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recombinant DNA technology

Qian Lu, Jihong Wang, Junshu Jiang, Shengnan Wang, Qilan Jia, Yue Wang, Weiping Li, Qin Zhou, Li Lv, Qingwei Li
BACKGROUND: The RGD-toxin protein Lj-RGD3 is a naturally occurring 118 amino acid peptide that can be obtained from the salivary gland of the Lampetra japonica fish. This unique peptide contains 3 RGD (Arg-Gly-Asp) motifs in its primary structure. Lj-RGD3 is available in recombinant form (rLj-RGD3) and can be produced in large quantities using DNA recombination techniques. The pharmacology of the three RGD motif-containing peptides has not been studied. This study investigated the protective effects of rLj-RGD3, a novel polypeptide, against ischemia/reperfusion-induced damage to the brain caused by middle cerebral artery occlusion (MCAO) in a rat stroke model...
2016: PloS One
Steven W Barger
Ask any neuroscientist to name the most profound discoveries in the field in the past 60 years, and at or near the top of the list will be a phenomenon or technique related to genes and their expression. Indeed, our understanding of genetics and gene regulation has ushered in whole new systems of knowledge and new empirical approaches, many of which could not have even been imagined prior to the molecular biology boon of recent decades. Neurochemistry, in the classic sense, intersects with these concepts in the manifestation of neuropeptides, obviously dependent upon the central dogma (the established rules by which DNA sequence is eventually converted into protein primary structure) not only for their conformation but also for their levels and locales of expression...
October 17, 2016: Journal of Neurochemistry
Kazutaka Kitaura, Tadasu Shini, Takaji Matsutani, Ryuji Suzuki
BACKGROUND: High-throughput sequencing of T cell receptor (TCR) genes is a powerful tool for analyses of antigen specificity, clonality and diversity of T lymphocytes. Here, we developed a new TCR repertoire analysis method using 454 DNA sequencing technology in combination with an adaptor-ligation mediated polymerase chain reaction (PCR). This method allows the amplification of all TCR genes without PCR bias. To compare gene usage, diversity and similarity of expressed TCR repertoires among individuals, we conducted next-generation sequencing (NGS) of TRA and TRB genes in peripheral blood mononuclear cells from 20 healthy human individuals...
October 11, 2016: BMC Immunology
L Fu, Y L Hou, X Ding, Y J Du, H Q Zhu, N Zhang, W R Hou
The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19...
August 30, 2016: Genetics and Molecular Research: GMR
Gangyi Chen, Juan Dong, Yi Yuan, Na Li, Xin Huang, Xin Cui, Zhuo Tang
Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites...
September 30, 2016: Scientific Reports
Gui-Fang Wang, Bing Qi, Lei-Lei Tu, Lian Liu, Guo-Cheng Yu, Jing-Xiang Zhong
AIM: To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS: Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP)...
2016: International Journal of Ophthalmology
Ema Romão, Francisco Morales-Yánez, Yaozhong Hu, Maxine Crauwels, Pieter De Pauw, Gholamreza Ghassanzadeh Hassanzadeh, Nick Devoogdt, Chloé Ackaert, Cécile Vincke, Serge Muyldermans
BACKGROUND: The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigen-binding fragments, also known as Nanobodies. METHODS: Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved...
September 23, 2016: Current Pharmaceutical Design
Guillaume Andrey, Malte Spielmann
Targeted mutagenesis is required to evaluate the function of DNA segments across the genome. In recent years the CRISPR/Cas9 technology has been widely used for functional genome studies and is partially replacing classical homologous recombination methods in different aspects. CRISPR/Cas9-derived tools indeed allow the production of a wide-range of engineered mutations: from point mutations to large chromosomal rearrangements such as deletions, duplications and inversions. Here we present a protocol to engineer Embryonic Stem Cells (ESC) with desired mutations using transfection of custom-made CRISPR/Cas9 vectors...
2017: Methods in Molecular Biology
Zeyu Chen, Rong Guo, Jianghong Xu, Chuangjun Qiu
This study evaluated the immunogenicity and protective immunity of a Hemophilus influenzae b (Hib) polysaccharide conjugate vaccine with the pneumococcal surface adhesin A (PsaA) protein carrier in young mice. The Hib polysaccharide was conjugated with the rPsaA protein carrier, which was produced using recombinant DNA technology. A total of 15 young mice aged 3 weeks to 5 weeks were immunized with the conjugate vaccine, and another 15 young mice of the same age were immunized with the licensed Hib-tetanus toxoid (TT) vaccine...
September 20, 2016: Frontiers of Medicine
Lynn C Thomason, Nina Costantino, Donald L Court
UNLABELLED: Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions...
2016: MBio
Begoña Miras-Moreno, Ana Belén Sabater-Jara, M A Pedreño, Lorena Almagro
Phytosterols are a kind of plant metabolite belonging to the triterpene family. These compounds are essential biomolecules for human health, and so they must be taken from foods. β-Sitosterol, campesterol, and stigmasterol are the main phytosterols found in plants. Phytosterols have beneficial effects on human health since they are able to reduce plasma cholesterol levels and have antiinflammatory, antidiabetic, and anticancer activities. However, there are many difficulties in obtaining them, since the levels of these compounds produced from plant raw materials are low and their chemical synthesis is not economically profitable for commercial exploitation...
