scholarly journals Generation of mutant pigs by lipofection-mediated genome editing in embryos

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maki Hirata ◽  
Manita Wittayarat ◽  
Zhao Namula ◽  
Quynh Anh Le ◽  
Qingyi Lin ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Large Scale ◽  
Gene Editing ◽  
Time Window ◽  
Mode Of Delivery ◽  
Practical Method ◽  
Ribonucleoprotein Complexes ◽  
Mammalian Embryos ◽  
Specialized Equipment ◽  
Reliable Methods

AbstractThe specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.

204 Efficient editing of porcine parthenogenetic zygotes by electroporation of Cas9 ribonucleoprotein complexes

2019 ◽  
Vol 31 (1) ◽  
pp. 227
Author(s):  
F. L. Ongaratto ◽  
P. Rodriguez-Villamil ◽  
U. Ganbaatar ◽  
C. De Frutos ◽  
S. Solin ◽  
...  
Keyword(s):  
Large Scale ◽  
Gene Editing ◽  
The Other ◽  
Scale Production ◽  
Efficient System ◽  
Ribonucleoprotein Complexes ◽  
Group 4 ◽  
Group 2 ◽  
And Control ◽  
Group 1

Gene editing by microinjection is an efficient system to produce mutant livestock; however, microinjection is time-consuming and requires special skill, limiting its use for large-scale production of gene-edited animals. Therefore, the aim of this study was to develop a system to deliver guide (g)RNA/Cas9/ribonucleoprotein (RNP) by electroporation into parthenogenic porcine zygotes. For experiment 1, we delivered gRNA/Cas9 RNP (250ng μL−1 of each), targeting GATA4 using 2 electroporation conditions. Group 1 (n=130): 20V, 3ms, ×2 pulses, 1 repeat; group 2 (n=102): 20V, 1ms, ×2 pulses, 2 repeats; and Control (n=96): parthenogenic zygotes, no electroporation. For experiment 2, we delivered gRNA/Cas9 RNP (250ng μL−1 of each) targeting ROSA26 by electroporation with 4 conditions compared with delivery of RNP by microinjection: group 1 (n=17): 20V, 3ms, ×1 pulses, 1 repeat; group 2 (n=49): 20V, 3ms, ×3 pulses, 1 repeat; group 3 (n=64): 30V, 3ms, ×1 pulses, 1 repeat; group 4 (n=61): 30V, 3ms, ×3 pulses, 1 repeat; group 5 (n=120): zygotes microinjected with Cas9/ROSA26 sgRNA (25/25ng μL−1), and Control (n=76): parthenogenic zygotes, no electroporation. The electroporated zygotes were cultured in porcine zygote medium-3 (PZM-3) with controlled atmosphere, and development was evaluated on Day 2 (cleavage) and Day 7 (blastocyst rate). Gene editing was evaluated on embryos (blastocyst and morulas) by PCR and Sanger sequencing of amplicons including the RNP target site. Data were compared using chi-squared test, and differences were considered significant at P<0.05. Cleavage rates in experiment 1 were similar for the control (86/96; 89.5%), group 1 (94/102; 92.1%), and group 2 (119/130; 91.5%). Blastocyst rates were higher for the control (46/96; 47%) than for the other groups (P<0.01). However, for the treated groups, the blastocyst rates were similar, group 1 (19/102; 9.2%) and group 2 (12/130; 18.6%). Furthermore, the non-homologous end joining (NHEJ) efficiency was similar for groups 1 (14/18; 77.7%) and 2 (14/17; 82.3%). In experiment 2, the cleavage (53/76; 69%) and blastocyst rates (30/76; 39%) were significantly higher for the control than for the treated groups (P<0.01). Among the groups, the lower cleavage and blastocyst rates were for group 4 (20/61; 32.7% and 3/61; 4.9%, respectively) compared with the other electroporation and microinjection groups (P<0.03). However, NHEJ efficiency was higher for electroporation groups 2 (6/8; 75%), 3 (17/17; 100%), and 4 (2/2; 100%) compared with microinjection (2/15; 13%). In conclusion, electroporation of Cas9/RNP is an efficient alternative to microinjection for gene editing in porcine zygotes.

