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  • 1
    Electronic Resource
    Electronic Resource
    Stable Long Term Germ-Line Transmission of Transgene Integration Sites in Mice (1999)
    Springer
    Transgenic research 8 (1999), S. 1-8 
    Details
    ISSN: 1573-9368
    Keywords: gene construct ; gene transfer ; heritability ; marker gene ; pigmentation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008824028100
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  • 2
    Electronic Resource
    Electronic Resource
    Gene transfer into canine myoblasts (1999)
    Springer
    Cytotechnology 30 (1999), S. 181-189 
    Details
    ISSN: 1573-0778
    Keywords: culture ; dog ; Duchenne dystrophy ; gene transfer ; satellite cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have developed and characterized cultures of healthy and dystrophic canine myoblasts for the evaluation of various gene transfer protocols. The number of desmin-positive myoblasts was elevated (〉〉80%) in cultures of myoblasts obtained from different muscle territories, the diaphragm muscle giving rise to the purest cultures. Myoblasts from dogs turned out to be a very convenient source of well transfectable and transducible cells. Transfection with plasmid DNA allowed efficient transgene expression (50% of β-galactosidase positive cells and about 375 ng luciferase/mg protein after transfection with a calcium phosphate-precipitated plasmid). Infection with high concentrations of adenoviral and retroviral vectors allowed transgene (β-galactosidase or mini-dystrophin) detection in about 75 to 90% of the canine cells. Therefore, primary dog myoblast cultures represent a useful in vitro model for viral and non-viral gene delivery, as well as for functional evaluation and cell grafting with applications in genetic diseases, vaccination or production of circulating therapeutic proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008026913715
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  • 3
    Electronic Resource
    Electronic Resource
    Adenovirus-mediated Gene Transfer of a Truncated Form of Fibroblast Growth Factor Receptor Inhibits Growth of Glioma Cells both In Vitro and In Vivo (1999)
    Springer
    Journal of neuro-oncology 44 (1999), S. 195-203 
    Details
    ISSN: 1573-7373
    Keywords: adenovirus ; dominant negative ; fibroblast growth factor receptor ; gene transfer ; glioma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Basic fibroblast growth factor (FGF-2) and high affinity FGF receptor (FGFR) have been detected in the nucleus as well as the cytoplasm of many human gliomas, and are known to stimulate cellular proliferation and angiogenesis in the tumors. To investigate the effects of inactivation of FGFR on the growth of malignant gliomas, we constructed a replication-deficient recombinant adenovirus vector encoding a truncated form of chicken FGFR1 (AxCA Δ FR). AxCA Δ FR-infected cells were confirmed to express truncated FGFR protein by immunoblotting and FGF-2-dependent clonogenicity of NIH3T3 cells was suppressed by infection with this virus vector. Then human malignant glioma cell lines U-251MG and T98G, both of which have been reported to express FGF-2 and FGFR, were infected with AxCA Δ FR. These infected cells showed nuclear as well as cytoplasmic expression of a truncated FGFR protein. Proliferation rate and the ability to form colonies in soft agar of the cells infected with this virus vector were significantly suppressed compared with those of uninfected and lacZ-expressing adenovirus-infected cells. Moreover, intratumoral injection of AxCA Δ FR significantly suppressed the subcutaneous tumor growth of the glioma cells in nude mice. We concluded that inactivation of the cytoplasmic and nuclear FGFR using this truncated FGFR-expressing adenovirus vector can inhibit the growth of malignant gliomas both in vitro and in vivo.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006355014351
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  • 4
    Electronic Resource
    Electronic Resource
    Glycotargeting: Influence of the Sugar Moiety on Both the Uptake and the Intracellular Trafficking of Nucleic Acid Carried by Glycosylated Polymers (1999)
    Springer
    Bioscience reports 19 (1999), S. 125-132 
    Details
    ISSN: 1573-4935
    Keywords: Oligonucleotides ; gene transfer ; routing ; membrane lectins ; glycoconjugates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Nucleic acids (plasmids as well as oligonucleotides) used to specifically express or modulate the expression of a gene, must reach the cytosol and/or the nucleus. Several systems have been developed to increase their uptake and their efficiency. Glycosylated polylysines have been shown to specifically help nucleic acids to be taken up in cells expressing a given cell surface membrane lectin. However, it appeared that the efficiency of the imported nucleic acid was not directly related to the extent of the uptake. Indeed, some glycosylated polylysines bearing sugar moities which are poor ligands of the cell surface lectins of a given cell were found to be more efficient than those bearing better sugar ligands. The interpretation of this paradoxal result is discussed with regards to the nature of the compartment allowing the nucleic acid to cross the membrane and to be delivered in the cytosol on the one hand, and to the presence of intracellular lectins on the other hand.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1020114611517
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  • 5
    Electronic Resource
    Electronic Resource
    The S7 ribosomal protein gene is truncated and overlaps a cytochrome c biogenesis gene in pea mitochondria (1999)
    Springer
    Plant molecular biology 40 (1999), S. 91-97 
    Details
    ISSN: 1573-5028
    Keywords: cytochrome c biogenesis ; gene transfer ; mitochondria ; pea ; ribosomal protein ; soybean
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The pea mitochondrial genome contains a truncated rps7 gene lacking ca. 40 codons at its 5′ terminus. This single-copy sequence is immediately downstream of and slightly overlapping an actively transcribed and edited reading frame of 744 bp (designated ccb248) homologous to the bacterial helC gene which encodes a subunit of the ABC-type heme transporter involved in cytochrome c biogenesis. This region of mitochondrial DNA appears recombinogenic, and the carboxy-termini of helC-type proteins are predicted to vary in sequence and length among plants. Sequences corresponding to the 5′ coding region of rps7 were not detected elsewhere in the pea mitochondrial genome using wheat rps7 probes, and only a very short internal rps7 segment was observed in soybean mitochondrial DNA. The presence of rps7-homologous sequences in the nuclear genomes of pea and soybean is consistent with the recent transfer of a functional mitochondrial rps7 gene to the nucleus in certain plant lineages.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026499906338
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  • 6
    Electronic Resource
    Electronic Resource
    Mycoplasma-Mediated Uptake of the Exogenous Human BRCA1 Gene by Hatching Blastocysts (1999)
    Springer
    Journal of assisted reproduction and genetics 16 (1999), S. 546-550 
    Details
    ISSN: 1573-7330
    Keywords: breast cancer ; mycoplasma ; Ureaplasma urealyticum ; foreign DNA ; gene transfer ; transgenic
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Purpose: Biological vectors for cell transfection are mainly viral in origin, with inherent shortcomings. Mycoplasmas are ubiquitous organisms that traverse cells easily. The objective was to determine if Ureaplasma urealyticum (T-mycoplasma) would vector exogenous BRCA1 DNA into blastocysts. Methods: Hatching mouse blastocysts (N = 70) were incubated in the presence of either viable or dead Ureaplasma urealyticum at 37°C for 1 hr. The blastocysts were exposed to human BRCA1 DNA lacking homology in the mouse genome for 2 hr, followed by DNase-I treatment and wash. Polymerase chain reaction and agarose gel electrophoresis analysis of amplified products were performed. Results: The BRCA1 gene was detected in the blastocysts only when viable Ureaplasma was present. PCR analyses of control Ureaplasma and untreated blastocysts were negative. Conclusion: Viable Ureaplasma organisms were shown to mediate the uptake of DNA fragments into blastocysts, resulting in transgenic mouse blastocysts with a normal human BRCA1 exon 11 gene.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1020553322073
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  • 7
    Electronic Resource
    Electronic Resource
    Tissue culture and Agrobacterium-mediated transformation of watercress (1999)
    Springer
    Plant cell, tissue and organ culture 58 (1999), S. 171-176 
    Details
    ISSN: 1573-5044
    Keywords: Bacillus thuringiensis Brassicaceae ; gene transfer ; insect resistance ; plant regeneration ; Rorippa nasturtium-aquaticum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Adventitious shoot regeneration could be obtained from more than 80% of the calluses initiated from stem explants of watercress (Rorippa nasturtium-aquaticum) by using an induction medium and a shoot regeneration medium. The induction medium contained 1.15 μM 2,4-dichlorophenoxyacetic acid and 5 μM thidiazuron; the shoot regeneration medium was composed of 0.5 μM thidiazuron and 2.25 μM 6-benzylaminopurine. This regeneration procedure was incorporated into an Agrobacterium-mediated transformation procedure for gene transfer into watercress. Factors affecting transformation included preculture, selection agents, use of tobacco nurse cells, and the length of coculture. A transgenic line of watercress transformed with a wild-type Bacillus thuringiensis insecticidal gene, cry1Ia3, was not toxic to larvae of the diamondback moth (Plutella xylostella), presumably due to premature polyadenylation of the transcript encoded by this gene in the plant.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006399130272
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  • 8
    Electronic Resource
    Electronic Resource
    Intracellular fate and nuclear targeting of plasmid DNA (1999)
    Springer
    Cell biology and toxicology 15 (1999), S. 193-202 
    Details
    ISSN: 1573-6822
    Keywords: DNA ; gene transfer ; importin ; nuclear import ; nuclear localization signal
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract One of the major steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of the transfected cells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have developed two chemical strategies for covalent coupling of NLS peptides to plasmid DNA. One method involves a site-specific labeling of plasmid DNA by formation of a triple helix with an oligonucleotide–NLS peptide conjugate. After such modification with one NLS peptide per plasmid molecule, plasmid DNA remained fully active in cationic lipid-mediated transfection. In the other method, we randomly coupled 5–115 p-azidotetrafluorobenzyllissamine–NLS peptide molecules per plasmid DNA by photoactivation. Oligonucleotide–NLS and plasmid–lissamine–NLS conjugates interacted specifically with the NLS-receptor importin α. Plasmid–lissamine–NLS conjugates were not detected in the nucleus, after cytoplasmic microinjection. Plasmids did not diffuse from the site of injection and plasmid–lissamine–NLS conjugates appeared to be progressively degraded in the cytoplasm. The process of plasmid DNA sequestration/degradation stressed in this study might be as important in limiting the efficiency of nonviral gene transfer as the generally recognized entry step of plasmid DNA from the cytoplasm into the nucleus
