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  • 2015-2019
  • 1990-1994  (21)
  • 1955-1959
  • 1950-1954
  • 1940-1944
  • 1991  (21)
  • Agrobacterium
  • genetic engineering
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell reports 10 (1991), S. 208-212 
    ISSN: 1432-203X
    Keywords: Agrobacterium ; mediated transformation ; Pepino ; pKIWI110 ; Regeneration ; Solanum muricatum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (β-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-203X
    Keywords: Agrobacterium ; Tumour induction ; Dendranthema grandiflora ; Polymerase chain reaction ; ß-Glucuronidase ; Neomycine phosphotransferase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary To develop an Agrobacterium mediated transformation protocol for chrysanthemum we studied the transformation efficiency of commonly used A.tumefaciens strains on 14 genotypes by comparing tumour size and frequency. One genotype was analyzed in detail using 14 strains of both A.tumefaciens and A.rhizogenes. Only a few genotype/strain combinations resulted in significant tumour formation. Especially 0-type strains were highly efficient. An 0-type strain was used to transfer genes for neomycine phosphotransferase (NPT II) and ß-glucuronidase (GUS) to a susceptible cultivar. Transfer of the GUS gene was confirmed by using the Polymerase Chain Reaction (PCR).
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Journal of agricultural and environmental ethics 4 (1991), S. 101-107 
    ISSN: 1573-322X
    Keywords: agricultural bioethics ; bovine somatotropin ; ownership of germ plasm ; genetic engineering ; animal rights ; intellectual property rights
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Philosophy
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 16 (1991), S. 853-864 
    ISSN: 1573-5028
    Keywords: GUS ; UidA ; 35S promoter ; Agrobacterium ; plant cell transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S-β-glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-5028
    Keywords: storage protein ; genetic engineering ; transgenic plants ; zeins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chimeric genes composed of the β-phaseolin promoter, an α-zein coding sequence and its modified versions containing lysine codons, and a β-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. α-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa α-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an α-zein content that was approximately 0.003% of the total seed protein. The amount of α-zein in other transgenic plants varied between 1 × 10−4% and 1 × 10−5% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the α-zein protein. Polysomes translating α-zein mRNA isolated from tobacco seeds contained fewer ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized α-zein was degraded in tobacco seeds with a half-life of less than 1 hour.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 17 (1991), S. 837-851 
    ISSN: 1573-5028
    Keywords: Agrobacterium ; binary vector ; gene fusion ; β-glucuronidase ; T-DNA insertion ; transgenic tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A binary vector, pPRF120, was designed to detect T-DNA insertions within transcriptionlly active areas of the plant genome. Linked to the right-border repeat, the vector contains a promoterless β-glucuronidase (GUS) gene which can, upon integration into chromosomes, be activated by cis-acting regulatory elements. The vector also incorporates a chimeric marker gene conferring resistance to kanamycin to ensure recovery of gene fusions regardless of the extent of their tissue-specific or developmentally regulated expression, and to permit analysis of the frequency of plants which express the promoterless reporter. Approximately 1000 transgenic tobacco plants harboring pPRF120 were regenerated. Analysis of 52 individuals indicated that more than 80% contain single, intact copies of the T-DNA, regardless of their ability to express the promoterless GUS gene. Screening of leaf tissue from the 1000 pPRF120 transformants revealed that ca. 5% of the plants contained GUS activity. Fluorogenic and histological GUS assays were used to visualize and quantify tissue- and cell-specific gene expression. The potential usefulness of pPRF120 in comparison to other vectors designed to generate in vivo gene fusions is discussed.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1573-5028
    Keywords: Agrobacterium ; virulence gene ; VirA protein ; gene regulation ; sensor protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The induction of vir gene expression in different types of Agrobacterium strains shows different pH sensitivity profiles. The pH sensitivity pattern demonstrated by octopine Ti strains was similar to that of a supervirulent leucinopine Ti strain, whereas this was different from that shown by nopaline Ti strains and agropine Ri strains. Data are given which indicate that these differences are due to different properties of the virA genes of these wild types. An exceptional case was formed by strains with the limited-host-range plasmid pTiAG57 which showed AS-dependent vir induction only if reduced inoculum sizes were used and the temperature was 28°C or below.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1573-5028
    Keywords: Agrobacterium ; crown gall ; poplar ; tree transformation ; wild cherry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Micropropagated shoots of three forest tree species, poplar (Populus tremula × P.alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra × J. regia), were inoculated each with six different wild-typeAgrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with anAgrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carryingneo (kanamycin resistance) anduidA (β-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to expressneo anduidA genes. These results suggest that wild-typeAgrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1573-5028
    Keywords: Agrobacterium ; haemagglutination ; lectin (Pisum) ; protein processing ; transgenic potato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating α and β subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1573-0778
    Keywords: genetic engineering ; Namalwa cells ; perfusion culture ; scaling-up ; serum-free medium ; stable production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979). Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 × 107 cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter™, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983). Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c). Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.