A Simple Synthesis of the Anticancer Drug Altretamine

2020 ◽  
Vol 17 (8) ◽  
pp. 628-630
Author(s):  
Vu Binh Duong ◽  
Pham Van Hien ◽  
Tran Thai Ngoc ◽  
Phan Dinh Chau ◽  
Tran Khac Vu
Keyword(s):  
Aqueous Solution ◽  
Anticancer Drug ◽  
Potassium Hydroxide ◽  
Large Scale ◽  
Synthesis Method ◽  
Practical Method ◽  
Cyanuric Chloride ◽  
Simple Synthesis ◽  
High Yield ◽  
One Step

A simple and practical method for the synthesis on a large scale of altretamine (1), a wellknown antitumor drug, has been successfully developed. The synthesis method involves the conversion of cyanuric chloride (2) into altretamine (1) by dimethylamination of 2 with an aqueous solution of 40% dimethylamine and potassium hydroxide in 1, -dioxan 4in one step to give altretamine (1) in high yield.

scholarly journals Cyanobacteria—From the Oceans to the Potential Biotechnological and Biomedical Applications

Marine Drugs ◽  
2021 ◽  
Vol 19 (5) ◽  
pp. 241
Author(s):  
Shaden A. M. Khalifa ◽  
Eslam S. Shedid ◽  
Essa M. Saied ◽  
Amir Reza Jassbi ◽  
Fatemeh H. Jamebozorgi ◽  
...  
Keyword(s):  
Bioactive Compounds ◽  
Biomedical Applications ◽  
Large Scale ◽  
Biological Activities ◽  
Scale Production ◽  
Pharmacological Activities ◽  
Large Scale Production ◽  
Marine Products ◽  
Hiv 1 ◽  
Successful Phase

Cyanobacteria are photosynthetic prokaryotic organisms which represent a significant source of novel, bioactive, secondary metabolites, and they are also considered an abundant source of bioactive compounds/drugs, such as dolastatin, cryptophycin 1, curacin toyocamycin, phytoalexin, cyanovirin-N and phycocyanin. Some of these compounds have displayed promising results in successful Phase I, II, III and IV clinical trials. Additionally, the cyanobacterial compounds applied to medical research have demonstrated an exciting future with great potential to be developed into new medicines. Most of these compounds have exhibited strong pharmacological activities, including neurotoxicity, cytotoxicity and antiviral activity against HCMV, HSV-1, HHV-6 and HIV-1, so these metabolites could be promising candidates for COVID-19 treatment. Therefore, the effective large-scale production of natural marine products through synthesis is important for resolving the existing issues associated with chemical isolation, including small yields, and may be necessary to better investigate their biological activities. Herein, we highlight the total synthesized and stereochemical determinations of the cyanobacterial bioactive compounds. Furthermore, this review primarily focuses on the biotechnological applications of cyanobacteria, including applications as cosmetics, food supplements, and the nanobiotechnological applications of cyanobacterial bioactive compounds in potential medicinal applications for various human diseases are discussed.

scholarly journals GAKTpore: Stereological Characterisation Methods for Porous Foams in Biomedical Applications

Materials ◽  
2021 ◽  
Vol 14 (5) ◽  
pp. 1269
Author(s):  
Gareth Sheppard ◽  
Karl Tassenberg ◽  
Bogdan Nenchev ◽  
Joel Strickland ◽  
Ramy Mesalam ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Biomedical Applications ◽  
Large Scale ◽  
Collagen Scaffold ◽  
Porous Structures ◽  
Tissue Engineering Scaffolds ◽  
Biological Responses ◽  
Sem Micrograph ◽  
Cell Adhesion And Proliferation ◽  
Characterisation Methods

