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Hui-Ju Tsai, Ching-Ping Tseng
Multiple functions of platelets in various physiological and pathological conditions have prompted considerable attention on understanding how platelets are generated and activated. Of the adaptor proteins that are expressed in megakaryocytes and platelets, Disabled-2 (Dab2) has been demonstrated in the past decades as a key regulator of platelet signaling. Dab2 has two alternative splicing isoforms p82 and p59. However, the mode of Dab2's action remains to be clearly defined. In this review, we highlight the current understanding of Dab2 expression and function in megakaryocytic differentiation, platelet activation and integrin signaling...
2016: Thrombosis Journal
James West, W Warren Gill
Genome editing in large animals has tremendous practical applications, from more accurate models for medical research through improved animal welfare and production efficiency. Although genetic modification in large animals has a 30 year history, until recently technical issues limited its utility. The original methods - pronuclear injection and integrating viruses - were plagued with problems associated with low efficiency, silencing, poor regulation of gene expression, and variability associated with random integration...
June 2016: Journal of Equine Veterinary Science
Bingjie Shi, Juan Li, Xuanling Shi, Wenxu Jia, Yi Wen, Xiongbing Hu, Fengfeng Zhuang, Jianzhong Xi, Linqi Zhang
Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity and low cytotoxicity. We report here systematic design and characterization of twenty eight novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity and lower cytotoxicity compared to zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials...
October 3, 2016: Journal of Acquired Immune Deficiency Syndromes: JAIDS
Liezhen Fu, Luan Wen, Nga Luu, Yun-Bo Shi
Genome editing with designer nucleases such as TALEN and CRISPR/Cas enzymes has broad applications. Delivery of these designer nucleases into organisms induces various genetic mutations including deletions, insertions and nucleotide substitutions. Characterizing those mutations is critical for evaluating the efficacy and specificity of targeted genome editing. While a number of methods have been developed to identify the mutations, none other than sequencing allows the identification of the most desired mutations, i...
October 17, 2016: Scientific Reports
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October 15, 2016: Cancer Research
Ayman Eid, Magdy M Mahfouz
Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9)...
October 14, 2016: Experimental & Molecular Medicine
Zachary L Demorest, Andrew Coffman, Nicholas J Baltes, Thomas J Stoddard, Benjamin M Clasen, Song Luo, Adam Retterath, Ann Yabandith, Maria Elena Gamo, Jeff Bissen, Luc Mathis, Daniel F Voytas, Feng Zhang
BACKGROUND: The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability - a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of trans-fatty acids, which have been associated with cardiovascular disease...
October 13, 2016: BMC Plant Biology
František Liška, Renata Peterková, Miroslav Peterka, Vladimír Landa, Václav Zídek, Petr Mlejnek, Jan Šilhavý, Miroslava Šimáková, Vladimír Křen, Colby G Starker, Daniel F Voytas, Zsuzsanna Izsvák, Michal Pravenec
Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases)...
2016: PloS One
Zhenyu He, Kehkooi Kee
Gene targeting and editing is an essential tool for both basic research and clinical application such as gene therapy. Several endonucleases have been invented to fulfill these purposes, including zinc finger nucleases, TALEN, and CRISPR/Cas9. Although all of these systems can target DNA sequence with high efficiency, they also exert off-target effects and genotoxicity. The off-target effects might not hinder their usage in animal models because the correctly targeted cells can be selected for further studies...
2017: Methods in Molecular Biology
Barun Mahata, Kaushik Biswas
Precise and targeted genome editing using Transcription Activator-Like Effector Endonucleases (TALENs) has been widely used and proven to be an extremely effective and specific knockout strategy in both cultured cells and animal models. The current chapter describes a protocol for the construction and generation of TALENs using serial and hierarchical digestion and ligation steps, and using the synthesized TALEN pairs to achieve locus-specific targeted gene editing in mammalian cell lines using a modified clonal selection strategy in an easy and cost-efficient manner...
2017: Methods in Molecular Biology
Chew-Li Soh, Danwei Huangfu
The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system...
2017: Methods in Molecular Biology
Lisa Rank, Sebastian Veith, Eva C Gwosch, Janine Demgenski, Magdalena Ganz, Marjolijn C Jongmans, Christopher Vogel, Arthur Fischbach, Stefanie Buerger, Jan M F Fischer, Tabea Zubel, Anna Stier, Christina Renner, Michael Schmalz, Sascha Beneke, Marcus Groettrup, Roland P Kuiper, Alexander Bürkle, Elisa Ferrando-May, Aswin Mangerich
Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure-function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells...
September 29, 2016: Nucleic Acids Research
Mo Chen, Qin Wang, Gang-Hua Zhu, Peng Hu, Yuan Zhou, Tian Wang, Ruo-Sha Lai, Zi-An Xiao, Ding-Hua Xie
The TPRN gene encodes taperin, which is prominently present at the taper region of hair cell stereocilia. Mutations in TPRN have been reported to cause autosomal recessive nonsyndromic deafness 79(DFNB 79). To investigate the role of taperin in pathogenesis of hearing loss, we generated TPRN knockout mice using TALEN technique. Sanger sequencing confirmed an 11 bp deletion at nucleotide 177-187 in exon 1 of TPRN, which results in a truncated form of taperin protein. Heterozygous TPRN(+/-) mice showed apparently normal auditory phenotypes to their wide-type (WT) littermates...
