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Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation.

BACKGROUND: Parthenocarpy, or fruit development in the absence of fertilization, has been genetically engineered in eggplant and in other horticultural species by using the DefH9-iaaM gene. The iaaM gene codes for tryptophan monoxygenase and confers auxin synthesis, while the DefH9 controlling regions drive expression of the gene specifically in the ovules and placenta. A previous greenhouse trial for winter production of genetically engineered (GM) parthenocarpic eggplants demonstrated a significant increase (an average of 33% increase) in fruit production concomitant with a reduction in cultivation costs. RESULTS: GM parthenocarpic eggplants have been evaluated in three field trials. Two greenhouse spring trials have shown that these plants outyielded the corresponding untransformed genotypes, while a summer trial has shown that improved fruit productivity in GM eggplants can also be achieved in open field cultivation. Since the fruits were always seedless, the quality of GM eggplant fruits was improved as well. RT-PCR analysis demonstrated that the DefH9-iaaM gene is expressed during late stages of fruit development. CONCLUSIONS: The DefH9-iaaM parthenocarpic gene is a biotechnological tool that enhances the agronomic value of all eggplant genotypes tested. The main advantages of DefH9-iaaM eggplants are: i) improved fruit productivity (at least 30-35%) under both greenhouse and open field cultivation; ii) production of good quality (marketable) fruits during different types of cultivation; iii) seedless fruit with improved quality. Such advantages have been achieved without the use of either male or female sterility genes.

Food Industry↗

Delivery of neuroactive compounds to the brain: potential utility of genetically modified cells.

Several methods for chronic delivery of compounds to the central nervous system (CNS) now exist. Peripheral drug administration is generally safest, but not always effective. If direct CNS delivery of a substance is required, then CNS implantation of drug-delivery systems or grafting of various cell types to the brain can be performed, although none of these interventions are yet of consistent, proven benefit in Alzheimer's disease and other neurodegenerative disorders. Grafting of genetically modified cells to the brain may be an alternative delivery system of some substances to the CNS.

Brain Diseases↗

Estimation of the minimum uncertainty of DNA concentration in a genetically modified maize sample candidate certified reference material.

Homogeneity testing and the determination of minimum sample mass are an important part of the certification of reference materials. The smallest theoretically achievable uncertainty of certified concentration values is limited by the concentration distribution of analyte in the different particle size fractions of powdered biological samples. This might be of special importance if the reference material is prepared by dry mixing, a dilution technique which is used for the production of the new and third generation of genetically modified (GMO) plant certified reference materials. For the production of dry mixed PMON 810 maize reference material a computer program was developed to calculate the theoretically smallest uncertainty for a selected sample intake. This model was used to compare three differently milled maize samples, and the effect of dilution on the uncertainty of the DNA content of GMO maize was estimated as well. In the case of a 50-mg sample mass the lowest achievable standard deviation was 2% for the sample containing 0.1% GMO and the minimum deviation was less than 0.5% for the sample containing 5% GMO.

DNA↗

Variability in the benzodiazepine response of serotonin 5-HT1A receptor null mice displaying anxiety-like phenotype: evidence for genetic modifiers in the 5-HT-mediated regulation of GABA(A) receptors.

Benzodiazepines (BZs) acting as modulators of GABA(A) receptors (GABA(A)Rs) are an important group of drugs for the treatment of anxiety disorders. However, a large inter-individual variation in BZ sensitivity occurs in the human population with some anxiety disorder patients exhibiting diminished sensitivity to BZ and reduced density of GABA(A)Rs. The mechanism underlying BZ treatment resistance is not known, and it is not possible to predict whether an anxiety patient will respond to BZ. 5-hydroxytryptamine1A receptor (5-HT1AR) null mice (R-/-) on the Swiss-Webster (SW) background reproduce several features of BZ-resistant anxiety; they exhibit anxiety-related behaviors, do not respond to BZ, have reduced BZ binding, and have decreased expression of the major GABA(A)R subunits alpha1 and alpha2. Here, we show that R-/- mice on the C57Bl6 (B6) background also have anxiety phenotype, but they respond to BZ and have normal GABA(A)R subunit expression. This indicates that the 5-HT1AR-mediated regulation of GABA(A)R alpha subunit expression is subject to genetic modification. Hybrid SW/B6-R-/- mice also exhibit BZ-resistant anxiety, suggesting that SW mice carry a genetic modifier, which mediates the effect of the 5-HT1AR on the expression of GABA(A)Ralpha subunits. In addition, we show that this genetic interaction in SW mice operates early in postnatal life to influence the expression of GABA(A)R alpha subunits at the transcriptional level. These data indicate that BZ-resistant anxiety results from a developmental arrest of GABA(A)R expression in SW-R-/- mice, and a similar mechanism may be responsible for the BZ insensitivity of some anxiety patients.