In tissue engineering, scaffolds are a key component that possess a highly elaborate pore structure. Careful characterisation of such porous structures enables the prediction of a variety of large-scale biological responses. In this work, a rapid, efficient, and accurate methodology for 2D bulk porous structure analysis is proposed. The algorithm, “GAKTpore”, creates a morphology map allowing quantification and visualisation of spatial feature variation. The software achieves 99.6% and 99.1% mean accuracy for pore diameter and shape factor identification, respectively. There are two main algorithm novelties within this work: (1) feature-dependant homogeneity map; (2) a new waviness function providing insights into the convexity/concavity of pores, important for understanding the influence on cell adhesion and proliferation. The algorithm is applied to foam structures, providing a full characterisation of a 10 mm diameter SEM micrograph (14,784 × 14,915 px) with 190,249 pores in ~9 min and has elucidated new insights into collagen scaffold formation by relating microstructural formation to the bulk formation environment. This novel porosity characterisation algorithm demonstrates its versatility, where accuracy, repeatability, and time are paramount. Thus, GAKTpore offers enormous potential to optimise and enhance scaffolds within tissue engineering.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals Hydrology influences breeding time in the white-throated dipper

BMC Ecology ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Anna L. K. Nilsson ◽  
Thomas Skaugen ◽  
Trond Reitan ◽  
Jan Henning L’Abée-Lund ◽  
Marlène Gamelon ◽  
...  
Keyword(s):  
Climate Change ◽  
River Discharge ◽  
Large Scale ◽  
Time Window ◽  
River System ◽  
Open Water ◽  
Local Conditions ◽  
Environmental Indices ◽  
Groundwater Levels ◽  
Breeding Time

Abstract Background Earlier breeding is one of the strongest responses to global change in birds and is a key factor determining reproductive success. In most studies of climate effects, the focus has been on large-scale environmental indices or temperature averaged over large geographical areas, neglecting that animals are affected by the local conditions in their home ranges. In riverine ecosystems, climate change is altering the flow regime, in addition to changes resulting from the increasing demand for renewable and clean hydropower. Together with increasing temperatures, this can lead to shifts in the time window available for successful breeding of birds associated with the riverine habitat. Here, we investigated specifically how the environmental conditions at the territory level influence timing of breeding in a passerine bird with an aquatic lifestyle, the white-throated dipper Cinclus cinclus. We relate daily river discharge and other important hydrological parameters, to a long-term dataset of breeding phenology (1978–2015) in a natural river system. Results Dippers bred earlier when winter river discharge and groundwater levels in the weeks prior to breeding were high, and when there was little snow in the catchment area. Breeding was also earlier at lower altitudes, although the effect dramatically declined over the period. This suggests that territories at higher altitudes had more open water in winter later in the study period, which permitted early breeding also here. Unexpectedly, the largest effect inducing earlier breeding time was territory river discharge during the winter months and not immediately prior to breeding. The territory river discharge also increased during the study period. Conclusions The observed earlier breeding can thus be interpreted as a response to climate change. Measuring environmental variation at the scale of the territory thus provides detailed information about the interactions between organisms and the abiotic environment.

Multi-Layer Construction Process for Fabricating Electrospun Fiber Embedded Microfluidic Devices

2015 ◽  
Author(s):  
Karen Chang Yan ◽  
John Sperduto ◽  
Michael Rossini ◽  
Michael Sebok
Keyword(s):  
Biomedical Applications ◽  
Electrospun Fiber ◽  
Microfluidic Devices ◽  
Construction Process ◽  
Preliminary Results ◽  
Device Optimization ◽  
Specialized Equipment ◽  
Microfabrication Techniques

Microfluidic devices are widely used in biomedical applications owing to their inherent advantages. Microfabrication techniques are common methods for fabricating microfluidic devices, which require specialized equipment. This paper presents a multi-layer construction process for producing microfluidic devices via integrating two accessible fabrication techniques — hydrogel molding, a microfabrication-free method, and electrospinning (ES). The formed microchannels were examined via analyzing micrographs. Preliminary results demonstrate the feasibility of the method and potential for incorporating complex channels and device optimization.

