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1
Blank, Robert H. "Politics and Genetic Engineering." Politics and the Life Sciences 11, no. 1 (February 1992): 81–85. http://dx.doi.org/10.1017/s0730938400017226.
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THE PURPOSE OF THIS short note is to update readers on recent developments in, and political implications of, genetic engineering, a critical tool in the expanding field of biotechnology. Based on new understanding of the mechanisms of DNA, molecular biologists are now able to chemically cut genes or sets of genes from one organism and splice them into the DNA of another. This is called recombinant DNA (rDNA) technology. Although these techniques were first applied to bacteria and yeasts, in the last decade researchers have made remarkable strides in putting foreign genes into more complex plants and animals.
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Dodds, John H., and Jesse M. Jaynes. "Crop Plant Genetic Engineering: Science Fiction to Science Fact." Outlook on Agriculture 16, no. 3 (September 1987): 111–15. http://dx.doi.org/10.1177/003072708701600303.
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Recombinant DNA technology covers a wide range of biochemical techniques used to cut, splice, and move DNA from one organism to another. Genetic engineering began as a basic scientific study to learn more about gene expression and gene structure in bacteria. In the last 10 years the techniques of recombinant DNA technology have moved from the university research laboratory to the industrial production level. The techniques are applicable to all organisms and studies have been made of the genomes of viruses, bacteria, yeasts, animals, and plants. It is the latter, genetic engineering of plants, which is covered in this article.
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Bogomolova, E. G., P. M. Kopeykin, and A. A. Tagaev. "Genetic engineering approaches to the development of modern therapeutics." Medical academic journal 20, no. 3 (September 15, 2020): 49–60. http://dx.doi.org/10.17816/maj34092.
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The classic approach to production of protein-based therapeutics is their isolation from natural sources. This approach was associated with a number of difficulties, such as collecting the primary material from natural sources, isolating and purifying the protein, and its standardizing. With the development of recombinant DNA technology, itbecame possible to obtain large quantities of protein preparations lacking any contaminations. Human insulin produced using recombinant DNA technology is the first commercial therapeutic obtained by this way. Due to the rapid development of genetic engineering technologies, a large number of proteins have been obtained inEscherichia colicells. In recent years, the approach for the development of drugs based on DNA molecules containing genes encoding therapeutic proteins has been developing more actively. Today, many scientists believe in the prospects of application of DNA vaccines. The ease of production, stability, the ability to mimic natural infections and elicit appropriate immune responses make this vaccine platform extremely attractive. Delivery and targeting of immunologically relevant cells are major tasks for maximizing the immunogenicity of DNA vaccines. Several different approaches that are currently being used to achieve this goal are discussed in this review. Pharmaceuticals based on nucleic acids have a number of undeniable advantages. The main options for prophylactic RNA vaccines, the methods used to deliver RNA to the cell, and methods for increasing the effectiveness of RNA vaccines are discussed. Usage of therapeutic drugs based on protein molecules and low molecular weight compounds is complicated by the fact that they cannot be targeted at a specific gene or its protein product, responsible for the occurrence of the disease. Action of nucleic acids can be directly directed to a particular DNA region in order to edit its nucleotide sequence. This method allows to correct a genetic defect, eliminating the cause of the disease. The principles of gene therapy and the successes achieved in this area are discussed. This review summarizes current achievements in the development of drugs based on recombinant proteins and nucleic acids.
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Ljubijankic, Goran. "Genetic engineering: from a clone to a protein." Genetika 34, no. 2-3 (2002): 73–84. http://dx.doi.org/10.2298/gensr0203073l.
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October 15, 1980, was a date when the highest increase of the share price of one company was recorded on the world most powerful New York change - rates of shares of the company Genetech jumped by more than 2.5 times in only 20 minutes. This event was a grand entrance of a new technology into the global economy and it irreversibly established a brand new status of biology within the development of civilization. Genetech is one of the first companies within the field of molecular biotechnology that accomplished such an enormous commercial success no more than seven years upon the last discoveries in the series of scientific findings that had provided formulation of a new technology designated as genetic engineering or a recombinant DNA technology. Today, 20 years later, it can be rightfully claimed that expectations of the new technology are fulfilled: even if the qualitative shift it provided to fundamental research is disregarded, its direct commercial effects are very convincing - the total value of sold products produced by this technology exceeded the sum of 60 billions dollars in 2000. Let us cite market parameters of only some products manufactured by the application of genetic engineering: annual global consumption of recombinant human insulin amounts to approximately 4.6 tons only in the industrialized part of the world, while the annual sale of recombinant human erythropoietin, interferon's and a human growth hormone amount to 2, i.e. 1.5 billions dollars, respectively.
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Duster, Troy. "Cline's Recombinant DNA Experiment as Political Rashomon." Politics and the Life Sciences 6, no. 1 (August 1987): 15–18. http://dx.doi.org/10.1017/s0730938400002677.
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Robin and Markle's “Let No One Split Asunder: Controversy in Human Genetic Engineering” is a notable contribution to the sociology of science and a clarification of some important social and political issues in the current controversy around the new recombinant DNA technologies. Their four-fold taxonomy of levels of inquiry and analysis is a theoretical advance. It is also a device for illuminating, decoding, deciphering, and finally for bettering our understanding of what happened structurally in the Cline case. Much of the contemporary work in the sociology of science touches on a few, or even several of these dimensions, but because the analytic categories are frequently (if inadvertently) run together, one is often burdened with a confusing vault between levels. (Few authors make the analytic distinctions of different levels as do Robin and Markle, so it is hardly surprising that they would “run them together.”)
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ZOLLER, M. "New recombinant DNA methodology for protein engineering." Current Biology 2, no. 9 (September 1992): 510. http://dx.doi.org/10.1016/0960-9822(92)90692-4.
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MUYSSON, DAVID J., and ANN M. VERRINDER GIBBINS. "THE ALTERATION OF MILK CONTENT BY GENETIC ENGINEERING AND RECOMBINANT DNA-MEDIATED SELECTION TECHNIQUES." Canadian Journal of Animal Science 69, no. 3 (September 1, 1989): 517–27. http://dx.doi.org/10.4141/cjas89-064.
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Since the advent of gene cloning technology, suggestions have been made of specific alterations that could be engineered into the genomes of cattle, sheep or goats to alter their milk composition. These alterations could affect milk protein characteristics or quantity in ways which might be of benefit to milk processing and dairy product manufacturing industries. Other changes could improve the suitability of bovine milk for human consumption. In addition, animals could be engineered to synthesize valuable foreign proteins, such as pharmaceuticals, to be secreted in milk. In this paper, we evaluate these suggestions critically, taking into account current understanding of milk composition and processing, as well as recent information concerning the structure and regulation of genes coding for proteins involved in determining milk content. All these suggested alterations depend on successful production of transgenic animals capable of efficiently expressing introduced genes. The view is examined that some improvements in type or amount of certain milk proteins might better be achieved by conventional breeding practices, or by breeding systems that would rely on recombinant DNA techniques for methods of selection. Key words: Casein, beta-lactoglobulin, α-lactalbumin, plasmin, transgenic, bovine, ovine, lactose
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Biagetti, Marco, Francesca Vitellozzi, and Carla Ceoloni. "Physical mapping of wheat-Aegilops longissima breakpoints in mildew-resistant recombinant lines using FISH with highly repeated and low-copy DNA probes." Genome 42, no. 5 (October 1, 1999): 1013–19. http://dx.doi.org/10.1139/g98-172.
