scholarly journals An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue

2020 ◽  
Author(s):  
Kyle Miller ◽  
Alan Eggenberger ◽  
Keunsub Lee ◽  
Fei Liu ◽  
Minjeong Kang ◽  
...  
Keyword(s):  
Internal Control ◽  
Gene Expression Analysis ◽  
Transient Gene Expression ◽  
Cell Counting ◽  
The Novel ◽  
Analysis Method ◽  
Animal Cells ◽  
A Cell ◽  
Quantitative Analyses ◽  
Biolistic Delivery

Abstract Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT®-2020. In addition, the same approach is applied to test the efficacy of CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.

scholarly journals An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle Miller ◽  
Alan L. Eggenberger ◽  
Keunsub Lee ◽  
Fei Liu ◽  
Minjeong Kang ◽  
...  
Keyword(s):  
Internal Control ◽  
Gene Expression Analysis ◽  
Transient Gene Expression ◽  
Cell Counting ◽  
The Novel ◽  
Analysis Method ◽  
Animal Cells ◽  
A Cell ◽  
Quantitative Analyses ◽  
Biolistic Delivery

AbstractBiolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel device enables a strategy of incorporating an internal control into each sample, which significantly decreases variance of the results. The cell counting software further reduces errors and increases throughput. The utility of this new platform is demonstrated by optimizing conditions for delivering DNA using the commercial transfection reagent TransIT-2020. In addition, the same approach is applied to test the efficacy of multiple gRNAs for CRISPR-Cas9-mediated gene editing. The novel combination of the bombardment device and analysis method allows simultaneous comparison and optimization of parameters in the biolistic delivery. The platform developed here can be broadly applied to any target samples using biolistics, including animal cells and tissues.

scholarly journals Pathological Features of Diabetic Retinopathy in Spontaneously Diabetic Torii Fatty Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yoshiaki Tanaka ◽  
Rina Takagi ◽  
Takeshi Ohta ◽  
Tomohiko Sasase ◽  
Mina Kobayashi ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Diabetic Retinopathy ◽  
Cell Counting ◽  
Sprague Dawley ◽  
Sd Rats ◽  
Pathologic Features ◽  
A Cell ◽  
Quantitative Analyses ◽  
Retinal Folds

Objective. The Spontaneously Diabetic Torii (SDT) fatty rat, established by introducing the fa allele (obesity gene) of the Zucker fatty rat into the SDT rat genome, is a new model of obese type 2 diabetes. We studied the pathologic features of diabetic retinopathy (DR) in this animal. Methods. The eyes of SDT fatty, SDT (controls), and Sprague Dawley (SD) rats (normal controls) were enucleated at 8, 16, 24, 32, and 40 weeks of age (n=5‐6 for each rat type at each age). The retinal thicknesses, numbers of retinal folds, and choroidal thicknesses were evaluated. Immunostaining for glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) was performed. Quantitative analyses of the immunopositive regions were performed using a cell-counting algorithm. Results. The retinas tended to be thicker in the SDT fatty rats and SDT rats than in the SD rats; the choroids tended to be thicker in the SDT fatty rats than in the SD rats. The retinal folds in the SDT fatty rats developed earlier and were more severe than in the SDT rats. Quantitative analyses showed that the GFAP- and VEGF-positive regions in the retinas of the SDT fatty rats were significantly larger than those of the SDT rats. Conclusions. SDT fatty rats developed more severe DR earlier than the SDT rats. The SDT fatty rats might be useful as a type 2 diabetes animal model to study DR.

Automated cell counting of astrocytes on patterned substrates containing aliphatic and charged properties

1995 ◽  
Vol 53 ◽  
pp. 974-975
Author(s):  
W. Shain ◽  
H. Ancin ◽  
H.C. Craighead ◽  
M. Isaacson ◽  
L. Kam ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Attachment ◽  
Culture Method ◽  
Cell Counting ◽  
Data Sets ◽  
Nuclear Staining ◽  
Double Positive ◽  
A Cell ◽  
Wafer Test ◽  
Cell Densities