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007693805849
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  • 9
    Electronic Resource
    Electronic Resource
    A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight, Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity (1999)
    Springer
    Pharmaceutical research 16 (1999), S. 1273-1279 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; cytotoxicity ; polyethylenimine ; polyfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency. Methods. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and 13C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promoter as reporter gene system. Results. LMW-PEI (Mw 11′900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw l′616′OOO D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition. Conclusions. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1014861900478
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  • 10
    Electronic Resource
    Electronic Resource
    Evolution of gene transfer in bacteria (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 1-6 
    Details
    ISSN: 1573-0972
    Keywords: Bacteria ; conjugation ; DNA ; evolution ; gene transfer ; transduction ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The transfer of genetic information by transformation, conjugation and transduction in bacteria occurs frequently in nature. These diverse gene transfer mechanisms in bacteria are the result of evolution and are not linked to reproduction as in eukaryotic organisms. In this review, gene transfer in bacteria will be considered from an evolutionary perspective.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008830914223
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  • 11
    Electronic Resource
    Electronic Resource
    Prospects for gene therapy of insulin-dependent diabetes mellitus (1998)
    Springer
    Diabetologia 41 (1998), S. 1401-1409 
    Details
    ISSN: 1432-0428
    Keywords: Keywords Beta-cell lines ; conditional transformation ; gene transfer ; glucose-regulated promoters ; immunomodulation ; insulin biosynthesis ; insulin secretion ; islet regeneration ; proinsulin processing ; transplantation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The application of gene therapy to Type I (insulin-dependent) diabetes mellitus awaits improvements in gene transfer technologies and the development of better tools for accurate diagnosis of pre-diabetic people. Identification of the most promising candidate genes for gene transfer requires further elucidation of the molecular events involved in beta-cell autoimmune destruction, islet ontogeny and differentiation, and beta-cell function. This review outlines a number of possible targets for gene therapy in Type I diabetes, which could help prevent the autoimmune damage to islets, induce islet regeneration, and restore insulin production through engineering of self non-beta cells or beta-cell transplantation. It also evaluates their potential merits and drawbacks. [Diabetologia (1998) 41: 1401–1409]
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001250051085
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  • 12
    Electronic Resource
    Electronic Resource
    Transduction of non-dividing adult human pancreatic beta cells by an integrating lentiviral vector (1998)
    Springer
    Diabetologia 41 (1998), S. 736-739 
    Details
    ISSN: 1432-0428
    Keywords: Keywords Lentiviral vector ; retrovirus ; human islet beta-cell ; gene transfer ; transplantation.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Pancreatic islet cells are terminally differentiated endocrine cells and are refractory to stable infection by retroviral vectors, which require the breakdown of the nuclear membrane during cell division in order to insert the transgene into the host cell genome. Thus, attempts to render beta-cell allografts less immunogenic have had to rely on stable transfection of surrogate cells. Similarly, this problem has precluded the development of conditionally immortalized human beta cells for clinical allotransplantation. In this report, we demonstrate that adult human islet beta cells can be transduced by a new three-plasmid integrating lentiviral vector with an efficiency of 62 ± 1.8 % at a multiplicity of infection (MOI) of 2.5 in vitro. This work makes genetic engineering of adult human pancreatic beta cells possible for the first time, allowing strategies to render beta-cell allografts non-immunogenic to be optimized and to creating conditionally immortalized human beta cells for clinical transplantation. [Diabetalogia (1998) 41: 736–739]
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001250050977
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  • 13
    Electronic Resource
    Electronic Resource
    Treatment of relapsed Hodgkin's disease using EBV-specific cytotoxic T cells (1998)
    Springer
    Annals of oncology 9 (1998), S. 129-132 
    Details
    ISSN: 1569-8041
    Keywords: cytotoxic T lymphocyte (CTL) ; dendritic cell ; Epstein-Barr virus (EBV) ; EBV-associated lymphoproliferative disease (EBV-LPD) ; gene-marking ; gene transfer ; Hodgkin's disease ; immunotherapy ; latent membrane protein 2 (LMP2)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Donor-derived Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) are successful in the prevention and treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) in allogeneic bone marrow transplant (BMT) recipients [1, 2]. This finding prompted us to use a similar approach to the treatment of relapsed EBV-positive Hodgkin's disease [3]. Autologous EBV-specific CTL lines could be generated on the first or second attempt from 11 of 15 patients with Hodgkin's disease. Peripheral blood TCR ζ-chain levels were low, but increased in the activated CTL lines. Three patients have received gene-marked autologous CTL. The first two patients experienced alleviation of stage B symptoms and a drop in peripheral blood EBV load. However, this situation reversed between 6 and 12 weeks after infusion, when chemotherapy and radiation were reinstated. Both patients eventually progressed and died. The third patient had a pleural effusion, which increased after CTL infusion. Analysis of the pleural effusion revealed both tumor cells and levels of marker gene over 100 fold greater than in peripheral blood. The infused CTL line showed activity against LMP2. The patient initially improved and then remained stable for over eight months after CTL infusion, but now has progressive disease. We currently are evaluating methods for introducing the LMP2 gene into dendritic cells and using these to present LMP2 to autologous T cells. Using both retrovirus and herpesvirus vectors to express LMP2 in dendritic cells, LMP2-specific CTL were successfully generated from individuals who were EBV-seronegative or who were non-responsive to LMP2 when presented on autologous LCL. In future protocols, LMP2-specific CTL will be used for treatment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008496509406
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  • 14
    Electronic Resource
    Electronic Resource
    An A/G-rich motif in the rat fibroblast growth factor-2 gene confers enhancer activity on a heterologous promoter in neonatal rat cardiac myocytes (1998)
    Springer
    Molecular and cellular biochemistry 188 (1998), S. 169-176 
    Details
    ISSN: 1573-4919
    Keywords: FGF-2 ; transcription ; gene transfer ; HSV-thymidine kinase promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We have cloned the rat fibroblast growth factor-2 (FGF-2) promoter region including 1058 base pairs (bp) of 5′-flanking DNA. Complete sequencing of this promoter region revealed a 74 bp domain between nucleotides -793 and -720 that was greater than 97% A/G-rich. A repeat of the sequence 5′-AGGGAGGG-3′ separated by 11 bp was located at the core of this domain. A 37 bp A/G-rich oligonucleotide containing these AGGG-repeat sequences was synthesised, and tested for function on a minimal herpes simplex virus thymidine kinase (TK) promoter, fused to the firefly luciferase gene (TKp.luc), in transiently transfected neonatal rat cardiac myocytes. Promoter activity was stimulated ~3 fold in the presence of AGGG-repeat sequences. This effect was neither tissue or species-specific since TK promoter activity was increased ~11 fold in both rat and human glial tumor cells. Four specific complexes (C14) were detected between neonatal rat heart nuclear proteins and the 37 bp A/G-rich oligonucleotide by gel mobility shift assay. Competition with excess unlabelled 37 bp A/G-rich oligonucleotide revealed that two complexes represented very high affinity/specificity interactions (C2 〉 C4) while C1 and C3 were of lower affinity. As a result, competition with up to a 25 fold molar excess of 37 bp A/G-rich oligonucleotide led to the loss of C2 and C4, and a corresponding and transient increase in the levels of C1 and C3, which themselves were reduced with more competitor oligonucleotide. The AGGG-repeat resembles the 5′-gGGGAGGG-3′ sequence previously implicated in the response of the atrial natriuretic factor promoter to the α-adrenergic agonist, phenylephrine. Although an additional 1.5 fold increase in TK promoter activity was detected in the presence of the 37 bp A/G-rich oligonucleotide with phenylephrine treatment of transfected myocytes, this effect was not statistically significant. Furthermore, there was no difference in the gel mobility shift (C14) pattern obtained with the 37 bp A/G-rich oligonucleotide and nuclear protein isolated from neonatal rat cardiac myocytes grown in the presence or absence of norepinephrine. These data suggest that the A/G rich sequences in the rat FGF-2 gene 5′-flanking DNA, including the AGGG-repeat, are able to confer stimulatory activity on a promoter in a tissue- and species-independent manner, but alone are not able to induce a significant phenylephrine response in neonatal rat cardiac myocytes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006886307083
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  • 15
    Electronic Resource
    Electronic Resource
    Transient Transfection of Oligodendrocyte Progenitors by Electroporation (1998)
    Springer
    Neurochemical research 23 (1998), S. 421-426 
    Details
    ISSN: 1573-6903
    Keywords: Oligodendrocytes ; electroporation ; gene transfer ; transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The transient transfection of transgenes into oligodendrocytes offers an important tool for studying the function of proteins during myelin formation. Currently established procedures, however, have generally resulted in low survival rates and low levels of uptake of the transgene into primary oligodendrocyte progenitors. We describe an electroporation method which yields transient transfection of oligodendrocyte progenitors of up to 10–15% of the surviving cells, and provides approximately 104 surviving, transfected cells per electroporation reaction. In recent applications transgene expression persisted as the transfected progenitors progressed through subsequent stages of the oligodendrocyte lineage. This technique is expected to facilitate the study of the function of key proteins and lipids during the development of primary cultured oligodendrocytes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1022426021173
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  • 16