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Fluorescence in situ hybridization (FISH) with multiple probes, consisting of highly repeated DNA sequences (pSc119.2 and pAs1) and of a low-copy, 3BS-specific RFLP sequence (PSR907), enabled determination of the physical position of the wheat-alien breakpoints (BPs) along the 3BS and 3DS arms of common wheat recombinant lines. These lines harbour 3SlS Aegilops longissima segments containing the powdery mildew resistance gene Pm13. In all 3B recombinants, the wheat-Aegilops longissima physical BPs lie within the interval separating the two most distal of the three pSc119.2 3BS sites. In all such recombinants a telomeric segment, containing the most distal of the pSc119.2 3BS sites, was in fact replaced by a homoeologous Ae. longissima segment, marked by characteristic pSc119.2 hybridization sites. Employment of the PSR907 RFLP probe as a FISH marker allowed to resolve further the critical region in the various 3B recombinant lines. Three of them, like the control common wheat, exhibited between the two most distal pSc119.2 sites a single PSR907 FISH site, which was missing in a fourth recombinant line. The amount of alien chromatin can thus be estimated to represent around 20% of the recombinant arm in the three former lines and a maximum of 27% in the latter. A similar physical length was calculated for the alien segment contained in three 3D recombinants, all characterized by the presence of the Ae. longissima pSc119.2 sites distal to the nearly telomeric pAs1 sites of normal 3DS. Comparison between the FISH-based maps and previously developed RFLP maps of the 3BS-3SlS and 3DS-3SlS arms revealed substantial differences between physical and genetic map positions of the wheat-alien BPs and of molecular markers associated with the critical chromosomal portions.Key words: wheat-alien recombinants, chromosome engineering, fluorescence in situ hybridization, highly repeated and low-copy DNA probes, physical versus genetic maps.
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Bernstein, R. L., and Sheldon Krimsky. "Genetic Alchemy, the Social History of the Recombinant DNA Controversy." Leonardo 18, no. 2 (1985): 121. http://dx.doi.org/10.2307/1577892.
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Tirrell, David A., Maurille J. Fournier, and Thomas L. Mason. "Genetic Engineering of Polymeric Materials." MRS Bulletin 16, no. 7 (July 1991): 23–28. http://dx.doi.org/10.1557/s0883769400056505.
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Polymerization reactions are generally divided into two broad classes: step growth or polycondensation reactions (examples would include the synthesis of polyamides and polyesters), and chain growth processes such as those used to prepare polyethylene or polystyrene. These processes are illustrated schematically in Figure 1.The statistical nature of step and chain growth polymerization processes ensures that the products of such reactions must be heterogeneous. Conventional polymeric materials thus consist of mixtures of chains, often characterized by relatively broad distributions of chain length or composition. In many materials applications, this kind of molecular heterogeneity is advantageous since it suppresses crystallization and helps to preserve desirable properties such as optical clarity or elasticity. On the other hand, synthetic developments that afford improved control of macromolecular architecture have had profound impact on materials science and technology. As examples, one can cite the discovery of Ziegler-Natta polymerization, now used to prepare billions of pounds per year of crystalline polyolefins, or the development of living anionic polymerization of olefins, which led directly to block copolymers and the commercially important thermoplastic elastomers.The advent of recombinant DNA methods has provided a basis for developing polymeric materials characterized by essentially absolute uniformity of chain length, sequence, and stereochemistry. This article outlines the principles governing the cloning and expression of artificial genes, and examines the potential role of artificial proteins in polymer materials science.
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Roberts, Robert. "Impact for molecular biology in cardiology." American Journal of Physiology-Lung Cellular and Molecular Physiology 261, no. 4 (October 1, 1991): L8—L14. http://dx.doi.org/10.1152/ajplung.1991.261.4.l8.
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The recent development and application of the techniques of recombinant DNA and molecular biology ignited an explosion in biomedical research, which has been embraced by medicine. However, cardiology as a subspecialty has been slower in adopting these techniques, in part because the heart is a nonproliferating organ and in part because it was not easily accessible until recently. The techniques of recombinant DNA were not possible until the 1970s. In that decade four major discoveries occurred that launched molecular biology into the 21st century. These seminal contributions were 1) the discovery and application of specific restriction endonucleases, 2) the discovery of reverse transcriptase, 3) the development of the cloning technique, and 4) the ability to rapidly sequence nucleic acids. The techniques of recombinant DNA offer several unique advantages over existing scientific disciplines, such as the abilities: 1) to perform in vivo structure-function analysis, 2) to genetically engineer drugs, 3) to perform diagnostic in situ hybridization, 4) to isolate genes responsible for hereditary disorders, and 5) to understand the genetic regulation of cardiac growth. These techniques are discussed in their application to cardiac disorders, including the development of new recombinant molecules for the treatment of coronary thrombosis and the potential to modulate the cardiac growth response to various forms of injury such as myocardial infarction and hypertension. cardiac growth; genetic engineering; molecular genetics; structure function analysis
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Roberts, Robert. "Impact for molecular biology in cardiology." American Journal of Physiology-Heart and Circulatory Physiology 261, no. 4 (October 1, 1991): 8–14. http://dx.doi.org/10.1152/ajpheart.1991.261.4.8.
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The recent development and application of the techniques of recombinant DNA and molecular biology ignited an explosion in biomedical research, which has been embraced by medicine. However, cardiology as a subspecialty has been slower in adopting these techniques, in part because the heart is a nonproliferating organ and in part because it was not easily accessible until recently. The techniques of recombinant DNA were not possible until the 1970s. In that decade four major discoveries occurred that launched molecular biology into the 21st century. These seminal contributions were 1) the discovery and application of specific restriction endonucleases, 2) the discovery of reverse transcriptase, 3) the development of the cloning technique, and 4) the ability to rapidly sequence nucleic acids. The techniques of recombinant DNA offer several unique advantages over existing scientific disciplines, such as the abilities: 1) to perform in vivo structure-function analysis, 2) to genetically engineer drugs, 3) to perform diagnostic in situ hybridization, 4) to isolate genes responsible for hereditary disorders, and 5) to understand the genetic regulation of cardiac growth. These techniques are discussed in their application to cardiac disorders, including the development of new recombinant molecules for the treatment of coronary thrombosis and the potential to modulate the cardiac growth response to various forms of injury such as myocardial infarction and hypertension. cardiac growth; genetic engineering; molecular genetics; structure function analysis
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Stryjewska, Agnieszka, Katarzyna Kiepura, Tadeusz Librowski, and Stanisław Lochyński. "Biotechnology and genetic engineering in the new drug development. Part I. DNA technology and recombinant proteins." Pharmacological Reports 65, no. 5 (September 2013): 1075–85. http://dx.doi.org/10.1016/s1734-1140(13)71466-x.
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Losos, Jan K., David H. Evans, and Ann M. Verrinder Gibbins. "Targeted modification of the complete chicken lysozyme gene by poxvirus-mediated recombination." Biochemistry and Cell Biology 83, no. 2 (April 1, 2005): 230–38. http://dx.doi.org/10.1139/o05-025.
