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PhiC31

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https://www.readbyqxmd.com/read/29133827/blastocyst-formation-rate-and-transgene-expression-are-associated-with-gene-insertion-into-safe-and-non-safe-harbors-in-the-cattle-genome
#1
Milad Khorramian Ghahfarokhi, Kianoush Dormiani, Ali Mohammadi, Farnoosh Jafarpour, Mohammad Hossein Nasr-Esfahani
Integration target site is the most important factor in successful production of transgenic animals. However, stable expression of transgene without disturbing the function of the host genome depends on promoter methylation, transgene copy number and transcriptional activity in integration regions. Recently, new genome-editing tools have made much progress, however little attention has been paid to the identification of genomic safe harbors. The aim of the present study was to evaluate the effect of insertion site, promoter and copy number of transgene on the production of embryos from cattle fibroblast cells following somatic cell nuclear transfer (SCNT)...
November 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28815494/use-of-the-dice-dual-integrase-cassette-exchange-system
#2
Alfonso P Farruggio, Mital S Bhakta, Michele P Calos
When constructing transgenic cell lines via plasmid DNA integration, precise targeting to a desired genomic location is advantageous. It is also often advantageous to remove the bacterial backbone, since bacterial elements can lead to the epigenetic silencing of neighboring DNA. The least cumbersome method to remove the plasmid backbone is recombinase-mediated cassette exchange (RMCE). RMCE is accomplished by arranging recombinase sites in the genome and in a donor plasmid such that a recombinase can both integrate the donor plasmid and excise its bacterial backbone...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28814578/genetic-human-prion-disease-modelled-in-prp-transgenic-drosophila
#3
Alana M Thackray, Alzbeta Cardova, Hanna Wolf, Lydia Pradl, Ina Vorberg, Walker S Jackson, Raymond Bujdoso
Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrP(Sc), an abnormal isomer of the normal host protein PrP(C), in the brain of affected individuals. PrP(Sc) is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity...
September 20, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28802644/rapid-characterization-of-the-cho-platform-cell-line-and-identification-of-pseudo-attp-sites-for-phic31-integrase
#4
Narges Damavandi, Mozhgan Raigani, Atefeh Joudaki, Fatemeh Davami, Sirous Zeinali
The Chinese Hamster Ovary (CHO) cell lines, applicable to post-translational modifications, are preferred systems for biopharmaceutical protein production. In this study, by using the Jump-In™ TI™ technology which employs PhiC31 and R4 bacteriophage recombinases, a platform CHO-K1 cell line containing a R4-attP site was generated. Here, a combination of Quantitative Fluorescent-Polymerase Chain Reaction (QF-PCR) and semi-random, two-step PCR (ST-PCR), was performed to feature the platform cell clones. Our results show that QF-PCR and ST-PCR, can be utilized for efficient and accelerated cell line characterization...
December 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28400295/phic31-integrase-can-improve-the-efficiency-of-different-construct-designs-for-monoclonal-antibody-expression-in-cho-cells
#5
Maryam Ahmadi, Fereidoun Mahboudi, Samira Ahmadi, Saeedeh Ebadat, Fatemeh Nematpour, Mohammad Reza Akbari Eidgahi, Fatemeh Davami
OBJECTIVES: Several types of expression vectors have been used for recombinant protein expression in Chinese hamster ovary cells (CHO) which usually result in variable and unstable levels of expression. METHODS AND RESULTS: In this study, we have compared the mAb0014 expression level of single ORF/IRES vector and dual ORF vector in the presence and absence of phiC31 integrase targeting system. Both expression vectors contain an elongation factor 1α (EF1α) promoter upstream of LC and harboring an attB site...
June 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28280212/genetic-and-transgenic-reagents-for-drosophila-simulans-d-mauritiana-d-yakuba-d-santomea-and-d-virilis
#6
David L Stern, Justin Crocker, Yun Ding, Nicolas Frankel, Gretchen Kappes, Elizabeth Kim, Ryan Kuzmickas, Andrew Lemire, Joshua D Mast, Serge Picard
Species of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. However, studies in these species have been limited by a paucity of genetic and transgenic reagents. Here, we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein (EYFP) gene expressed in the eyes and a ϕC31 attP site-specific integration site...
