transgene techniques

Wusheng Liu, Mary R Rudis, Matthew H Cheplick, Reginald J Millwood, Jian-Ping Yang, Christine A Ondzighi-Assoume, Garrett A Montgomery, Kellie P Burris, Mitra Mazarei, Jonathan D Chesnut, Charles Neal Stewart
A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates...
November 14, 2019: Plant Cell Reports
Gabriela Bortolança Chiarotto, Luciana Politti Cartarozzi, Matheus Perez, Natalia Perussi Biscola, Aline Barroso Spejo, Fernanda Gubert, Marcondes França Junior, Rosália Mendez-Otero, Alexandre Leite Rodrigues de Oliveira
BACKGROUND: The development of new therapeutic strategies to treat amyotrophic lateral sclerosis (ALS) is of utmost importance. The use of cyclic nitroxides such as tempol may provide neuroprotection and improve lifespan. We investigated whether tempol (50 mg/kg) presents therapeutic potential in SOD1G93A transgenic mice. METHODS: Tempol treatment began at the asymptomatic phase of the disease (10th week) and was administered every other day until week 14, after which it was administered twice a week until the final stage of the disease...
November 14, 2019: Journal of Neuroinflammation
Doori Park, Ik-Young Choi, Nam-Soo Kim
BACKGROUND: Various kinds of transposable elements (TEs) constitute high proportions of eukaryotic genomes. Although most of these TEs are not actively mobile, genome stress can induce mobilization of dormant TEs. Transgenic plants undergo tissue culture and subsequent whole-plant regeneration, which can cause genomic stress and in turn induce mobilization of inactive TEs. OBJECTIVES: To investigate the activation of transposable elements on the genome wide of the GM plant...
November 12, 2019: Genes & Genomics
Renkai Ge, Ke Chen, Yi Cheng, Yue Dai
The mesencephalic locomotor region (MLR) is an essential area for initiation of locomotion. Its functional roles and circuits underlying locomotion have been studied intensively in many species. Studies suggest that cuneiform nucleus and pedunculopontine nucleus (PPN) are two core regions in the MLR for locomotion. However, it remains unclear about cellular components and morphological and intrinsic membrane properties of the neurons in these regions, especially the serotonergic neurons. Using neonatal ePet-EYFP transgenic mice and immunofluorescent technique, we demonstrated existence of 5-HT neurons in the MLR and discovered that 5-HT neurons distributed mainly in the caudal PPN...
November 12, 2019: Experimental Brain Research. Experimentelle Hirnforschung. Expérimentation Cérébrale
Ying Yan, Megan E Williamson, Rebecca J Davis, Anne A Andere, Christine J Picard, Maxwell J Scott
For genetic approaches for controlling insect pests such as the sterile insect technique (SIT), it is advantageous to release only males as females are ineffective as control agents and they consume about 50% of the diet. Here we developed tetracycline-repressible Lucilia cuprina transgenic strains in which adult females were fully fertile and viable on a diet that lacked tetracycline and all of their female offspring died at the embryo stage. The transgenic strains are an improvement over the strains we developed previously, which had the disadvantage that adult females on diet without tetracycline were sterile and died prematurely...
November 12, 2019: Molecular Genetics and Genomics: MGG
Rakhshanda Mushtaq, Khurram Shahzad, Zahid Hussain Shah, Hameed Alsamadany, Hind A S Alzahrani, Yahya Alzahrani, Tahir Mujtaba, Zaheer Ahmed, Shahid Mansoor, Aftab Bashir
Cotton production is widely effected by Cotton Leaf Curl Virus (CLCuV) in world posing serious losses to cotton yield.The CRT genes from CLCuV resistant G. arboreum and CLCuV susceptible G. hirsutum were cloned and sequenced to know the differences of protein composition in both species. Molecular techniques were used to isolate full length putative biotic stress resistance genes from G. arboreum besides the analysis of identified novel genes in model plant tobacco (Nicotiana tabacum) for resistance to cotton leaf curl disease complex...
November 8, 2019: Journal of Virological Methods
Elizabeth A Pietruczyk, Terilyn K L Stephen, Simon Alford, Sarah E Lutz
This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat...
October 23, 2019: Journal of Visualized Experiments: JoVE
Zhanqi Dong, Jiangqiong Long, Liang Huang, Zhigang Hu, Peng Chen, Nan Hu, Ning Zheng, Xuhua Huang, Cheng Lu, Minhui Pan
The microsporidian Nosema bombycis is an obligate intracellular parasitic fungus that causes devastating disease in sericulture. To date, no efficient biotechnological method to inhibit the proliferation of microspores has been established. Here, we developed a powerful genetic engineering technique involving microsporidia-inducible genome editing in transgenic silkworm that confers resistance to N. bombycis. This system includes an HSP70 promoter-induced expression of the Cas9 protein line and a target BmATAD3A gene line...
