IL1,/CIITApIV promoter (Ad-IL1,/CIITApIV-Luc). The reporter vector or adenovirus was transfected to C57Bl/6 myeloid dendritic cells (DCs), RAW264.7, and Hep G2 to study the in vitro characteristics of this hybrid promoter. An inflammation model was prepared by injecting lipopolysaccharide (LPS) into Balb/c mice intraperitoneally (i.p.), and infected with Ad-IL1,/CIITApIV-Luc or Ad-CMV-Luc to study the in vivo characteristics of the IL1,/CIITApIV promoter. Results The IL1,/CIITApIV hybrid promoter has pronounced promoter activity, broad-range responsiveness to cytokines or LPS, and can be rechallenged after first induction. In the inflammation model, IL1,/CIITApIV could drive hepatic luciferase expression increasedly rapidly after LPS challenge and in a LPS dose-dependent manner. Conclusions Using the IL1,/CIITApIV hybrid promoter in gene transfer vectors may make it possible to produce transgenic proteins in vivo in direct relationship with the intensity and duration of an individual's status. By providing endogenously controlled production of transgenic proteins, this approach might limit the severity of autoimmune or inflammatory response without interfering with the beneficial components of host defense and immunity. Copyright © 2006 John Wiley & Sons, Ltd. [source] Lentiviral gene delivery to CNS by spinal intrathecal administration to neonatal miceTHE JOURNAL OF GENE MEDICINE, Issue 4 2006Elena Fedorova Abstract Background Direct injection of lentivectors into the central nervous system (CNS) mostly results in localized parenchymal transgene expression. Intrathecal gene delivery into the spinal canal may produce a wider dissemination of the transgene and allow diffusion of secreted transgenic proteins throughout the cerebrospinal fluid (CSF). Herein, we analyze the distribution and expression of LacZ and SEAP transgenes following the intrathecal delivery of lentivectors into the spinal canal. Methods Four weeks after intrathecal injection into the spinal canal of newborn mice, the expression of the LacZ gene was assessed by histochemical staining and by in situ polymer chain reaction (PCR). Following the spinal infusion of a lentivector carrying the SEAP gene, levels of enzymatically active SEAP were measured in the CSF, blood serum, and in brain extracts. Results Intrathecal spinal canal delivery of lentivectors to newborn mice resulted in patchy, widely scattered areas of ,-gal expression mostly in the meninges. The transduction of the meningeal cells was confirmed by in situ PCR. Following the spinal infusion of a lentivector carrying the SEAP gene, sustained presence of the reporter protein was detected in the CSF, as well as in blood serum, and brain extracts. Conclusions These findings indicate that intrathecal injections of lentivectors can provide significant levels of transgene expression in the meninges. Unlike intracerebral injections of lentivectors, intrathecal gene delivery through the spinal canal appears to produce a wider diffusion of the transgene. This approach is less invasive and may be useful to address those neurological diseases that benefit from the ectopic expression of soluble factors impermeable to the blood-brain barrier. Copyright © 2006 John Wiley & Sons, Ltd. [source] | ||||||||||