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recombinant DNA technology

Yan-Yan Ma, Jing-Zhe Li, Er-Ning Gao, Dan Qian, Ju-Ying Zhong, Chang-Zhen Liu
Gene editing is a kind of technologies that makes precise modification to the genome. It can be used to knock out/in and replace the specific DNA fragment, and make accurate gene editing on the genome level. The essence of the technique is the DNA sequence change with use of non homologous end link repair and homologous recombination repair, combined with specific DNA target recognition and endonuclease.This technology has wide range of development prospects and high application value in terms of scientific research, agriculture, medical treatment and other fields...
January 2017: Zhongguo Zhong Yao za Zhi, Zhongguo Zhongyao Zazhi, China Journal of Chinese Materia Medica
Pan Liu, Tomokazu Souma, Andrew Zu-Sern Wei, Xueying Xie, Xunrong Luo, Jing Jin
In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera...
September 6, 2017: Journal of Visualized Experiments: JoVE
Lauren M Oldfield, Peter Grzesik, Alexander A Voorhies, Nina Alperovich, Derek MacMath, Claudia D Najera, Diya Sabrina Chandra, Sanjana Prasad, Vladimir N Noskov, Michael G Montague, Robert M Friedman, Prashant J Desai, Sanjay Vashee
Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells...
September 19, 2017: Proceedings of the National Academy of Sciences of the United States of America
Brian Lovett, Raymond John St Leger
Microbial insect pathogens offer an alternative means of pest control with potential to wean our heavy reliance on chemical pesticides. Insect pathogenic fungi play an important natural role in controlling disease vectors and agricultural pests. Most commercial products employ Ascomycetes in the genera Metarhizium and Beauveria. However, their utilization has been limited by inconsistent field results due to sensitivity to abiotic stresses and naturally low virulence. Other naturally occurring biocontrol agents also face these hurdles to successful application, but the availability of complete genomes and recombinant DNA technologies have facilitated design of multiple fungal pathogens with enhanced virulence and stress resistance...
September 14, 2017: Pest Management Science
Xianjun Lai, James C Schnable, Zhengqiao Liao, Jie Xu, Gengyun Zhang, Chuan Li, Erliang Hu, Tingzhao Rong, Yunbi Xu, Yanli Lu
BACKGROUND: Maize was originally domesticated in a tropical environment but is now widely cultivated at temperate latitudes. Temperate and tropical maize populations have diverged both genotypically and phenotypically. Tropical maize lines grown in temperate environments usually exhibit delayed flowering, pollination, and seed set, which reduces their grain yield relative to temperate adapted maize lines. One potential mechanism by which temperate maize may have adapted to a new environment is novel transposable element insertions, which can influence gene regulation...
September 6, 2017: BMC Genomics
Yuyong Wu, Lili You, Shengchun Li, Meiqi Ma, Mengting Wu, Lixin Ma, Ralph Bock, Ling Chang, Jiang Zhang
Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps...
2017: Frontiers in Plant Science
Meili Liu, Yanxi Luo, Zhengrong Zhao, Hui Yan
Molecular cloning is one of the most important and widely used technologies in molecular biology research. Generally, the target DNA fragment and the vector are separately digested by restriction enzyme, then purified and recovered, and then ligated with DNA-ligase. For some very short gene fragments (<300 bp), the recovery efficiency of the purified fragment is very low after digestion and cleavage, leading to the difficulty in its inserting into the expression vector. To address this issue, we developed a cloning method based on restoration of antibiotic resistance in constructing recombinant plasmid, which proved highly efficient in cloning very short gene fragments...
July 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Zhenlin Fan, Jian Song, Tuchen Guan, Xiuxiu Lv, Jingyan Wei
The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNA(UTu)) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321...
September 12, 2017: ACS Synthetic Biology
Shirin Eyvazi, Bahram Kazemi, Mojgan Bandehpour, Siavoush Dastmalchi
CD24 is a heavy glycosylated protein that is overexpressed in numerous cancer and cancer stem cells. It is involved in the development, invasion, and metastasis of the cancer cells. CD24 can be considered in targeted cancer therapy as a new target. Here, phage display technology was used for the identification of novel scFv antibodies against CD24. To do so, the CD24 protein was expressed and purified from a stable transgenic CHO cell line. The cells were developed by using a CD24 encoding construct, which targets the 18S rRNA gene...
September 1, 2017: Immunology Letters
Ekaterina G Frank, Mary P McLenigan, John P McDonald, Donald Huston, Samantha Mead, Roger Woodgate
The cDNA encoding human DNA polymerase ι (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies led to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase ι (polι) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified polι as a 740 amino acid protein...
August 19, 2017: DNA Repair
Lmar Babrak, Jeffery A McGarvey, Larry H Stanker, Robert Hnasko
Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibodies (rAb). This determination can be achieved by sequence analysis of immunoglobulin (Ig) transcripts obtained from a monoclonal antibody (MAb) producing hybridoma and subsequent expression of a rAb. However the polyploidy nature of a hybridoma cell often results in the added expression of aberrant immunoglobulin-like transcripts or even production of anomalous antibodies which can confound production of rAb...
October 2017: Molecular Immunology
Micol Falabella, Linqing Sun, Justin Barr, Andressa Z Pena, Erin E Kershaw, Sebastien Gingras, Elena A Goncharova, Brett A Kaufman
CRISPR-Cas9 based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by non-homologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR-editing in founder mice...