    Type of Medium: Electronic Resource
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    Sociological forum 6 (1991), S. 551-565 
    ISSN: 1573-7861
    Keywords: agriculture ; capitalism ; development ; world economy ; Third World ; genetic engineering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Sociology
    Notes: Abstract Frederick Buttel was one of the pioneers in studying the social impacts of biotechnology, claiming originally that it will involve profound changes in social structure. Recently Buttel turned around his argument proposing that, rather than revolutionary, biotechnology is more a “substitutionist” technological form to be applied to declining sectors of the economy than an “epoch-making” technology. This paper provides both external and internal critiques of Buttel's new position based on the concept of the “third technological revolution,” looking at the impact of new technologies as a global and interrelated phenomenon, and not on an individual case-by-case basis. The concluding section suggests the necessity of bringing into the analysis those living in the Third World: 60% of this population lives from agriculture and will be affected by the deployment of agricultural biotechnologies, whether through “substitutionism” or through totally new products.
    Type of Medium: Electronic Resource
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  • 12
    ISSN: 1617-4623
    Keywords: Agrobacterium ; Cis-acting DNA element ; Cross-talk ; DNA-protein interaction ; Positive regulator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Transcription of a virulence gene on the hairyroot-inducing plasmid A4, which is induced by plant factors in Agrobacterium tumefaciens, was also activated by phosphate limitation in both A. tumefaciens and Escherichia coli. The starting site of RNA synthesized under the two inducing conditions was the same, and an identical promoter was responsible for both inducible expressions. The response of the virulence gene to phosphate limitation did not require the positive regulator VirG for the virulence regulon, but depended entirely on the presence of PhoB protein, the positive regulator for the phosphate regulon. The DNA signal upstream of the virulence gene, which is targeted by the VirG protein, was recognized by the E. coli PhoB protein in vitro. These results indicate that cross-talk between the two regulons occurred during the recognition of a DNA signal by the regulatory protein.
    Type of Medium: Electronic Resource
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 230 (1991), S. 302-309 
    ISSN: 1617-4623
    Keywords: VirG ; Agrobacterium ; vir activation ; Supervirulence ; pTiBo542
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The virG gene of the Agrobacterium tumefaciens Ti plasmid pTiBo542 has previously been reported to elicit stronger vir gene expression than its counterpart in the pTiA6 plasmid, a property we call the “superactivator” phenotype. The DNA sequence of the pTiBo542 virG gene was determined and compared to that of the pTiA6 gene. The DNA sequences of these genes differ at 16 positions: two differences are in the promoter regions, 12 are in the coding regions, and two are in the 3′ untranslated regions. The 3′ end of the pTiA6 virG gene also contains a probable insertion sequence that is not found downstream of the pTiBo542 gene. The base pair differences in the two coding regions result in only two amino acid differences, both in the amino-terminal halves of the proteins. Five hybrid virG genes were constructed and used to activate the expression of a virB::lacZ gene fusion. Differences in the coding regions of these genes accounted for most of the superactivator phenotype, while differences at the promoter and 3′ untranslated regions also contributed. These findings suggest that the properties of these VirG proteins and their quantities are important for vir gene induction, and also suggest a long-term selective pressure for mutations contributing to differences between these two genes.
    Type of Medium: Electronic Resource
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  • 14
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 230 (1991), S. 24-27 
    ISSN: 1617-4623
    Keywords: Agrobacterium ; Ti plasmid ; Tn5mob ; Chromosome ; Repressor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Virulence functions associated with the oncogenicity of Agrobacterium tumefaciens are encoded by vir genes contained in six major operons located on the Ti plasmid. The virC and virD operons encode functions responsible for host range and T-intermediate processing. These two operons are regulated positively by the product of virG and negatively by the product of the chromosomal gene ros, which encodes a 15.5 kDa repressor. To determine the location of the ros gene we have constructed A. tumefaciens HFR strains, using transposon Tn5mob to mobilize the ros locus, and used them to map the location of ros relative to auxotrophic loci. Tight linkage was found between ros, his-34 and his-19. A linkage map is presented showing the location of ros relative to other known chromosomal genes associated with virulence functions.
    Type of Medium: Electronic Resource
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  • 15
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 24 (1991), S. 91-95 
    ISSN: 1573-5044
    Keywords: Agrobacterium ; regeneration ; Ribes nigrum ; tissue culture ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transformation of the black currant cv. Ben More was achieved by utilising the binary vector system of Agrobacterium tumefaciens. This system involved the inoculation of peeled internodal stem segments with A. tumefaciens strain LBA4404 containing the binary vector PBI121.X with the marker genes Betaglucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Shoot regeneration occurred on nutrient media based on M&S salts. Transformation was confirmed by the fluorogenic assay procedure which determined that the GUS gene had been transferred into the plant material and was being expressed. Concurrent transfer of the NPTII gene into the plant material was also confirmed with a ‘dot blot’ assay on selected GUS positive plantlets.