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We have developed a novel ex vivo system for the rapid one-step targeted modification of large eucaryotic DNA sequences. The highly recombinant environment resulting from infection of rabbit cornea cells with the Shope fibroma virus was exploited to mediate precise modifications of the complete chicken lysozyme gene domain (21.5 kb). Homologous recombination was designed to occur between target DNA (containing the complete lysozyme gene domain) maintained in a λ bacteriophage vector and modified targeting DNA maintained in a plasmid. The targeting plasmids were designed to transfer exogenous sequences (for example, β-galactosidase α-complement, green fluorescent protein, and hydrophobic tail coding sequences) to specific sites within the lysozyme gene domain. Cotransfection of the target phage and a targeting plasmid into Shope fibroma virus infected cells resulted in the poxvirus-mediated transfer of the modified sequences from plasmid to phage. Phage DNA (recombinant and nonrecombinant) was then harvested from the total cellular DNA by packaging into λ phage particles and correct recombinants were identified. Four different gene-targeting pairings were carried out, and from 3% to 11% of the recovered phages were recombinant. Using this poxvirus-mediated targeting system, four different regions of the chicken lysozyme gene domain have been modified precisely by our research group overall with a variety of inserts (6–971 bp), deletions (584–3000 bp), and replacements. We have never failed to obtain the desired recombinant. Poxvirus-mediated recombination thus constitutes a routine, rapid, and remarkably efficient genetic engineering system for the precise modification of large eucaryotic gene domains when compared with traditional practices.Key words: chicken lysozyme, gene targeting, homologous recombination, poxvirus, avian bioreactor.
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CHURCH, R. B., F. J. SCHAUFELE, and K. MECKLING. "EMBRYO MANIPULATION AND GENE TRANSFER IN LIVESTOCK." Canadian Journal of Animal Science 65, no. 3 (September 1, 1985): 527–38. http://dx.doi.org/10.4141/cjas85-064.
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In the past few years significant progress has been made in manipulation of reproduction and in development of genetic engineering techniques which can be applied to animal species. Artificial insemination and embryo transfer are now used widely in the livestock industry. The advent of non-surgical embryo collection and transfer, embryo freezing and splitting along with estrus synchronization has allowed the industry to move from the laboratory to the farm. Embryo manipulation now involves embryo splitting to produce monozygotic twins, in vitro fertilization, cross-species fertilization, embryo sexing, and chimeric production of tetraparental animals among others. Advances in recombinant DNA, plasmid construction and embryo manipulation technologies allow the production of genetically engineered animals. The application of recombinant DNA technology involves the isolation and manipulation of desired genes which have potential for significant changes in productivity in genetically engineered livestock. Recombinant DNA constructs involve the coupling of promoter, enhancer, regulatory and structural DNA sequences to form a "fusion gene" which can then be multiplied, purified, assayed and expressed in cell culture prior to being introduced into an animal genome. Such DNA gene constructs are readily available for many human and mouse genes. However, they are not readily available for livestock species because the detailed molecular biology has not yet been established in these species. Gene transfer offers a powerful new tool in animal research. Transfer of genes into the bovine genome has been accomplished. However, successful directed expression of these incorporated genes has not been achieved to date. New combinations of fusion genes may be an effective way of producing transgenic domestic animals which show controlled expression of the desired genes. Embryo manipulation and genetic engineering in livestock species is moving rapidly. The problems being addressed at present in numerous laboratories will result in enhanced livestock production in the not too distant future. Key words: Embryo transfer, embryo manipulation, transgenic livestock, genetic engineering, gene transfer, monozygotic twins
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Dulal, Kalpana, Benjamin Silver, and Hua Zhu. "Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones." Journal of Biomedicine and Biotechnology 2012 (2012): 1–10. http://dx.doi.org/10.1155/2012/357147.
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Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.
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Luo, Yumei, Zikai Chen, Detu Zhu, Haitao Tu, and Shen Quan Pan. "Yeast Actin-Related Protein ARP6 Negatively RegulatesAgrobacterium-Mediated Transformation of Yeast Cell." BioMed Research International 2015 (2015): 1–11. http://dx.doi.org/10.1155/2015/275092.
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The yeasts, includingSaccharomyces cerevisiaeandPichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values.Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related proteinARP6as a negative regulator of AMT.ARP6is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting thatARP6might regulate AMT via SWR-C. Moreover, knockout ofARP6led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.
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Franke, C. A., C. M. Rice, J. H. Strauss, and D. E. Hruby. "Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants." Molecular and Cellular Biology 5, no. 8 (August 1985): 1918–24. http://dx.doi.org/10.1128/mcb.5.8.1918.
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The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.
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Franke, C. A., C. M. Rice, J. H. Strauss, and D. E. Hruby. "Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants." Molecular and Cellular Biology 5, no. 8 (August 1985): 1918–24. http://dx.doi.org/10.1128/mcb.5.8.1918-1924.1985.
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The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.
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Sandoval-Villegas, Nicolás, Wasifa Nurieva, Maximilian Amberger, and Zoltán Ivics. "Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering." International Journal of Molecular Sciences 22, no. 10 (May 11, 2021): 5084. http://dx.doi.org/10.3390/ijms22105084.
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Transposons are mobile genetic elements evolved to execute highly efficient integration of their genes into the genomes of their host cells. These natural DNA transfer vehicles have been harnessed as experimental tools for stably introducing a wide variety of foreign DNA sequences, including selectable marker genes, reporters, shRNA expression cassettes, mutagenic gene trap cassettes, and therapeutic gene constructs into the genomes of target cells in a regulated and highly efficient manner. Given that transposon components are typically supplied as naked nucleic acids (DNA and RNA) or recombinant protein, their use is simple, safe, and economically competitive. Thus, transposons enable several avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture comprising the generation of pluripotent stem cells, the production of germline-transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species and therapy of genetic disorders in humans. This review describes the molecular mechanisms involved in transposition reactions of the three most widely used transposon systems currently available (Sleeping Beauty, piggyBac, and Tol2), and discusses the various parameters and considerations pertinent to their experimental use, highlighting the state-of-the-art in transposon technology in diverse genetic applications.
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Madison, Lara L., and Gjalt W. Huisman. "Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic." Microbiology and Molecular Biology Reviews 63, no. 1 (March 1, 1999): 21–53. http://dx.doi.org/10.1128/mmbr.63.1.21-53.1999.
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SUMMARY Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.
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Fernandez, L., M. L. Marin, S. Langa, R. Martin, C. Reviriego, A. Fernandez, M. Olivares, J. Xaus, and J. M. Rodriguez. "A Novel Genetic Label for Detection of Specific Probiotic Lactic Acid Bacteria." Food Science and Technology International 10, no. 2 (April 2004): 101–8. http://dx.doi.org/10.1177/1082013204043761.
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A novel method for genetic labelling of specific lactic acid bacteria strains was developed. The approach implied the transformation of the hosts with a plasmid containing a heterologous DNA fragment. The sequence of a DNA fragment that has been used to label a variety of genetically modified (GM) soya was used to design a forward primer and three reverse primers yielding PCR products recognisable by their sizes. Stability of the recombinant plasmid in the transformed strains was studied by PCR, and the results varied significantly depending on the strain. To test the usefulness of the DNA label to study in vivo properties of probiotic bacteria, such as viability after transit through the digestive tract, mice were orally inoculated with a genetically-labelled Enterococcus faecium strain. Later, their faeces were aseptically collected and the genetically-labelled strain was detected among the colonies that grew on MRS agar.
