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Plant transformation by microinjection techniques 总被引:4,自引:0,他引:4
Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking. 相似文献
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Kost Benedikt; Galli Alessandro; Potrykus Ingo; Neuhaus Gunther 《Journal of experimental botany》1995,46(9):1157-1167
An efficient system has been established that allows well controlledDNA microinjection into tobacco (Nicotiana tabacum) mesophyllprotoplasts with partially regenerated cell walls and subsequentanalysis of transient as well as stable expression of injectedreporter genes in particular targeted cells or derived clones.The system represents an effective tool to study parametersimportant for the successful transformation of plant cells bymicroinjection and other techniques. Protoplasts were immobilizedin a very thin layer of medium solidified with agarose or alginate.DNA microinjection was routinely monitored by coinjecting FITC-dextranand aimed at the cytoplasm of target cells. The injection procedurewas optimized for efficient delivery of injection solution intothis compartment. Cells were found to be at the optimal stagefor microinjection about 24 h after immobilization in solidmedium. Embedded cells could be kept at this stage for up to4 d by incubating them at 4 C in the dark. Within 1 h successfuldelivery of injection, solution was routinely possible into2040 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913,a plasmid containing the neo (neomycin phosphotransferase II)gene, stably transformed, paromomycin-resistant clones couldbe recovered through selection. Transgenic tobacco lines havebeen established from such clones. Injection solutions containingpSHI913 at a concentration of either 50 µg ml1or 1 mg ml1 have been tested. With 1 mg ml1 plasmidDNA the percentage of resistant clones per successfully injectedcell was determined to be about 3.5 times higher. Incubationof embedded protoplasts at 4C before microinjection was foundto reduce the percentage of resistant clones obtained per injectedcell Protoplasts were immobilized above a grid pattern and the locationof injected cells was recorded by Polaroid photography. Thefate of particular targeted cells could be observed. Isolationand individual culture of clones derived from injected cellswas possible. Following cytoplasmic coinjection of FITC-dextranand 1 mg ml1 plasmid DNA on average about 20% of thetargeted cells developed into microcalli and roughly 50% ofthese calli were stably transformed. Transient expression ofthe firefly luciferase gene (Luc) was nondestructively analysed24 h after injection of pAMLuc. Approximately 50% of the injectedcells that were alive at this time point expressed the Luc genetransiently. Apparently, stable integration of the injectedgenes occurred in essentially all transiently expressing cellsthat developed into clones. Key words: DNA microinjection, firefly luciferase, FITCdextran, Nicotiana tabacum, protoplast transformation 相似文献
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Treatment of mouse spleen cells with a rabbit anti-mouse brain (RAMB) antiserum markedly suppressed antibody-dependent cell-mediated cytotoxicity (ADCC) on trinitrophenyl-coupled sheep erythrocyte targets. This inhibitory activity of RAMB antiserum was complement independent, absorbable with mouse brain tissue, and appeared to be separable from the anti-Thy-1 activity of this serum. Absorption studies indicated that various T- and B-lymphocyte cell lines as well as macrophage-like cell lines are not able to absorb the inhibitory activity of RAMB antiserum. In contrast, thymocytes and spleen cells, as well as the neural cell line, PC12, a chromocytoma derived from rat adrenal medulla, were capable of absorbing the inhibitory activity to some extent, suggesting that antigens characteristic for ADCC effector cells can be found on these cell populations. 相似文献
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Gunther Ott Lubos Arnold Jiri Smrt Michal Sobkowski Stefan Limmer Hans-Peter Hofmann 《Nucleosides, nucleotides & nucleic acids》2013,32(5):1069-1085
Abstract Dimethylaminomethylene was applied as the protecting group for the exocyclic amino groups of adenosine and guanosine in the automated chemical synthesis of oligoribonucleotides on a polymer bound support. The dimethyl-aminomethylene protecting group can be removed at room temperature under conditions where the concomitant loss of the 2′-protection group can be excluded. The transformation of 2′-O-(t-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl) protected nucleosides to 3′-H-phosphonates yields synthons, well suited for the automated chemical synthesis of oligoribonucleotides. Using these H-phosphonate monomers, a coupling time of two minute: is sufficient to obtain average coupling yields of more than 98 %. Synthesized RNA is recognized as a substrate in an enzymatic reaction, forms the expected secondary structures and is suitable for NMR structural investigations. 相似文献
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In a comparative proteome analysis of peripheral blood mononuclear cells (PBMCs), we analyzed 130 two-dimensional gels obtained from 33 healthy control individuals and 32 patients diagnosed with rheumatoid arthritis (RA). We found 16 protein spots that are deregulated in patients with RA and, using peptide mass fingerprinting and Western blot analyses, identified these spots as belonging to 9 distinct proteins. A hierarchical clustering procedure organizes the study subjects into two main clusters based on the expression of these 16 protein spots, one that contains mostly healthy control individuals and the other mostly RA patients. The majority of the proteins differentially expressed in RA patients when compared with healthy controls can be detected as protein fragments in PBMCs obtained from RA patients. This set of deregulated proteins includes several factors that have been shown to be autoantigens in autoimmune diseases. 相似文献