Prolonged liver-specific transgene expression by a non-primate lentiviral vector

Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease.     

Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions

Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks, mediated by the accumulation of Cry1B and Cry1Aa toxin, respectively, which was comparable with that afforded by the spraying of chemical insecticides on control plants. The wound-induced A9.1 line exhibited a satisfactory level of protection, with a notably low level of penetration of SSB larvae in the stems, but higher external symptoms than constitutive lines, probably due to the time lag to benefit from the protective effect of Cry1B.     

Enhanced protection against fatal mycobacterial infection in SCID beige mice by reshaping innate immunity with IFN-gamma transgene.

Humans with immune-compromised conditions such as SCID are unable to control infection caused by normally nonpathogenic intracellular pathogens such as Mycobacterium bovis bacillus Calmette-Guérin. We found that SCID beige mice lacking both lymphocytes and NK cells had functionally normal lung macrophages and yet a selectively impaired response of type 1 cytokines IFN-gamma and IL-12, but not TNF-alpha, during M. bovis bacillus Calmette-Guérin infection. These mice succumbed to such infection. A repeated lung gene transfer strategy was designed to reconstitute IFN-gamma in the lung, which allowed investigation of whether adequate activation of innate macrophages could enhance host defense in the complete absence of lymphocytes. IFN-gamma transgene-based treatment was initiated 10 days after the establishment of mycobacterial infection and led to increased levels of both IFN-gamma and IL-12, but not TNF-alpha, in the lung. Lung macrophages were activated to express increased MHC molecules, type 1 cytokines and NO, and increased phagocytic and mycobactericidal activities. Activation of innate immunity markedly inhibited otherwise uncontrollable growth of mycobacteria and prolonged the survival of infected SCID hosts. Thus, our study proposes a cytokine transgene-based therapeutic modality to enhance host defense in immune-compromised hosts against intracellular bacterial infection, and suggests a central effector activity played by IFN-gamma-activated macrophages in antimycobacterial cell-mediated immunity.     

Potential protection of skin by acute UVA irradiation--from cellular to animal models

The UVA (320-380 nm) component of sunlight has oxidizing properties which may be deleterious to skin cells and tissue but can also lead to the strong up-regulation of the heme-catabolizing enzyme, heme oxygenase-1. This enzyme has well-established antioxidant actions in cells as well as anti-inflammatory properties in mammals. There is also evidence from rodent models that this enzyme is responsible for the UVA-mediated protection against UVB-induced immunosuppression that occurs in skin. The relevance of these findings to acute and chronic effects of sunlight including skin carcinogenesis is currently under investigation as are the potential implications for sunlight protection in humans.     

Epitope-dependent inhibition of T cell activation by the Ea transgene: an explanation for transgene-mediated protection from murine lupus

A high level expression of the Ea(d) transgene encoding the I-E alpha-chain is highly effective in the suppression of lupus autoantibody production in mice. To explore the possible modulation of the Ag-presenting capacity of B cells as a result of the transgene expression, we assessed the ability of the transgenic B cells to activate Ag-specific T cells in vitro. By using four different model Ag-MHC class II combinations, this analysis revealed that a high transgene expression in B cells markedly inhibits the activation of T cells in an epitope-dependent manner, without modulation of the I-E expression. The transgene-mediated suppression of T cell responses is likely to be related to the relative affinity of peptides derived from transgenic I-E alpha-chains (Ealpha peptides) vs antigenic peptides to individual class II molecules. Our results support a model of autoimmunity prevention based on competition for Ag presentation, in which the generation of large amounts of Ealpha peptides with high affinity to I-A molecules decreases the use of I-A for presentation of pathogenic self-peptides by B cells, thereby preventing excessive activation of autoreactive T and B cells.     

UV-induced skin cancer in a hairless mouse model

Ultraviolet (UV) radiation is a very common carcinogen in our environment, but epidemiological data on the relationship between skin cancers and ambient solar UV radiation are very restricted. In hairless mice the process of UV carcinogenesis can be studied in depth. Experiments with this animal model have yielded quantitative data on how tumor development depends on dose, time and wavelength of the UV radiation. In combination with epidemiological data, these experimental results can be transposed to humans. Comparative studies on molecular, cellular and physiological changes in mouse and man can further our fundamental understanding of UV carcinogenesis in man. This is likely to improve risk assessments such as those related to a stratospheric ozone depletion, and to yield well-targeted intervention schemes, e.g. prescribing a specific drug or diet, for high-risk individuals.     

通过转基因改善家畜奶的品质

通过大量的研究报道,综述了转基因技术在改善家畜奶品质方面的研究概况,如提高奶中κ,αsl-,αs2-和β－酪蛋白含量,增加溶菌酶含量,生产人乳铁蛋白和乳白蛋白,降低乳糖含量,使其品质更接近人奶。     

The induction of skeletal muscle hypertrophy by a ski transgene is promoter-dependent

The chicken c-ski gene expresses at least three alternatively spliced messages. Transgenic mice expressing proteins from cDNA corresponding to two of these messages (FB27 and FB29) under the control of a murine sarcoma virus (MSV) long terminal repeat (LTR) express the transgene in skeletal muscle and develop a muscular phenotype. Both a biologically active form of c-ski and the MSV LTR are required for the development of the muscular phenotype. The normal c-ski gene linked to two other tissue-specific promoters failed to induce muscle growth in transgenic mice, as did an inactive mutant of c-ski expressed under the control of the MSV LTR.     

A new opaque variant of maize by a single dominant RNA-interference-inducing transgene

In maize, alpha-zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD alpha-zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants.     

Bacteriorhodopsin Is a Powerful Light-Driven Proton Pump

The activity of bacteriorhodopsin was investigated with Halobacterium halobium cell envelopes, which lack cytoplasmic constituents. It was found that the physiological concentration of magnesium ion greatly enhanced the light-induced pH change; under optimal conditions, the pH change of the external medium was as large as 3.5 pH units, even though the volume fraction of the envelope vesicles was as low as 0.01. This pH change is about three times larger than the largest change reported thus far. This same effect was observed with transition metal ions, but not with other alkaline divalent cations. That is, divalent cations that formed hydroxides below pH 10 were effective in enhancing the light-induced pH change. This result suggests that some divalent cations acted as buffers against a large increase in the internal pH, and that the internal pH was an important factor in determining the activity of bacteriorhodopsin. It was also shown that a high level of the proton-pump activity was maintained in a wide range of external pHs, at least between 4.5 and 9.4.