April 3, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28266580/cre-lox-recombinase-mediated-cassette-exchange-for-reversible-site-specific-genomic-targeting-of-the-disease-vector-aedes-aegypti
#7
Irina Häcker, Robert A Harrell Ii, Gerrit Eichner, Kristina L Pilitt, David A O'Brochta, Alfred M Handler, Marc F Schetelig
Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects...
March 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28195163/targeted-gene-knock-in-by-crispr-cas-ribonucleoproteins-in-porcine-zygotes
#8
Ki-Eun Park, Anne Powell, Shelley E S Sandmaier, Chan-Mi Kim, Alan Mileham, David M Donovan, Bhanu P Telugu
The domestic pig is an important "dual purpose" animal model for agricultural and biomedical applications. There is an emerging consensus in the biomedical community for the use of large animal models such as pigs to either serve as an alternative, or complement investigations from the mouse. However, the use of pig has not proven popular due to technical difficulties and time required in generating models with desired genetic modifications. In this regard, the ability to directly modify the genome in the zygote and generate edited animals is highly desirable...
February 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28165856/sechsp70-as-a-tool-to-approach-amyloid-%C3%AE-42-and-other-extracellular-amyloids
#9
Lorena De Mena, Deepak Chhangani, Pedro Fernandez-Funez, Diego E Rincon-Limas
Self-association of amyloidogenic proteins is the main pathological trigger in a wide variety of neurodegenerative disorders. These aggregates are deposited inside or outside the cell due to hereditary mutations, environmental exposures or even normal aging. Cumulative evidence indicates that the heat shock chaperone Hsp70 possesses robust neuroprotection against various intracellular amyloids in Drosophila and mouse models. However, its protective role against extracellular amyloids was largely unknown as its presence outside the cells is very limited...
July 3, 2017: Fly
https://www.readbyqxmd.com/read/28139886/genomic-integration-of-the-full-length-dystrophin-coding-sequence-in-duchenne-muscular-dystrophy-induced-pluripotent-stem-cells
#10
Alfonso P Farruggio, Mital S Bhakta, Haley du Bois, Julia Ma, Michele P Calos
The plasmid vectors that express the full-length human dystrophin coding sequence in human cells was developed. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, providing challenges for cloning and plasmid production in Escherichia coli. The authors expressed dystrophin from the strong, widely expressed CAG promoter, along with co-transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E...
April 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/28053117/gb3-0-a-platform-for-plant-bio-design-that-connects-functional-dna-elements-with-associated-biological-data
#11
Marta Vazquez-Vilar, Alfredo Quijano-Rubio, Asun Fernandez-Del-Carmen, Alejandro Sarrion-Perdigones, Rocio Ochoa-Fernandez, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez
Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation...
February 28, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/27727435/pronuclear-injection-based-targeted-transgenesis
#12
Samantha L P Schilit, Masato Ohtsuka, Rolen M Quadros, Channabasavaiah B Gurumurthy
Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models. While efficient, the injected DNA integrates randomly into the genome, leading to potential disruption of endogenous genes or regulatory elements, variation in copy number, or integration into heterochromatic regions that inhibit transgene expression. A recently developed method addresses such pitfalls of traditional transgenesis by targeting the transgene to predetermined sites in the genome that can safely harbor exogenous DNA...
October 11, 2016: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/27604579/evaluating-the-efficiency-of-phic31-integrase-mediated-monoclonal-antibody-expression-in-cho-cells
#13
Maryam Ahmadi, Fereidoun Mahboudi, Mohammad Reza Akbari Eidgahi, Reza Nasr, Fatemeh Nematpour, Samira Ahmadi, Saeedeh Ebadat, Mojtaba Aghaeepoor, Fatemeh Davami
Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO-S cells...
November 2016: Biotechnology Progress
https://www.readbyqxmd.com/read/27493490/evaluation-of-an-hprt-luciferase-reporter-gene-on-a-mammalian-artificial-chromosome-in-response-to-cytotoxicity
#14
Takeshi Endo, Natsumi Noda, Yasushi Kuromi, Kenji Kokura, Yasuhiro Kazuki, Mitsuo Oshimura, Tetsuya Ohbayashi
BACKGROUND: Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene, and has been used as an internal control for real-time quantitative RT-PCR and various other methods of gene expression analysis. To evaluate the Hprt mRNA levels as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its response to cytotoxicity. METHODS: We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouse Hprt promoter fused with green-emitting luciferase (SLG) coding sequence...
