scholarly journals A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars

2019 ◽  
Vol 12 ◽  
pp. 01019 ◽  
Author(s):  
S. Sabbadini ◽  
L. Capriotti ◽  
C. Limera ◽  
O. Navacchi ◽  
G. Tempesta ◽  
...  
Keyword(s):  
Marker Gene ◽  
Marker Genes ◽  
Wine Grape ◽  
Thompson Seedless ◽  
Multiple Virus ◽  
Almost All ◽  
New Breeding Techniques ◽  
Breeding Techniques ◽  
Selection Of

Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most of Vitis viniferacultivars have high susceptibility to fungal and viral diseases therefore, new breeding techniques (e.g. Cisgenesis, RNAi and gene editing) offer the possibility to introduce new clones of the main cultivars with increased diseases resistance, in order to reduce environmental impact and improve quality in the intensive wine grape industry. This study is finalized to develop efficient in vitro regeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim, in vitro regeneration protocols based on the production of meristematic bulks (Mezzetti et al., 2002) were optimized for different grapevine cultivars (Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks (1103 Paulsen, and 110 Richter). The meristematic bulks were then used as explants for Agrobacteriummediated genetic transformation protocols, by comparing the use of NPTII and e-GFP as marker genes. Results confirmed the efficiency of meristematic bulks as the regenerating tissue to produce new modified plants in almost all the above genotypes. The highest regeneration efficiency in some genotypes allowed the selection of stable modified lines/calli with only the use of e-GFP marker gene. This protocol can be applied in the use of MYB marker gene for the production of cisgenic lines. Genotypes having the highest regeneration and transformation efficiency were also used for transformation experiments using a hairpin gene construct designed to silence the RNA-dependent RNA polymerase (RpRd) of the GFLV and GLRaV3, which would induce multiple virus resistances, and the Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 to control B. cinerea infection.

scholarly journals Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus

2007 ◽  
Vol 88 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Nele Tamberg ◽  
Valeria Lulla ◽  
Rennos Fragkoudis ◽  
Aleksei Lulla ◽  
John K. Fazakerley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Semliki Forest Virus ◽  
Cultured Cells ◽  
Marker Gene ◽  
Virus Production ◽  
Marker Genes ◽  
Replicase Gene ◽  
Marker Virus

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.

scholarly journals Pilot investigation on the dose-dependent impact of irradiation on primary human alveolar osteoblasts in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna-Klara Amler ◽  
Domenic Schlauch ◽  
Selin Tüzüner ◽  
Alexander Thomas ◽  
Norbert Neckel ◽  
...  
Keyword(s):  
Bone Cells ◽  
Marker Gene ◽  
Marker Genes ◽  
Dependent Manner ◽  
Current Standard ◽  
Functional Changes ◽  
Standard Procedures ◽  
The Impact ◽  
Dose Dependent

AbstractRadiotherapy of head and neck squamous cell carcinoma can lead to long-term complications like osteoradionecrosis, resulting in severe impairment of the jawbone. Current standard procedures require a 6-month wait after irradiation before dental reconstruction can begin. A comprehensive characterization of the irradiation-induced molecular and functional changes in bone cells could allow the development of novel strategies for an earlier successful dental reconstruction in patients treated by radiotherapy. The impact of ionizing radiation on the bone-forming alveolar osteoblasts remains however elusive, as previous studies have relied on animal-based models and fetal or animal-derived cell lines. This study presents the first in vitro data obtained from primary human alveolar osteoblasts. Primary human alveolar osteoblasts were isolated from healthy donors and expanded. After X-ray irradiation with 2, 6 and 10 Gy, cells were cultivated under osteogenic conditions and analyzed regarding their proliferation, mineralization, and expression of marker genes and proteins. Proliferation of osteoblasts decreased in a dose-dependent manner. While cells recovered from irradiation with 2 Gy, application of 6 and 10 Gy doses not only led to a permanent impairment of proliferation, but also resulted in altered cell morphology and a disturbed structure of the extracellular matrix as demonstrated by immunostaining of collagen I and fibronectin. Following irradiation with any of the examined doses, a decrease of marker gene expression levels was observed for most of the investigated genes, revealing interindividual differences. Primary human alveolar osteoblasts presented a considerably changed phenotype after irradiation, depending on the dose administered. Mechanisms for these findings need to be further investigated. This could facilitate improved patient care by re-evaluating current standard procedures and investigating faster and safer reconstruction concepts, thus improving quality of life and social integrity.