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Tariq, Safaa. "The Case of Qualitative Genetics in Fish." International Journal Papier Advance and Scientific Review 1, no. 1 (August 30, 2020): 14–20. http://dx.doi.org/10.47667/ijpasr.v1i1.8.
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Character inheritance or heredity is the inheritance of traits from parents to their children (offspring). The event or process by which a cell of an organism tends to become or possess the characteristics of its parents. The discovery of recombinant DNA opened up developments in genetic engineering. Genetic engineering technology provides a lot of hope for us in various areas of need including gene therapy efforts. Inheritance is the traits or traits of living things that are passed down from generation to generation or passed down from parent to offspring. The traits of a living being are inherited through male sex cells and female sex cells. Normal pigmented goldfish are mated with yellow-striped goldfish on the dorsal spine to produce 100% fish with yellow lines on the dorsal spine.
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Panke, Sven, Juan M. Sánchez-Romero, and Víctor de Lorenzo. "Engineering of Quasi-Natural Pseudomonas putida Strains for Toluene Metabolism through anortho-Cleavage Degradation Pathway." Applied and Environmental Microbiology 64, no. 2 (February 1, 1998): 748–51. http://dx.doi.org/10.1128/aem.64.2.748-751.1998.
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ABSTRACT To construct a bacterial catalyst for bioconversion of toluene and several alkyl and chloro- and nitro-substituted derivatives into the corresponding benzoates, the upper TOL operon of plasmid pWW0 ofPseudomonas putida was fully reassembled as a single gene cassette along with its cognate regulatory gene, xylR. The corresponding DNA segment was then targeted to the chromosome of aP. putida strain by using a genetic technique that allows deletion of all recombinant tags inherited from previous cloning steps and leaves the otherwise natural strain bearing exclusively the DNA segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes, and further metabolism of benzoate through the housekeepingortho-ring cleavage pathway of the catechol intermediate.
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Humeniuk, H. B., M. Z. Mosula, I. B. Chen, and N. M. Drobyk. "James Watson’s genius (dedicated to the 90th birth anniversary)." Faktori eksperimental'noi evolucii organizmiv 22 (September 9, 2018): 363–67. http://dx.doi.org/10.7124/feeo.v22.976.
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The scientific and organizational activities of the worldwide known scientist in the field of molecular biology James Dewey Watson were described in this article. 55 years ago James Watson and Francis Crick made one of the key discoveries of the twentieth century. They have found that DNA has a double helix structure. This discovery was based on the X-rays patterns obtained by Maurice Wilkson and Rosalind Franklin. Subsequently, this DNA model had been proved, and J. Watson and F. Crick were awarded with the Nobel Prize in Physiology or Medicine in 1962. Since, our knowledge of the main molecule of life has been greatly expanded. A significant flowering of molecular genetics has began: synthesis of RNA and DNA in vitro, decoding of genetic code, recombinant DNA technology, genetic engineering, sequencing of genomes and post genomic technologies. James Watson is one of the authors of the cell biology classic textbook “Molecular Biology of the Cell”. In addition, he has developed the current areas of molecular biology such as molecular oncology and molecular neurobiology. Today genomes of different animals and humans have been decoded and the functions of many genes have been determined. But at present still unknown how the DNA starts and how it affects the work of the organs and the organism as a system.
Keywords: James Watson, DNA structure, Nobel Prize in physiology or medicine, Molecular Biology of the Cell.
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Curd, Martin. "Commentary on “…Let No One Split Asunder: Controversy in Human Genetic Engineering”." Politics and the Life Sciences 6, no. 1 (August 1987): 13–15. http://dx.doi.org/10.1017/s0730938400002665.
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Though not the first attempt to ameliorate a human genetic disease by modifying somatic cells, the experiments performed by Dr. Martin J. Cline on two women suffering from thalassemia (one in Jerusalem, the other in Naples) were the first and still the only ones to use rDNA techniques.1 In some respects the criticism and censure of Cline by the NIH resembles the case of Dr. Ian T. Kennedy.2 Both Cline and Kennedy were found guilty of having violated the NIH Guidelines for Research Involving Recombinant DNA Molecules, but there is a crucial difference between the two cases. Cline attempted to implant genetically altered cells into human beings. Consequently, his work involved moral issues concerning the use of human subjects in biomedical experiments which go beyond the usual concerns over safety and control in rDNA research. In fact, I shall argue that the rDNA aspect of the Cline affair was relatively minor compared with the moral reasoning which guided the actions and judgments of Cline and his critics. Despite its importance for understanding the behavior of the participants, this ethical dimension does not receive adequate emphasis in Robin and Markle's account of the Cline episode within the framework of their four sociological perspectives (substantive, network, organizational, societal).
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Mayer, Ian, and Ewen McLean. "Bioengineering and biotechnological strategies for reduced waste aquaculture." Water Science and Technology 31, no. 10 (May 1, 1995): 85–102. http://dx.doi.org/10.2166/wst.1995.0366.
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While recent years have witnessed dramatic advances in the reduction of aquaculture waste production, primarily due to advances in feed technology, the co-implementation of new bioengineering and biotechnological strategies are vital for alleviating the environmental impact of the rapidly expanding global aquaculture industry. The deployment of a new generation of automated feeding devices, and continued advances in recirculation technologies for land-based systems are amongst the more significant bioengineering advances that have resulted in reduced waste production. Advances in feed technologies will continue to play a pivotal role in the reduction of aquculture waste. Further, the advent of modern recombinant DNA technologies now allows for the economic production of a variety of feed supplements, most notably microbial phytases. Other, often overlooked, biotechnological strategies for achieving improved growth and conversion efficiencies include such physiological modifications as sustained exercise and compensatory growth. Somewhat more controversial biotechnological methods which may be beneficial in reduced waste management include endocrine manipulations and genetic engineering. Again, recent advances in recombinant DNA and transgenic technologies have also led to renewed interest in these strategies.
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Frow, Emma. "From “Experiments of Concern” to “Groups of Concern”: Constructing and Containing Citizens in Synthetic Biology." Science, Technology, & Human Values 45, no. 6 (October 25, 2017): 1038–64. http://dx.doi.org/10.1177/0162243917735382.
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Synthetic biology represents a recent and explicit attempt to make biology easier to engineer, and through this to open up the design space of genetic engineering to a wider range of practitioners (including, but not limited to, engineers). Proponents of this approach emphasize the standardization of practices as key to successful biological engineering; yet, meaningful transatlantic differences are emerging with respect to the constitution of key concerns and the governance of synthetic biology in the United States (US) and the United Kingdom (UK). In this article, I tease out how national approaches to governing synthetic biology are being framed against different salient past experiences with recombinant DNA technology. In the US, the governance of synthetic biology is consistently articulated in relation to the early days of recombinant DNA technology and the self-governance mechanisms pioneered in response to Asilomar. In the UK, more recent experiences with genetically modified crops provide the overarching imaginary against which governance initiatives are being proposed. I suggest that these differing sociotechnical imaginaries have implications for how new “groups of concern” are being defined in relation to synthetic biology and how measures to contain perceived risks are being pursued in the US and Britain.
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Kaliyeva, Makhabbat, and Alexander Shustov. "DEVELOPMENT OF AUTONOMOUSLY REPLICATING VIRAL RNA TO EXPRESS THE RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR." CBU International Conference Proceedings 3 (September 19, 2015): 398–404. http://dx.doi.org/10.12955/cbup.v3.629.
