Showing papers on "Transgene published in 1998"
TL;DR: In 1953 Medawar pointed out that survival of the genetically disparate (allogeneic) mammalian conceptus contradicts the laws of tissue transplantation and suppresses T cell activity and defends itself against rejection.
Abstract: In 1953 Medawar pointed out that survival of the genetically disparate (allogeneic) mammalian conceptus contradicts the laws of tissue transplantation. Rapid T cell-induced rejection of all allogeneic concepti occurred when pregnant mice were treated with a pharmacologic inhibitor of indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme expressed by trophoblasts and macrophages. Thus, by catabolizing tryptophan, the mammalian conceptus suppresses T cell activity and defends itself against rejection.
2,499 citations
TL;DR: The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host.
Abstract: In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3' long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.
1,962 citations
TL;DR: The life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone.
Abstract: An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.
1,510 citations
TL;DR: Results show that successful generation of MHC class II‐restricted, OVA‐specific αβTCR transgenic mice was dependent upon combining cDNA‐ and genomic DNA‐based constructs for expression of the respective α‐ and β‐chains of the TCR.
Abstract: We describe the generation of ovalbumin (OVA)-specific, MHC class II-restricted alpha beta T cell receptor (TCR) transgenic mice. Initial attempts at generating these transgenic mice utilized heterologous regulatory elements to drive the expression of cDNA genes encoding the separate alpha- and beta-chains of the TCR. Unexpectedly, T cells bearing the transgenic alpha beta TCR failed to emerge from the thymus in these mice, although the transgenes did modify endogenous TCR expression. However, subsequent modification of the approach which enabled expression of the TCR beta-chain under the control of its natural regulatory elements generated mice whose peripheral T cells expressed the transgenic TCR and were capable of antigen-dependent proliferation. These results show that successful generation of MHC class II-restricted, OVA-specific alpha beta TCR transgenic mice was dependent upon combining cDNA- and genomic DNA-based constructs for expression of the respective alpha- and beta-chains of the TCR.
1,462 citations
TL;DR: The development of AD-like pathology is substantially enhanced when a P51 mutation, which causes a modest increase in Aβ42(43), is introduced into Tg2576-derived mice, and both doubly and singly transgenic mice showed reduced spontaneous alternation performance in a “Y” maze before substantial Aβ deposition was apparent.
Abstract: Genetic causes of Alzheimer's disease (AD) include mutations in the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (P52) genes1. The mutant APPK670N,M67M transgenic line, Tg2576, shows markedly elevated amyloid β-protein (AP) levels at an early age and, by 9–12 months, develops extracellular AD-type Ap deposits in the cortex and hippocampus2. Mutant PS1 transgenic mice do not show abnormal pathology, but do display subtly elevated levels of the highly amyloidogenic 42- or 43-amino acid peptide Aβ342(43) (ref. 3). Here we demonstrate that the doubly transgenic progeny from a cross between line Tg2576 and a mutant PS1M46L transgenic line develop large numbers of fibrillar Aβ deposits in cerebral cortex and hippocampus far earlier than their singly transgenic Tg2576 litter-mates. In the period preceding overt Aβ deposition, the doubly transgenic mice show a selective 41% increase in Aβ42(43) in their brains. Thus, the development of AD-like pathology is substantially enhanced when a P51 mutation, which causes a modest increase in Aβ42(43), is introduced into Tg2576-derived mice. Remarkably, both doubly and singly transgenic mice showed reduced spontaneous alternation performance in a “Y” maze before substantial Aβ deposition was apparent. This suggests that some aspects of the behavioral phenotype in these mice may be related to an event that precedes plaque formation.
1,410 citations
TL;DR: It is found that a single injection of tamoxifen into pregnant mice induced Cre-mediated recombination within the embryonic central nervous system, thereby activating expression of a reporter gene.
1,279 citations
TL;DR: It is shown that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants.
Abstract: Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.
1,228 citations
TL;DR: Analysis of embryos carrying different combinations of these alleles revealed requirements for Fgf8 gene function during gastrulation, as well as cardiac, craniofacial, forebrain, midbrain and cerebellar development.