November 9, 2019: Applied Microbiology and Biotechnology
Yuan Li, Xiang Mao, Xianyi Zhou, Yuting Su, Xiangyu Zhou, Kaituo Shi, Shasha Zhao
BACKGROUND: Neural differentiation from embryonic stem cells (ESCs) is an excellent model for elucidating the key mechanisms involved in neurogenesis, and also provides an unlimited source of progenitors for cell-based nerve regeneration. However, the existing protocols such as small molecule substances, 3D matrix, co-culture technique and transgenic method, are complicated and difficult to operate, thus are limited by laboratory conditions. Looking for an easy-to-operate protocol with easily gained material and high induction efficiency has always been a hot issue in neuroscience research...
November 7, 2019: Journal of Neuroscience Methods
Marcos E Farías, Mariela M Marani, Darío Ramírez, Ana M Niebylski, N Mariano Correa, Patricia G Molina
A new polyclonal antibody that recognizes the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS), which provides resistance to glyphosate in soybean (Roundup Ready®, RR soybean), was produced. New Zealand rabbits were injected with a synthetic peptide (Pc_312-324, (PEP)) present in the soybean CP4-EPSPS protein. The anti-PEP antibodies production was evaluated by electrophoresis (SDS-PAGE) and an enzyme-linked immunosorbent assay (ELISA) was developed in order to study their specificity. The ELISA showed that the polyclonal antibody was specific to PEP...
October 4, 2019: Bioelectrochemistry
Hiroshi Kiyonari, Yasuhide Furuta
The 15th Transgenic Technology (TT) Meeting of the International Society for Transgenic Technologies (ISTT) was held for the first time in Japan in April of 2019 (TT2019). Delegates from around the world, including researchers, technicians and trainees, spent an exciting 4 days, engaging in scientific and technical discussions, information sharing and networking. In the era of genome editing, CRISPR technologies prevailed as popular subjects of discussion throughout the meeting on topics ranging from their applications in the mouse and growing number of other research and industrial animal species, technical challenges in the production of genome-edited animals, to ethical considerations in biomedical research...
November 8, 2019: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Dustin Cram, Manoj Kulkarni, Miles Buchwaldt, Nandhakishore Rajagopalan, Pankaj Bhowmik, Kevin Rozwadowski, Isobel A P Parkin, Andrew G Sharpe, Sateesh Kagale
BACKGROUND: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement. DESCRIPTION: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat...
November 6, 2019: BMC Plant Biology
Ioannis Exarchos, Anna A Rogers, Lauren M Aiani, Robert E Gross, Gari D Clifford, Nigel P Pedersen, Jon T Willie
Despite commercial availability of software to facilitate sleep-wake scoring of electroencephalography (EEG) and electromyography (EMG) in animals, automated scoring of rodent models of abnormal sleep, such as narcolepsy with cataplexy, has remained elusive. We optimize two machine-learning approaches, supervised and unsupervised, for automated scoring of behavioral states in orexin/ataxin-3 transgenic mice, a validated model of narcolepsy type 1, and additionally test them on wild-type mice. The supervised learning approach uses previously labeled data to facilitate training of a classifier for sleep states, whereas the unsupervised approach aims to discover latent structure and similarities in unlabeled data from which sleep stages are inferred...
November 6, 2019: Sleep
Aya Imafuku, Masatoshi Oka, Yoei Miyabe, Sachiko Sekiya, Kosaku Nitta, Tatsuya Shimizu
Renal fibrosis is one of the largest global health care problems, and microvascular (MV) injury is important in the development of progressive fibrosis. Although conventional cell therapy suppresses kidney injury via the role of vasoprotective cytokines, the effects are limited due to low retention of administered cells. We recently described that transplantation of hepatocyte growth factor (HGF)-transgenic mesothelial cell sheets showed a remarkable cell survival and strong therapeutic effects in a rat renal fibrosis model...