    Electronic Resource
    Electronic Resource
    Transfer of plasmid RP4 in the spermosphere and rhizosphere of barley seedling (1998)
    Springer
    Antonie van Leeuwenhoek 73 (1998), S. 69-77 
    Details
    ISSN: 1572-9699
    Keywords: spermosphere ; rhizosphere ; bulk soil ; gene transfer ; seed coating
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transfer of plasmid RP4 to indigenous bacteria in bulk soil could only be detected in soil with nutrient amendment. Lack of physiological active donor and recipient cells was apparently one of the limiting factors in un-amended bulk soil. Plasmid transfer was detected both in the spermosphere and rhizosphere of barley seedlings. Transfer occured from seed coated donor bacteria (i) to introduced recipient bacteria and (ii) to indigenous bacteria present in soil. Plasmid transfer was also detected from donor bacteria introduced to the soil to seed coated recipient bacteria. Transfer efficiencies in the rhizosphere were significantly below the transfer efficiencies obtained in the spermosphere. The transfer efficiencies detected in the barley spermosphere were among the highest reported from any natural environment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1000661115753
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  • 17
    Electronic Resource
    Electronic Resource
    Envelope and Long Terminal Repeat Sequences of an Infectious Murine Leukemia Virus from a Human SCLC Cell Line: Implications for Gene Transfer (1998)
    Springer
    Virus genes 17 (1998), S. 157-168 
    Details
    ISSN: 1572-994X
    Keywords: xenotropic endogenous MuLV ; gene transfer ; Bxv-1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Development of methods for gene transfer into specific cell types or tissues is important for experimental research as well as clinical therapeutical approaches. We report here the cloning and characterization of the envelope (env) gene and the U3 region of a retrovirus from an infected human Small Cell Lung Cancer (SCLC) cell line. The replication of this murine retrovirus is also fully supported by other lung cancer cell lines of different histological origin. We present evidence that a long terminal repeat (LTR)-β-galactosidase (β-Gal) reporter construct performed as well as an analogous cytomegalovirus (CMV) promoter β-Gal construct in the human lung epithelial cell line A549 and in the human larynx carcinoma cell line HEp2.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008020808314
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  • 18
    Electronic Resource
    Electronic Resource
    An extended deletion map of the Lr19 translocation and modified forms (1998)
    Springer
    Euphytica 103 (1998), S. 95-102 
    Details
    ISSN: 1573-5060
    Keywords: gene transfer ; physical mapping ; RFLPs ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A physical deletion map of the Lr19 translocated chromosome segment was extended by mapping three additional Thinopyrum RFLP loci. The relative locations of the marker loci on the translocated segment were determined as: centromere, Sd1, Xpsr165, Xpsr105, Xpsr129, XcsIH81-1, Xwg380, Xmwg2062, Lr19, Wsp-D1, Sr25/Y. Various recombinants, putative recombinats and mutants of the Lr19 segment were also characterised with respect to the additional markers.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018370718296
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  • 19
    Electronic Resource
    Electronic Resource
    Cationic lipid-mediated gene transfer: Analysis of cellular uptake and nuclear import of plasmid DNA (1998)
    Springer
    Cell biology and toxicology 14 (1998), S. 95-104 
    Details
    ISSN: 1573-6822
    Keywords: DNA ; cellular uptake ; nuclear import ; microinjection ; gene transfer ; cationiclipid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cationic lipids are widely used for gene transfer in vitro and show promise as vectors for in vivo gene therapy applications. However, there is limited understanding of the cellular mechanisms involved in nonviral gene transfer. We investigated two major steps that could be limiting barriers to cationic lipid-mediated gene transfer in vitro. We used a fluorescent plasmid to study the cellular uptake and the intracellular fate of lipoplexes during in vitro transfection of fibroblast cells and found that 100% of the cells take up lipoplexes. The intracellular staining observed with lipoplexes was clearly different from that obtained with endocytosed fluorescent dextran. This suggests that cells readily take up lipoplexes by a mechanism that could be different from endocytosis in our conditions. However, the escape of DNA from intracellular vesicles could be a major limiting barrier to gene transfer. Direct injection of plasmid DNA into the nucleus and cytoplasm of cells indicated that DNA traffic from the cytoplasm to the nucleus might be also an important limiting step.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007425803756
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  • 20
    Electronic Resource
    Electronic Resource
    Target Specific Optimization of Cationic Lipid-Based Systems for Pulmonary Gene Therapy (1998)
    Springer
    Pharmaceutical research 15 (1998), S. 1340-1347 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; airways ; cationic lipids ; surface charge ; co-lipid content ; topology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung. Methods. The effect of varying the surface and colloidal properties of cationic lipid-based gene delivery systems was assessed by intratracheal instillation into rats. An expression plasmid encoding chloramphenicol acetyl transferase (CAT) was used to measure transgene expression. Results. Cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and topology of the plasmid, were found to significantly affect transgene expression. Complexation with lipids was found to have a protective effect on DNA integrity in bronchoalveolar lavage fluid (BALF). DNA complexed with lipid showed enhanced persistence in rat lungs as measured by quantitative polymerase chain reaction. Conclusions. Fluorescence microscopy analysis indicated that the instilled formulation reaches the lower airways and alveolar region. Data also suggests cationic lipid-mediated gene expression is primarily localized in the lung parenchyma and not infiltrating cells isolated from the BALF.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1011933117509
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    Electronic Resource
    Electronic Resource
    Chitosan-Based Vector/DNA Complexes for Gene Delivery: Biophysical Characteristics and Transfection Ability (1998)
    Springer
    Pharmaceutical research 15 (1998), S. 1332-1339 
    Details
    ISSN: 1573-904X
    Keywords: gene therapy ; gene transfer ; cationic polymer ; chitosan ; polyethylenimine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. Methods. Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. Results. In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50−100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time, from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. Conclusions. Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1011981000671
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  • 22
    Electronic Resource
    Electronic Resource
    Synergistic Antitumor Interaction of Human Monocyte Chemotactant Protein-1 Gene Transfer and Modulator for Tumor-Infiltrating Macrophages (1998)
    Springer
    Pharmaceutical research 15 (1998), S. 685-689 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; colon 26 ; monocyte chemotactic and activating factor ; chemokine ; biological response modulater
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. In order to evaluate the possibility of synergistic antitumor gene therapy by the gene delivery of monocyte chemotactant protein-1 (MCP-1/MCAF/IE), the effect of a biological response modulater for macrophages on tumor progression of gene transfected tumor cells was studied. Methods. Cachexia-inducing adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid or MCP-1 cDNA. Results. The production of MCP-1 reached 70-80 ng/ml in vitro when transfectant cells were cultured at a cell density of 1 × 105 cells/ml for 3 days. Transfection of MCP-1 cDNA did not affect the growth ratein vitro. Also, MCP-1-transfectants formed tumors after intra-footpad inoculation similar in size to the parental cells. The number of infiltrating macrophages in the primary tumor of the transfectant rapidly increased from the 3rd to 5th day after inoculation as revealed by immunohistochemical staining using an antibody against mouse macrophages. An earlier, greater, but no longer-lasting increase in tumor-infiltrating macrophages was induced in tumors by MCP-1 transfection was compared to that induced by the parent cells. On the 10th day after the inoculation, the tumor-infiltrating macrophages in mice inoculated MCP-1 transfectants were decreased to a level similar to that of the parent cells. Groups of mice were treated intraperitoneally with LPS at different times after the inoculation. Tumor cells producing high levels of MCP-1 were significantly lysed by macrophages treated with LPS, whereas parental or control transfected cells were not. Conclusions. Combination immunotherapy can provide a rationale for the application of MCP-1 treatment to increase immunological responses to cancer.
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    URL: http://dx.doi.org/10.1023/A:1011906600304
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  • 23
    Electronic Resource
    Electronic Resource
    Cell cycle dependence of retroviral transduction: An issue of overlapping time scales (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 272-281 
    Details
    ISSN: 0006-3592
    Keywords: gene transfer ; retrovirus ; cell cycle ; intracellular stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant retroviruses are currently used as gene delivery vehicles for the purpose of gene therapy. It is generally believed that the efficiency of retroviral transduction depends on the cell cycle status of the target cells. However, it has been reported that this is not the case for the transduction of human and murine fibroblasts, in contrast to other cell types such as lymphocytes. The predictions of a mathematical model that we constructed, offer an explanation of this contradiction, based on the dynamics of the underlying processes of target cell growth and the intracellular decay of retroviral vectors. The model suggests that the utility of synchronization experiments, that are usually employed to study cell cycle specificity, is severely limited when the time scales of the above kinetic events are comparable to each other. The predictions of the model also suggest the use of retroviral vectors as cell cycle markers, as an alternative way to detect cell cycle dependence of retroviral transduction. This method obviates the need for cell synchronization and therefore, it does not perturb the cell cycle or interfere with the life cycle of retroviral vectors. Moreover, it does not depend on the intracellular stability of retroviral vectors. Our results show that in contrast to previously reported results, transduction of murine fibroblasts is cell cycle dependent, and they are consistent with the current notion that mitosis is the phase that confers transduction susceptibility. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:272-281, 1998.
    Additional Material: 7 Ill.