Amygdala↗

Extension of optic nerve fibers on genetically modified cells producing brain-derived neurotrophic factor.

Since brain-derived neurotrophic factor (BDNF) promotes the survival of retinal ganglion cells (RGCs), we transfected a rat BDNF cDNA into rat fibroblasts, and retinal fragments of rat embryos were cultured on cell monolayers of these cells. Retinal fragments showed enhanced neurite extension on BDNF-transfected cells compared with that on control cells. The degree of the neurite extension, however, decreased depending upon the embryonic stages. These results suggest that fibroblasts genetically modified to produce BDNF might be a promoter of neurite extension by RGCs, but this does not apply to the RGCs of late embryonic stages.

Animals↗

Nonmyeloablative conditioning followed by transplantation of genetically modified HLA-matched peripheral blood progenitor cells for hematologic malignancies in patients with acquired immunodeficiency syndrome.

To assess the safety and efficacy of nonmyeloablative allogeneic transplantation in patients with HIV infection, a clinical protocol was initiated in patients with refractory hematologic malignancies and concomitant HIV infection. The results from the first 2 patients are reported. The indications for transplantation were treatment-related acute myelogenous leukemia and primary refractory Hodgkin disease in patients 1 and 2, respectively. Only patient 1 received genetically modified cells. Both patients tolerated the procedure well with minimal toxicity, and complete remissions were achieved in both patients, but patient 2 died of relapsed Hodgkin disease 12 months after transplantation. Patient 1 continues in complete remission with undetectable HIV levels and rising CD4 counts, and with both the therapeutic and control gene transfer vectors remaining detectable at low levels more than 2 years after transplantation. These results suggest that nonmyeloablative allogeneic transplantation in the context of highly active antiretroviral therapy is feasible in patients with treatment-sensitive HIV infection.

Acquired Immunodeficiency Syndrome↗

The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen.

The T-helper 1 (Th1) immune reaction is most important in dendritic cell (DC)-based immunotherapy. Interleukin 12 (IL-12) and granulocyte macrophage colony-stimulating factor (GM-CSF) play a pivotal role in inducing Th1 and cytotoxic T lymphocyte (CTL) responses. In this study, DCs expressing the natural tumor antigen gp70 of BALB/c-derived CT26 were adenovirally transduced with the IL-12 gene and/or GM-CSF gene, and it was examined whether vaccinations using these genetically engineered DCs can induce strong therapeutic antitumor immunity. Mice were immunized once by subcutaneous (s.c.) injection with genetically modified DCs. The cytotoxic activity of splenocytes against CT26 was assayed in a 51Cr-release assay 14 days after immunization. The therapeutic efficacy of the vaccination was examined in s.c. tumor models. The cytotoxic activity of CTLs against CT26 in mice immunized with DCs expressing gp70 (DC-AxCAgp70) was significantly augmented by co-transduction with the GM-CSF/IL-12 gene (p<0.0001) and remarkably reduced by the depletion of CD4+ or CD8+ cells (p<0.01). The cytotoxic activity against CT26 of the plain spleen cells in mice immunized with DC-AxCAgp70/GM-CSF/IL-12 was significantly higher than that in mice immunized with DC-AxCAgp70 (p<0.0001), and this activity decreased to almost 50% upon the depletion of NK cells. Vaccinations using DC-AxCAgp70/GM-CSF/IL-12 or DC-AxCAgp70/IL-12 could elicit potent therapeutic immunity in s.c. tumor models; tumor-free mice were observed in these vaccination groups. However, there was no significant difference between these two groups. A vaccination therapy using DCs co-transduced with the TAA gene and Th 1-type cytokine genes, especially the IL-12 gene, is ideal for immunotherapy in terms of the activation of DCs, NK cells, CD4+ T cells and CD8+ T cells, and may be useful in the clinical application of a cancer vaccine therapy.

Adenoviridae↗

Insulin-like growth factor-II/mannose 6 phosphate receptors facilitate the matrix effects of latent transforming growth factor-beta1 released from genetically modified keratinocytes in a fibroblast/keratinocyte co-culture system.