Neural protheses have potential to restore nervous system functions lost by trauma or disease. Nanofabrication extends this approach to implants for stimulating and recording from single or small groups of neurons in the spinal cord and brain; however, tissue compatibility is a major limitation to their practical application. We are using a cell culture method for quantitatively measuring cell attachment to surfaces designed for nanofabricated neural prostheses.Silicon wafer test surfaces composed of 50-μm bars separated by aliphatic regions were fabricated using methods similar to a procedure described by Kleinfeld et al. Test surfaces contained either a single or double positive charge/residue. Cyanine dyes (diIC18(3)) stained the background and cell membranes (Fig 1); however, identification of individual cells at higher densities was difficult (Fig 2). Nuclear staining with acriflavine allowed discrimination of individual cells and permitted automated counting of nuclei using 3-D data sets from the confocal microscope (Fig 3). For cell attachment assays, LRM5 5 astroglial cells and astrocytes in primary cell culture were plated at increasing cell densities on test substrates, incubated for 24 hr, fixed, stained, mounted on coverslips, and imaged with a 10x objective.

scholarly journals Modeling and Simulation of Thermo-Fluid-Electrochemical Ion Flow in Biological Channels

2015 ◽  
Vol 3 (1) ◽  
Author(s):  
Riccardo Sacco ◽  
Fabio Manganini ◽  
Joseph W. Jerome
Keyword(s):  
Driving Forces ◽  
The Novel ◽  
Thermodynamical Equilibrium ◽  
Finite Element Scheme ◽  
Equilibrium Conditions ◽  
Solution Map ◽  
Ion Flow ◽  
Stabilized Finite Element ◽  
A Cell ◽  
Biological Channels

AbstractIn this articlewe address the study of ion charge transport in the biological channels separating the intra and extracellular regions of a cell. The focus of the investigation is devoted to including thermal driving forces in the well-known velocity-extended Poisson-Nernst-Planck (vPNP) electrodiffusion model. Two extensions of the vPNP system are proposed: the velocity-extended Thermo-Hydrodynamic model (vTHD) and the velocity-extended Electro-Thermal model (vET). Both formulations are based on the principles of conservation of mass, momentum and energy, and collapse into the vPNP model under thermodynamical equilibrium conditions. Upon introducing a suitable one-dimensional geometrical representation of the channel,we discuss appropriate boundary conditions that depend only on effectively accessible measurable quantities. Then, we describe the novel models, the solution map used to iteratively solve them, and the mixed-hybrid flux-conservative stabilized finite element scheme used to discretize the linearized equations. Finally,we successfully apply our computational algorithms to the simulation of two different realistic biological channels: 1) the Gramicidin-A channel considered in [12]; and 2) the bipolar nanofluidic diode considered in [45].

scholarly journals EVALUASI PENERAPAN SISTEM PENGENDALIAN INTERN PERSEDIAAN BARANG DAGANGAN PADA TOKO MAHKOTA DIESEL MANADO

2017 ◽  
Vol 12 (01) ◽  
Author(s):  
Juwita Natalia Samuna ◽  
Harijanto Sabijono ◽  
Stanley Kho Walandouw
Keyword(s):  
Control System ◽  
Qualitative Analysis ◽  
Internal Control ◽  
Spare Parts ◽  
Accounting System ◽  
Control Environment ◽  
Analysis Method ◽  
Serial Number ◽  
Internal Control System ◽  
Control Procedures

Internal control system is a way or system that can prevent the occurrence of irregularities in the company. Internal control system of the inventory aims to protect the company's assets from theft. The research object is taken at the Manado Diesel Crown Store is one of the stores in Manado that runs the business by selling machines and spare parts. This study aims to evaluate the effectiveness of the application of internal control system merchandise inventory at Diesel Manado.The method of analysis used in this research is descriptive qualitative analysis method. Based on the results of internal control inventory of merchandise inventory at Manado Diesel Crown Store, then in the control environment that occurs in the company, there is a double position between the sales and the warehouse. In the accounting system, the documents used are not serial number printed. In control procedures, authorization system is only on certain transactions.Keywords: internal control system, merchandise inventory

scholarly journals Cell-Tak coating may cause mis-normalization of Seahorse metabolic flux data

2021 ◽  
Author(s):  
Michal Šíma ◽  
Stanislava Martínková ◽  
Anežka Kafková ◽  
Jan Pala ◽  
Jan Trnka
Keyword(s):  
Metabolic Flux ◽  
Cell Number ◽  
Cell Counting ◽  
Fluorescent Detection ◽  
Data Normalization ◽  
Tissue Samples ◽  
Flux Data ◽  
Research Areas ◽  
Healthy State ◽  
A Cell