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Recombinant human granulocyte colony-stimulating factor (G-CSF) produced in cultured mammalian cells undergoes proper posttranslational modifications and, thereby, possesses better pharmacological properties in comparison with the homologous protein expressed in bacteria. Biopharmaceuticals derived from cell culture tend to be expensive because of lower yields compared to bacterially expressed competitors and numerous issues with the scalability of production. Particular limitation of scalability pertains to delivery of expression vectors to the cell culture. Natural and efficient way to deliver foreign DNA or RNA to cell is a viral infection. We intended to develop the viral genome capable of G-CSF expression.Objectives: To develop autonomously replicating viral RNA capable of heterologous expression of the G-CSF in cultured mammalian cells.Methods: Genetic engineering, cell culture, and virology.Results: Viable genome of the Venezuelan equine encephalomyelitis virus (VEE) was constructed. The G-CSF gene was synthesized de novo. Gene cassette GFP-2A-G-CSF was constructed for simultaneous expression of the fluorescent marker of viral replication (GFP) and the product of interest. Recombinant viral genome VEE/GFP-2A-G-CSF was assembled; its viability was confirmed upon transfection to the BHK-21 cells.Conclusion: Genetic engineering of the cDNA copies of the genomes of RNA viruses provides excessive opportunities to develop mammalian expression systems. We achieved production of recombinant proteins in cultured mammalian cells transfected with the VEE viral RNA carrying the inserted genes of marker protein and G-CSF.
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Hobden, A. N., and T. J. R. Harris. "The impact of biotechnology and molecular biology on the pharmaceutical industry." Proceedings of the Royal Society of Edinburgh. Section B. Biological Sciences 99, no. 1-2 (1992): 37–45. http://dx.doi.org/10.1017/s0269727000013038.
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Synopsis:Biotechnology had its initial impact on the pharmaceutical industry well before the perceived time. The use of fermentation technology to produce antibiotics was a cornerstone for the development of the industry. This event was both before cloning (BC) and before DNA (rather than after DNA – AD). Even now the antibiotic market, which is worth over 10 billion U.S. dollars a year, is the most valuable segment of the total market, (c.200 billion dollars per year). Nevertheless the impact of biotechnology in drug discovery was until recently perceived solely to be the use of recombinant DNA techniques to produce therapeutic proteins and modified versions of them by protein engineering.There are several other places where genetic engineering is influencing drug discovery. The expression of recombinant proteins in surrogate systems (e.g. in E. coli, yeast or via baculovirus infection or in mammalian cells) provides materials for structure determination (e.g. HIV protease) and structure/function studies (e.g. various receptors). Recombinant DNA techniques are influencing assay technology by allowing access to proteins in sufficient quantity for high throughput screening.In addition, screening organisms can be constructed where a particular protein function can be measured in a microorganism by complementation or via reporter gene expression.Transgenic animals also illustrate the power of the technology for drug discovery. Not only will transgenic rats and mice be used as models of disease but also for efficacy and toxicological profiling. What is learned in transgenic rodents may well set the scene for somatic cell gene therapy in humans.
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Zuo, Peijun, and A. Bakr M. Rabie. "Novel Method of Cell-Free In Vitro Synthesis of the Human Fibroblast Growth Factor 1 Gene." Journal of Biomedicine and Biotechnology 2010 (2010): 1–5. http://dx.doi.org/10.1155/2010/971340.
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Recombinant DNA projects generally involve cell-based gene cloning. However, because template DNA is not always readily available, in vitro chemical synthesis of complete genes from DNA oligonucleotides is becoming the preferred method for cloning. This article describes a new, rapid procedure based onTaqpolymerase for the precise assembly of DNA oligonucleotides to yield the complete human fibroblast growth factor 1 (FGF1) gene, which is 468 bp long and has a G+C content of 51.5%. The new method involved two steps: (1) the design of the DNA oligonucleotides to be assembled and (2) the assembly of multiple oligonucleotides by PCR to generate the whole FGF1 gene. The procedure lasted a total of only 2 days, compared with 2 weeks for the conventional procedure. This method of gene synthesis is expected to facilitate various kinds of complex genetic engineering projects that require rapid gene amplification, such as cell-free whole-DNA library construction, as well as the construction of new genes or genes that contain any mutation, restriction site, or DNA tag.
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Rabino, Isaac. "Ethical Debates in Genetic Engineering: U.S. Scientists' Attitudes on Patenting, Germ-Line Research, Food Labeling, and Agri-Biotech Issues." Politics and the Life Sciences 17, no. 2 (September 1998): 147–63. http://dx.doi.org/10.1017/s0730938400012132.
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A 1995 survey of 1,257 scientists working in the field of recombinant DNA research indicates wide areas of agreement as well as some noteworthy divisions when it comes to such thorny questions as patenting, germ-line research, food labeling, and biodiversity. In general, the scientists surveyed approve of patenting living organisms that result from rDNA research, but vary significantly on what should be patentable. They advocate human germ-line therapy, yet have reservations about using it for any but serious diseases. They oppose mandatory labeling of biologically engineered food products, but understand that the public has a right to know and advocate openness. Finally, they favor development of genetically modified crops, but recognize potential threats to biodiversity and maintain that publicly funded researchers should be legally obligated to consider the potential environmental effects of their research. Some clear differences arise between scientists working in industry and those in academia and between men and women.
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Niamsiri, Nuttawee, Soazig C. Delamarre, Young-Rok Kim, and Carl A. Batt. "Engineering of Chimeric Class II Polyhydroxyalkanoate Synthases." Applied and Environmental Microbiology 70, no. 11 (November 2004): 6789–99. http://dx.doi.org/10.1128/aem.70.11.6789-6799.2004.
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ABSTRACT PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1 Po ) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB−4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1 Po genes that produced more PHA than the native phaC1 Po . Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (M w) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.
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Creager, Angela N. H. "Recipes for recombining DNA: A history of Molecular Cloning: A Laboratory Manual." BJHS Themes 5 (2020): 225–43. http://dx.doi.org/10.1017/bjt.2020.5.
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AbstractLaboratory instructions and recipes are sometimes edited into books with a wide circulation. Even in the late twentieth century, publications of this nature remained influential. For example, protocols from a 1980 summer course on gene cloning at Cold Spring Harbor Laboratory provided the basis for a bestselling laboratory manual by Tom Maniatis, Ed Fritsch and Joe Sambrook. Not only did the Molecular Cloning: A Laboratory Manual become a standard reference for molecular biologists (commonly called the ‘bible’), but also its recipes and clear instructions made gene cloning and recombinant DNA technologies accessible to non-specialists. Consequently, this laboratory manual contributed to the rapid spread of genetic-engineering techniques throughout the life sciences, as well as in industry. As is often the case with how-to books, however, finding a way to update methods in this rapidly changing field posed a challenge, and various molecular-biology reference books had different ways of dealing with knowledge obsolescence. This paper explores the origins of this manual, its publication history, its reception and its rivals – as well as the more recent migration of such laboratory manuals to the Internet.
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Hay, Irene, Marie-Josée Morency, and Armand Séguin. "Assessing the persistence of DNA in decomposing leaves of genetically modified poplar trees." Canadian Journal of Forest Research 32, no. 6 (June 1, 2002): 977–82. http://dx.doi.org/10.1139/x02-017.