October 28, 2016: Biochemical and Biophysical Research Communications
Arslan Akmammedov, Tomonori Katsuyama, Renato Paro
Owing to their modular and highly specific DNA recognition mode, transcription activator-like effector nucleases (TALENs) have been rapidly adopted by the scientific community for the purpose of generating site-specific double-strand breaks (DSBs) on a DNA molecule. A pair of TALENs can be used to produce random insertions or deletions of various lengths via nonhomologous end-joining or together with a homologous donor DNA to induce precise sequence alterations by homologous recombination (HR). Here, we describe a method for TALEN assembly (easyT) and a strategy for genome engineering via HR...
2016: Methods in Molecular Biology
Gang Li, Pierre Cunin, Di Wu, Dorothée Diogo, Yu Yang, Yukinori Okada, Robert M Plenge, Peter A Nigrovic
Understanding the implications of genome-wide association studies (GWAS) for disease biology requires both identification of causal variants and definition of how these variants alter gene function. The non-coding triallelic dinucleotide polymorphism CCR6DNP is associated with risk for rheumatoid arthritis, and is considered likely causal because allelic variation correlates with expression of the chemokine receptor CCR6. Using transcription activator-like effector nuclease (TALEN) gene editing, we confirmed that CCR6DNP regulates CCR6...
September 2016: PLoS Genetics
Mert Yanik, Brigitte Müller, Fei Song, Jacqueline Gall, Franziska Wagner, Wolfgang Wende, Birgit Lorenz, Knut Stieger
In vivo genome editing represents an emerging field in the treatment of monogenic disorders, as it may constitute a solution to the current hurdles in classic gene addition therapy, which are the low levels and limited duration of transgene expression. Following the introduction of a double strand break (DSB) at the mutational site by highly specific endonucleases, such as TALENs (transcription activator like effector nucleases) or RNA based nucleases (clustered regulatory interspaced short palindromic repeats - CRISPR-Cas), the cell's own DNA repair machinery restores integrity to the DNA strand and corrects the mutant sequence, thus allowing the cell to produce protein levels as needed...
September 10, 2016: Progress in Retinal and Eye Research
Hiroshi Asahara
MicroRNAs (miRNAs), which are small (~21 nucleotides) non-coding RNAs, are important players in endochondral ossification, articular cartilage homeostasis, and arthritis pathogenesis. Comprehensive and genetic analyses of cartilage-specific or cartilage-related miRNAs have provided new information on cartilage development, homeostasis, and related diseases. State-of-the-art combinatorial approaches, including transcription-activator like effector nuclease (TALEN)/clustered regularly interspaced short palindromic repeats (CRISPR) technique for targeting miRNAs and high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation for identifying target messenger RNAs, should be used to determine complex miRNA networks and miRNA-dependent cartilage regulation...
August 2016: Journal of Bone Metabolism
Matthew J O'Brien, Margaret R Moran, Joyce W Tang, Maria C Vargas, Mary Talen, Laura J Zimmermann, Ronald T Ackermann, Namratha R Kandula
PURPOSE: The purpose of this study was to explore how adults with prediabetes perceive their risk of developing diabetes and examine their preferences for evidence-based treatment options to prevent diabetes. METHODS: A qualitative study was conducted in 2 large Midwest primary care practices, involving in-depth semistructured interviews with 35 adult patients with prediabetes. RESULTS: This ethnically diverse (77% nonwhite) sample of middle-aged primary care patients exhibited multiple diabetes risk factors...
September 12, 2016: Diabetes Educator
Francisco Martin, Sabina Sánchez-Hernández, Alejandra Gutiérrez-Guerrero, Javier Pinedo-Gomez, Karim Benabdellah
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 endonuclease (Cas9) derived from bacterial adaptive immune systems is a revolutionary tool used in both basic and applied science. It is a versatile system that enables the genome of different species to be modified by generating double strand breaks (DSBs) at specific locations. However, all of the CRISPR/Cas9 systems can also produce DSBs at off-target sites that differ substantially from on-target sites. The generation of DSBs in locations outside the intended site can produce mutations that need to be carefully monitored, especially when using these tools for therapeutic purposes...
2016: International Journal of Molecular Sciences
Sabine Gogolok, Claudia Garcia-Diaz, Steven M Pollard
Transcription activator-like effectors (TALEs) contain modular programmable DNA binding domains. Fusing TALEs with effector domains creates synthetic transcription factors (TALE-TFs) or nucleases (TALENs), enabling precise gene manipulations. The construction of TALEs remains challenging due to their repetitive sequences. Here we report a simple TALE assembly reaction (STAR) that enables individual laboratories to generate multiple TALEs in a facile manner. STAR uses an isothermal assembly ('Gibson assembly') that is labour- and cost-effective, accessible, rapid and scalable...
2016: Scientific Reports
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