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  • 24
    Electronic Resource
    Electronic Resource
    Use of a hammerhead ribozyme with cationic liposomes to reduce leukocyte type 12-lipoxygenase expression in vascular smooth muscle (1997)
    Springer
    Molecular and cellular biochemistry 172 (1997), S. 47-57 
    Details
    ISSN: 1573-4919
    Keywords: smooth muscle ; gene transfer ; DNA ; RNA ; ribozyme ; liposome ; lipoxygenase ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Chemically synthesized hammerhead-type ribozymes targeted against the porcine leukocyte-type 12-lipoxygenase (LO) have been developed and studied. One chimeric ribozyme consists of DNA in the non-enzymatic portions, and RNA in the enzymatic core as well as two phosphorothioate internucleotide linkages at 3′ terminus. The second ribozyme consists of ribonucleotide sequences generated by in vitro transcription. In this chapter we describe methodologies to first analyze the ribozyme catalytic activity in vitro by studying cleavage of target RNA in vitro. The subsequent sections will describe how to target the catalytic ribozyme and deliver it to porcine vascular smooth muscle cells (PVSMC) by a liposome-mediated method. Finally ways to evaluate its activity to inhibit expression of the 12-LO mRNA will be presented. These results demonstrate the feasibility of using ribozymes as novel candidates for therapeutic agents to block specific gene expression in vascular cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006855219018
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  • 25
    Electronic Resource
    Electronic Resource
    Transfer of macromolecules into living adult cardiomyocytes by microinjection (1997)
    Springer
    Molecular and cellular biochemistry 172 (1997), S. 103-109 
    Details
    ISSN: 1573-4919
    Keywords: adult ventricular cardiomyocytes ; microinjection ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Among techniques commonly used to deliver bioactive molecules into living cells, microinjection is a very efficient method. Microinjection has been used extensively for gene transfer into different cell types. We applied the microinjection technique to the adult rat ventricular cardiac muscle cells (AVC) in primary culture and optimized microinjection parameters and the appropriate cell culture conditions. We also optimized the use of particular agents (i.e. 2,3-butanedione monoxime, verapamil) for the prevention of the cell damage caused by the micropuncture. We obtained the expression of a CMV-β-galactosidase reporter gene in up to 20% of the injected cells with efficient maintenance of long term cell viability. Under our experimental conditions direct microinjection is a very advantageous technique to transfer macromolecules into living adult cardiac muscle cells and a powerful system to study and manipulate the biochemistry and molecular biology of the cardiac myocyte.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006867621743
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  • 26
    Electronic Resource
    Electronic Resource
    DNA transfer into vascular smooth muscle using fusigenic Sendai virus (HJV)-liposomes (1997)
    Springer
    Molecular and cellular biochemistry 172 (1997), S. 3-12 
    Details
    ISSN: 1573-4919
    Keywords: smooth muscle ; gene transfer ; DNA ; fibroblast growth factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Manipulation of the genetic machinery of cells both in vitro and in vivo is becoming an ever more important means of elucidating pathways of molecular and cellular biochemistry. In addition, gene therapy has been proposed as a novel and potentially powerful treatment for both inherited and acquired diseases. Successful gene transfer and gene blockade generally depend on high efficiency delivery of exogenous DNA or RNA into living cells, and much effort has therefore been focused on the development of methods for achieving this delivery in a safe and effective manner. We describe here our application of fusigenic Sendai virus (HVJ)-liposome technology toward the effective delivery of DNA into vascular smooth muscle cells (VSMC) in cell culture. Cellular uptake and intracellular distribution of oligodeoxynucleotide (ODN) after transfection with HVJ-liposome complexes was characterized using fluorescent (FITC)-labeled ODN, and the biologic effect of HVJ-liposome mediated transfection was demonstrated via inhibition of DNA synthesis in cultured VSMC using antisense ODN against basic fibroblast growth factor.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006801807206
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  • 27
    Electronic Resource
    Electronic Resource
    Novel methods for adenovirus-mediated gene transfer to blood vessels in vivo (1997)
    Springer
    Molecular and cellular biochemistry 172 (1997), S. 37-46 
    Details
    ISSN: 1573-4919
    Keywords: gene transfer ; gene expression ; adenovirus ; blood vessel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Adenovirus-mediated gene transfer is a promising method for studies of vascular biology and potentially for gene therapy. Intravascular approaches for gene transfer to blood vessels in vivo generally require interruption of blood flow and have several limitations. We have used two alternative approaches for gene transfer to blood vessels in vivo using perivascular application of vectors. First, replication-deficient adenovirus expressing nuclear-targeted bacterial b-galactosidase was injected into cerebrospinal fluid via the cisterna magna of rats. Leptomeningeal cells over the major arteries were efficiently transfected, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. When viral suspension was injected with the rat in a lateral position, the reporter gene was expressed extensively on the ipsilateral surface of the brain. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, positioning of the head may ‘target’ specific regions of the brain. Second, vascular gene delivery was accomplished by perivascular injection of virus in peripheral vessels. Injection of the adenoviral vector within the periarterial sheath of monkeys resulted in gene transfer to the vessel wall that was substantial in magnitude although limited to cells in the adventitia. Approximately20% of adventitial cells expressed the transgene, with no gene transfer to cells in the intima or media. These approaches may provide alternative approaches for gene transfer to blood vessels, and may be useful for studies of vascular biology and perhaps vascular gene therapy.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006803202179
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  • 28
    Electronic Resource
    Electronic Resource
    Expression of fibroblast growth factor receptor-1 in rat heart H9c2 myoblasts increases cell proliferation (1997)
    Springer
    Molecular and cellular biochemistry 176 (1997), S. 89-97 
    Details
    ISSN: 1573-4919
    Keywords: cell division ; gene transfer ; mitogenic response
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Basic fibroblast growth factor (FGF-2) plays an important role in myocardial growth and development and in particular cardiac myocyte proliferation. FGF-2 exerts its effects by binding to cell surface receptors (FGFR-1) of the tyrosine kinase family. We have detected the presence of both long and short isoforms of FGFR-1 in embryonic and adult mouse heart. In this report, we have examined the ability of long and short FGFR-1 isoforms to signal a mitogenic response. Assessment of RNA from rat myoblast H9c2 cells by reverse transcriptase-polymerase chain reaction and RNA blotting revealed that they were deficient in transcripts corresponding to long and short FGFR-1 species. Hybrid genes containing the cDNAs coding for long and short FGFR-1 isoforms directed by the myosin light chain-2 promoter and simian virus 40 enhancer sequences, were used to transiently transfect H9c2 cells. Total tyrosine phosphorylation was increased 2.0 and 2.6 fold in H9c2 cells transfected with the long and short FGFR-1 isoforms, respectively, compared to 'control' transfected H9c2 cells. This was accompanied by a 2.1 and 2.0 fold increase in DNA synthesis, as measured by tritiated thymidine incorporation, in H9c2 cells expressing the long and short FGFR-1 isoforms, respectively. To assess effects on proliferation, H9c2 cells were stably transfected with the myosin light chain-2/FGFR-1 cDNA genes. The rate of proliferation was increased 1.6 and 3.1 fold in H9c2 cells stably expressing the long and short FGFR-1 isoforms, respectively, compared to 'control' H9c2 cells. In contrast to non transfected H9c2 cells, treatment of H9c2 cells stably expressing long FGFR-1 with FGF-2 for 24 h resulted in a slight increase (1.3 fold, p 〈 0.02) in cell number. However, a greater response (1.5 fold, p 〈 0.0005) was observed with H9c2 cells stably expressing short FGFR-1 after treatment with FGF-2. These results suggest that both long and short FGFR-1 isoforms are capable of signalling a mitogenic response.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006879029333
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  • 29
    Electronic Resource
    Electronic Resource
    Adenovirus-mediated gene transfer using in-situ perfusion of the liver graft (1997)
    Springer
    Transplant international 10 (1997), S. 202-206 
    Details
    ISSN: 1432-2277
    Keywords: Key words Gene transfer ; adenovirus ; liver transplantation ; Adenovirus ; gene transfer ; liver transplantation ; Liver transplantation ; adenovirus ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract To establish an efficient technique for adenovirus-mediated gene transfer in liver transplantation, we evaluated the in situ perfusion of liver grafts. The grafts were perfused in situ with 1 × 1010 of E1-deleted, replication-defective adenoviral vectors encoding the LacZ gene driven by the human CMV promoter, either through the hepatic artery (group 1) or the portal vein (group 2). Group 3 animals served as negative controls; their liver grafts were perfused with lactated Ringer's solution through the portal vein. PCR confirmed the presence of viral DNA in every graft perfused with viral vectors. In X-gal staining, positive staining was observed almost exclusively at the portal triad in group 1, whereas in group 2 minimal staining was observed, predominantly in the parenchymal area. Protein production from the transfected gene was confirmed by a functional protein assay; the values were 0.16 % ± 0.07 % liver protein in group 1, 0.13 % ± 0.02 % in group 2, and 0.007 % ± 0.0003 % in group 3 on postoperative day 2. In conclusion, in situ perfusion of the viral vectors through the hepatic artery resulted in an effective expression of the transfected gene, predominantly at the portal triad.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001470050042
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  • 30
    Electronic Resource
    Electronic Resource
    Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone (Haliotis divorsicolor suportexta) (1997)
    Springer
    Transgenic research 6 (1997), S. 85-95 
    Details
    ISSN: 1573-9368
    Keywords: abalone ; gene transfer ; sperm-electroporation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We investigated gene transfer in abalone via electroporated sperm. The mobility of sperm electroporated either in seawater or in marine invertebrate physiological solution was as good as that of the control group. The fertilization rate reached as high as 94.7--99.6% (93.0-- 99.7% for the control group) when 200 eggs were fertilized by 106 or 107 sperm treated with electroporation at 10 kV and 27 pulses for six cycles. Moreover, the fertilization rate of sperm electroporated in the presence of foreign DNA (opAFP-2000CAT) ranging from 0.1 to 3.2 μg and at voltages ranging from 2 to 10 kV, at 27 or 211 pulses for six or 12 cycles showed no differences from the control sperm. After DNase digestion, the genome of the electroporated sperm was analysed by polymerase chain reaction, and it was shown that a 138-bp product was amplified, corresponding to the transgene's amplification product. Southern blotting also showed that a positive band located at the same position as that of opAFP-2000CAT was found in the electroporated sperm after DNase treatment. Analysis by PCR of the genome isolated from a trochophore-stage abalone larva, derived from sperm electroporated with 3.2 g opAFP- 2000CAT, showed the existence of foreign DNA in 13 out of 20 examined samples (65%). The integration of the transferred DNA into the genome of transgenic abalone was also shown by Southern blot analysis. Furthermore, CAT activity was positive for the experimental larvae, but the level of CAT expression was lower than that of larvae derived from sperm electroporated with pCAT- Control vector, driven by SV40 promoter and enhancer sequences. These results demonstrate the potential for the use of sperm as mass gene transfer strategy in marine mollusks such as abalone
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018413318223
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    Electronic Resource
    Electronic Resource
    Expression of cryIA(c) gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod-borer (Heliothis armigera) larvae (1997)
    Springer
    Transgenic research 6 (1997), S. 177-185 
    Details
    ISSN: 1573-9368
    Keywords: cryIA(c) gene ; gene transfer ; toxic to pod-borers ; transgenicchickpea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two strains of chickpea (Cicer arietinum L.) ICCV-1 and ICCV-6, were used for transgenic plant generation. Embryo axis of mature seed devoid of the root meristem and the shoot apex was used as experimental material. The explants were cultured in medium containing MS macro salts, 4 × MS micro salts, B5 vitamins, 3.0 mg l−1 BAP, 0.004 mg l−1 NAA, 30 mg l−1 sucrose and cultured at 26 °C in dark, 24 h prior to bombardment. Gene delivery to the explants was carried out using a Bio-Rad Biolistic 1000/He particle gun. A chimaeric, truncated bacterial cryIA(c) gene construct was developed for plant expression with the CaMV35S promoter, nos terminator, an initiatory kozak sequence and a translational enhancer (STAR-P) sequence of tobacco mosaic virus. This cryIA(c) gene was cotransferred with a plasmid containing nptII gene as the selection marker. Transgenic kanamycin resistant chickpea plants were obtained through multiple shoot formation and repeated selection of the bombarded explants. Molecular analyses of the transformants revealed the presence of the transferred functional cryIA(c) gene in plant. Insect feeding assay indicated that the expression level of the cryIA(c) gene was inhibitory to the development of the feeding larvae of Heliothis armigera Hubner, the chickpea pod-borer
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    URL: http://dx.doi.org/10.1023/A:1018433922766
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  • 32
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    Electronic Resource
    The application of gene transfer techniques to marine resource management: recent advances, problems and future directions (1997)
    Springer
    Hydrobiologia 352 (1997), S. 263-278 
    Details
    ISSN: 1573-5117
    Keywords: transgenic fish ; gene transfer ; growth enhancement ; lopifection ; particle bombardment ; electroporation ; fish sperm ; fish embryos
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Recent advantages in transgenic fish research are reviewed, with special reference to the methods for gene transfer. These include microinjection, electroporation, particle bombardment, and lipofection. The success and problems associated with each of these methods, and the possible applications of transgenic fish research to aquaculture are discussed.
