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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
Maryam Ahmadi, Fereidoun Mahboudi, Mohammad Reza Akbari Eidgahi, Reza Nasr, Fatemeh Nemat Pour, Samira Ahmadi, Saeedeh Ebadat, Mojtaba Aghaee Poor, 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...
September 7, 2016: Biotechnology Progress
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
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
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
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
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...
2016: International Journal of Molecular Sciences
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
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
Hamid Dolatshad, Daniel Biggs, Rebeca Diaz, Nicole Hortin, Christopher Preece, Benjamin Davies
For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost...
December 2015: Mammalian Genome: Official Journal of the International Mammalian Genome Society
Yan Luo, Yongsheng Wang, Jun Liu, Hui Lan, Minghao Shao, Yuan Yu, Fusheng Quan, Yong Zhang
Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs)...
October 2015: Transgenic Research
Sonal Nagarkar-Jaiswal, Steven Z DeLuca, Pei-Tseng Lee, Wen-Wen Lin, Hongling Pan, Zhongyuan Zuo, Jiangxing Lv, Allan C Spradling, Hugo J Bellen
Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue...
2015: ELife
Masato Ohtsuka, Hiromi Miura, Keiji Mochida, Michiko Hirose, Ayumi Hasegawa, Atsuo Ogura, Ryuta Mizutani, Minoru Kimura, Ayako Isotani, Masahito Ikawa, Masahiro Sato, Channabasavaiah B Gurumurthy
BACKGROUND: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI...
2015: BMC Genomics
K Dormiani, H Mir Mohammad Sadeghi, H Sadeghi-Aliabadi, K Ghaedi, M Forouzanfar, H Baharvand, M H Nasr-Esfahani
Targeted integration of a therapeutic gene at specific loci in safe genomic regions by a non-viral vector can restore the function of the damaged gene. This approach also minimizes the potential genotoxic effects of transferred DNA. In this study, we have developed a non-viral vector that functions according to site-specific recombination (SSR). The vector contained a bacterial backbone and puromycin resistance gene (pur(r)), a β-globin expressing cassette and an attB recombination site. We used phiC31 integrase to insert a copy of the vector into specific genomic locations of a human hematopoietic cell line...
August 2015: Gene Therapy
Rajagopal N Aravalli, John D Belcher, Clifford J Steer
The liver plays a major role in many inherited and acquired genetic disorders. It is also the site for the treatment of certain inborn errors of metabolism that do not directly cause injury to the liver. The advancement of nucleic acid-based therapies for liver maladies has been severely limited because of the myriad untoward side effects and methodological limitations. To address these issues, research efforts in recent years have been intensified toward the development of targeted gene approaches using novel genetic tools, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats as well as various nonviral vectors such as Sleeping Beauty transposons, PiggyBac transposons, and PhiC31 integrase...
June 2015: Liver Transplantation
Dingpei Long, Weijian Lu, Yuli Zhang, Lihui Bi, Zhonghuai Xiang, Aichun Zhao
We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected...
2015: Scientific Reports
Ana Vanessa Vieira Oliveira, Gabriela Araújo Silva, Daniel C Chung
Gene transfer efficiency and expression stability are key factors to a successful gene therapy approach. In the present work we have developed a combined system for gene transfer that integrates well established non-viral polymeric vectors based on chitosan particles with the properties of phiC31-integrase that promotes a relatively non-immunogenic, site-specific integration, with sustained gene expression. Simultaneously, to overcome one of the major limitations in adeno-associated virus mediated gene transfer--the delivery of large genes--we have tested the capacity of our non-viral vectors to incorporate a large (8 Kb) transgene...
April 2015: Acta Biomaterialia
Roberta Sessa Stilhano, Priscila Keiko Matsumoto Martin, Suely Maymone de Melo, Vivian Yochiko Samoto, Giovani Bravin Peres, Yara Maria Correa da Silva Michelacci, Flavia Helena da Silva, Vanessa Gonçalves Pereira, Vania D'Almeida, Adriana Taveira da Cruz, Miriam Galvonas Jasiulionis, Sang Won Han
BACKGROUND: Mucopolysaccharidose type I (MPSI) is a lysosomal monogenic disease caused by mutations in the gene for α- L-iduronidase (IDUA). MPSI patients need a constant supply of IDUA to alleviate progression of the disease. IDUA gene transfer using integrative vectors might provide a definitive solution and support advancement to clinical trials, although studies have not yet been satisfactory. To achieve a stable IDUA gene expression in vivo, phiC31 was tested in the present study...
January 2015: Journal of Gene Medicine
Vahid Bahrambeigi, Nafiseh Ahmadi, Stefan Moisyadi, Johann Urschitz, Rasoul Salehi, Shaghayegh Haghjooy Javanmard
BACKGROUND: TRAIL and IFNγ are promising anti-cancer cytokines and it has been shown that IFNγ may sensitize cancer cells to TRAIL. Adipose derived mesenchymal stem cells (ADSCs) are attractive vehicles for delivering anti-cancer agents. In this study, we evaluated the therapeutic potential of PhiC31 (φC31) recombinase and/or piggyBac transposase (pBt) modified ADSCs expressing either TRAIL, IFNγ, or co-expressing TRAIL/IFNγ in mouse models of melanoma. METHODS: The expression and bioactivity of mouse IFNγ and TRAIL in φC31 and pBt modified cells were confirmed...
2014: Molecular Cancer
José S Meza, Francisco Díaz-Fleischer, Lázaro R Sánchez-Velásquez, Cristina Silvia Zepeda-Cisneros, Alfred M Handler, Marc F Schetelig
Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens...
2014: PloS One
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