scholarly journals Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

2020 ◽  
Vol 21 (7) ◽  
pp. 2627
Author(s):  
Olivier Gröninger ◽  
Samuel Hess ◽  
Dirk Mohn ◽  
Elia Schneider ◽  
Wendelin Stark ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Calcium Phosphate ◽  
Amorphous Calcium Phosphate ◽  
Marker Gene ◽  
Ion Concentration ◽  
Marker Genes ◽  
Calcium Phosphate Nanoparticles ◽  
Cell Commitment

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell–material interaction, the material–medium interaction was also important for the stem cell commitment here, affecting the cell–medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

scholarly journals Bioactivity of vitamin E

2006 ◽  
Vol 19 (2) ◽  
pp. 174-186 ◽  
Author(s):  
Regina Brigelius-Flohé
Keyword(s):  
Vitamin E ◽  
Molecular Basis ◽  
Antioxidant Properties ◽  
The Body ◽  
The Novel ◽  
Transfer Protein ◽  
Almost All ◽  
Selection Of

More than 80 years after the discovery of the essentiality of vitamin E for mammals, the molecular basis of its action is still an enigma. From the eight different forms of vitamin E, only α-tocopherol is retained in the body. This is in part due to the specific selection of RRR-α-tocopherol by the α-tocopherol transfer protein and in part by its low rate of degradation and elimination compared with the other vitamers. Since the tocopherols have comparable antioxidant properties and some tocotrienols are even more effective in scavenging radicals, the antioxidant capacity cannot be the explanation for its essentiality, at least not the only one. In the last decade, a high number of so-called novel functions of almost all forms of vitamin E have been described, including regulation of cellular signalling and gene expression. α-Tocopherol appears to be most involved in gene regulation, whereas γ-tocopherol appears to be highly effective in preventing cancer-related processes. Tocotrienols appear to be effective in amelioration of neurodegeneration. Most of the novel functions of individual forms of vitamin E have been demonstrated in vitro only and require in vivo confirmation. The distinct bioactivities of the various vitamers are discussed, considering their metabolism and the potential functions of metabolites.

Clonal lifespan of Paramecium tetraurelia: effect of selection on its extension and use of fissions for its determination

1987 ◽  
Vol 88 (1) ◽  
pp. 129-138
Author(s):  
Y. Takagi ◽  
T. Nobuoka ◽  
M. Doi
Keyword(s):  
Marked Effect ◽  
Marker Genes ◽  
Paramecium Tetraurelia ◽  
Random Events ◽  
The Mean ◽  
Human Diploid Cells ◽  
Diploid Cells ◽  
Poor Nutrition ◽  
Selection Of

Paramecia cells, like human diploid cells cultured in vitro, provide a useful model system for understanding the mechanism that limits division potential. The reported maxima of the clonal lifespan of Paramecium tetraurelia fall into two ranges: from 220 to 258 fissions and from 310 to 325 fissions. We found that neither the selection of vigorous lines nor the cryptic occurrence of autogamy offers a plausible explanation for the much longer lifespans in the latter range. We found the sporadic occurrence of very long clonal lifespans, such as 330 fissions, without selection and autogamy. Selection, which was evaluated by using different methods to maintain lines, had little effect on the extension of the maximal clonal lifespan, whereas it did have a marked effect on the extension of the mean clonal lifespan. Autogamy, which was checked with two closely linked marker genes, was frequent, but only during the period when lines were terminating. Statistical analysis on the mean clonal lifespans of two groups of subclones cultured at 25 and 20 degrees C or with rich and poor nutrition showed that the clonal lifespan that was fission-related under favourable conditions tended to dissociate from fissions under less favourable conditions. We discuss mechanisms that determine the clonal lifespan, programmed events contributing to the maximum clonal lifespan and random events contributing to the mean clonal lifespan.

scholarly journals Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Snehasish Sarkar ◽  
Souri Roy ◽  
Sudip K. Ghosh
Keyword(s):  
Insect Pests ◽  
Marker Gene ◽  
Pigeon Pea ◽  
Small Subunit ◽  
Grain Legume ◽  
Marker Genes ◽  
35S Promoter ◽  
Selectable Marker Genes ◽  
Insect Resistant

AbstractPigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.

scholarly journals Short-Term Cryopreservation and Thawing have Minimal Effects on Plasmodium Falciparum Ex Vivo Invasion Profile

2021 ◽  
Author(s):  
Laty G. Thiam ◽  
Felix Ansah ◽  
Makhtar Niang ◽  
Gordon Awandare ◽  
Yaw Aniweh
Keyword(s):  
Ex Vivo ◽  
Clinical Isolates ◽  
Short Term ◽  
Culture Adaptation ◽  
Erythrocyte Invasion ◽  
Laboratory Facilities ◽  
Reliable Mechanism ◽  
Almost All ◽  
Selection Of

Abstract Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. Our findings indicate that natural P. falciparum infections mostly occur as polyclonal infections. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

scholarly journals Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma

2016 ◽  
Vol 174 (3) ◽  
pp. 343-353 ◽  
Author(s):  
Matthias Kroiss ◽  
Dietmar Plonné ◽  
Sabine Kendl ◽  
Diana Schirmer ◽  
Cristina L Ronchi ◽  
...  
Keyword(s):  
Er Stress ◽  
Adrenocortical Carcinoma ◽  
Marker Gene ◽  
Index Patient ◽  
Cholesterol Content ◽  
Serum Lipoprotein ◽  
Marker Genes ◽  
Stress Marker ◽  
High Concentrations

ObjectiveOral mitotane (o,p′-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC). AimSerum mitotane concentrations >14 mg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p′-dichlorodiphenylacetic acid (o,p′-DDA) and o,p′-dichlorodiphenyldichloroethane (o,p′-DDE).DesignLipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI–H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins.ResultsChyle of the index patient contained 197 mg/ml mitotane, 53 mg/ml o,p′-DDA, and 51 mg/l o,p′-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction.DiscussionMitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p′-DDE was similarly distributed, but 87.9±4.2% of o,p′-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI–H295 cells and reduced ER stress marker gene expression.ConclusionMitotane absorption involves chylomicron binding. High concentrations of o,p′-DDA and o,p′-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.

scholarly journals Selection of a marker gene to construct a reference library for wetland plants, and the application of metabarcoding to analyze the diet of wintering herbivorous waterbirds

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2345 ◽  
Author(s):  
Yuzhan Yang ◽  
Aibin Zhan ◽  
Lei Cao ◽  
Fanjuan Meng ◽  
Wenbin Xu
Keyword(s):  
Marker Gene ◽  
Pcr Amplification ◽  
Food Composition ◽  
Wetland Plants ◽  
Marker Genes ◽  
Amplification Efficiency ◽  
Reference Library ◽  
Bean Goose ◽  
Dna Metabarcoding ◽  
Almost All

Food availability and diet selection are important factors influencing the abundance and distribution of wild waterbirds. In order to better understand changes in waterbird population, it is essential to figure out what they feed on. However, analyzing their diet could be difficult and inefficient using traditional methods such as microhistologic observation. Here, we addressed this gap of knowledge by investigating the diet of greater white-fronted gooseAnser albifronsand bean gooseAnser fabalis, which are obligate herbivores wintering in China, mostly in the Middle and Lower Yangtze River floodplain. First, we selected a suitable and high-resolution marker gene for wetland plants that these geese would consume during the wintering period. Eight candidate genes were included:rbcL,rpoC1,rpoB,matK,trnH-psbA,trnL (UAA),atpF-atpH, andpsbK-psbI. The selection was performed via analysis of representative sequences from NCBI and comparison of amplification efficiency and resolution power of plant samples collected from the wintering area. ThetrnL gene was chosen at last with c/h primers, and a local plant reference library was constructed with this gene. Then, utilizing DNA metabarcoding, we discovered 15 food items in total from the feces of these birds. Of the 15 unique dietary sequences, 10 could be identified at specie level. As for greater white-fronted goose, 73% of sequences belonged toPoaceaespp., and 26% belonged toCarexspp. In contrast, almost all sequences of bean goose belonged toCarexspp. (99%). Using the same samples, microhistology provided consistent food composition with metabarcoding results for greater white-fronted goose, while 13% ofPoaceaewas recovered for bean goose. In addition, two other taxa were discovered only through microhistologic analysis. Although most of the identified taxa matched relatively well between the two methods, DNA metabarcoding gave taxonomically more detailed information. Discrepancies were likely due to biased PCR amplification in metabarcoding, low discriminating power of current marker genes for monocots, and biases in microhistologic analysis. The diet differences between two geese species might indicate deeper ecological significance beyond the scope of this study. We concluded that DNA metabarcoding provides new perspectives for studies of herbivorous waterbird diets and inter-specific interactions, as well as new possibilities to investigate interactions between herbivores and plants. In addition, microhistologic analysis should be used together with metabarcoding methods to integrate this information.

203 ESTABLISHMENT OF AN IN VITRO SCREENING ASSAY FOR DEVELOPMENTAL TOXICITY USING MOUSE EMBRYOID BODIES

2014 ◽  
Vol 26 (1) ◽  
pp. 216
Author(s):  
Y. Choi ◽  
E. B. Jeung
Keyword(s):  
Developmental Toxicity ◽  
Marker Gene ◽  
Embryoid Bodies ◽  
Toxic Chemicals ◽  
Penicillin G ◽  
Marker Genes ◽  
High Dose ◽  
Germ Layers ◽  
Hes Cells

Toxicity is generally referred to as the degree to which a substance can damage an organism. According to numerous reports, determination of toxicity through an in vitro system is an economical and ethical method, compared with an in vivo system using animals. The hES cells can differentiate into three germ layers and the unique feature of hES cells from the three germ layers via the EB formation adds good insight in understanding human developmental biology in vitro. Evaluation of developmental toxicity of a compound on early embryo states can therefore be based on its effect on EB formation. In this study, we used mouse embryonic stem cells (mESC) to investigate the effects of several developmental toxic chemicals on the formation of EBs. We used the EB hanging drop method and tested five toxic chemicals; cytosine arabinoside, dexamethasone, hydroxyurea, indomethacin, 5-fluorouracil, and two negative controls; ascorbic acid and penicillin G. We demonstrated a significant reduction of EB size after treatment with a high dose of each chemical. We evaluated cell toxicity by performing measurements of cell viability after treatment with each chemical. Expression of apoptosis-related genes (p53, Sirt1, p21, Puma, Noxa, Mdm2) and pluripotency marker genes (Oct4, Nanog, Sox2, ZFP206) and differentiation marker genes, such as endoderm (HNF4, AFP), mesoderm (T-brachy), and ectoderm (Pax6) were determined by quantitative real-time PCR. The chemicals induced abnormal differentiation and apoptosis. We confirmed apoptotic positive cells by TUNEL assay. In addition, we also demonstrate the reduction of the size of EBs through expression of apoptosis-related marker genes (p53, Caspase-3, PARP) and necrosis-related marker gene (HMGB1). The results obtained demonstrate that EBs can be used as an in vitro model for testing developmental toxicity.

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