scholarly journals Agrobacterium tumefaciens-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—Euglena gracilis

2021 ◽  
Vol 22 (12) ◽  
pp. 6299
Author(s):  
Ina Becker ◽  
Binod Prasad ◽  
Maria Ntefidou ◽  
Viktor Daiker ◽  
Peter Richter ◽  
...  
Keyword(s):  
Euglena Gracilis ◽  
Nuclear Genome ◽  
Pcr Amplification ◽  
Foot And Mouth Disease ◽  
Marker Genes ◽  
Nuclear Transformation ◽  
Gus Gene ◽  
Gene Encoding ◽  
Mouth Disease Virus ◽  
Resistant Cells

Euglena gracilis (E. gracilis) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of E. gracilis mediated by Agrobacterium (A. tumefaciens). E. gracilis was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. A. tumefaciens-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gus gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gus gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (hptII) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of Euglena with the hptII-2A–gus gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when A. tumefaciens LBA4404 (A600 = 1.0) and E. gracilis (A750 = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer E. gracilis for producing high-value products and fundamental studies.

scholarly journals Genetic Transformation of Iris germanica Mediated by Agrobacterium tumefaciens

1999 ◽  
Vol 124 (6) ◽  
pp. 575-580 ◽  
Author(s):  
Zoran Jeknic ◽  
Stephen P. Lee ◽  
Joel Davis ◽  
Richard C. Ernst ◽  
Tony H.H. Chen
Keyword(s):  
Southern Blot Analysis ◽  
Selectable Marker ◽  
Nuclear Genome ◽  
Suspension Cultures ◽  
Marker Genes ◽  
Gus Gene ◽  
Gene Encoding ◽  
Selectable Marker Genes ◽  
Future Improvement ◽  
Iris Germanica

A protocol was developed for production of transgenic iris plants (Iris germanica L. `Skating Party') from regenerable suspension cultures via Agrobacterium-mediated transformation. We tested a series of selection agents, and identified hygromycin and geneticin as the most suitable for selecting transformed iris cells. Suspension cultures of iris were cocultured for 3 days with A. tumefaciens LBA 4404(pTOK233) carrying an intron-interrupted uidA (GUS) gene encoding β-glucuronidase, and hpt (hygromycin) and nptII (geneticin) selectable marker genes. Hygromycin- or geneticin-resistant calli having GUS enzyme activity were identified and used to induce plant regeneration. More than 300 morphologically normal transgenic iris plants were obtained in ≈6 months. About 80% of the transformants were GUS-positive and NPTII-positive (paromomycin-resistant). Integration of transgenes into the nuclear genome of iris plants was confirmed by Southern blot analysis. We have, therefore, developed an efficient A. tumefaciens-mediated transformation system for Iris germanica, which will allow future improvement of this horticulturally important ornamental monocot via genetic engineering.

scholarly journals Study of the genetic heterogeneity of SAT-2 foot-and-mouth disease virus in sub-Saharan Africa with specific focus on East Africa

2007 ◽  
Vol 74 (4) ◽  
Author(s):  
M. Sahle ◽  
R.M. Dwarka ◽  
E.H. Venter ◽  
W. Vosloo
Keyword(s):  
East Africa ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Sub Saharan Africa ◽  
Gene Encoding ◽  
Western Africa ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Sub Saharan ◽  
Unrelated Strain

The epidemiology of serotype SAT-2 foot-and-mouth disease was investigated in sub-Saharan Africa by phylogenetic analysis using the 1D gene encoding the major antigenic determinant. Fourteen genotypes were identified of which three are novel and belong to East Africa, bringing the total number of genotypes for that region to eight. The genotypes clustered into three lineages that demonstrated surprising links between East, southern and south-western Africa. One lineage was unique to West Africa. These results established numerous incursions across country borders in East Africa and long term conservation of sequences for periods up to 41 years. Ethiopia, Kenya and Uganda have all experienced outbreaks from more than one unrelated strain, demonstrating the potential for new introductions. The amount of variation observed within this serotype nearly equalled that which was found between serotypes; this has severe implications for disease control using vaccination.

scholarly journals Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Seo-Yong Lee ◽  
Yeo-Joo Lee ◽  
Rae-Hyung Kim ◽  
Jeong-Nam Park ◽  
Min-Eun Park ◽  
...  
Keyword(s):  
Vaccine Strain ◽  
Antigenic Variation ◽  
Foot And Mouth Disease ◽  
Vaccine Virus ◽  
Mouth Disease ◽  
Economic Losses ◽  
Gene Encoding ◽  
Virus Challenge ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACT There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV. IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

scholarly journals Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.