Abstract: We describe a strategy for generating an allelic series of mutations at a given locus that requires the production of only one targetted mouse line. The ‘allelogenic’ mouse line we produced carries a hypomorphic allele of Fgf8, which can be converted to a null allele by mating to ere transgenic animals. The hypomorphic allele can also be reverted to wild-type by mating the allelogenic mice to flp transgenic animals, thereby generating a mouse line suitable for Cre-induced tissue-specific knockout experiments. Analysis of embryos carrying different combinations of these alleles revealed requirements for Fgf8 gene function during gastrulation, as well as cardiac, craniofacial, forebrain, midbrain and cerebellar development.
1,046 citations
TL;DR: The finding that the VEGF promoter of nontransformed cells is strongly activated by the tumor microenvironment points to a need to analyze and understand stromal cell collaboration in tumor angiogenesis.
972 citations
TL;DR: Use of the loxCre system of site-specific recombination is described to generate transgenic mouse lines in which different numbers of a transgene are present at the same chromosomal location, thereby eliminating the contribution of position effects and allowing analysis of the effect of copy number alone on transGene silencing.
Abstract: In both plants1–3 and Drosophila melanogastei4,5. expression from a transgenic locus may be silenced when repeated trans-gene copies are arranged as a concatameric array. This repeat-induced gene silencing is frequently manifested as a decrease in the proportion of cells that express the transgene, resulting in a variegated pattern of expression. There is also some indication that, in transgenic mammals, the number of transgene copies within an array can exert a repressive influence on expression, with several mouse studies reporting a decrease in the level of expression per copy as copy number increases6–8. However, because these studies compare different sites of transgene integration as well as arrays with different numbers of copies, the expression levels observed may be subject to varying position effects as well as the influence of the multicopy array. Here we describe use of the loxCre system of site-specific recombination to generate transgenic mouse lines in which different numbers of a transgene are present at the same chromosomal location, thereby eliminating the contribution of position effects and allowing analysis of the effect of copy number alone on transgene silencing. Reduction in copy number results in a marked increase in expression of the transgene and is accompanied by decreased chromatin compaction and decreased methylation at the transgene locus. These findings establish that the presence of multiple homologous copies of a transgene within a concatameric array can have a repressive effect upon gene expression in mammalian systems.
927 citations
TL;DR: It is reported that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing.
Abstract: Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5′-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.
TL;DR: Precise DNA rearrangements and genetic switches can be efficiently generated in a straightforward manner using Cre recombinase and are likely to have a profound impact on developmental biology and the generation of useful animal models of human disease.
TL;DR: It is demonstrated that the P1/HC-Pro polyprotein encoded by tobacco etch virus functions as a suppressor of PTGS, which reveals that plant viruses can condition enhanced susceptibility within a host through interdiction of a potent defense response.
TL;DR: Chromosome analysis revealed that most metaphase‐arrested Rad51− cells carried isochromatid‐type breaks, indicating that Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.
Abstract: Yeast rad51 mutants are viable, but extremely sensitive to gamma-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells. We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci. Upon inhibition of the RAD51 transgene, Rad51- cells accumulated in the G2/M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51- cells carried isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.
TL;DR: Systemic, posttranscriptional silencing of transgenes in Nicotiana benthamiana was initiated in localized regions of the plant by introduction of transgene-homologous DNA fragments, including those without a promoter.
TL;DR: It is concluded that polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and that transgene expression in the nucleus is prevented by complexation with cationIC lipids but not with cATIONic polymers.
TL;DR: It is suggested that enteric glia play an essential role in maintaining the integrity of the bowel and suggest that their loss or dysfunction may contribute to the cellular mechanisms of inflammatory bowel disease.
TL;DR: PrP knockout mice expressing PrPs with amino-proximal deletions caused severe ataxia and neuronal death limited to the granular layer of the cerebellum as early as 1-3 months after birth, suggesting that these truncated PrPs may be nonfunctional and compete with some other molecule with a PrP-like function for a common ligand.
