scholarly journals Chitosan Combined with Poly-L-arginine as Efficient, Safe, and Serum-Insensitive Vehicle with RNase Protection Ability for siRNA Delivery

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Samarwadee Plianwong ◽  
Praneet Opanasopit ◽  
Tanasait Ngawhirunpat ◽  
Theerasak Rojanarata
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Sirna Delivery ◽  
Weight Ratio ◽  
Deterrent Effect ◽  
Egfp Gene ◽  
Rnase Protection ◽  
Low Cytotoxicity ◽  
Green Fluorescent ◽  
Positively Charged

Chitosan (CS) combined with poly-L-arginine (PLA) was formulated and evaluated for its performance to deliver siRNA to HeLa cells expressing enhanced green fluorescent protein (EGFP). Compared with the formulations using single polymer in which the polyplexes were completely formed at the weight ratio of >20 : 1 for CS/siRNA or 1 : 1 for PLA/siRNA, the combination of CS and PLA could reduce the amounts of the polymers required for the complete complexation with siRNA, thereby forming positively charged, nanosized polyplex at the weight ratio of CS/PLA/siRNA of 5 : 0.5: 1. In addition, while the transfection efficiency of CS/siRNA and PLA/siRNA was very low at physiological pH (7.4), CS/PLA/siRNA at the optimal weight ratio of 5 : 0.5 : 1 satisfactorily silenced the endogenous EGFP gene at pH 7.4 as well as at pH 6.4 without the deterrent effect from serum. The combined polymers could protect siRNA from RNase degradation over a period of at least 6 h. Furthermore, MTT assay results demonstrated that CS/PLA/siRNA complexes showed acceptably low cytotoxicity with 75% cell viability. Therefore, CS combined with PLA is easy to prepare, safe, and promising for use as an efficient siRNA delivery vehicle.

scholarly journals Cationic Polybutyl Cyanoacrylate Nanoparticles for DNA Delivery

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Jinghua Duan ◽  
Yangde Zhang ◽  
Wei Chen ◽  
Chengrong Shen ◽  
Mingmei Liao ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Hepg2 Cells ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Electrophoretic Analysis ◽  
Dna Encoding ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Low Cytotoxicity ◽  
Green Fluorescent

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we used polybutyl cyanoacrylate (PBCA) and chitosan to prepare PBCA nanoparticles (NPs) by emulsion polymerization and prepared NP/DNA complexes through the complex coacervation of nanoparticles with the DNA. The object of our work is to evaluate the characterization and transfection efficiency of PBCA-NPs. The NPs have a zeta potential of 25.53 mV at pH 7.4 and size about 200 nm. Electrophoretic analysis suggested that the NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed that the NPs exhibit a low cytotoxicity to human hepatocellular carcinoma (HepG2) cells. Qualitative and quantitative analysis of transfection in HepG2 cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by digital fluorescence imaging microscopy system and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 hours. Quantitative results from FACS showed that PBCA-NPs were significantly more effective in transfecting HepG2 cells after 72 hours postincubation. The results of this study suggested that PBCA-NPs have favorable properties for nonviral delivery.

scholarly journals Non-Covalent Associates of siRNAs and AuNPs Enveloped with Lipid Layer and Doped with Amphiphilic Peptide for Efficient siRNA Delivery

2018 ◽  
Vol 19 (7) ◽  
pp. 2096 ◽  
Author(s):  
Julia Poletaeva ◽  
Ilya Dovydenko ◽  
Anna Epanchintseva ◽  
Kseniya Korchagina ◽  
Dmitrii Pyshnyi ◽  
...  
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Sirna Delivery ◽  
Chemical Properties ◽  
Specific Gene ◽  
Delivery Vehicle ◽  
Lipid Layer ◽  
A Cell ◽  
Green Fluorescent

Elaboration of non-viral vehicles for delivery of therapeutic nucleic acids, in particular siRNA, into a cell is an actively growing field. Gold nanoparticles (AuNPs) occupy a noticeable place in these studies, and various nanoconstructions containing AuNPs are reported. We aimed our work to the rational design of AuNPs-based siRNA delivery vehicle with enhanced transfection efficiency. We optimized the obtaining of non-covalent siRNAs-AuNPs cores: ionic strength, temperature and reaction time were determined. Formation of cores was confirmed using gel electrophoresis. Stable associates were prepared, and then enveloped into a lipid layer composed of phosphatidylcholine, phosphatidylethanolamine and novel pH-sensitive lipidoid. The constructions were modified with [Str-(RL)4G-NH2] peptide (the resulting construction). All intermediate and resulting nanoconstructions were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) to control their physico-chemical properties. To examine the biological effect of the delivery vehicle, green fluorescent protein (GFP)-expressing human embryonic kidney (HEK) Phoenix cells were incubated with the resulting construction containing anti-GFP siRNA, with the siRNA effect being studied by flow cytometry and confocal microscopy. Transfection of the cells with the resulting construction reduced the GFP fluorescence as efficiently as Lipofectamin 3000. Thus, siRNA vehicle based on non-covalently bound siRNA-AuNP core and enveloped into a lipid layer provides efficient delivery of siRNA into a cell followed by specific gene silencing.

scholarly journals Generation of a Recombinant Cytomegalovirus for Expression of a Hantavirus Glycoprotein

2003 ◽  
Vol 77 (22) ◽  
pp. 12203-12210 ◽  
Author(s):  
Albert A. Rizvanov ◽  
Albert G. M. van Geelen ◽  
Sergey Morzunov ◽  
Elmer W. Otteson ◽  
Charlotte Bohlman ◽  
...  
Keyword(s):  
Recombinant Virus ◽  
Fluorescent Protein ◽  
Deer Mice ◽  
Anamnestic Response ◽  
Egfp Gene ◽  
Virus Challenge ◽  
Virus Particles ◽  
Green Fluorescent ◽  
Human Cmv ◽  
A Site