1989 ◽  
Vol 109 (6) ◽  
pp. 2589-2601 ◽  
Author(s):  
K L Kindle ◽  
R A Schnell ◽  
E Fernández ◽  
P A Lefebvre
Keyword(s):  
Enzyme Activity ◽  
Nitrate Reductase ◽  
Plasmid Dna ◽  
Reductase Activity ◽  
Nuclear Genome ◽  
Nuclear Transformation ◽  
Wild Type ◽  
Gene Encoding ◽  
Photosynthetic Organism ◽  
Low Efficiency

We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using micro-projectile bombardment to introduce the gene encoding nitrate reductase into a nit1 mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit+ phenotype were stable through mitosis and meiosis, even in the absence of selection. nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit+ strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected. With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.

scholarly journals Development of Reverse Transcription-PCR (Oligonucleotide Probing) Enzyme-Linked Immunosorbent Assays for Diagnosis and Preliminary Typing of Foot-and-Mouth Disease: a New System Using Simple and Aqueous-Phase Hybridization

2000 ◽  
Vol 38 (12) ◽  
pp. 4604-4613 ◽  
Author(s):  
Soren Alexandersen ◽  
Morag A. Forsyth ◽  
Scott M. Reid ◽  
Graham J. Belsham
Keyword(s):  
Reverse Transcription ◽  
Pcr Amplification ◽  
Foot And Mouth Disease ◽  
Variable Region ◽  
Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Mouth Disease Virus ◽  
Foot And Mouth

A reverse transcription-PCR (RT-PCR)–enzyme-linked immunosorbent assay system that detects a relatively conserved region within the RNA genome of all seven serotypes of foot-and-mouth disease virus (FMDV) has been developed. The high specificity of the assay is achieved by including a rapid hybridization step with a biotin-labeled internal oligonucleotide. The assay is highly sensitive, fast, and easy to perform. A similar assay, based on a highly variable region of the FMDV genome and employing a single asymmetric RT-PCR and multiple hybridization oligonucleotides, was developed to demonstrate the method's ability to type FMDV. Based on our theoretical and practical knowledge of the methodology, we predict that similar assays are applicable to diagnosis and strain differentiation in any system amenable to PCR amplification.

Biophysical Measurement of Molecular Weight of Foot-and-Mouth Disease RNA Fragments

1974 ◽  
Vol 32 ◽  
pp. 262-263
Author(s):  
Sydney S. Breese ◽  
Howard L. Bachrach
Keyword(s):  
Chemical Properties ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Distilled Water ◽  
Bovine Plasma ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Carbon Coated ◽  
Rna Fragments ◽  
Physical And Chemical

Continuing studies on the physical and chemical properties of foot-and-mouth disease virus (FMDV) have included electron microscopy of RNA strands released when highly purified virus (1) was dialyzed against demlneralized distilled water. The RNA strands were dried on formvar-carbon coated electron microscope screens pretreated with 0.1% bovine plasma albumin in distilled water. At this low salt concentration the RNA strands were extended and were stained with 1% phosphotungstic acid. Random dispersions of strands were recorded on electron micrographs, enlarged to 30,000 or 40,000 X and the lengths measured with a map-measuring wheel. Figure 1 is a typical micrograph and Fig. 2 shows the distributions of strand lengths for the three major types of FMDV (A119 of 6/9/72; C3-Rezende of 1/5/73; and O1-Brugge of 8/24/73.

High resolution surface structure of foot-and-mouth disease virus

1978 ◽  
Vol 36 (2) ◽  
pp. 376-377
Author(s):  
S. S. Breese ◽  
H. L. Bachrach
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Surface Structure ◽  
Disease Virus ◽  
Potassium Phosphate ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Physical Measurements ◽  
Mouth Disease Virus ◽  
Foot And Mouth

Models for the structure of foot-and-mouth disease virus (FMDV) have been proposed from chemical and physical measurements (Brown, et al., 1970; Talbot and Brown, 1972; Strohmaier and Adam, 1976) and from rotational image-enhancement electron microscopy (Breese, et al., 1965). In this report we examine the surface structure of FMDV particles by high resolution electron microscopy and compare it with that of particles in which the outermost capsid protein VP3 (ca. 30, 000 daltons) has been split into smaller segments, two of which VP3a and VP3b have molecular weights of about 15, 000 daltons (Bachrach, et al., 1975).Highly purified and concentrated type A12, strain 119 FMDV (5 mg/ml) was prepared as previously described (Bachrach, et al., 1964) and stored at 4°C in 0. 2 M KC1-0. 5 M potassium phosphate buffer at pH 7. 5. For electron microscopy, 1. 0 ml samples of purified virus and trypsin-treated virus were dialyzed at 4°C against 0. 2 M NH4OAC at pH 7. 3, deposited onto carbonized formvar-coated copper screens and stained with phosphotungstic acid, pH 7. 3.

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