TL;DR: It is demonstrated that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas, and EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways.
Abstract: The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30%–50% of human gliobastoma multiforme (GBM). These mutations are associated usually with deletions of the INK4a–ARF locus, which encodes two gene products (p16INK4a and p19ARF) involved in cell-cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis, using avian retroviral vectors to transfer a mutant EGFR gene to glial precursors and astrocytes in transgenic mice expressing tv-a, a gene encoding the retrovirus receptor. TVA, under control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas. These lesions occur more frequently with gene transfer to mice expressing tv-a from the progenitor-specific nestin promoter than to mice expressing tv-a from the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, suggesting that tumors arise more efficiently from immature cells in the glial lineage. Furthermore, EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways. We have produced these combinations by simultaneously infecting tv-a transgenic mice with vectors carrying cdk4 and EGFR or by infecting tv-a transgenic mice bearing a disrupted INK4a–ARF locus with the EGFR-carrying vector alone. Moreover, EGFR-induced gliomagenesis does not occur in conjunction with p53 deficiency, unless the mice are also infected with a vector carrying cdk4. The gliomagenic combinations of genetic lesions required in mice are similar to those found in human gliomas.
TL;DR: It is demonstrated that overexpression of two MYB genes from Antirrhinum represses phenolic acid metabolism and lignin biosynthesis in transgenic tobacco plants.
Abstract: MYB-related transcription factors are known to regulate different branches of flavonoid metabolism in plants and are believed to play wider roles in the regulation of phenylpropanoid metabolism in general. Here, we demonstrate that overexpression of two MYB genes from Antirrhinum represses phenolic acid metabolism and lignin biosynthesis in transgenic tobacco plants. The inhibition of this branch of phenylpropanoid metabolism appears to be specific to AmMYB308 and AmMYB330, suggesting that they recognize their normal target genes in these transgenic plants. Experiments with yeast indicate that AmMYB308 can act as a very weak transcriptional activator so that overexpression may competitively inhibit the activity of stronger activators recognizing the same target motifs. The effects of the transcription factors on inhibition of phenolic acid metabolism resulted in complex modifications of the growth and development of the transgenic plants. The inhibition of monolignol production resulted in plants with at least 17% less lignin in their vascular tissue. This reduction is of importance when designing strategies for the genetic modification of woody crops.
TL;DR: These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector and aid in the development of nonviral gene delivery systems with increased efficiency.
Abstract: Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.
TL;DR: A review of transgene-induced silencing phenomena in plants and the involvement of RNA was hypothesized to explain post-transcriptional silencing in plants, fungi and nematodes.
Abstract: The recent development of gene transfer methods for almost all eukaryotes has revealed that transgenes can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene, thus limiting the potential application of genetic transformation. Despite this limitation, transgene-induced gene silencing events were considered originally as anecdotal phenomena. However, as more and more similarities were found between transgene-induced gene silencing and natural epigenetic phenomena, considerable interest has been devoted to this subject (for recent reviews see Depicker and Van Montagu, 1997; Stam et al., 1997b). Epigenetics is commonly defined as ‘the study of mitotically and/or meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence’ (Russo et al., 1996). For a long time, DNA was considered as the only target for epigenetic modifications. Epigenetic changes corresponding to changes in chromatin structure and affecting transcription have been reported in almost all eukaryotes: yeast, fungi, Drosophila, plants and mammals (Dorer, 1997; Foss and Selker, 1991; Rossignol and Faugeron, 1994; Ye and Signer, 1996). However, recent studies have suggested that, besides DNA, other molecules can be modified in a manner that resembles epigenetic DNA changes. First, it was shown that proteins can be converted into molecules of aberrant conformation called prions in yeast and mammals (Lacroute, 1971; Prusiner, 1982). More recently, the involvement of RNA was hypothesized to explain post-transcriptional silencing in plants, fungi and nematodes (Cogoni et al., 1996; Fire et al., 1998; Napoli et al., 1990). This review will focus on transgene-induced silencing phenomena in plants. The number of copies of a transgene that integrate into the genome of a transformed plant and
TL;DR: A mouse embryonic stem (ES) cell lines expressing EGFP is established, which can be propagated in culture, reintroduced into mice, or induced to differentiate in vitro, while still maintaining ubiquitous EGFP expression.