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    URL: http://dx.doi.org/10.1023/A:1003000127867
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  • 33
    Electronic Resource
    Electronic Resource
    Self-Assembling DNA-Lipid Particles for Gene Transfer (1997)
    Springer
    Pharmaceutical research 14 (1997), S. 190-196 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; cationic lipid ; DNA complexes ; hydrophobic effect
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. We have demonstrated that a heteromolecular complex consisting of cationic lipids and DNA can be prepared and isolated (1). Cationic lipids bind DNA through electrostatic interactions. However, when sufficient lipids are bound to DNA the physical and chemical properties of the complex are governed by hydrophobic effects. Here we describe an approach where this hydrophobic complex is used as an intermediate in the preparation of lipid-DNA particles (LDPs). Methods. The approach relies on the generation of mixed micelles containing the detergent, n-octyl β-D-glucopyranoside (OGP), the cationic lipid, N-N-dioleoyl-N, N-dimethylammonium chloride (DODAC), and selected zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) or egg sphingomyelin (SM). Results. When these micelles were prepared at low detergent concentrations (20 mM OGP) and combined with pCMVβ DNA, LDPs spontaneously formed. The mean diameter of these particles as measured by quasielastic light scattering was 55−70 nm, a result that was confirmed by negative stain electron microscopy. Further characterization of these LDPs showed that DNA within the particles was inaccessible to the small fluorochrome TO-PRO-1 and protected against DNase I degradation. LDPs could also be prepared in high concentrations of OGP (100 mM), however particles formed only after removal of OGP by dialysis. Particles formed in this manner were large (〉2000nm) and mediated efficient transfection of Chinese hamster ovary cells. Transfection activity was greater when the lipid composition used consisted of SM/ DODAC. Small particles (〈100nm) prepared of SM/DODAC were, however, inefficient transfecting agents. Conclusions. We believe that LDP formation is a consequence of the molecular forces that promote optimal hydrocarbon-hydrocarbon interactions and elimination of the hydrocarbon-water interface.
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    URL: http://dx.doi.org/10.1023/A:1012000711033
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  • 34
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    Electronic Resource
    In Vivo Gene Transfer by Intravenous Administration of Stable Cationic Lipid/DNA Complex (1997)
    Springer
    Pharmaceutical research 14 (1997), S. 742-749 
    Details
    ISSN: 1573-904X
    Keywords: cationic lipid ; DNA ; in vivo ; gene transfer ; alkaline phosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. A stable cationic lipid/DNA complex has been developed for in vivo gene transfer. The formulation capitalizes on a previously described procedure to obtain stable lipid/DNA complexes for in vitro gene transfer (1). Methods. Conditions for DNA/lipid complex formation were modified to yield a DNA concentration of 1 mg/ml. Heat stable alkaline phosphatase (AP) under a CMV promoter was used as a reporter gene. Results. The resulting complex was completely insensitive to serum inactivation. Tail vein injection of a 80 μg DNA into Balb C mice yielded significant levels of reporter enzyme activity in the lung, heart, spleen, muscle, and liver. Less AP activity was observed in the kidney. No AP activity was observed in blood, bone marrow or brain. A titration of the lipid (DOSPA) to DNA-nucleotide ratio showed the optimal molar ratio for in vivo gene transfer to be 1/1. Using this ratio in a dose response study showed approximately 80 μg of DNA/mouse yielded the highest level of gene expression. Using this dose at a 1/1 lipid to DNA nucleotide ratio, the time course for alkaline phosphatase activity was determined. Maximal AP activity was observed 24 hours after injection for all tissues. By day 5, the activity dropped approximately 10 fold for all tissues. By day 7, residual activity was detected in the lung, heart, and muscle. Histology of the lung showed both interstitial and endothelial cells to be transfected. In all other tissues, however, endothelial cells were the only transfected cell type. Conclusions. These results demonstrate that reformulation of an existing cationic lipid can result in the formation of a stable lipid/DNA complex, which is able to reproducibly transfect lung, heart, spleen, and liver upon intravenous administration.
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    URL: http://dx.doi.org/10.1023/A:1012146305040
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    The Absence of Accessible Vitronectin Receptors in Differentiated Tissue Hinders Adenoviral-Mediated Gene Transfer to the Intestinal Epithelium In Vitro (1997)
    Springer
    Pharmaceutical research 14 (1997), S. 1216-1222 
    Details
    ISSN: 1573-904X
    Keywords: adenoviral vector ; Caco-2 ; gene transfer ; integrin ; intestinal epithelium ; vitronectin receptor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished. Methods. Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium. Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces. Internalization receptors for adenoviral uptake were decteted by a fluorescence-labeled vitronectin antibody. Gene expression was studied by using the β-galactosidase reporter gene. All experiments were done on undifferentiated and differentiated Caco-2 cells. Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension. Gene expression was tested after reseeding the cells into dishes. Results. The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells. Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors. Conclusions. These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors. If these receptors can be exposed in differentiated epithelium, transduction can be made more efficicient. Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.
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    URL: http://dx.doi.org/10.1023/A:1012163025455
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  • 36
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    Non-Polarized Secretion of Mouse Interferon-β from Gene-Transferred Human Intestinal Caco-2 Cells (1997)
    Springer
    Pharmaceutical research 14 (1997), S. 483-485 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; interferon-β ; Caco-2 cells ; non-polarized secretion ; gene therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. The intestinal epithelium is considered to be a feasible target for somatic gene therapy. To this end, Caco-2 cells derived from human colon carcinoma were transfected with a mouse interferon-β (IFN-β) expression vector and several stable sublines were established; this hetero-specific cytokine allows unexpected cellular effects to be avoided. Using the highest mouse IFN-β-producing sublines, the mode of IFN secretion was examined. Methods. The secretion polarity of mouse IFN-β in its gene-transduced Caco-2 sublines was studied in a bicameral culture system in which the chambers were separated by microporous filters. Results. Mouse IFN-β was secreted to the same extent from both apical and basolateral surfaces of the transduced cells regardless of cell aging. Conclusions. These results suggest that in the intestinal epithelium exogenous gene products such as IFNs can be delivered to both the luminal and blood sides in vivo. Thus, the intestinal epithelium may be suitable for systemic and local delivery of therapeutic proteins by gene transfer.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1012151616910
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  • 37
    Electronic Resource
    Electronic Resource
    Gene therapy strategies for carcinoma of the breast (1997)
    Springer
    Breast cancer research and treatment 44 (1997), S. 93-114 
    Details
    ISSN: 1573-7217
    Keywords: cancer gene therapy ; gene transfer ; breast carcinoma ; molecular therapeutics ; molecular chemotherapy ; immunotherapy ; loss of heterozygosity (LOH) ; oncogenes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005761723853
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  • 38
    Electronic Resource
    Electronic Resource
    Isolation and analysis of different subpopulations of normal human breast epithelial cells early after their infection with a retroviral vector encoding a cell surface marker (1997)
    Springer
    Breast cancer research and treatment 44 (1997), S. 153-165 
    Details
    ISSN: 1573-7217
    Keywords: gene transfer ; human breast epithelial cells ; retrovirus ; selectable marker
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The use of gene transfer procedures has greatlyfacilitated the investigation of cell lineage relationships andother developmental processes in a variety of primarytissues. In this report we describe the infectionand selection of primary human breast epithelial cellsusing retroviral vectors (Jzen-HSA-NEO and MSCV-HSA.NEO) containing thecomplete 228 bp coding sequence of a murinecell surface marker (Heat Stable Antigen, HSA) aswell as the neomycin resistance (neor) gene. Expressionof the transduced HSA gene was detectable usingeither flow cytometry or immunohistochemistry after staining infectedcells with an anti-murine HSA-specific antibody (M1/69). Expressionof the transduced neor gene conferred resistance toG418. In initial experiments with the MCF-7 breastcancer cell line, continued expression of both markerswas demonstrated in a high proportion of cellsfor at least 4 weeks after their infectionby positive M1/69 staining of cells that hadbeen selected by prior incubation in G418. Evidenceof gene transfer to early stage (〈 9days old) primary cultures of normal human breastepithelial cells (15 experiments with cells from 12normal individuals) was also obtained using an infectionprotocol in which these cells were exposed tohelper-free viral supernatants (2 incubations, 4 to 6hr each) after being subcultured for 12 to18 hr to increase their rate of proliferation.The presence of 5–50% (mean=26%) HSA+ cells was demonstrated in these experiments within 5days after their infection and the HSA+populationsincluded both myoepithelial and luminal phenotypes. The transduced(HSA+) cells within both of these subpopulations couldalso be separately isolated by FACS and subcultured.These results should provide an important starting pointfor future studies of genetically modified or markedprimary human breast epithelial cell populations.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005713419023
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  • 39
    Electronic Resource
    Electronic Resource
    Immunotherapy I: Cytokine gene transfer strategies (1997)
    Springer
    Cancer and metastasis reviews 16 (1997), S. 421-432 
    Details
    ISSN: 1573-7233
    Keywords: cytokine ; tumor ; gene transfer ; immunotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The cytokine approach to gene therapy of cancer stems from early studies of direct, repeated injection of recombinant cytokines at the tumor site, and extension of the bystander effect that enables a few cytokine gene transduced cells in a tumor to bring about its total destruction. This effect can be extended through the immune system, since cytokine-activated regression of a small mass of tumor cells can afford systemic protection. Transduced cells used as a vaccine provide a local concentration of both cytokine and tumor antigens. Cytokines sustain antigen uptake and presentation by increasing the immunogenic potential of the environment through the recruitment of antigen presenting cells and leukocytes, and activation of a cascade of events which amplify and tone up the efficacy of a vaccine. The promises and difficulties of this approach are discussed by considering what is still missing from experimental studies and what can best be done as soon as possible in animals and humans to reach compelling conclusions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005980418533