Metabolic flux investigations of cells and tissue samples are a rapidly advancing tool in diverse research areas. Reliable methods of data normalization are crucial for an adequate interpretation of results and to avoid a misinterpretation of experiments and incorrect conclusions. The most common methods for metabolic flux data normalization are to cell number, DNA and protein. Data normalization may be affected by a variety of factors, such as density, healthy state, adherence efficiency, or proportional seeding of cells. The mussel-derived adhesive Cell-Tak is often used to immobilize poorly adherent cells. Here we demonstrate that this coating may strongly affect the fluorescent detection of DNA leading to an incorrect and highly variable normalization of metabolic flux data. Protein assays are much less affected and cell counting can virtually completely remove the effect of the coating. Cell-Tak coating also affects cell shape in a cell line-specific manner and may change cellular metabolism. Based on these observations we recommend cell counting as a gold standard normalization method for Seahorse metabolic flux measurements with protein content as a reasonable alternative.

scholarly journals Pengaruh Transparansi, Kompetensi, Dan Sistem Akuntansi Keuangan Terhadap Akuntabilitas Pengelolaan Keuangan Daerah Dengan Penerapan Sistem Pengendalian Internal Pemerintah Sebagai Variabel Moderasi

2021 ◽  
Vol 4 (2) ◽  
pp. 419-433
Author(s):  
Ria Herlina ◽  
Taufeni Taufik ◽  
Azwir Nasir
Keyword(s):  
Control System ◽  
Internal Control ◽  
Financial Management ◽  
Financial Accounting ◽  
Accounting System ◽  
Analysis Method ◽  
Accounting Systems ◽  
Finance Management ◽  
Internal Control System ◽  
The Government

This study aims to examine the effect of transparency, competency, financial accounting systems on the accountability of regional financial management with the government's internal control system as a moderating variable with a case study in Indragiri Hulu Regency. The population of this study were all employees in all OPDs in Indragiri Hulu Regency as many as 45 OPDs so that a sample of 180 respondents was obtained from this population using the purposive sampling method. The data analysis method used in this research is the quantitative analysis method with WarpPLS version 6.0 as data processing software. The results show that transparency, competency, and financial accounting systems affect the accountability of regional financial management, the implementation of the government internal control system can moderate the effect of transparency and financial accounting systems on the accountability of regional financial management, and the implementation of the government internal control system cannot moderate the effect of competency on the accountability of regional finance management. Keywords: Transparency, Competency, Financial Accounting System, The Accountability of Regional Financial Management, Implementation of The Government Internal Control System

scholarly journals THE TIME OF SYNTHESIS AND THE CONSERVATION OF MITOSIS-RELATED PROTEINS IN CULTURED HUMAN AMNION CELLS

1967 ◽  
Vol 34 (1) ◽  
pp. 97-110 ◽  
Author(s):  
Jesse E. Sisken ◽  
Elaina Wilkes
Keyword(s):  
Time Lapse ◽  
Major Effect ◽  
Human Amnion ◽  
Daughter Cells ◽  
Low Concentrations ◽  
Associated Proteins ◽  
A Cell ◽  
Quantitative Analyses ◽  
Related Proteins ◽  
Kinetics Of

p-Fluorophenylalanine (PFPA), an analogue of phenylalanine which may be incorporated into proteins, increases the duration of mitosis. In the present experiments, based upon quantitative analyses of time-lapse cinemicrographic films, brief treatments of cells with PFPA are shown to affect the duration of metaphase in only those cells which enter division during or shortly after treatment. The offspring of cells with prolonged metaphases also tend to have prolonged metaphases. Analyses of the kinetics of the appearance of prolonged metaphases indicate that some protein specifically associated with mitosis is synthesized primarily during a period which corresponds closely to G2. The manner in which the defect is passed on to daughter cells indicates that the protein involved is conserved and reutilized by daughter cells for their subsequent divisions. Comparable experiments performed with low concentrations of puromycin indicate that the major effect of PFPA is due to its incorporation into protein rather than its ability to inhibit protein synthesis. The fact that puromycin-induced effects can also be passed on to daughter cells is interpreted to mean that cells make only specific amounts of some mitosis-associated proteins and that if a cell "inherits" a deficiency in such protein it is not able to compensate for the deficiency.

scholarly journals Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2331 ◽  
Author(s):  
Qianqian Zhang ◽  
Wei Liu ◽  
Yingli Cai ◽  
A-Feng Lan ◽  
Yinbing Bian
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Expression Analysis ◽  
Internal Control ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Stable Gene ◽  
Experimental Conditions ◽  
The Stability ◽  
Vacuolar Protein

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

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