August 31, 2017: G3: Genes—Genomes—Genetics
Jean-Félix Dallery, Nicolas Lapalu, Antonios Zampounis, Sandrine Pigné, Isabelle Luyten, Joëlle Amselem, Alexander H J Wittenberg, Shiguo Zhou, Marisa V de Queiroz, Guillaume P Robin, Annie Auger, Matthieu Hainaut, Bernard Henrissat, Ki-Tae Kim, Yong-Hwan Lee, Olivier Lespinet, David C Schwartz, Michael R Thon, Richard J O'Connell
BACKGROUND: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. RESULTS: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7...
August 29, 2017: BMC Genomics
Linnea Nilebäck, Dimple Chouhan, Ronnie Jansson, Mona Widhe, Biman B Mandal, My Hedhammar
Natural silk is easily accessible from silkworms and can be processed into different formats suitable as biomaterials and cell culture matrixes. Recombinant DNA technology enables chemical-free functionalization of partial silk proteins through fusion with peptide motifs and protein domains, but this constitutes a less cost-effective production process. Herein, we show that natural silk fibroin (SF) can be used as a bulk material that can be top-coated with a thin layer of the recombinant spider silk protein 4RepCT in fusion with various bioactive motifs and domains...
September 20, 2017: ACS Applied Materials & Interfaces
Amir Hossein Mohseni, Mohammad Soleimani, Keivan Majidzadeh-A, Sedigheh Taghinezhad-S, Hossein Keyvani
Background: Human tissue-type plasminogen activator (t-PA) is a key protease of the trypsin family. It catalyzes the activation of zymogen plasminogen to the fibrin-degrading proteinase, plasmin, leading to digestion of fibrin clots. The recombinant enzyme produced by recombinant technology issued to dissolve blood clots in treatment of various human diseases such as coronary artery thrombosis, pulmonary embolism, acute ischemic stroke (AIS). Pichia pastoris expression system is a unique system for the production of high level of recombinant proteins...
August 27, 2017: Asian Pacific Journal of Cancer Prevention: APJCP
Birhanu M Kinfu, Maike Jahnke, Mareike Janus, Volkan Besirlioglu, Michael Roggenbuck, Richard Meurer, Ljubica Vojcic, Martin Borchert, Ulrich Schwaneberg, Jennifer Chow, Wolfgang R Streit
The exciting promises of functional metagenomics for the efficient discovery of novel biomolecules from nature are often hindered by factors associated with expression hosts. Aiming to shift functional metagenomics to a host independent innovative system, we here report on the cloning, heterologous expression, and reconstitution of an RNA polymerase (RNAP) from the thermophilic Geobacillus sp. GHH01 and in vitro transcription thereafter. The five genes coding for RNAP subunits, a house keeping sigma factor and two transcription elongation factors were cloned and over expressed as His6 -tagged and/ or tag-free proteins...
August 26, 2017: Biotechnology and Bioengineering
Dirk Grimm, Hildegard Büning
Recombinant gene delivery vectors derived from naturally occurring or genetically engineered Adeno-associated viruses (AAV) have taken center stage in human gene therapy, fueled by rapidly accumulating and highly encouraging clinical data. Nonetheless, it has also become evident that the current generation of AAV vectors will require improvements in transduction potency, antibody evasion and cell specificity, to realize their full potential and to widen applicability in larger patient cohorts. Fortunately, in the recent past, the field has seen a flurry of exciting new developments that enhance our understanding of AAV vector biology including virus-host interactions and/or that expand our arsenal of technologies for AAV capsid design and evolution...
August 23, 2017: Human Gene Therapy
Miaorong Huang, Ruiai Chen, Guangcai Ren
The keratinase (kerA) gene from Bacillus licheniformis PWD-1 was expressed and purified in insect cells. First, the sequence encoding Ker-His-Flag was designed based on the amino acid sequence of the protein and peptide and codon optimization in order to ensure the high expression in insect cells. In the next step, the synthetic DNA was inserted into the pUC57 vector and then sub-cloned in the pFastBac™-1 donor vector by BamHI/HindIII restriction sites. The constructed vector was transformed to E. coli DH10Bac™ cell to generate recombinant bacmid carrying Ker-His-Flag...
2017: PloS One
Jan Kok, Lieke A van Gijtenbeek, Anne de Jong, Sjoerd B van der Meulen, Ana Solopova, Oscar P Kuipers
Lactococcus lactis is a major microbe. This lactic acid bacterium (LAB) is used worldwide in the production of safe, healthy, tasteful and nutritious milk fermentation products. Its huge industrial importance has led to an explosion of research on the organism, particularly since the early 1970s. The upsurge in the research on L. lactis coincided not accidentally with the advent of recombinant DNA technology in these years. The development of methods to take out and re-introduce DNA in L. lactis, to clone genes and to mutate the chromosome in a targeted way, to control (over)expression of proteins and, ultimately, the availability of the nucleotide sequence of its genome and the use of that information in transcriptomics and proteomics research have enabled to peek deep into the functioning of the organism...
August 1, 2017: FEMS Microbiology Reviews
Bjoern Petersen
In the last decade, the research community has witnessed a blooming of targeted genome editing tools and applications. Novel programmable DNA nucleases such as zinc finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs) and the clustered regularly interspaced short palindromic repeats/Cas9 system (CRISPR/Cas9) possess long recognition sites and are capable of cutting DNA in a very specific manner. These DNA nucleases mediate targeted genetic alterations by enhancing the DNA mutation rate via induction of double-strand breaks at a predetermined genomic site...
August 2017: Reproduction in Domestic Animals, Zuchthygiene
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