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  • 40
    Electronic Resource
    Electronic Resource
    Introduction of new traits into cotton through genetic engineering: insect resistance as example (1997)
    Springer
    Euphytica 96 (1997), S. 163-166 
    Details
    ISSN: 1573-5060
    Keywords: Agrobacterium tumefaciens ; Bacillus thuringiensis ; cotton ; gene transfer ; Gossypium hirsutum ; insect resistance ; protease inhibitors ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The main goal of gene transfer into cotton is the development of insect-resistant varieties. The stakes are important since cotton protection against insects uses almost 24% of the world's chemical insecticides market, which is not without consequences on the environment. The first approach was to introduce and express in the cotton genome, genes from the bacterium Bacillus thuringiensis (B.t.) which produces entomopathogenic toxins. The development of an efficient Agrobacterium tumefaciens mediated transformation system was the first step. The expression of B.t. genes was studied and synthetic genes more adapted to a plant genome have been constructed. Studies on their expression in cotton is underway. The second focus was to develop strategies that would minimize the risks of inducing insect resistance. The main approach is to associate several genes coding for entomopathogenic proteins with different modes of action. Genes encoding protease inhibitors were chosen. One possibility is to associate a B.t. gene and a gene encoding a protease inhibitor. Several protease inhibitors were tested in artificial diets on major pests of cotton. The corresponding genes have been introduced into the cotton genome. These various orientations of the research program will be presented.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1002906308304
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  • 41
    Electronic Resource
    Electronic Resource
    Adenovirus-mediated gene transfer using ex vivo perfusion of the heart graft (1996)
    Springer
    Surgery today 26 (1996), S. 624-628 
    Details
    ISSN: 1436-2813
    Keywords: gene transfer ; adenovirus ; heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A replication-deficient adenovirus was used for ex vivo gene transfer into rat heart grafts under conditions simulating clinical transplantation. The adenoviral vector, AdHCMVsp1LacZ, containing an expression cassette of Escherichiae coli lacZ, was used to perfuse heart grafts during cold ischemia before transplantation. Heart grafts were perfused with University of Wisconsin (UW) solution containing either 0 pfu, 5×1010 pfu, or 1×1011 pfu of viral vector, and were preserved for either 2 or 4 h and then transplanted into syngeneic recipients. The animals were killed at 1, 7, and 14 days after transplantation. The infection rate was assessed by histochemical staining for β-galactosidase. Using polymerase chain reaction (PCR), viral DNA presence was confirmed in every graft perfused with viral vectors. The protein production from the transfected gene was confirmed by a functional protein assay. An efficient gene transfer was achieved with an infection rate of 1%–1.5% for all cardiac myocytes, as assessed by 5-bromo-4-chloro-indolyl-β-d-galactopyranoside (X-gal) staining. All studies were negative in the control grafts. Gene expression persisted for at least 10 days after transplantation. We thus conclude that an efficient adenovirus-mediated gene transfection and expression of gene products can be achieved in ex vivo perfusion of the heart graft during cold preservation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00311668
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  • 42
    Electronic Resource
    Electronic Resource
    Cancer and gene therapy (1996)
    Springer
    Annals of hematology 73 (1996), S. 207-218 
    Details
    ISSN: 1432-0584
    Keywords: Key words Cancer ; gene therapy ; gene transfer ; review
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The authors review the current literature pertaining to gene therapy as related to human cancer, covering gene therapy strategies, techniques of gene transfer, and targeted gene delivery. Gene therapy has various applications. Many technical obstacles in gene delivery need to be overcome. The safe delivery and expression of a gene to its destination are essential for clinical use. A greater understanding of the genetic basis of cancer and tumor immunology is necessary before the goal of cancer gene therapy can be fully realized.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002770050231
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  • 43
    Electronic Resource
    Electronic Resource
    Expression of the murine wild-type tyrosinase gene in transgenic rabbits (1996)
    Springer
    Transgenic research 5 (1996), S. 405-411 
    Details
    ISSN: 1573-9368
    Keywords: breeding control ; colour marker ; gene transfer ; pigmentation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The tyrosinase gene is known to be essential for melanization and has been shown to rescue pigmentation in albino mice. Previously we have described the strict copy-number-dependent expression of a murine wild-type tyrosinase gene construct over several generations in transgenic mice. In this study, we analysed the same gene construct as a marker gene for the transmission and expression of transgenes in rabbits. Using an albino hybrid strain, we produced transgenic rabbits expressing the murine tyrosinase gene. Strict correlation between integration and expression of the transgene and stable germline transmission of the integrated gene construct according to the Mendelian pattern of inheritance was observed. Thus, breeding control was facilitated by simple phenotypic examination of the transgenic animals. In contrast to mice transgenic for the same gene construct, tyrosinase-transgenic rabbits showed a greater variety in hue, intensity and extent of coat pigmentation, which is caused by the diversity in the loci affecting the melanization. Benefits and limitations of tyrosinase as a marker gene for the detection of homozygous individuals in the albino hybrid strain used are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01980205
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  • 44
    Electronic Resource
    Electronic Resource
    Selection of transgenic flax plants is facilitated by spectinomycin (1996)
    Springer
    Transgenic research 5 (1996), S. 131-137 
    Details
    ISSN: 1573-9368
    Keywords: Agrobacterium tumefaciens ; hypocotyl ; gene transfer ; flax ; neomycin ; spectinomycin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regeneration of transformed flax shoots after inoculation withAgrobacterium tumefaciens carrying a binary vector with either a neomycin phosphotransferase (nptII) gene and a β-glucuronidase (GUS) reporter gene or a spectinomycin resistance gene was examined. Hypocotyls from 4-day-old seedlings were inoculated with either of the twoA. tumefaciens strains. Selection and regeneration were achieved on a medium containing 0.1 μM thidiazuron, 0.01 μM napthalene acetic acid, 100 mgl−1 kanamycin sulphate or spectinomycin sulphate and 300 mgl−1 cefotaxime. Use of different neomycins for the selection of transformed tissues did select transformed calli but not transformed shoots either directly or via a callus phase. Selection based on spectinomycin resistance allowed the growth of transformed shoots. Transgenic shoots were rooted on a medium containing 100 mgl−1 spectinomycin sulphate. Integration of the spectinomycin resistance gene into the flax genome was confirmed by Southern blot hybridizations and spectinomycin resistance was shown to be inherited as a dominant Mendeliant trait. Therefore, spectinomycin resistance is more suitable for genetic engineering of flax than aminoglycoside resistance.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01969431
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  • 45
    Electronic Resource
    Electronic Resource
    Mammalian artificial chromosomes: A review (1996)
    Springer
    Cytotechnology 21 (1996), S. 253-261 
    Details
    ISSN: 1573-0778
    Keywords: Centromeres ; telomeres ; molecular vectors ; gene transfer ; gene therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A mammalian artificial chromosome (MAC) may be assembled through the juxtapposition of three kinds of DNA elements: a centromere, several DNA replication origins, and two telomeric repeats. The resulting structure should be able to carry and express one or more selected genes (transgenes), introduced for specific purposes. The minimal length is unknown, but may be of several Mb. Of its basic elements, the telomeres may present lesser problems, in view of their simple composition and organization. Centromeres could be an issue, given their many unknowns. Mammalian DNA replication origins are at present poorly characterized, but it is expected that at least one may be contained within the MAC components, especially the transgene. Their overall assembly may require a combination of in vivo and in vitro approaches. A promising strategy aims at constructing two telomeric arms of a MAC, one of which may include the transgene. The two novel arms could acquire a functional centromere through recombination with the two arms of a resident chromosome. Alternatively, if the two telomeric constructs are also endowed with properly placed and oriented centromeric sequences, a centromere may be rescued in vivo by homologous recombination with the external parts of the centromere of the resident chromosome. Positive selection for the artificial arms and counterselection against the resident arms should facilitate the assembly process. The assembly of such construct would not change the ploidy number of the host cell. After loading of a transgene, however, the resulting MAC may be isolated and transferred into an expression cell, where it may represent a novel chromosomal element. In this case untoward effects to the host cell may derive from an ensuing dosage effect for the transgene(s) rather than from the presence of a MAC per se. A MAC may contribute to a deeper understanding of the structural requirements for chromosomal function and evolution as well as the mechanism of chromatin formation. It should also help in the development of second generation vectors for transfer of Mb-long DNA sequences, as required for properly regulated mammalian gene function as well as, possibly, for therapy.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365348
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  • 46
    Electronic Resource
    Electronic Resource
    A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice (1996)
    Springer