    Type of Medium: Electronic Resource
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  • 16
    Electronic Resource
    Electronic Resource
    Springer
    European journal of epidemiology 7 (1991), S. 207-212 
    ISSN: 1573-7284
    Keywords: Rickettsia ; Intracellular parasite ; genetic engineering ; molecular biology ; rickettsial genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Our understanding of the biology of the rickettsiae, organisms that are the archetype of the obligate intracytoplasmic bacterial parasites, remains muddy and fragmentary. For example although we all appreciate that the rickettsiae can exploit their unique environment, the host cell cytoplasm, but are unable to grow axenically, the basis of this fact is still one of microbiology's central mysteries. It is unfortunate, but true, that because of the inherent difficulty of working within this system, progress on the answers to such questions will be slow and laborious. However, with the application of molecular biological methods, that is, the powerful modern approaches of genetics and biochemistry, the rickettsiology community has the realistic prospect that this field is far from being at a stand-still and that significant increases in our comprehension of the fundamental problems of rickettsial biology are occurring and will continue to occur at ever accelerating rates. Some examples, both in terms of scientific conclusions and technical approaches, of the progress made in recent years and expectations for the near future will be presented.
    Type of Medium: Electronic Resource
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  • 17
    ISSN: 1573-5044
    Keywords: Agrobacterium ; leaf explant ; mesophyll protoplast ; regeneration ; selective agent ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Agrobacterium tumefaciens strains harbouring plasmid vectors pBCAT1, pVU1011 or pMON806 were used to transform leaf explants of Nicotiana tabacum cultivars ‘Delgold’ and ‘Candel’, N. debneyi, and N. rustica var. NRT. Transgenic plants resistant to the selective agents kanamycin, hygromycin or methotrexate were regenerated and used as sources of leaf mesophyll protoplasts. Protoplasts divided and regenerated plants in the presence of selective agents at levels inhibitory to protoplasts of non-transformed plants. Cross-resistance of protoplasts to more than one selective agent was not observed in this study which suggests that this approach may lead to an efficient interspecific somatic hybrid selection system.
    Type of Medium: Electronic Resource
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  • 18
    Electronic Resource
    Electronic Resource
    Springer
    Plant cell, tissue and organ culture 27 (1991), S. 129-139 
    ISSN: 1573-5044
    Keywords: Agrobacterium ; auxins ; cytokinins ; roots ; Solanum tuberosum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A novel and versatile in vitro assay system is presented which has considerable scope for accurate description of environmentally and/or genetically induced changes in the architecture of intact plant root systems. Use of the assay system is exemplified by the analysis of potato plants with visibly distinct root systems. These were obtained by growth of normal and transgenic potato plants, transformed with Agrobacterium Tcyt and rol genes on standard culture medium and on media supplemented with NAA and zeatin. Consequences for parameters such as root weight, root length, percentage secondary roots and root density distribution with depth were quantified. The logarithmic regression: log10(ϱ)=A+Bd, in which ϱ is the root density (root weight per media volume) at depth d, proved useful in quantifying the decline of root density with depth by the single slope parameter B. From the calculated relations for the various root systems, and their root weights, profiles of their comparable root weight distributions were drawn. A number of features and drawbacks, and possible uses of the assay system are discussed.
    Type of Medium: Electronic Resource
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  • 19
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 38 (1991), S. 296-303 
    ISSN: 0006-3592
    Keywords: ethanol ; genetic engineering ; Escherichia coli ; lignocellulose ; xylose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The conversion of xylose to ethanol by recombinant Escherichia coli has been investigated in pH-controlled batch fermentations. Chemical and environmental parameters were varied to determine tolerance and to define optimal conditions. Relatively high concentrations of ethanol (56 g/L) were produced from xylose with excellent efficiencies. Volumetric productivities of up to 1.4 g ethanol/L h were obtained. Productivities, yields, and final ethanol concentrations achieved from xylose with recombinant E. coli exceeded the reported values with other organisms. In addition to xylose, all other sugar constituents of biomass (glucose, mannose, arabinose, and galactose) were efficiently converted to ethanol by recombinant E. coli. Unusually low inocula equivalent to 0.033 mg of dry cell weight/L were adequate for batch fermentations. The addition of small amounts of calcium, magnesium, and ferrous ions stimulated fermentation. The inhibitory effects of toxic compounds (salts, furfural, and acetate) which are present in hemicellulose hydrolysates were also examined.
    Additional Material: 5 Ill.
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  • 20
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 261-267 
    ISSN: 0730-2312
    Keywords: tissue transplantation ; catecholamines ; dopamine ; L-DOPA ; genetic engineering ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In animal models, grafts derived from several different tissues, principally fetal substantia nigra and adrenal medulla from young adults, have been found to be effective in alleviating some of the manifestations of lesions of the substantia nigra. It has been suggested that these grafts function by diffusely secreting dopamine, by exerting trophic effects on the host brain, or by producing a new innervation of the host corpus striatum. Evidence for each of these modes of action is briefly reviewed. Several brain tissue transplantation techniques have been described. Each of these techniques has significant limitations in animal models. The significance of these limitations for human application is described, and possibilities for improving the efficacy of brain tissue transplantation in animal models and for human application are discussed.
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  • 21
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 268-272 
    ISSN: 0730-2312
    Keywords: hematopoiesis ; stem cell ; perfusion ; hematopoietic growth factor ; genetic engineering ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM-CSF and IL-3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.
    Additional Material: 1 Ill.
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