Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery

Nanomedicine ◽  
2021 ◽  
Author(s):  
Akbar Hasanzadeh ◽  
Fatemeh Radmanesh ◽  
Elaheh Sadat Hosseini ◽  
Iman Hashemzadeh ◽  
Jafar Kiani ◽  
...  
Keyword(s):  
Carbon Dots ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
High Serum ◽  
Spectroscopic Techniques ◽  
Delivery Vector ◽  
One Step ◽  
Diphenyl Tetrazolium Bromide ◽  
Green Fluorescent

Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials & methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.

A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

2005 ◽  
Vol 342 (2) ◽  
pp. 341-344 ◽  
Author(s):  
Dineshkumar H. Dandekar ◽  
Manish Kumar ◽  
Jayashree S. Ladha ◽  
Krishna N. Ganesh ◽  
Debashis Mitra
Keyword(s):  
Green Fluorescent Protein ◽  
Quantitative Method ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Green Fluorescent

scholarly journals Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

2018 ◽  
Vol 10 (4) ◽  
pp. 12
Author(s):  
Mahipal Singh ◽  
Xiaoling Ma
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Genetically Engineered ◽  
Dermal Fibroblasts ◽  
Biologically Active ◽  
Cell Type ◽  
Gfp Expression ◽  
Primary Fibroblasts ◽  
Species Specific ◽  
Green Fluorescent

Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression. 

Ultrasonic one-step synthesis of biocompatible yellow-green fluorescent carbon dots

2013 ◽  
Vol 43 (7) ◽  
pp. 895-900 ◽  
Author(s):  
YuanFang LI ◽  
JiaJia ZHENG ◽  
Hui LIU ◽  
XiaoXi YANG ◽  
ChengZhi HUANG
Keyword(s):  
Carbon Dots ◽  
One Step ◽  
Green Fluorescent ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

scholarly journals Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nagasundarapandian Soundrarajan ◽  
Hye-sun Cho ◽  
Byeongyong Ahn ◽  
Minkyung Choi ◽  
Le Minh Thong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Green Fluorescent

Universal Correlation and Mechanism for the Antibacterial Activity of Silver Nanoparticles

2009 ◽  
Vol 1209 ◽  
Author(s):  
Georgios A. Sotiriou ◽  
Adrian Camenzind ◽  
Frank Krumeich ◽  
Andreas Meyer ◽  
Sven Panke ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Silver Clusters ◽  
Flame Spray ◽  
X Ray Diffraction ◽  
X Ray ◽  
One Step ◽  
Green Fluorescent ◽  
Universal Correlation ◽  
Nanostructured Silica ◽  
Made In

AbstractSilver clusters (4-150 nm) anchored on nanostructured silica particles (300-400 m2/g) with closely controlled Ag content and size were made in one-step by scalable flame spray pyrolysis of Ag-nitrate and hexamethyldisiloxane containing solutions. Composite Ag/SiO2 nanoparticles were characterized by S/TEM, EDX spectroscopy, X-ray diffraction, N2 adsorption. The activity of such nanoparticles against the Gram negative bacterium Escherichia coli was investigated by monitoring the recombinantly synthesized green fluorescent protein. It is shown that higher Ag content particles exhibit a stronger antibacterial effect.

scholarly journals Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

2018 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Green Fluorescent Protein ◽  
Cost Effectiveness ◽  
Genome Editing ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
C Elegans ◽  
Multiple Loci ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

scholarly journals Foreign Gene Expression in the Mouse Cauda Epididymis is Regulated by Androgens

2018 ◽  
Vol 2 (4) ◽  
pp. 671-678
Author(s):  
Pedro Esponda
Keyword(s):  
Gene Transfer ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Female Reproductive Tract ◽  
Foreign Gene ◽  
Total Period ◽  
Green Fluorescent ◽  
In Vivo Gene Transfer

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

scholarly journals Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

2000 ◽  
Vol 84 (09) ◽  
pp. 460-467 ◽  
Author(s):  
M. L. M. Lamfers ◽  
M. J. Wijnberg ◽  
J. M. Grimbergen ◽  
L. G. M. Huisman ◽  
M. C. Aalders ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Receptor Binding ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Adenoviral Gene Transfer ◽  
Amino Terminal ◽  
Green Fluorescent

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

scholarly journals Novel Histone-Based DNA Carrier Targeting Cancer-Associated Fibroblasts

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1695
Author(s):  
Alexey Kuzmich ◽  
Olga Rakitina ◽  
Dmitry Didych ◽  
Victor Potapov ◽  
Marina Zinovyeva ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Growth Factor Receptor ◽  
Surface Protein ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Cancer Associated Fibroblasts ◽  
Histone H2a ◽  
Green Fluorescent

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRβ). In this study, we fused histone H2A with PDGFRβ-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRβ-positive and PDGFRβ-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRβ-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRβ-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRβ-positive tumor stromal cells.

Fluorescent reporters for the ubiquitin–proteasome system

2005 ◽  
Vol 41 ◽  
pp. 113-128 ◽  
Author(s):  
Florian A. Salomons ◽  
Lisette G.G.C. Verhoef ◽  
Nico P. Dantuma
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ubiquitin Proteasome System ◽  
Fluorescent Reporters ◽  
Cellular Processes ◽  
The Status ◽  
Ubiquitin Proteasome ◽  
Green Fluorescent ◽  
Specific Degradation

Regulated turnover of proteins in the cytosol and nucleus of eukaryotic cells is primarily performed by the ubiquitin–proteasome system (UPS). The UPS is involved in many essential cellular processes. Alterations in this proteolytic system are associated with a variety of human pathologies, such as neurodegenerative diseases, cancer, immunological disorders and inflammation. The precise role of the UPS in the pathophysiology of these diseases, however, remains poorly understood. Detection of UPS aberrations has been a major challenge because of the complexity of the system. Most studies focus on various aspects of the UPS, such as substrate recognition, ubiquitination, deubiquitination or proteasome activity, and do not provide a complete picture of the UPS as an integral system. To monitor the efficacy of the UPS, a number of reporter substrates have been developed based on fluorescent proteins, such as the green fluorescent protein and its spectral variants. These fluorescent UPS reporters contain specific degradation signals that target them with high efficiency and accuracy for proteasomal degradation. Several studies have shown that these reporters can probe the functionality of the UPS in cellular and animal models and provide us with important information on the status of the UPS under various conditions. Moreover, these reporters can aid the identification and development of novel anti-cancer and anti-inflammatory drugs based on UPS inhibition.

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