transformation methods
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Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations.
Lymfocytární transformační test nebo test blastické transformace či proliferační aktivity lymfocytů patří mezi in vitro vyšetřovací metody v imunologii, zaměřené na hodnocení specifické buněčné imuni ty u pacientů s imunodeficiencemi, autoimunitními, infekčními a nád orovými onemocněními. O jeho využití v klinické praxi se v poslední době vedou polemiky vzhledem k celé řadě technických a koncepčních faktorů, které mohou ovlivnit výsledky. Proto je v některých oblastech imun ologické diagnostiky nahrazován jinými metodami (vyšetření aktivačn ích znaků lymfocytů, stanovení produkce intracelulárních cytokinů). Propraco vání a validace lymfocytárního transformačního testu však můž e přispět k lepší diagnostice některých alergických a infekčních onemocnění.
The lymphocyte transformation test or the test of lymphocyte transformation or proliferation activity of lymphocytes belongs to in vitro diagnos- tic methods in immunology for evaluation of specific cellular imunity in patiens with immunodeficiences, autoimmune, infectious and tumor diseases. In the last time, its use in clinical practice is discussed because of many technical and conceptual factors affectin g results. Accordingly, this test is often replaced by other methods (detection of lymphocyte activation markers, detection of production of intracellu lar cytokines). Therefore the modification and validation of the lymphocyte transf ormation test can contribute to improvement of diagnostics of some allergic and infectious diseases.
In this study, the Agrobacterium tumefaciens-mediated transformation method for Ganderma weberianum has been established. Driven by the cauliflower mosaic virus (CaMV) 35S promoter, the hygromycin phosphotransferase (hpt), β-glucuronidase (uidA), and enhanced green fluorescent protein (egfp) genes have been efficiently expressed in transgenic mycelia and spores. The transformation system was composed of the growing mycelia, A. tumefaciens strain GV3101, and the expression vector pBI-H1, harboring the CaMV 35S promoter and selective hpt marker. The genetic transformation of G. weberianum was achieved through co-cultivation of Agrobacterium lawn and fungal mycelia at 28 °C on yeast extract agar (YEA) medium. Stable genetic transformants were obtained through successive hygromycin B selections and single spore isolation. Over 80 % of transformants showed genetic stability even after ten rounds of subculturing. The simple and efficient genetic transformation method is a useful tool for molecular genetics analyses and gene manipulation of G. weberianum.
- MeSH
- Agrobacterium tumefaciens genetika metabolismus MeSH
- Ganoderma genetika MeSH
- genetické techniky * MeSH
- genetické vektory genetika metabolismus MeSH
- transformace genetická * MeSH
- zelené fluorescenční proteiny MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
In myelodysplastic syndromes (MDSs), the evolution of risk for disease progression or death has not been systematically investigated despite being crucial for correct interpretation of prognostic risk scores. In a multicenter retrospective study, we described changes in risk over time, the consequences for basal prognostic scores, and their potential clinical implications. Major MDS prognostic risk scoring systems and their constituent individual predictors were analyzed in 7212 primary untreated MDS patients from the International Working Group for Prognosis in MDS database. Changes in risk of mortality and of leukemic transformation over time from diagnosis were described. Hazards regarding mortality and acute myeloid leukemia transformation diminished over time from diagnosis in higher-risk MDS patients, whereas they remained stable in lower-risk patients. After approximately 3.5 years, hazards in the separate risk groups became similar and were essentially equivalent after 5 years. This fact led to loss of prognostic power of different scoring systems considered, which was more pronounced for survival. Inclusion of age resulted in increased initial prognostic power for survival and less attenuation in hazards. If needed for practicability in clinical management, the differing development of risks suggested a reasonable division into lower- and higher-risk MDS based on the IPSS-R at a cutoff of 3.5 points. Our data regarding time-dependent performance of prognostic scores reflect the disparate change of risks in MDS subpopulations. Lower-risk patients at diagnosis remain lower risk whereas initially high-risk patients demonstrate decreasing risk over time. This change of risk should be considered in clinical decision making.
- MeSH
- časové faktory MeSH
- Kaplanův-Meierův odhad MeSH
- lidé MeSH
- myelodysplastické syndromy mortalita MeSH
- nádorová transformace buněk patologie MeSH
- rizikové faktory MeSH
- senioři MeSH
- Světová zdravotnická organizace MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
Red rot of sugarcane caused by the hemi-biotrophic fungal pathogen, Colletotrichum falcatum, is a major threat to sugarcane cultivation in many tropical countries such as India, Bangladesh, and Pakistan. With the accumulating information on pathogenicity determinants, namely, effectors and pathogen-associated molecular patterns (PAMPs) of C. falcatum, it is of paramount importance to decipher the functional role of these molecular players that may ultimately decide upon the outcome of sugarcane-C. falcatum interaction. Since C. falcatum is a multinucleated filamentous fungus, the conventional Agrobacterium-mediated transformation method could not be effectively utilized for targeted manipulation of genomic DNA. Hence, we developed a highly efficient protoplast-based transformation method for the virulent pathotype of C. falcatum - Cf671, which involves isolation of protoplast, polyethylene glycol (PEG)-mediated transformation, and regeneration of transformed protoplasts into hyphal colonies. In this study, germinating conidiospores of Cf671 were treated with different enzyme-osmoticum combinations, out of which 20 mg/mL lysing enzyme with 5 mg/mL β-glucanase in an osmoticum of 1.2 mol/L MgSO4 yielded maximum number of viable protoplasts. The resultant protoplasts were transformed with pAsp shuttle vector. Transformed protoplasts were regenerated into hyphal colonies under hygromycin selection and observed for GFP fluorescence. This protocol resulted in a transformation efficiency of > 130 transformants per μg of plasmid DNA. This method of transformation is rapid, simple, and more efficient for gene knockout, site-directed mutagenesis, ectopic expression, and other genetic functional characterization experiments in C. falcatum, even with large vectors (> 10 kb) and can also be applied for other filamentous fungi.