DISPERSION MODELS FOR EMISSIONS FROM AGRICULTURAL SOURCES G.-J. MEJER and K.-H. KRAUSE Institut fiir landtechnische Grundlagenforschung der Bundesforschungsanstalt fiir Landwirtschaft Summary The aim of dispersion models is the prediction of atmospheric dilution of pollutants in order to prevent or avoid nuisance. Established dispersion models, designed for the large scale of industrial air pol­ lution have to be modified to the small scale of agricultural pol­ lutions. An experimental setup is described to measure atmospheric dilution of tracer gas under agricultural conditions. The experimental results deliver the data base to identify the parameters of the models. For undisturbed airflow modified Gaussian models are applicable. For the consideration of obstacles more sophisticated models are necessary. 1. INTRODUCTION The aim of dispersion models is to develop reliable methods for calcu­ lating the atmospheric dilution of airborne pollutants under practical conditions. One application in agriculture is the determination of that distance, at which i.g. odouriferous pollutants of an animal farm are diluted in the atmosphere to a concentration below a certain threshold, in order to allow the farmer a profitable production and likewise to prevent odour nuisance from the neighbourhood. Another application is the prediction of the effectiveness of changes in the emission source configuration, in order to reduce the odour nuisance in the existent vicinity. That could help to avoid expensive misinvestments. In air pollution control it is useful! to subdivide this large problem into three main divisions /1/, fig. 1:

1986 ◽  
pp. 113-113
Keyword(s):  
Large Scale ◽  
Small Scale ◽  
Air Pollution Control ◽  
Dispersion Models ◽  
Tracer Gas ◽  
Gaussian Models ◽  
Airborne Pollutants ◽  
Animal Farm ◽  
Reliable Methods

scholarly journals Introduction of Surface Loops as a Tool for Encapsulin Functionalization.

2021 ◽  
Author(s):  
Sandra Michel-Souzy ◽  
Naomi M. Hamelmann ◽  
Sara Zarzuela-Pura ◽  
Jos M. J. Paulusse ◽  
Jeroen J. L. M. Cornelissen
Keyword(s):  
Biomedical Applications ◽  
Large Scale ◽  
Thermotoga Maritima ◽  
Scale Production ◽  
Structure Property ◽  
Protein Subunit ◽  
Protein Cages ◽  
Notable Increase ◽  
The Stability ◽  
Surface Loops

Encapsulin based protein cages are nanoparticles with different biomedical applications, such as targeted drug delivery or imaging agents. These particles are biocompatible and can be produced in bacteria, allowing large scale production and protein engineering. In order to use these bacterial nanocages in different applications, it is important to further explore the potential of their surface modification and optimize their production. In this study we design and show new surface modifications of the Thermotoga maritima (Tm) and Brevibacterium linens (Bl) encapsulins. Two new loops on Tm encapsulin with a His-tag insertion after the residue 64 and the residue 127, and the modification of the C-terminal on Bl encapsulin, are reported. The multi-modification of the Tm encapsulin enables up to 240 different functionalities on the cage surface, resulting from 4 potential modifications per protein subunit. We furthermore report an improved protocol giving a better stability and providing a notable increase of the production yield of the cages. Finally, we tested the stability of different encapsulin variants over a year and the results show a difference in stability arising from the tag insertion position. These first insights in the structure-property relationship of encapsulins, with respect to the position of a function loop, allow for further study of the use of these protein nanocages in biomedical applications.

Decentralized MRAC for Large-Scale Interconnected Systems With State and Input Delays by Integrators Inclusion

2017 ◽  
Vol 139 (9) ◽  
Author(s):  
Seyed Hamid Hashemipour ◽  
Nastaran Vasegh ◽  
Ali Khaki Sedigh
Keyword(s):  
Large Scale ◽  
Practical Method ◽  
Open Loop ◽  
Loop System ◽  
Input Delay ◽  
Time Varying ◽  
Delay Compensation ◽  
State And Input Delays ◽  
Input Delays ◽  
Model Reference Adaptive

This paper investigates the problem of decentralized model reference adaptive control (MRAC) for a class of large-scale systems with time-varying delays in the interconnected terms and state and input delays. The upper bounds of interconnection terms with time-varying delays and external disturbances are assumed to be completely unknown. By integrators inclusion, a dynamic input delay compensator is established for input delay compensation and it is used as a practical method for state calculation x(t + R). Also, a method is presented for a class of decentralized feedback controllers, which can evolve the closed-loop system error uniformly bounded stable. As a numerical example, the proposed technique is applied to an unstable open-loop system to show the feasibility and effectiveness of the method.

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