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DNA transformation of forest tree species is now a striking reality and offers the possibility to generate transgenic trees with useful new characteristics. However, it is important to make the proper environmental assessment of these transgenic trees when established in field trials. For instance, the DNA released into the soil by decaying leaves and roots from the transgenic trees may become available for incorporation by soil microbes. The objective of this study was to investigate the persistence of recombinant plant marker genes in decomposing transgenic poplar leaf material. We studied the stability of the DNA encoding the neomycin phosphotransferase II resistance marker used in tree genetic engineering. DNA persistence in the environment was determined by placing transgenic poplar leaves in permeable bags that were located on weeds, on the soil, and below the soil and left under natural conditions on the site of a field trial for up to 12 months. This work is the first quantitative analysis of tree DNA stability in a natural forest environment. Our data indicate that fragments of the genetically modified DNA are not detectable in the field for more than 4 months.
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Romer, Tomasz E. "The Role of Recombinant Human Growth Hormone Biosimilars in the Management of Growth Disorders." European Endocrinology 05 (2009): 47. http://dx.doi.org/10.17925/ee.2009.05.00.47.
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Since ancient times plant and animal tissues have been used as medicines. In the 20th century growth hormone as a purified extraction from human pituitaries was still used to treat growth disorders. Since the genetic engineering of host cells became possible, a new generation of medicines obtained using recombinant DNA (rDNA) technology has emerged. These medicines have been named ‘biopharmaceuticals’. The first biopharmaceutical growth hormones were patented in the 1980s, so already over two decades of clinical experience support the development of a new, off-patent growth hormone preparation obtained by rDNA technology. The European Medicines Agency (EMEA) has put in place a centralised procedure for the approval of new biopharmaceuticals. This procedure includes testing comparability with a reference product and demands post-approval pharmacovigilance. Omnitrope® was the first off-patent recombinant human growth hormone (rhGH) approved on the basis of the biosimilar pathway; it underwent a very demanding approval procedure in 2006 and is now used for several indications in Europe, the US, Canada, Japan, Australia and other countries where it has received marketing approval.
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Klein, Reinhild, Douglas M. Templeton, and Michael Schwenk. "Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)." Pure and Applied Chemistry 86, no. 10 (October 21, 2014): 1573–617. http://dx.doi.org/10.1515/pac-2013-1028.
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Abstract This report discusses the history and mechanisms of vaccination of humans as well as the engineering of therapeutic antibodies. Deeper understanding of the molecular interactions involved in both acquired and innate immunity is allowing sophistication in design of modified and even synthetic vaccines. Recombinant DNA technologies are facilitating development of DNA-based vaccines, for example, with the recognition that unmethylated CpG sequences in plasmid DNA will target Toll-like receptors on antigen-presenting cells. Formulations of DNA vaccines with increased immunogenicity include engineering into plasmids with “genetic adjuvant” capability, incorporation into polymeric or magnetic nanoparticles, and formulation with cationic polymers and other polymeric and non-polymeric coatings. Newer methods of delivery, such as particle bombardment, DNA tattooing, electroporation, and magnetic delivery, are also improving the effectiveness of DNA vaccines. RNA-based vaccines and reverse vaccinology based on gene sequencing and bioinformatic approaches are also considered. Structural vaccinology is an approach in which the detailed molecular structure of viral epitopes is used to design synthetic antigenic peptides. Virus-like particles are being designed for vaccine deliveries that are based on structures of viral capsid proteins and other synthetic lipopeptide building blocks. A new generation of adjuvants is being developed to further enhance immunogenicity, based on squalene and other oil–water emulsions, saponins, muramyl dipeptide, immunostimulatory oligonucleotides, Toll-like receptor ligands, and lymphotoxins. Finally, current trends in engineering of therapeutic antibodies including improvements of antigen-binding properties, pharmacokinetic and pharmaceutical properties, and reduction of immunogenicity are discussed. Taken together, understanding the chemistry of vaccine design, delivery and immunostimulation, and knowledge of the techniques of antibody design are allowing targeted development for the treatment of chronic disorders characterized by continuing activation of the immune system, such as autoimmune disorders, cancer, or allergies that have long been refractory to conventional approaches.
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Jin, Yu Fen, Yan Lei Li, Yan Hua, Xiao Gang Zhang, and Ting Yu. "Expression of Recombinant Human Cytomegalovirus Fusion Proteins pp150-pp65 Fragments and its Application." Advanced Materials Research 634-638 (January 2013): 1313–18. http://dx.doi.org/10.4028/www.scientific.net/amr.634-638.1313.
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Objective To evaluate the effectiveness of prokaryotic expression of fusion proteins pp150-pp65 of human cytomegalovirus (hCMV) for its application as antigen, the fusion protein of pp150-pp65 were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column for preparing the colloidal gold kit. Methods Using DNA from HCMV strain as template, the genes encoding pp150 and pp65 protein fragment were amplified by PCR technique, respectively. After confirmed by DNA sequence analysis, the recombinant plasmid pET28a-pp150-pp65 was transformed into E.Coil.BL21(DE3) and induced to express with IPTG. The expressed fusion protein was characterized by SDS-PAGE and western blot after purified, then we used the purified fusion protein to develop combined detection kit of IgM/IgG antibody against HCMV (colloidal gold method) with Beijing Innovita Bio-tech Co., Ltd for detecting the samples, compared with the imported kits (ELISA). Results The gene of fusion fragment pp150-pp65 was correctly amplified and the recombinant vector was successfully constructed. The purified protein with the molecular weight of 45KD had good antigenicity by western blot. The protein was subjected to assay with an ELI SA capture kit in its specific and sensitive assay based on colloidal gold nanoparticles, testing of 600 serum samples indicated that this kit had a sensitivity of 92.7%;, specificity of 83.1%, crude consistency o f 90.2%, compared to the imported HCMV-IgG kit; the sensitivity of the kit was 88.1%, specificity was 89.2%, coarse consistency was 88.5%, compared to the imported HCMV-IgM kit; Conclusion In this experiment, the HCMV antigen with high purity and specificity (pp150-pp65 recombinant protein) was prepared effectively through genetic engineering technology. Compared to imported reagents, the colloidal gold kit consisting of fusion protein in a capture assay had high sensitivity and specificity. Preliminary clinical use warrants further development and use of this kit. Furthermore, it provides a technological basis for detection of HCMV in different stages of clinical infection.
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Sugasawa, Takehito, Kai Aoki, Koichi Watanabe, Koki Yanazawa, Tohru Natsume, Tohru Takemasa, Kaori Yamaguchi, et al. "Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR." Genes 10, no. 6 (June 8, 2019): 436. http://dx.doi.org/10.3390/genes10060436.
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With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.
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Ausubel, Frederick M. "Tracing My Roots: How I Became a Plant Biologist." Annual Review of Genetics 52, no. 1 (November 23, 2018): 1–20. http://dx.doi.org/10.1146/annurev-genet-120417-031722.