    Pharmaceutical research 13 (1996), S. 1896-1901 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; MIP-lα ; chemokine ; protective immunity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein lα (hu-MIP-lα), murine-macrophage inflammatory protein lα (mu-MIP-lα), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-lα, mu-MIP-lα, or hu-IL-8 expression vector. The production of hu-MIP-1α reached 〉1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 105 cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-lα, mu-MIP-lα, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-lα and mu-MIP-lα. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-lα showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-lα gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-lα gene might be useful as an effective therapy for the treatment of certain tumors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1016057830271
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  • 47
    Electronic Resource
    Electronic Resource
    New Cationic Lipid Formulations for Gene Transfer (1996)
    Springer
    Pharmaceutical research 13 (1996), S. 1856-1860 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; gene therapy ; cationic lipid ; transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To develop appropriate dosage forms of DNA for gene delivery. Methods. 3β[N-(N′, N′ dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) was mixed either with Tween 80 alone, or with additional lipid components including castor oil and phosphatidylcholine (PC) or dioleoylphosphatidylethanolamine (DOPE) to make different lipid formulations. The particle size and the physical stability of the formulations upon mixing with plasmid DNA containing the luciferase cDNA were examined using laser light scattering measurement. The transfection activity of the DNA/lipid complexes was tested in presence or absence of serum using a cell culture system. Results. We demonstrated that many favorable properties as a gene carrier could be achieved by formulating DNA into new dosage forms using Tween 80 as the major emulsifier. Compared to the cationic liposomes, these new formulations transfected different cell lines with an equivalent or higher efficiency. Not only are they resistant to serum, but also form stable DNA complexes which could be stored for longer periods of time without losing transfection activity. Conclusions. Cationic lipids formulated into different lipid formulations using Tween 80 as a surfactant appeared to have more favorable physical and biological activities than traditional cationic liposomes as a carrier for gene delivery.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1016041326636
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  • 48
    Electronic Resource
    Electronic Resource
    Effect of Non-Ionic Surfactants on the Formation of DNA/Emulsion Complexes and Emulsion-Mediated Gene Transfer (1996)
    Springer
    Pharmaceutical research 13 (1996), S. 1642-1646 
    Details
    ISSN: 1573-904X
    Keywords: emulsions ; gene transfer ; transfection ; gene therapy ; non-ionic surfactant ; cationic lipid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To study the structure-function relationship of non-ionic surfactants in emulsion-mediated gene delivery. Methods. Four different types of non-ionic surfactants including Tween, Span, Brij and pluronic copolymers were used as co-emulsifiers for preparation of emulsions composed of Castor oil, dioleoylphosphatidylethanolamine (DOPE) and 3β[N-(N′, N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol). The effect of different surfactants on the formation of DNA/emulsion complexes and transfection activity were analyzed using plasmid DNA containing luciferase cDNA as a reporter gene. Results. Non-ionic surfactants containing branched polyoxyethylene chains as the hydrophilic head group were more effective in preventing the formation of large DNA/emulsion complexes than those containing one or no polyoxyethylene chain. All emulsion formulations except those containing Brij 700 exhibited high activity in transfecting mouse BL-6 cells in the absence of serum. In the presence of serum, however, transfection activity of each formulation varied significantly. Emulsions containing Tween, Brij 72, pluronic F68 and F127 demonstrated increased activity in transfecting cells in the presence of 20% serum. In contrast to emulsions containing Span, long chain polyoxyethylene of Brij showed decreased transfection activity. The particle size of the DNA/emulsion complexes and their ability to transfect cells are dependent on the concentration of non-ionic surfactant in the formulation. Conclusions. The structure of the hydrophilic head group of the non-ionic surfactants in the emulsion is important in determining how DNA molecules interact with emulsions and the extent to which DNA is transferred inside the cell.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1016480421204
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  • 49
    Electronic Resource
    Electronic Resource
    Immunotherapy I: Cyclosine gene transfer strategies (1996)
    Springer
    Cancer and metastasis reviews 15 (1996), S. 317-328 
    Details
    ISSN: 1573-7233
    Keywords: cytokine ; tumor ; gene transfer ; immunotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The cytokine approach to gene therapy of cancer stems from early studies of direct, repeated injection of recombinant cytokines at the tumor site, and extension of the bystander effect that enables a few cytokine gene transduced cells in a tumor to bring about its total destruction. This effect can be extended through the immune system, since cytokine-activated regression of a small mass of tumor cells can afford systemic protection. Transduced cells used as a vaccine provide a local concentration of both cytokine and tumor antigens. Cytokines sustain antigen uptake and presentation by increasing the immunogenic potential of the environment through the recruitment of antigen presenting cells and leukocytes, and activation of a cascade of events which amplify and tone up the efficacy of a vaccine. The promises and difficulties of this approach are discussed by considering what is still missing from experimental studies and what can best be done as soon as possible in animals and humans to reach compelling conclusions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00046345
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  • 50
    Electronic Resource
    Electronic Resource
    Transgenic insect cells: mosquito cell mutants and the dihydrofolate reductase gene (1996)
    Springer
    Cytotechnology 20 (1996), S. 23-31 
    Details
    ISSN: 1573-0778
    Keywords: gene transfer ; transgenic insect cells ; Aedes albopticus ; dihydrofolate reductase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00350386
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  • 51
    Electronic Resource
    Electronic Resource
    Membrane adsorption characteristics determine the kinetics of flow-through transductions (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 260-270 
    Details
    ISSN: 0006-3592
    Keywords: retrovirus ; gene therapy ; gene transfer ; virus adsorption ; membranes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Retrovirus-mediated gene transfer is currently limited by random Brownian motion of the retrovirus. This limitation can be overcome by flowing the retrovirus solution through a porous membrane that supports the target cells, leading to a significant increase in the transduction efficiency. This procedure is termed “flow-through transduction.” In this study, we characterized the effects of the fluid flowrate and the influence that membrane characteristics have on the flow-through transduction procedure. The transduction efficiencies increased with flowrate until a plateau was reached at average flow velocities exceeding 0.3 cm/h for flow times of 3 to 4 h, using a collagen-coated depth (COL) membrane. A correlation between the optimal time for maximal gene transfer using flow-through transductions and the optimal time for maximal virus activity on the membrane was found, suggesting that the membrane adsorption capacity for virus determined the amount of gene transfer that could occur.Membrane adsorption characteristics were further investigated using two different membrane types: a tracketched polyester screen (PE) membrane and the COL membrane. Flow-through transductions using the PE and COL membranes showed that a high level of gene transfer could be attained using the COL membrane while the PE membrane gave much lower transduction efficiencies. The addition of the polycation polybrene (PB) changed these results markedly, making transductions achieved on the PE membrane similar in number to those obtained on the COL membrane. Since PB is believed to influence the virus adsorption to PE membrane, these results further support the conclusion that the increase in gene transfer achieved by the flow-through transduction procedure is due to virus adsorption to the membrane. The flow-through transduction procedure thus leads to co-localization of the viral vector and the target cell that in turn leads to a high transduction efficiency. © 1996 John Wiley & Sons, Inc.
    Additional Material: 9 Ill.
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  • 52
    Electronic Resource
    Electronic Resource
    Release of genetically modified micro-organisms into the environment (1996)
    Chichester : Wiley-Blackwell
    Journal of Chemical Technology AND Biotechnology 65 (1996), S. 5-14 
    Details
    ISSN: 0268-2575
    Keywords: GMOs ; environment ; release ; genetic engineering ; gene transfer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Biotechnology in general, and recombinant DNA technology in particular, has the capacity to change the health and wealth of every individual, but like other major advances in science and technology such as nuclear power and electronics it can also be exploited to mankind's detriment. For this reason and the fact that recombinant DNA technology involves altering the molecules encoding life itself, the subject has given rise to a high level of public debate. Centuries of experience with the release of conventional micro-organisms for sewage treatment, agriculture and food production have shown that the release of large numbers of foreign organisms into an environment does not necessarily cause ecological damage. In fact few of these organisms survive for long periods. The threat of horizontal gene transfer from recombinant organisms to indigenous ones is however very real and mechanisms exist whereby, at least theoretically, any genetically engineered trait can be transferred to any prokaryotic organism and many eukaryotic ones. The rapid spread of antibiotic-resistant organisms since the widespread introduction of antibiotics graphically demonstrates that such a threat is real. There have now been several experiments to determine the effect of environmental release of micro-organisms, both in enclosed areas and in unenclosed sites. Proposals for the use of genetically modified micro-organisms that contain specific gene deletions have had a relatively smooth passage through government approval agencies and public enquiries and some products have been approved for commercial use. In addition a strain of bakers' yeast with an altered control element has also been approved for commercial use in the UK. Genetically modified viruses, especially those containing foreign genes have not received such favourable treatment and have been the subject of heated national and international debate. Many microbiologists are convinced, however, that the use and release of carefully constructed genetically engineered organisms will result in significant benefit, but with little risk to the environment. Ecologists, however, are not so sanguine and in the current ‘green’ political atmosphere their opinions are very influential. Thus most developed countries now have in force a series of very cautious regulations and guidelines for the release of genetically modified micro-organisms.
