1,720,987 research outputs found
Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures
No full textHairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco-engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2–3 mg/L). N-glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant-typical complex structures for N. tabacum-derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT-derived counterpart. Both antibody glyco-formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co-infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor-targeting mAb H10 with a human-compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of ‘next generation’ human therapeutic antibodies. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei
FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy
No full textThe large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations. © 2013 Giorgi et al
Saponaria officinalis karyology and karyotype by means of image analyzer and atomic force microscopy
No full textThe aim of this work was to offer a contribution to the characterization of taxonomic entity of Saponaria officinalis (2n 5 28; an herbaceous perennial species; saporin, a type 1 Ribosome Inactivating Protein, is present in leaves and seeds) by a cytogenetic and karyomorphological approach. We investigated the karyotype's morphometry correlated with Stebbin's symmetric index; the same information has been used for computing the indices resemblance between chromosomes (REC), symmetric indices (SYI), and total form (TF%) which allow the comparison between species and evaluation of karyological evolution. Fluorescence intensities of the stained nuclei were measured by a flow cytometer and, for the first time, values for nuclear DNA content were estimated by comparing nuclei fluorescence intensities of the test population with those of appropriate internal DNA standards. Our study is also aimed to introduce chromosomal volumes, which were determined by atomic force microscopy (AFM), as novel karyomorphological parameter which could allow for chromosome discrimination especially when tiny ones are present. © 2008 Wiley-Liss, Inc
Spermidine inhibits cell cycle progression and induces developmental cell death in the primary root of maize seedlings
No full textInvolvement of polyamine oxidase in cell cycle regulation and developmental cell death in the primary root of maize seedlings
No full textUnderstanding plant reproductive systems through the combined evaluation of molecular markers and flow cytometry.
No full textDNA content differences in laboratory mouse strains determined by flow cytometry
No full textThe nuclear DNA content of seven mouse laboratory strains has been measured by flow cytometry. The differences observed between strains as well as those between sexes within the strain were all statistically significant. The highest DNA content (approximately 6.4 pg/female nucleus) was found in the Balb/c strain; the lowest (approximately 5.7 pg/male nucleus) in the C3H/he strain. The difference between sexes varied from 1.6% (in CD-1 mice) to 6.3% (in nude mice). The interest of these results is twofold. First, the mouse can now be used to study the adaptive significance of genome size variation, so far studied only in plants. Second, DNA content analysis can become a quick method for mouse strain identification
Inheritance of parental genomes in progenies of Poa pratensis L. from sexual and apomictic genotypes as assessed by RAPD markers and Flow Cytometry.
No full textMoving gene(s) responsible for the apomictic trait into crop plants that naturally reproduce through a sexual process would open up new areas in plant breeding and agricultural systems. Kentucky bluegrass (Poa pratensis L.) is one of the most important forage and turf grasses in temperate climates. It reproduces through facultative aposporous parthenogenesis, but the reproductive behaviour ranges naturally from nearly obligate apomixis to complete sexuality. In addition to apomictic reproduction, sexual hybridization may take place. Selfing may also occur, and occasionally reduced egg cells may develop through parthenogenesis generating (poly)haploids. The inheritance of parental genomes was assessed in Kentucky bluegrass progenies by employing RAPD markers in combination with flow cytometry (FCM). Nine progenies from different crosses carried out between completely sexual and highly apomictic genotypes were evaluated in order to probe the reproductive behaviour of the mother plants and to distinguish the different classes of aberrant plants. Not only were maternals and balanced BII hybrids recorded, but so were (poly)triploid BIII hybrids, selfs, and (poly)haploids. The application of these techniques demonstrated that FCM analysis accurately distinguishes the n, 2n, and 3n ploidy levels of progenies, and that RAPD markers unequivocally recognize progenies of apomictic and hybrid origin. The occurrence of aneusomaty was documented in one of the selected sexual genotypes, whose crossed progeny plants manifested two distinct classes of ploidy. The nomenclature BI was adopted to refer to hybrids with a hypodiploid nuclear condition. On the whole, the FCM analysis confirmed most of the RAPD data. The combined evaluation of DNA markers and DNA contents proved to be an efficient screening tool for scoring maternal plants, assessing the genetic origin of aberrant plants, and quantifying the inheritance of parental genomes in Kentucky bluegrass. Hybrid populations from sexual×apomictic matings that segregate for the mode of reproduction represent a valuable basis for attempting to identify molecular markers linked to the apomixis gene(s)
DNA content differences in laboratory mouse strains determined by flow cytometry
No full textThe nuclear DNA content of seven mouse laboratory strains has been measured by flow cytometry. The differences observed between strains as well as those between sexes within the strain were all statistically significant. The highest DNA content (approximately 6.4 pg/female nucleus) was found in the Balb/c strain; the lowest (approximately 5.7 pg/male nucleus) in the C3H/he strain. The difference between sexes varied from 1.6% (in CD-1 mice) to 6.3% (in nude mice). The interest of these results is twofold. First, the mouse can now be used to study the adaptive significance of genome size variation, so far studied only in plants. Second, DNA content analysis can become a quick method for mouse strain identification
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