This study was conducted to explore the mechanism of activation of transforming growth factor-beta1 (TGF-beta1) which is critical to its role in many physiological and pathological conditions. To date, almost all reports concerning TGF-beta1 activation delineated that release of mature TGF-beta1 from latency associated protein (LAP) is required for its activation. We report that latent TGF-beta1 (LTGF-beta1) released from TGF-beta1 genetically modified keratinocytes grown in the top chamber of a co-culture system functions as a fibrogenic factor through interaction with insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors of human dermal fibroblasts grown in the lower chamber of this system. Following successful transduction, the pLin-LTGF-beta1 vector was amplified in PA31 7 packaging cells which possess viral structural proteins for vector in the presence of neomycin. Conditioned medium derived from packaging cells containing competent viral particles was then used to transduce either keratinocytes or fibroblasts grown in the upper chamber of a co-culture system, in which a 0.4 microm porous membrane separates the two chambers. In this way, LTGF-beta1 produced by transduced cells in the upper chamber is released and diffuses into the lower chambers where dermal fibroblasts are grown. Conditioned medium from the lower chamber was removed 3 days later and used to evaluate the latency and bioactivity of TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) and mink lung (Mv1 Lu) epithelial growth inhibition assay. Cells were also harvested and used for RNA extraction. The results of these experiments showed that 1) the TGF-beta1-LAP complex, which was latent in traditionally used mink lung growth inhibition assay, directly modulated the expression of collagenase, type I, and type III collagen mRNA by dermal fibroblasts; 2) this stimulation was inhibited by M6P in a dose-dependent manner; 3) the TGF-beta1-LAP inhibits Mv1Lu epithelial cells only when this complex was incubated with cell membranes isolated from dermal fibroblasts; and 4) LTGF-beta1 activation seems to occur through a conformational alteration rather than by release of the mature TGF-beta1 from LAP in our co-cultured system. This conformational alteration seems to occur through the interaction of the TGF-beta1-LAP complex with the IGF-II/M6P receptors. Thus, the quantity of IGF-II/M6P receptors is important in cellular response to LTGF-beta1 in any physiological and pathological conditions.

Autocrine Communication↗

Expression of a novel gene product by transplants of genetically modified primary fibroblasts in the central nervous system.

Primary fibroblasts initiated from skin biopsies of Wistar rats were transfected with a plasmid that encodes the human growth hormone and the neomycin resistance genes. Cell clones selected for G418 resistance and expressing high levels of human growth hormone were propagated in vitro and subsequently transplanted into the cerebral cortex of adult allogeneic rats. Grafts were examined by immunocytochemistry at weekly intervals up to 2 months. Fibroblasts in the transplants survived and expressed growth hormone clearly up to 4 weeks, but showed reduced expression at 6 to 8 weeks. Significant levels of human growth hormone were also detected by radioimmunoassay in the serum of the host rats up to 1 month. The experiments demonstrate that primary fibroblasts can be genetically modified to deliver a new gene product into the central nervous system, and the gene product can pass the blood-brain barrier to enter the systemic circulation. This model illustrates the potential to introduce desired products into the brain through the genetic alteration of autologous primary fibroblasts.

Animals↗

Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye.

Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population. Laser photocoagulation, photodynamic therapy and excision of neovascular membranes have met with limited success. Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damaged retinal pigment epithelium following surgical removal of the membranes. We tested our hypothesis that the subretinal transplantation of genetically modified autologous IPE cells expressing biological therapeutics might be a promising strategy for the treatment of ARMD and other retinal disorders. Pigment epithelium-derived factor (PEDF) has strong antiangiogenic and neuroprotective activities in the eye. Subretinal transplantation of PEDF expressing IPE cells inhibited pathological choroidal neovascularization in rat models of laser-induced rupture of Bruch's membrane and of oxygen induced ischemic retinopathy. PEDF expressing IPE transplants also increased the survival and preserved rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration. These findings suggest a promising concept for the treatment of ARMD and other retinal disorders.

Adenoviridae↗

Grafting genetically modified cells to the brain: possibilities for the future.

Diagnostic and therapeutic approaches to disorders of the central nervous system (CNS) are particularly difficult to develop because of the relative inaccessibility of the mammalian brain to study and chemical treatment, the complexity and interconnectedness of CNS subsystems, and the profound and continued lack of fundamental understanding of the relationship between structure and function in the CNS. Neural grafting in the CNS has recently suggested a potential approach to CNS therapy through the selective replacement of cells lost as a result of disease or damage. Independently, studies aimed at direct genetic therapy in model systems have recently begun to suggest conceptually new approaches to the treatment of several kinds of human genetic disease, especially those caused by single-gene enzyme deficiencies. We suggest that a combination of these two approaches, namely the grafting into the CNS of genetically modified cells, may provide a new approach toward the restoration of some functions in the damaged or diseased CNS. We present evidence for the feasibility of this approach, including a description of some current techniques for mammalian cell gene transfer and CNS grafting, and several possible approaches to clinical applications.