November 1, 2019: Stem Cells Translational Medicine
Deshka S Foster, Alan T Nguyen, Malini Chinta, Ankit Salhotra, R Ellen Jones, Shamik Mascharak, Ashley L Titan, R Chase Ransom, Oscar L da Silva, Eliza Foley, Emma Briger, Michael T Longaker
Fluorescent proteins are used extensively in transgenic animal models to label and study specific cell and tissue types. Expression of these proteins can be imaged and analyzed using fluorescent and confocal microscopy. Conventional confocal microscopes cannot penetrate through tissue more than 4-6 μm thick. Tissue clearing procedures overcome this challenge by rendering thick specimens into translucent tissue. However, most tissue clearing techniques do not satisfactorily preserve expression of endogenous fluorophores...
October 31, 2019: Scientific Reports
Xiangchen Kong, Yifan Tu, Bingfeng Li, Longmei Zhang, Linxian Feng, Lixiang Wang, Lin Zhang, Huarong Zhou, Xianxin Hua, Xiaosong Ma
OBJECTIVE: Roux-en-Y gastric bypass surgery (RYGB) improves the first phase of glucose-stimulated insulin secretion (GSIS) in patients with type 2 diabetes. How it does so remains unclear. Farnesoid X receptor (FXR), the nuclear receptor of bile acids (BAs), is implicated in bariatric surgery. Moreover, the transient receptor potential ankyrin 1 (TRPA1) channel is expressed in pancreatic β-cells and involved in insulin secretion. We aimed to explore the role of BAs/FXR and TRPA1 in improved GSIS in diabetic rats after RYGB...
November 2019: Molecular Metabolism
Jiangshuo Su, Jiafu Jiang, Fei Zhang, Ye Liu, Lian Ding, Sumei Chen, Fadi Chen
Chrysanthemum ( Chrysanthemum morifolium Ramat.) is a leading flower with applied value worldwide. Developing new chrysanthemum cultivars with novel characteristics such as new flower colors and shapes, plant architectures, flowering times, postharvest quality, and biotic and abiotic stress tolerance in a time- and cost-efficient manner is the ultimate goal for breeders. Various breeding strategies have been employed to improve the aforementioned traits, ranging from conventional techniques, including crossbreeding and mutation breeding, to a series of molecular breeding methods, including transgenic technology, genome editing, and marker-assisted selection (MAS)...
2019: Horticulture Research
Satsuki Sato, Jinya Suzuki, Masamichi Hirose, Mika Yamada, Yasuo Zenimaru, Takahiro Nakaya, Mai Ichikawa, Michiko Imagawa, Sadao Takahashi, Shoichiro Ikuyama, Tadashi Konoshita, Fredric B Kraemer, Tamotsu Ishizuka
Atrial fibrillation (AF) is prevalent in patients with obesity and diabetes, and such patients often exhibit cardiac steatosis. Since the role of cardiac steatosis per se in the induction of AF has not been elucidated, the present study was designed to explore the relation between cardiac steatosis and AF. Transgenic (Tg) mice with cardiac-specific overexpression of perilipin 2 (PLIN2) were housed in the laboratory for more than 12 months before the study. Electron microscopy of the atrium of PLIN2-Tg mice showed accumulation of small lipid droplets around mitochondrial chains, and 5- to 9-fold greater atrial triacylglycerol (TAG) content compared to wildtype (Wt) mice...
October 29, 2019: American Journal of Physiology. Endocrinology and Metabolism
Xu-Jing Wang, Yu-Feng Dong, Xi Jin, Jiang-Tao Yang, Zhi-Xing Wang
Controlling transgene flow in China is important, as this country is part of the center of origin of rice. A gene-splitting technique based on intein-mediated trans-splicing represents a new strategy for controlling transgene flow via biological measures. In this study, the G2-aroA gene which provides glyphosate tolerance was split into an N-terminal and a C-terminal region, which were then fused to intein N and intein C of the Ssp DnaE intein, ultimately forming EPSPSn:In and Ic:EPSPSc fusion genes, respectively...
October 25, 2019: Transgenic Research
Jimeng Hu, Xiaobo Wu, Chen Yang, Khalid Rashid, Chenkai Ma, Mengbo Hu, Qiang Ding, Haowen Jiang
Prostate cancer (PCa) is one of the most common health-related issues in the male individuals of western countries. Icaritin (ICT) is a traditional Chinese herbal medicine that exhibits antitumor efficacy in variety of cancers including PCa. However, the precise function and detailed molecular mechanism of ICT in the regression of PCa remain unclear. Ubiquitin-conjugating enzyme E2C (UBE2C) is an anaphase-promoting complex/cyclosome (APC/C)-specific ubiquitin conjugating enzyme, which acts as an oncogene in PCa progression...
October 24, 2019: Cancer Medicine
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