June 2016: Yonago Acta Medica
https://www.readbyqxmd.com/read/27464802/testing-of-cis-regulatory-elements-by-targeted-transgene-integration-in-zebrafish-using-phic31-integrase
#15
Yavor Hadzhiev, Irene Miguel-Escalada, Darius Balciunas, Ferenc Müller
Herein we present several strategies for testing the function of cis-regulatory elements using the PhiC31 integrase system. Firstly, we present two different strategies to analyze the activity of candidate enhancer elements. Targeted integration of candidate enhancers into the same genomic location circumvents the variability-associated random integration and position effects. This method is suitable for testing of candidate enhancers identified through computational or other analyses a priori. Secondly, we present methodology for targeted integration of BACs into the same genomic location(s)...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27443928/contemporary-zebrafish-transgenesis-with-tol2-and-application-for-cre-lox-recombination-experiments
#16
A Felker, C Mosimann
Spatiotemporal transgene regulation by transgenic DNA recombinases is a central tool for reverse genetics in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. One of the most widespread technologies for this purpose is Cre recombinase-controlled lox site recombination that is attracting increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis provides a stable platform to integrate lox cassette transgenes, while the amenability of the zebrafish embryo to drug treatments makes the model an ideal candidate for tamoxifen-inducible CreERT2 experiments...
2016: Methods in Cell Biology
https://www.readbyqxmd.com/read/27382771/-construction-of-fat-1-eukaryotic-expression-vector-of-excision-markers-and-the-establishment-of-transgenic-sheep-cell-lines
#17
A Lima, Heping Zhu, Ruiyao Wang, Tao Yan, Xiaohu Su, Lu Li, Bingping Wang, Shunwendoule Na, Guichun Qi, Huanmin Zhou
In order to establish marker-free transgenic cell lines, we cloned Fat-1 gene, attB and Loxp sequences by PCR. Then we inserted these sequences to pN1-EGFP vector and got pEGFP-N1-Fat-1 expression vector. PhiC31 integrase mRNA which was generated by in vitro transcription and a pEGFP-N1-Fat-1 expression vector co-electroporated into sheep fetal fibroblasts, and then we got transgenic cell lines expressing green fluorescence. Prokaryotic expression and purification of Cre recombinant protein was performed. Cre recombinant protein was transducted into stably-transfected cell colonies...
February 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/27240344/targeted-gene-knockin-in-porcine-somatic-cells-using-crispr-cas-ribonucleoproteins
#18
Ki-Eun Park, Chi-Hun Park, Anne Powell, Jessica Martin, David M Donovan, Bhanu P Telugu
The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field...
May 26, 2016: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/27034267/generation-of-chickens-expressing-cre-recombinase
#19
Philip A Leighton, Darlene Pedersen, Kathryn Ching, Ellen J Collarini, Shelley Izquierdo, Roy Jacob, Marie-Cecile van de Lavoir
Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with β-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated. Cre recombinase activity was verified by mating Cre birds to birds carrying a floxed transgene. Floxed sequences were only excised in offspring from roosters that inherited the Cre recombinase but were excised in all offspring from hens carrying the Cre recombinase irrespective of the presence of the Cre transgene...
October 2016: Transgenic Research
https://www.readbyqxmd.com/read/26851519/pminerva-a-donor-acceptor-system-for-the-in-vivo-recombineering-of-scfv-into-igg-molecules
#20
M Batonick, M M Kiss, E P Fuller, C M Magadan, E G Holland, Q Zhao, D Wang, B K Kay, M P Weiner
Phage display is the most widely used method for selecting binding molecules from recombinant antibody libraries. However, validation of the phage antibodies often requires early production of the cognate full-length immunoglobulin G (IgG). The conversion of phage library outputs to a full immunoglobulin via standard subcloning is time-consuming and limits the number of clones that can be evaluated. We have developed a novel system to convert scFvs from a phage display vector directly into IgGs without any in vitro subcloning steps...
April 2016: Journal of Immunological Methods
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