TL;DR: The genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector is reported.
Abstract: Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000–10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.
TL;DR: It is indicated that vector-mediated transduction of dendritic cells is necessary for cellular immune responses to muscle gene therapy, a step which AAV avoids, providing a useful biological niche for its use in gene therapy.
Abstract: Immune responses to vector-corrected cells have limited the application of gene therapy for treatment of chronic disorders such as inherited deficiency states. We have found that recombinant adeno-associated virus (AAV) efficiently transduces muscle fibers in vivo without activation of cellular and humoral immunity to neoantigenic transgene products such as beta-galactosidase, which differs from the experience with recombinant adenovirus, where vibrant T-cell responses to the transgene product destroy the targeted muscle fibers. T cells activated following intramuscular administration of adenovirus expressing lacZ (AdlacZ) can destroy AAVlacZ-transduced muscle fibers, indicating a prior state of immunologic nonresponsiveness in the context of AAV gene therapy. Adoptive transfer of dendritic cells infected with AdlacZ leads to immune mediated elimination of AAVlacZ-transduced muscle fibers. AAVlacZ-transduced antigen-presenting cells fail to demonstrate beta-galactosidase activity and are unable to elicit transgene immunity in adoptive transfer experiments. These studies indicate that vector-mediated transduction of dendritic cells is necessary for cellular immune responses to muscle gene therapy, a step which AAV avoids, providing a useful biological niche for its use in gene therapy.
Patent•
19 Jun 1998TL;DR: In this paper, the authors proposed a method for modifying endogenous gene expression in a cell, tissue, or organ of a transgenic organism, in particular a transgene animal or plant.
Abstract: The present invention relates generally to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention provides novel synthetic genes and genetic constructs which are capable of repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto.
TL;DR: The generation of Tg mice expressing selected HIV-1 gene(s) revealed that nef harbors a major disease determinant, suggesting that Nef may play a critical role in human AIDS, independently of its role in virus replication.
TL;DR: Stratified epithelium contains a mitotically active basal layer of cells that cease proliferating, then migrate outwards and undergo terminal differentiation, and NF-κB activation in this tissue, in contrast to its role in other settings, is important for cellular growth inhibition.
Abstract: Stratified epithelium contains a mitotically active basal layer of cells that cease proliferating, then migrate outwards and undergo terminal differentiation. The control of this process, which is abnormal in cutaneous neoplasia and inflammation, is not well understood. In normal epidermis, NF-κB proteins were found to exist in the cytoplasm of basal cells and then to localize in the nuclei of suprabasal cells, suggesting a role for NF-κB in the switch from proliferation to growth arrest and differentiation. Functional blockade of NF-κB by expressing dominant-negative NF-κB inhibitory proteins in transgenic murine and human epidermis produced hyperplastic epithelium in vivo. Consistent with this, application of a pharmacologic inhibitor of NF-κB to intact skin induced epidermal hyperplasia. In contrast, overexpression of active p50 and p65 NF-κB subunits in transgenic epithelium produced hypoplasia and growth inhibition. These data suggest that spatially restricted NF-κB activation occurs in stratified epithelium and indicate that NF-κB activation in this tissue, in contrast to its role in other settings, is important for cellular growth inhibition.
TL;DR: Stress-induced overproduction of the P5CS enzyme and proline accumulation in transgenic rice plants showed an increase in biomass under salt-stress and water-stress conditions as compared to the non-transformed control plants.
TL;DR: HD-leptin delivery was associated with a significant improvement in associated safety/toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss in normal lean and ob/ob mice.
Abstract: Adenoviral (Ad)-mediated in vivo gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and/or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinase-based system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation E1-deleted Ad vector (Ad-leptin) in normal lean and ob/ob (leptin-deficient) mice. In contrast to evidence of liver toxicity, inflammation, and cellular infiltration observed with Ad-leptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety/toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications.