scholarly journals Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae

2020 ◽  
Vol 21 (3) ◽  
pp. 718 ◽  
Author(s):  
Ana Molina-Márquez ◽  
Marta Vila ◽  
Rocío Rengel ◽  
Emilio Fernández ◽  
Federico García-Maroto ◽  
...  
Keyword(s):  
Marker Gene ◽  
Gene Products ◽  
Rt Pcr ◽  
Resistance Marker ◽  
Selective Marker ◽  
Antibiotic Resistance Marker ◽  
Expression Plasmid ◽  
Simple Method ◽  
Independent Gene ◽  
Selection Of

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3′-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

2005 ◽  
Vol 32 (8) ◽  
pp. 671 ◽  
Author(s):  
Song Chen ◽  
Christopher A. Helliwell ◽  
Li-Min Wu ◽  
Elizabeth S. Dennis ◽  
Narayana M. Upadhyaya ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Marker Gene ◽  
Direct Repeat ◽  
Transgene Silencing ◽  
Selective Marker ◽  
Hairpin Rna ◽  
Transgenic Lines ◽  
Flanking Sequences ◽  
Dna Vector ◽  
Selection Of

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d / r) or inverted-repeat (i / r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i / r T-DNA insertions is a primary inducer of transgene silencing in plants.

scholarly journals Modified Cre-loxPRecombination in Aspergillus oryzae by Direct Introduction of Cre Recombinase for Marker Gene Rescue

2012 ◽  
Vol 78 (12) ◽  
pp. 4126-4133 ◽  
Author(s):  
Osamu Mizutani ◽  
Kazuo Masaki ◽  
Katsuya Gomi ◽  
Haruyuki Iefuji
Keyword(s):  
Aspergillus Oryzae ◽  
Selectable Marker ◽  
Marker Gene ◽  
Molecular Technique ◽  
Direct Introduction ◽  
Expression Plasmid ◽  
Simple Method ◽  
Gene Deletions ◽  
Content Type ◽  
Recombination System

ABSTRACTMarker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxPrecombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced intoAspergillus oryzaeprotoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced intoA. oryzaeprotoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked byloxPsites. By using this method, we readily constructed a marker gene-rescued strain lackingligDto optimize homologous recombination. Furthermore, we succeeded in integrative recombination at aloxPsite inA. oryzae. Thus, we developed a simple method to use the Cre-loxPrecombination system inA. oryzaeby direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.

scholarly journals Identification, Characterization, and Molecular Detection of Alfalfa mosaic virus in Potato

2006 ◽  
Vol 96 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
H. Xu ◽  
J. Nie
Keyword(s):  
Mosaic Virus ◽  
Gene Sequence ◽  
Alfalfa Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Simple Method ◽  
Cp Gene ◽  
Nucleotide Sequence Alignment

Alfalfa mosaic virus (AMV) was detected in potato fields in several provinces in Canada and characterized by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The identity of eight Canadian potato AMV isolates was confirmed by sequence analysis of their coat protein (CP) gene. Sequence and phylogenetic analysis indicated that these eight AMV potato isolates fell into one strain group, whereas a slight difference between Ca175 and the other Canadian AMV isolates was revealed. The Canadian AMV isolates, except Ca175, clustered together among other strains based on alignment of the CP gene sequence. To detect the virus, a pair of primers, AMV-F and AMV-R, specific to the AMV CP gene, was designed based on the nucleotide sequence alignment of known AMV strains. Evaluations showed that RT-PCR using this primer set was specific and sensitive for detecting AMV in potato leaf and tuber samples. AMV RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 tubers. Restriction analysis of PCR amplicons with SacI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by restriction fragment length polymorphism analysis may be a useful approach for screening potato samples on a large scale for the presence of AMV.

scholarly journals Selection of dancer member using simple additive weighting

2018 ◽  
Vol 7 (3) ◽  
pp. 1096 ◽  
Author(s):  
Hana Adela ◽  
Kamarul Azmi Jasmi ◽  
Bushrah Basiron ◽  
Miftachul Huda ◽  
Andino Maseleno
Keyword(s):  
Community Life ◽  
Simple Method ◽  
Unitary State ◽  
Simple Additive Weighting ◽  
Selection Of

Travel and dance form in Indonesia is closely related to the development of community life, both in terms of ethnic structure and within the scope of the unitary state. This study determines the criteria for selecting dancer members and how to apply the qualified Simple method. Based on predetermined criteria is the ability to dance physical flexibility, skilled, nimble, confident, have the ability, fill out the form, and certificate of achievement. From the results obtained values then V1, V2, V3, V4, V5 is a member of a qualified dancer and has a highest value with a score of 100 which was obtained from V2. 

scholarly journals Transoperative refusion: a simple and safe method in emergency surgery

2014 ◽  
Vol 41 (4) ◽  
pp. 292-296
Author(s):  
Luiz Carlos Buarque Gusmão ◽  
Sérgio Henrique Chagas Valoes ◽  
José da Silva Leitão Neto
Keyword(s):  
Cost Benefit ◽  
Blood Transfusions ◽  
Factor V ◽  
West Nile Fever ◽  
Clotting Factors ◽  
Blood Contamination ◽  
Simple Method ◽  
Cost Benefit Ratio ◽  
The Cost ◽  
Selection Of

The objective is to reinforce the importance of blood reinfusion as a cheap, safe and simple method, which can be used in small hospitals, especially those in which there is no blood bank. Moreover, even with the use of devices that perform the collection and filtration of blood, more recent studies show that the cost-benefit ratio is much better when autologous transfusion is compared with blood transfusions, even when there is injury to hollow viscera and blood contamination. It is known that the allogeneic blood transfusion carries a number of risks to patients, among them are the coagulation disorders mediated by excess enzymes in the conserved blood, and deficiency in clotting factors, mainly the Factor V, the proacelerin. Another factor would be the risk of contamination with still unknown pathogens or that are not investigated during screening for selection of donors, such as the West Nile Fever and Creutzfeldt-Jacob, better known as "Mad Cow" disease. Comparing both methods, we conclude that blood autotransfusion has numerous advantages over heterologous transfusion, even in large hospitals. We are not against blood transfusions, just do not agree that the patient's own blood is discarded without making sure there will be enough blood in stock to get him out of the hemorrhagic shock.

scholarly journals A multi-site cutting device implements efficiently the divide-and-conquer strategy in tumor sampling

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 1587 ◽  
Author(s):  
Jose I. Lopez ◽  
Jesus M. Cortes
Keyword(s):  
Synthetic Data ◽  
Daily Practice ◽  
Divide And Conquer ◽  
Intratumor Heterogeneity ◽  
Pathology Laboratory ◽  
Simple Method ◽  
French Fry ◽  
Histological Processing ◽  
Tumor Sampling ◽  
Selection Of

We recently showed that in order to detect intra-tumor heterogeneity a Divide-and-Conquer (DAC) strategy of tumor sampling outperforms current routine protocols. This paper is a continuation of this work, but here we focus on DAC implementation in the Pathology Laboratory. In particular, we describe a new simple method that makes use of a cutting grid device and is applied to clear cell renal cell carcinomas for DAC implementation. This method assures a thorough sampling of large surgical specimens, facilitates the demonstration of intratumor heterogeneity, and saves time to pathologists in the daily practice. The method involves the following steps: 1. Thin slicing of the tumor (by hand or machine), 2. Application of a cutting grid to the slices (e.g., a French fry cutter), resulting in multiple tissue cubes with fixed position within the slice, 3. Selection of tissue cubes for analysis, and finally, 4. Inclusion of selected cubes into a cassette for histological processing (with about eight tissue fragments within each cassette). Thus, using our approach in a 10 cm in-diameter-tumor we generate 80 tumor tissue fragments placed in 10 cassettes and, notably, in a tenth of time. Eighty samples obtained across all the regions of the tumor will assure a much higher performance in detecting intratumor heterogeneity, as proved recently with synthetic data.

scholarly journals Osteogenic differentiation potential and marker gene expression of different porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media

2020 ◽  
Author(s):  
Sangeetha Kannan ◽  
Jyotirmoy Ghosh ◽  
Sujoy K. Dhara
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Marker Gene ◽  
Microscopical Examination ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Basal Media ◽  
Selection Of

AbstractMultipotent porcine mesenchymal stem cells (pMSC) are indispensable for research and therapeutic use. Derivation and culture media might affect the selection of MSC subpopulation and thus the differentiation potential of cells. In this study we evaluated the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on porcine bone marrow MSC derivation; pre-differentiation expression of ALP, COL1A1, SPP1 and BGLAP osteogenic marker genes at passage 5 and 10 pMSC; and differentiation potential of passage 5 pMSC. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopical examination and calcium deposit in osteocytes was confirmed by Alizarin Red S staining. Results indicated media independent selection of different bone marrow MSC subpopulations with different surface marker gene expressions. Many pMSC subpopulations in different media had CD14+ expressing cells. We also observed basal media dependent changes in osteogenic markers expression and differentiation potential of pMSC. The αMEM/aDMEM media grown pMSC showed best osteogenic differentiation potential. We thus recommended the testing of αMEM/aDMEM mixed media in other species for pre-differentiation MSC culture that are intended for better osteogenic differentiation.SummaryPre-differentiation basal media influence osteogenic differentiation potential of mesenchymal stem cells (MSC). Among the tested media, αMEM/aDMEM was the best for pre-differentiation porcine MSC culture intending to use in osteogenesis.

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