September 28, 2016: Journal of Agricultural and Food Chemistry
Wubin Wang, Meifeng Liu, Yufeng Wang, Xuliang Li, Shixuan Cheng, Liping Shu, Zheping Yu, Jiejie Kong, Tuanjie Zhao, Junyi Gai
Annual wild soybean (Glycine soja Sieb. and Zucc.), the wild progenitor of the cultivated soybean [Glycine max (L.) Merr.], is valuable for improving the later. The construction of a linkage map is crucial for studying the genetic differentiation between these species, but marker density is the main factor limiting the accuracy of such a map. Recent advances in next-generation sequencing technologies allow for the generation of high-density linkage maps. Here, two sets of inter-specific recombinant inbred line populations, named NJIRNP and NJIR4P, composed of 284 and 161 lines, respectively, were generated from the same wild male parent, PI 342618B, and genotyped by restriction-site-associated DNA sequencing...
2016: Frontiers in Plant Science
Tim Heise, Chantal Mathieu
Manufacturers of insulin products for diabetes therapy have long sought ways to modify the absorption rate of exogenously administered insulins in an effort to better reproduce the naturally occurring pharmacokinetics of endogenous insulin secretion. Several mechanisms of protraction have been used in pursuit of a basal insulin for which a low injection frequency would provide tolerable and reproducible glucose control; these mechanisms have met with varying degrees of success. Before the advent of recombinant DNA technology, development focused on modifications to the formulation that increased insulin self-association, such as supplementation with zinc or the development of pre-formed precipitates using protamine...
September 4, 2016: Diabetes, Obesity & Metabolism
Seema Patel
Despite the advent of next-generation sequencing (NGS) technologies, sophisticated data analysis and drug development efforts, bacterial drug resistance persists and is escalating in magnitude. To better control the pathogens, a thorough understanding of their genomic architecture and dynamics is vital. Bacterial genome is extremely complex, a mosaic of numerous co-operating and antagonizing components, altruistic and self-interested entities, behavior of which are predictable and conserved to some extent, yet largely dictated by an array of variables...
August 30, 2016: Infection, Genetics and Evolution
Asim Azhar, Ejaj Ahmad, Qamar Zia, Mohammad Owais, Ghulam Md Ashraf
Therapeutic proteins are engineered proteins produced in the laboratory for pharmaceutical use. With the advent of recombinant DNA technology, the proteins can be generated in specific host cells under defined conditions. In the process of production of genetically engineered animals, the gene of interest can be added at a single cell stage to produce a cloned animal from genetically engineered cells. Several recombinant cytokines, clotting factors etc have been licensed and are currently being utilized for the treatment of cancer, infectious diseases, hemophilia, anemia, multiple sclerosis, and hepatitis B/C...
September 1, 2016: Current Protein & Peptide Science
Zohreh Sadat Badieyan, Taras Berezhanskyy, Maximilian Utzinger, Manish Kumar Aneja, Daniela Emrich, Reinhold Erben, Christiane Schüler, Philipp Altpeter, Mehrije Ferizi, Günther Hasenpusch, Carsten Rudolph, Christian Plank
Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection...
October 10, 2016: Journal of Controlled Release: Official Journal of the Controlled Release Society
Yuewen Zhao, Xiaojing Yang, Zongchao Jia, Robert L Reid, Pierre Leclerc, Frederick W K Kan
The mammalian oviduct synthesizes and secretes a major glycoprotein known as oviductin (OVGP1) which has been shown to interact with gametes and early embryos. We report here the use of recombinant DNA technology to produce, for the first time, the secretory form of human OVGP1 in HEK293 cells. HEK293 colonies stably expressing recombinant human OVGP1 (rHuOVGP1) were established by transfecting cells with an expression vector pCMV6-Entry constructed with OVGP1 cDNA. Large quantities of rHuOVGP1 were obtained from the stably transfected cells using the CELLSPIN cell cultivation system...
August 31, 2016: Reproduction: the Official Journal of the Society for the Study of Fertility
Yalan Zhou, Yanan Zong, Xiangdong Kong
CRISPR/Cas9 technology originated from type II CRISPR/Cas system, which is widely found in bacteria and equips them with acquired immunity against viruses and plasmids. CRISPR-associated protein Cas9 is a RNA-guided endonuclease, which can efficiently introduce double-strand breaks at specific sites and activate homologous recombination and/or non-homologous end joining mechanism for the repair of impaired DNA. Features such as easy-to-use, cost-effectiveness, multiple targeting ability have made it the third-generation genomic engineering tool following ZFNs and TALENs...
October 2016: Zhonghua Yi Xue Yi Chuan Xue za Zhi, Zhonghua Yixue Yichuanxue Zazhi, Chinese Journal of Medical Genetics
Zsolt Kelemen, Jonathan Przybyla-Toscano, Nicolas Tissot, Loïc Lepiniec, Christian Dubos
Yeast one-hybrid (Y1H) assay has been proven to be a powerful technique to characterize in vivo the interaction between a given transcription factor (TF), or its DNA-binding domain (DBD), and target DNA sequences. Comprehensive characterization of TF/DBD and DNA interactions should allow designing synthetic promoters that would undoubtedly be valuable for biotechnological approaches. Here, we use the ligation-independent cloning system (LIC) in order to enhance the cloning efficiency of DNA motifs into the pHISi Y1H vector...
2016: Methods in Molecular Biology
Anne Abot, Gregory Arnal, Lucas Auer, Adèle Lazuka, Delphine Labourdette, Sophie Lamarre, Lidwine Trouilh, Elisabeth Laville, Vincent Lombard, Gabrielle Potocki-Veronese, Bernard Henrissat, Michael O'Donohue, Guillermina Hernandez-Raquet, Claire Dumon, Véronique Anton Leberre
BACKGROUND: Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides through their production of a vast variety of Glycoside Hydrolases (GH). The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharides or glycans. RESULTS: This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database...
2016: BMC Genomics
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