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  • 53
    Electronic Resource
    Electronic Resource
    Aspects of cellular physiology that influence DNA-mediated gene transfer in NIH3T3 cells (1995)
    Springer
    Molecular and cellular biochemistry 145 (1995), S. 169-175 
    Details
    ISSN: 1573-4919
    Keywords: NIH3T3 cells ; cell physiology ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Cellular physiology has a significant influence on the efficiency of various gene transfer procedures, as shown by the fact that transfection efficiency varies dramatically among different cell lines. However, the aspects of cellular physiology which influence the transfection process remain substantially uncharacterized. In this study, NIH3T3 cells were treated with inhibitors of protein synthesis, DNA synthesis, and RNA synthesis to determine the importance of these processes in the calcium-phosphate transfection process. The results suggest that protein synthesis during the first 4 h after DNA addition enhances transfection. In contrast, inhibition of RNA synthesis has no effect on transfection during the first 24 h post-DNA addition. The DNA synthesis inhibitor results remain inconclusive due to a secondary inhibition of an unknown cellular factor. Secondly, agents that destabilize microtubules, microfilaments, and the golgi apparatus were used to determine whether these elements play a role in the transfection process. The results suggest that microtubules are not involved in the transfection process, microfilaments are important but not necessary for the transfection process, and a functional golgi apparatus is essential early in the transfection process. These studies provide a foundation from which further investigations into the cellular processes involved in the uptake and expression of exogenous DNA can proceed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00935489
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  • 54
    Electronic Resource
    Electronic Resource
    Ganciclovir mediated regression of rat brain tumors expressing the herpes simplex virus thymidine kinase imaged by magnetic resonance (1995)
    Springer
    Journal of neuro-oncology 24 (1995), S. 259-265 
    Details
    ISSN: 1573-7373
    Keywords: brain tumor ; glioma ; thymidine kinase ; Herpes simplex virus ; ganciclovir ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Using a rat C6 brain tumor model, we studied the antitumor effects of Herpes simplex virus type 1 thymidine kinase (HSV-tk) gene transfer followed by ganciclovir treatment. C6 glioma cells were transfectedin vitro with the HSV-tk gene, and tested for their sensitivity to ganciclovir. Although there was no surviving cell at a 30 μM ganciclovir concentration, unmodified C6 cells were not affected by the drug. Forin vivo experiments, intracerebral tumors were induced in rats by stereotactic injection of 104 HSV-tk-modified C6 cells. Ten days later, the animals were treated with intraperitoneal injections of ganciclovir for 21 days. The tumors evolution was evaluated by high resolution magnetic resonance imaging. In 33% of the rats, the signal intensity of the tumors became heterogeneous, with development of highly hyperintense areas, and a complete tumor regression was subsequently noted. Histological examination of successfully treated tumors revealed progressive necrosis with formation of cysts. The survival time of the HSV-tk/ganciclovir treated animals was consistently increased, all rats surviving more than 30 days and 33% of them being still alive after 80 days.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01052842
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  • 55
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    Electronic Resource
    Vascular applications of human gene therapy (1995)
    Springer
    Journal of thrombosis and thrombolysis 1 (1995), S. 299-302 
    Details
    ISSN: 1573-742X
    Keywords: vascular smooth muscle cell ; atherosclerosis ; gene transfer ; liposomes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Complexing recombinant DNA with cationic liposomes is a convenient means of introducing foreign genes into cells (lipofection) and could potentially form the basis for genetically modifying diseased blood vessels in patients. The mechanism of lipofection is incompletely understood, but it is recognized that the degree of successful gene transfer is highly dependent on cell type. We have transfected primary cultures of human vascular smooth muscle cells with a plasmid expressing either firefly luciferase (Luc) or nuclearlocalized β-galactosidase (NL-β-gal). Cells were derived from either normal human internal mammary arteries, fragments of primary atherosclerotic plaque, or fragments of restenotic lesion. Concurrent lipofection of rabbit vascular smooth muscle cells and NIH 3T3 cells was performed as well. Compared with NIH 3T3 cells, expression in human vascular smooth muscle cells was markedly reduced: In cells derived from internal mammary artery, Luc expression, normalized for protein content, was 123-fold lower than in NIH 3T3 cells, while the proportion of cells expressing NL-β-gal was 30-fold lower. Luc expression in cells derived from restenotic tissue was significantly greater than from cells derived from primary plaque. Within a given population of cells, the mitotic index of cells expressing the recombinant gene was significantly higher than the mitotic index for the total population of cells (p 〈 0.05). Finally, cotransfection experiments, in which lipofection of smooth muscle cells was performed using genes for NL-β-gal and for human growth hormone, showed that among positive transfectants a high proportion of cells (23–36%) coexpressed both genes. Thus, the efficiency of successful lipofection in human vascular smooth muscle cells in vitro is low. Transfection appears to be preferentially facilitated in cells derived from restenotic tissue and specific properties of smooth muscle cells, including growth rates, appear to be critical for successful transfection. Further elucidation of cell properties that promote transfection is required to augment the efficiency of liposome-mediated gene transfer in human vascular cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01060740
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  • 56
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    Electronic Resource
    Human MCAF Gene Transfer Enhances the Metastatic Capacity of a Mouse Cachectic Adenocarcinoma Cell Line in Vivo (1995)
    Springer
    Pharmaceutical research 12 (1995), S. 1598-1604 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; metastasis ; colon 26 ; monocyte chemotactic and activating factor ; chemokine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To evaluate the effect of monocyte chemotactic and activating factor (MCAF/MCP-1/JE) on tumor progression and metastasis. Methods. Cachexia-inducing adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid or MCAF expression vector. Spontaneous lung metastases were determined in mouse. Results. The production of MCAF reached 0.4 ng/ml in vitro when transfectant cells were cultured at a cell density of 5 × 104 cells/ml for 3 days. Transfection of MCAF expression vector did not affect the growth rate in vitro. Also, after MCAF-transfection, the size of tumors after intra-footpad inoculation was similar to that of the parental cells. When the primary tumors were resected on the 10th day after inoculation, the incidence of spontaneous lung metastasis was less than 20% in both cells. The number of endothelial cells in the primary tumor rapidly increased from the 10th to the 14th day after inoculation, as revealed by immunohistochemical staining. In accordance with enhanced angiogenesis, the incidence rates of spontaneous metastasis increased when the primary tumors were resected on the 14th day after inoculation. Moreover, the spontaneous lung metastases were augmented in the animals injected with MCAF-transfectants compared to those injected with parental cells with a concomitant increase of angiogenesis. Conclusions. These results suggest that MCAF may augment the metastatic potential by modulating tumor associated angiogenesis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1016276613684
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  • 57
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    Electronic Resource
    Retrovirally mediated wild-type p53 restores S-phase modulation without inducing WAF1 mRNA in breast carcinoma cells containing mutant p53 (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 59 (1995), S. 537-544 
    Details
    ISSN: 0730-2312
    Keywords: cell cycle ; tumor suppressor gene ; p21 ; Cip 1 ; Sdi 1 ; Pic 1 ; gene transfer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The mechanism of negative growth regulation by the nuclear phosphoprotein p53 in breast cancer cells may rely on its role as a transcriptional activator of cell cycle-related genes. We have tested this hypothesis using retrovirally transduced wild-type (wt) p53 in breast cancer cell lines containing homozygously endogenous mutant (mt) p53. Restoring the expression of wt p53, the percentage of cells in S phase was reduced, G1/S transition was slowed, and progression through S was restrained. The fraction of cells with a flattened “Cdk-minus” phenotype increased 5- to 10-fold. High constitutive mRNA expression of the cyclin-Cdk inhibitor WAF1 in MDAMB231 cells was not induced upon restored wt p53 expression suggesting a p53-independent pathway in the regulation of WAF1 mRNA expression. Wt p53 acted trans-dominantly in the presence of accumulating mt p53 and installed a modulation of G1/S transition and S phase progression independent of WAF1 expression. © 1995 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240590413
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  • 58
    Electronic Resource
    Electronic Resource
    Shoot apical meristems as a target for gene transfer by microballistics (1955)
    Springer
    Euphytica 85 (1955), S. 45-51 
    Details
    ISSN: 1573-5060
    Keywords: meristem ; shoot apex ; ballistic microtargeting ; gene transfer ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The classical approach of gene transfer to a given plant species delivers the foreign gene to transformable cells and then puts the effort into generating plants. This approach is very difficult in many important crop plants, including cereals, and the results of regeneration are very genotype-dependent. In contrast, we use regenerable cells and try to transform them. Shoot apical meristems provide a tissue which regenerates in situ a fertile plant for most given genotypes or species. Transformation of meristem cells may lead to transgenic sectors in chimeras. These sectors may contribute to the gametes and, thus, to transgenic offspring, which then should be homohistonts and not sectorial chimeras like their parents. Our model plant for these studies is wheat. Microtargeting is a ballistic approach which is particularly suitable for the controlled delivery of microprojectiles to meristem cells in situ (Sautter et al., 1991). We summarize in this paper our experience with ballistic microtargeting of transgenes to wheat shoot apical meristem cells in situ.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023929
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  • 59
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    Electronic Resource
    Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants (1955)
    Springer
    Euphytica 85 (1955), S. 81-88 
    Details
    ISSN: 1573-5060
    Keywords: gene transfer ; Hordeum vulgare ; neomycin phosphotransferase II ; particle bombardment ; transgenic barley
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for β-glucuronidase (GUS). Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T0). This chimeric plant passed the transferred nptII gene to its T1 progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T0 spikes and 15 T1 offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T2 and T3 plants were produced by isolating embryos from green grains of transgenic T1 and T2 plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T2 offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed. Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023933
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  • 60
    Electronic Resource
    Electronic Resource
    The sulphur-rich Brazil nut 2S albumin is specifically formed in transgenic seeds of the grain legume Vicia narbonensis (1955)
    Springer
    Euphytica 85 (1955), S. 181-192 
    Details
    ISSN: 1573-5060
    Keywords: Vicia narbonensis ; gene transfer ; gene expression ; seeds ; 2S albumin ; methionine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Epicotyl explants were co-cultivated with Agrobacterium tumefaciens EHA101 to transfer a chimeric 2S albumin gene construct carried in the binary Ti plasmid vectors pGSGLUC1 or pGA472 into the grain legume Vicia narbonensis. This gene encoding the sulphur-rich Brazil nut albumin was under the control of either the CaMV 35S promoter which permits gene expression in all organs, or the Vicia faba legumin B4 promoter which elicits seed-specific gene expression. After callus formation and selection for kanamycin resistance, somatic embryos were induced which, in the case of transformation with the vector pGSGLUC1, were screened for GUS activity. Embryos that produced GUS were in addition analysed for 2S albumin formation. Selected transgenic embryos were cloned by multiple shoot regeneration. Rooted and fertile plants were obtained by grafting transgenic shoots on the appropriate seedlings. R1 and R2 generations were raised and analysed for GUS as well as 2S albumin gene expression. Expression of the 35S promoter/2S albumin gene fusion took place in all organs of the transgenic plants including the cotyledons of seeds, whereas seed-specific gene expression was found in transformants with the legumin promoter/2S albumin gene fusion. The 2S albumin accumulated in the 2S protein fraction of transgenic seeds and its primary translation product was processed into the 9 and 3 kDa polypeptide chains. The foreign protein was localised in the protein bodies of the grain legume. Analysis of the R2 plants indicated Mendelian inheritance of the 2S albumin gene. In homozygous V. narbonensis plants the amounts of 2S albumin were twice that present in the corresponding heterozygous plants. Whereas only low level formation of the foreign protein was achieved if the gene was under the control of the 35S promoter, approximately 3.0% of the soluble seed protein was 2S albumin if seed-specific gene expression was directed by the legumin B4 promoter. Some of these transformants exhibited a three-fold increase in the methionine content of the salt-soluble protein fraction extracted from seeds.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023947
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  • 61
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    Electronic Resource
    Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment (1955)
    Springer
    Euphytica 85 (1955), S. 13-27 
    Details
    ISSN: 1573-5060
    Keywords: gene transfer ; crop species ; particle bombardment ; transgenic plants ; cereals ; legumes ; woody plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The limiting component in the creation of transgenic crops has been the lack of effective means to introduce foreign genes into elite germplasm. However, the development of novel direct DNA transfer methodology, by-passing limitations imposed by Agrobacterium-host specificity and cell culture constraints, has allowed the engineering of almost all major crops, including formerly recalcitrant cereals, legumes and woody species. The creation of transgenic rice, wheat, maize, barley, oat, soybean, phaseolus, peanut, poplar, spruce, cotton and others, in an efficient and in some cases, variety-independent fashion, is a significant step towards the routine application of recombinant DNA methodology to the improvement of most important agronomic crops. In this review we will focus on key elements and advantages of particle bombardment technology in order to evaluate its impact on the accelerated commercialization of products based on agricultural biotechnology and its utility in studying basic plant developmental processes and function through transgenesis. Fundamental differences between conventional gene transfer methods, utilizing Agrobacterium vectors or protoplast/suspension cultures, and particle bombardment will be discussed in depth.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023926
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