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My trajectory to becoming a plant biologist was shaped by a complex mix of scientific, political, sociological, and personal factors. I was trained as a microbiologist and molecular biologist in the late 1960s and early 1970s, a time of political upheaval surrounding the Vietnam War. My political activism taught me to be wary of the potential misuses of scientific knowledge and to promote the positive applications of science for the benefit of society. I chose agricultural science for my postdoctoral work. Because I was not trained as a plant biologist, I devised a postdoctoral project that took advantage of my microbiological training, and I explored using genetic technologies to transfer the ability to fix nitrogen from prokaryotic nitrogen-fixing species to the model plant Arabidopsis thaliana with the ultimate goal of engineering crop plants. The invention of recombinant DNA technology greatly facilitated the cloning and manipulation of bacterial nitrogen-fixation ( nif) genes, but it also forced me to consider how much genetic engineering of organisms, including human beings, is acceptable. My laboratory has additionally studied host–pathogen interactions using Arabidopsis and the nematode Caenorhabditis elegans as model hosts.
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Tasca, Francesca, Qian Wang, and Manuel A. F. V. Gonçalves. "Adenoviral Vectors Meet Gene Editing: A Rising Partnership for the Genomic Engineering of Human Stem Cells and Their Progeny." Cells 9, no. 4 (April 13, 2020): 953. http://dx.doi.org/10.3390/cells9040953.
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Gene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and differentiation capabilities consent studying cellular differentiation processes, screening small-molecule drugs, modeling human disorders, and testing regenerative medicines. To integrate gene editing and stem cell technologies, there is a critical need for achieving efficient delivery of the necessary molecular tools in the form of programmable DNA-targeting enzymes and/or exogenous nucleic acid templates. Moreover, the impact that the delivery agents themselves have on the performance and precision of gene editing procedures is yet another critical parameter to consider. Viral vectors consisting of recombinant replication-defective viruses are under intense investigation for bringing about efficient gene-editing tool delivery and precise gene-editing in human cells. In this review, we focus on the growing role that adenoviral vectors are playing in the targeted genetic manipulation of human stem cells, progenitor cells, and their differentiated progenies in the context of in vitro and ex vivo protocols. As preamble, we provide an overview on the main gene editing principles and adenoviral vector platforms and end by discussing the possibilities ahead resulting from leveraging adenoviral vector, gene editing, and stem cell technologies.
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Gann, Alexander, and Jean Beggs. "Noreen Elizabeth Murray CBE. 26 February 1935 — 12 May 2011." Biographical Memoirs of Fellows of the Royal Society 60 (January 2014): 349–74. http://dx.doi.org/10.1098/rsbm.2014.0009.
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Noreen Murray was one of the architects of the recombinant DNA revolution that transformed the study of biology from the early 1970s. Her particular prowess for genetic manipulation of bacteria and their phage was critical in developing the bacteriophage lambda vectors that were a vital part of the early genetic engineering toolbox. Her skill as a microbial geneticist had earlier become apparent through her work on genetic recombination and complementation in the fungus Neurospora , especially as a postdoctoral researcher at Stanford where her work brought her to the attention of some of the giants of early molecular biology. Back in the UK, first at Cambridge and then, for the bulk of her career, at Edinburgh, she produced a remarkable body of work focused on uncovering the mechanisms and biology of restriction enzymes, and their adaptation as tools underpinning modern biological research and the rise of the biotechnology industry. Much of this work was done in collaboration with her husband Ken Murray FRS, whose biographical memoir accompanies this one. Together they were known not only for the quality of their research but also for their vast generosity both on a personal level and on a larger canvas through their philanthropy.
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Nalluri, Nirmala, and Vasavi Rama Karri. "Recent advances in genetic manipulation of crops: A promising approach to address the global food and industrial applications." Plant Science Today 7, no. 1 (January 8, 2020): 70–92. http://dx.doi.org/10.14719/pst.2020.7.1.659.
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Continuous increase in world’s population demands high food production, which has become a major challenge to the humanity. When there is sufficient amount of nutritious food to all the people there will be no problem of food scarcity. So, to increase the food production, many countries are adopting strategies of genetic engineering to enhance the crop yield. Recombinant DNA technology can be a viable source to develop genetically modified crops with enhanced resistance and improved yields to fight against malnutrition and food scarcity. With this technology, selected traits can be inserted into the plant genome, unlike traditional plant breeding, where many characters of two different crops will be combined which may lead to genetic modification at an extensive level. Present review focuses on the methods of plant transformation and outlines the scope of genetic transformation for improved crop production by transferring selected genes for biotic and abiotic stress tolerance. In addition, current study also provides information about various genetically modified crops produced worldwide and their commercialization towards various biotechnological products like GM livestock, GM microorganisms, vaccines and industrial products like bio-plastic produced from the transgenic plants.
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Machado, Raul, A. J. Ribeiro, J. Padrão, D. Silva, A. Nobre, J. A. Teixeira, F. J. Arias, António M. Cunha, José C. Rodríguez-Cabello, and M. Casal. "Exploiting the Sequence of Naturally Occurring Elastin: Construction, Production and Characterization of a Recombinant Thermoplastic Protein-Based Polymer." Journal of Nano Research 6 (June 2009): 133–45. http://dx.doi.org/10.4028/www.scientific.net/jnanor.6.133.
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Genetic engineering was used to produce an elastin-like polymer (ELP) with precise amino acid composition, sequence and length, resulting in the absolute control of MW and stereochemistry. A synthetic monomer DNA sequence encoding for (VPAVG)20, was used to build a library of concatemer genes with precise control on sequence and size. The higher molecular weight polymer with 220 repeats of VPAVG was biologically produced in Escherichia coli and purified by hot and cold centrifugation cycles, based on the reversible inverse temperature transition property of ELPs. The use of low cost carbon sources like lactose and glycerol for bacteria cells culture media was explored using Central Composite Design approach allowing optimization of fermentation conditions. Due to its self-assembling behaviour near 33 °C stable spherical microparticles with a size ~ 1µm were obtained, redissolving when a strong undercooling is achieved. The polymer produced showed hysteresis behaviour with thermal absorbing/releasing components depending on the salt concentration of the polymer solution.
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Vo-Quang, Tuyen, Yves Malpiece, Dominique Buffard, P. Alexandre Kaminski, Dominique Vidal, and A. Donny Strosberg. "Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors." Bioscience Reports 5, no. 2 (February 1, 1985): 101–11. http://dx.doi.org/10.1007/bf01117056.
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This paper describes a new method of lasmid DNA purification which is fast and reliable enough for most purposes in recombinant DNA technology. The present method does not require the use of toxic chemicals such as phenol or ethidium bromide, costly ultra-centrifugation procedures or other processes which can modify the supercoiled structure of the plasmids, such as adsorption on glass fiber. This method is based on the principle of gel filtration chromatography, at low pressure (1 bar) or medium pressure (between 5 and 10 bars), using Sephacryl S1000 or Superose 6B. It permits recovery oI plasmids: (I) in preparative quantities (from 300 gg to 4 mg), (II) exempt from RNA, DNA and protein contamination, and (III) suitable for various common genetic engineering procedures immediately after purification. To test the reliability of the technique as well as the degree of purilication, the plasmids were used to construct thermoampliIiable vectors, carrying the tacUV5 promoter and the 5′ end of the β -gallactosidase gone with a single EcoRl site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins in Escherichia coli.
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Yomantas, Yurgis A. V., Irina L. Tokmakova, Natalya V. Gorshkova, Elena G. Abalakina, Svetlana M. Kazakova, Evgueni R. Gak, and Sergey V. Mashko. "Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in Methylophilus methylotrophus AS1 Cells Expressing the aroP-Encoded Transporter of Escherichia coli." Applied and Environmental Microbiology 76, no. 1 (October 30, 2009): 75–83. http://dx.doi.org/10.1128/aem.02217-09.