Brain↗

Genetically modified human embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson's disease.

Embryonic stem (ES) cells have great potential as a cell source for cell replacement therapy. To investigate the possibility of using ES cells as a carrier of therapeutic gene(s), human ES cells (MB03) were co-transfected with cDNAs coding for tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GTPCH I), then bulk-selected in the presence of neomycin and hygromycin-B. Successful transfection was confirmed by Western immunoblotting and RT-PCR. The genetically modified ES cells (bk-THGC) were found to produce a significant amount of L-dopa spontaneously and relieved apomorphine-induced asymmetric motor behavior by approximately 54% when grafted into striatum of 6-OHDA-denervated rat brain. The number of rotations, however, increased up to 176+/-18% in 6 weeks when PBS was used instead (sham-graft). Immunohistochemical stainings revealed that the grafted human ES cells survived and expressed TH for at least 6 weeks while the experiment was continued.

Animals↗

AHSP and beta-thalassemia: a possible genetic modifier.

The identification of defectives genes underlying inherited diseases has made it clear that patients with the same genotype can have variable clinical expression. Suggestions proposing that the protein AHSP, a alpha-globin specific chaperone could influence disease severity in patients with beta-thalassemia, an inherited disorder characterized by a quantitative deficiency of beta-globin genes. This article presents a review of the AHSP gene structure, function and expression. A discussion of the AHSP gene acknowledgements is presented with an overview of the possible genetic modifier function of AHSP on beta-thalassemia pathophysiology.

Blood Proteins↗

Disappearance of lysosomal storage in spleen and liver of mucopolysaccharidosis VII mice after transplantation of genetically modified bone marrow cells.

Mice homozygous for the gusmps allele lack beta-glucuronidase activity and provide a useful model for human Mucopolysaccharidosis type VII (MPS VII), also known as Sly syndrome. Bone marrow (BM) transplantation was shown to correct the metabolic defect and to increase the life span of diseased animals. We have used this murine model in a preclinical study aimed at evaluating whether the techniques currently available for gene transfer into large mammalian and human BM cells will provide efficient enzyme replacement therapy in MPS patients. Autologous BM was transplanted into deficient mice after retrovirus-mediated transfer of the human beta-glucuronidase cDNA. Conditioning of recipients was performed by a single sublethal irradiation of 4.5 Gy, giving rise to low donor engraftment. In recipient mice analyzed until 145 days after gene transfer, the percentage of genetically modified hematopoietic cells was less than 5%. Nevertheless, beta-glucuronidase enzyme activity was detectable in various organs, including the brain, and disappearance of lysosomal storage was obvious in the liver and spleen. These results show that the autologous transplantation of genetically engineered BM cells could be beneficial in MPS patients.

Animals↗

Induction of tolerance to self-antigens using genetically modified bone marrow cells.

The challenge of finding a lasting cure for autoimmune disease(s) has not been met. Although the use of systemic anti-inflammatory agents still dominates the treatment of these diseases, there is a push towards developing novel and more specific strategies. In addressing autoimmunity, there is the intrinsic need to understand the mechanisms that lead to the development and maintenance of immunological tolerance to self-antigens. Experimental evidence has shown that directed antigen expression in the thymus can induce immunological tolerance to that antigen. This forms the cornerstone of one strategy directed towards the cure of autoimmunity. In this strategy, individuals with autoimmune disease are transplanted with bone marrow stem cells that have been genetically modified and in this way allow expression of the self-antigen in the thymus.

Animals↗

Successful reconstitution of human hematopoiesis in the SCID-hu mouse by genetically modified, highly enriched progenitors isolated from fetal liver.

Highly purified CD34++CD38-Lin- hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli beta-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the beta-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38-Lin- cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.

Animals↗

Grafting of genetically modified human fetal fibroblasts to produce human butyrylcholinesterase in mice.

Human diploid fibroblast cultures were established from fetal skin tissue. Enzymic dissociation yielded cultures of higher growth capacity of fibroblasts than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. Transfer of recombinant human butyrylcholinesterase (BChE, EC 3.1.1.8) gene into primary human fibroblasts was achieved successfully using lipofection and retrovirus-mediated transfection. The analysis of drug-resistant colonies suggested the presence of the transcripted BChE mRNA in the cytoplasm of transfected cells. The secreted BChE protein in culture medium was assayed for enzyme activity using butyrylthiocholine as substrate. The genetically modified fibroblasts were mixed with rat tail collagen and transplanted subcutaneously and intraperitoneally to mice. Immunoreactive human BChE appeared in the plasma from the transplanted mice. reaching the top level at day 13. It was not present any longer in most of the mice 20 days later.

Animals↗