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ABSTRACT The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, which encodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Kmr marker, and electroporated into an aroP carrier recipient strain. This approach led to the construction of a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.
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Et al., Al-Dallee. "Expression of Recombinant Human Glucocerebrosidase Protein in Sunflowers." Baghdad Science Journal 16, no. 1 (March 11, 2019): 0018. http://dx.doi.org/10.21123/bsj.16.1.0018.
Full textAbstract:
Molecular farming has become one of the most significant implementations of modern biotechnology to generate modified plant crops to produce medicinal proteins. Agrobacterium is one plant genetic engineering tool that integrates genes of interest inside a host plant. In recent years, the need to produce recombinant proteins as therapeutics has growing rapidly, and human glucocerebrosidase is one of the proteins that is need to treat disease. In this study, specific primers were designed to amplify Hu-GBA1 gene from constructed pGEM-GBA plasmid which was cloned into the plant expression vector pCAMBIA1304. The generated recombinant pCAMBIA1304-GBA plasmid was used to transform A. tumefaciens LBA4404 and applied for transformation of sunflower cotyledon explants. Colony PCR technique was used to confirm the presence of Hu-GBA1 gene in transformed A. tumefaciens. Agrobacterium containing pCAMBIA1304-GBA was suspended in Infection Medium (IM) supplement with 200 mM acetosyringone. A bacterial suspension was used to transform sunflower cotyledons. After infection, cotyledons were co-cultivated in Co-cultivation medium (CCM), supplied with 200 mM acetosyringone without antibiotics. The cotyledons were then transferred to selection media containing 7.5 mg/L Hygromycin and 250 mg/L Cefotaxime and grown for additional 14 days at 25℃ in photoperiod of 16h L/8h D. The transformed sunflower cotyledons were successfully generated complete plant with used 6-Benzylaminopurine and Naphthalene acetic acid as growth hormones. The presence of the Hu-GBA1 gene in the genomic DNA of transgenic sunflower plant was proven by PCR as a band of 1561bp size. The GBA mRNA expression in modified sunflowers was detected by qRT-PCR compared with control GBA mRNA. Enzyme Linked Immunoassay was done on crude recombinant protein that extracted from transformed sunflower using Human Glucosylceramide ELISA Kit, the Elisa test results confirmed the production of recombinant glucocerebrosidase and the concentration of crude recombinant enzyme extracted from transformed sunflower with GBA1 gene was 0.45 ng/µl
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Et al., Al-Dallee. "Expression of Recombinant Human Glucocerebrosidase Protein in Sunflowers." Baghdad Science Journal 16, no. 1 (March 11, 2019): 0018. http://dx.doi.org/10.21123/bsj.2019.16.1.0018.
Full textAbstract:
Molecular farming has become one of the most significant implementations of modern biotechnology to generate modified plant crops to produce medicinal proteins. Agrobacterium is one plant genetic engineering tool that integrates genes of interest inside a host plant. In recent years, the need to produce recombinant proteins as therapeutics has growing rapidly, and human glucocerebrosidase is one of the proteins that is need to treat disease. In this study, specific primers were designed to amplify Hu-GBA1 gene from constructed pGEM-GBA plasmid which was cloned into the plant expression vector pCAMBIA1304. The generated recombinant pCAMBIA1304-GBA plasmid was used to transform A. tumefaciens LBA4404 and applied for transformation of sunflower cotyledon explants. Colony PCR technique was used to confirm the presence of Hu-GBA1 gene in transformed A. tumefaciens. Agrobacterium containing pCAMBIA1304-GBA was suspended in Infection Medium (IM) supplement with 200 mM acetosyringone. A bacterial suspension was used to transform sunflower cotyledons. After infection, cotyledons were co-cultivated in Co-cultivation medium (CCM), supplied with 200 mM acetosyringone without antibiotics. The cotyledons were then transferred to selection media containing 7.5 mg/L Hygromycin and 250 mg/L Cefotaxime and grown for additional 14 days at 25℃ in photoperiod of 16h L/8h D. The transformed sunflower cotyledons were successfully generated complete plant with used 6-Benzylaminopurine and Naphthalene acetic acid as growth hormones. The presence of the Hu-GBA1 gene in the genomic DNA of transgenic sunflower plant was proven by PCR as a band of 1561bp size. The GBA mRNA expression in modified sunflowers was detected by qRT-PCR compared with control GBA mRNA. Enzyme Linked Immunoassay was done on crude recombinant protein that extracted from transformed sunflower using Human Glucosylceramide ELISA Kit, the Elisa test results confirmed the production of recombinant glucocerebrosidase and the concentration of crude recombinant enzyme extracted from transformed sunflower with GBA1 gene was 0.45 ng/µl
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Fujiwara, Ayako, Katsuhiro Kawato, Saori Kato, Kiyoshi Yasukawa, Ryota Hidese, and Shinsuke Fujiwara. "Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR." Applied and Environmental Microbiology 82, no. 10 (March 11, 2016): 3022–31. http://dx.doi.org/10.1128/aem.04116-15.
Full textAbstract:
ABSTRACTDNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, theEuryarchaeota-specific helicase EshA (TK0566) from the hyperthermophilic archaeonThermococcus kodakarensis(Tk-EshA) was obtained as a recombinant form, and its enzymatic properties were examined.Tk-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5′ overhung, 3′ overhung, and blunt end) at 50°C.Tk-EshA unwound forked and 3′ overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers whenTk-EshA was added to a PCR mixture. To examine the effect ofTk-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence ofTk-EshA. In contrast, noise DNAs were eliminated in the presence ofTk-EshA. Noise reduction byTk-EshA was also confirmed whenTaqDNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, α type) were used for PCR. Misamplified bands were also eliminated duringtoxAgene amplification fromPseudomonas aeruginosaDNA, which possesses a high GC content (69%).Tk-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs duringtoxAamplification.Tk-EshA is a useful tool to reduce noise DNAs for accurate PCR.IMPORTANCEPCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostableEuryarchaeota-specific helicase (Tk-EshA) was included in the PCR mixture. The addition ofTk-EshA has reduced noise DNAs in PCR.
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Maity and Mishra. "Statistically Designed Medium Reveals Interactions between Metabolism and Genetic Information Processing for Production of Stable Human Serum Albumin in Pichia pastoris." Biomolecules 9, no. 10 (October 4, 2019): 568. http://dx.doi.org/10.3390/biom9100568.
Full textAbstract:
Human serum albumin (HSA), sourced from human serum, has been an important therapeutic protein for several decades. Pichia pastoris is strongly considered as an expression platform, but proteolytic degradation of recombinant HSA in the culture filtrate remains a major bottleneck for use of this system. In this study, we have reported the development of a medium that minimized proteolytic degradation across different copy number constructs. A synthetic codon-optimized copy of HSA was cloned downstream of α–factor secretory signal sequence and expressed in P. pastoris under the control of Alcohol oxidase 1 promoter. A two-copy expression cassette was also prepared. Culture conditions and medium components were identified and optimized using statistical tools to develop a medium that supported stable production of HSA. Comparative analysis of transcriptome data obtained by cultivation on optimized and unoptimized medium indicated upregulation of genes involved in methanol metabolism, alternate nitrogen assimilation, and DNA transcription, whereas enzymes of translation and secretion were downregulated. Several new genes were identified that could serve as possible targets for strain engineering of this yeast.