ABSTRACT A cytomegalovirus (CMV) was isolated from its natural host, Peromyscus maniculatus, and was designated Peromyscus CMV (PCMV). A recombinant PCMV was constructed that contained Sin Nombre virus glycoprotein G1 (SNV-G1) fused in frame to the enhanced green fluorescent protein (EGFP) gene inserted into a site homologous to the human CMV UL33 (P33) gene. The recombinant CMV was used for expression and immunization of deer mice against SNV-G1. The results of the study indicate that P. maniculatus could be infected with as few as 10 virus particles of recombinant virus. Challenge of P. maniculatus with either recombinant or wild-type PCMV produced no overt pathology in infected animals. P. maniculatus immunized with recombinant virus developed an antibody response to SNV and EGFP. When rechallenged with recombinant virus, animals exhibited an anamnestic response against SNV. Interestingly, a preexisting immune response against PCMV did not prevent reinfection with recombinant PCMV.

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

Oligonucleotide Preparation Approach for Assembly of DNA Synthons

2019 ◽  
Vol 24 (6) ◽  
pp. 556-568
Author(s):  
Aleksei V. Yantsevich ◽  
Veronika V. Shchur ◽  
Sergey A. Usanov
Keyword(s):  
Fluorescent Protein ◽  
Solid Phase ◽  
Base Pairs ◽  
Egfp Gene ◽  
Standard Facility ◽  
Synthetic Dna ◽  
Pcr Product ◽  
Green Fluorescent ◽  
Inside Out ◽  
Spe Purification

An effective oligonucleotide preparation approach for the thermodynamically balanced, inside-out (TBIO) PCR-based assembly of long synthetic DNA molecules (synthons) is described in the current work. We replaced the necessity to purify individual oligonucleotides with just one purification procedure per approximately 500 base pairs (bp) of duplex DNA. So for an enhanced green fluorescent protein (EGFP) gene of 717 bp, we synthesized 24 oligonucleotides with a length of 50 bases and performed just two solid-phase extraction (SPE) purification procedures. It was found that the capacity of ZipTip microextractors, usually used for sample desalting in proteomics, perfectly corresponds to the gene synthesis scale (40–60 pmol). The robustness of the approach was validated with a 65-mer oligonucleotide design of the same gene. The modification of the oligonucleotide concentration gradient from the original TBIO scheme substantially increased the purity of the PCR product. We proposed a mechanism for the formation of supramolecular structures, which often occur during TBIO assembly. By using the proposed workflow, any laboratory with a standard facility for molecular biology manipulation, a 16-channel oligonucleotide synthesizer, and a conventional thermocycler has the ability to prepare one gene with a length of about 700 bp per day.

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. 

Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 719-726 ◽  
Author(s):  
Nicole Faust ◽  
Florencio Varas ◽  
Louise M. Kelly ◽  
Susanne Heck ◽  
Thomas Graf
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Neutrophil Granulocytes ◽  
Egfp Gene ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Myelomonocytic Cells ◽  
Green Fluorescent

Abstract Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

scholarly journals Covalently tethering siRNA to hydrogels for localized, controlled release and gene silencing

2019 ◽  
Vol 5 (8) ◽  
pp. eaax0801 ◽  
Author(s):  
Minh Khanh Nguyen ◽  
Cong Truc Huynh ◽  
Alex Gilewski ◽  
Samantha E. Wilner ◽  
Keith E. Maier ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Sirna Delivery ◽  
Hydrolytic Degradation ◽  
Nucleic Acid Delivery ◽  
Covalent Bonds ◽  
Disulfide Linkages ◽  
Green Fluorescent ◽  
Interfering Rna ◽  
Transfection Agent

Small interfering RNA (siRNA) has found many applications in tissue regeneration and disease therapeutics. Effective and localized siRNA delivery remains challenging, reducing its therapeutic potential. Here, we report a strategy to control and prolong siRNA release by directly tethering transfection-capable siRNA to photocrosslinked dextran hydrogels. siRNA release is governed via the hydrolytic degradation of ester and/or disulfide linkages between the siRNA and hydrogels, which is independent of hydrogel degradation rate. The released siRNA is shown to be bioactive by inhibiting protein expression in green fluorescent protein–expressing HeLa cells without the need of a transfection agent. This strategy provides an excellent platform for controlling nucleic acid delivery through covalent bonds with a biomaterial and regulating cellular gene expression, which has promising potential in many biomedical applications.

scholarly journals Effect of 5-lipoxygenase on the development of pulmonary hypertension in rats

2004 ◽  
Vol 286 (5) ◽  
pp. H1775-H1784 ◽  
Author(s):  
John E. Jones ◽  
Jennifer L. Walker ◽  
Yanli Song ◽  
Norbert Weiss ◽  
Wellington V. Cardoso ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Critical Role ◽  
Vascular Endothelial Cell ◽  
Weight Ratio ◽  
Right Heart Catheterization ◽  
Heart Catheterization ◽  
Green Fluorescent ◽  
The Right

5-Lipoxygenase (5-LO) and its downstream leukotriene products have been implicated in the development of pulmonary hypertension. In this study, we examined the effects of 5-LO overexpression in rat lungs on pulmonary hypertension using a recombinant adenovirus expressing 5-LO (Ad5-LO). Transthoracic echocardiography and right heart catheterization data showed that 5-LO overexpression in the lung did not cause pulmonary hypertension in normal rats; however, it markedly accelerated the progression of pulmonary hypertension in rats treated with monocrotaline (MCT). An increase in pulmonary artery pressure occurred earlier in the rats treated with MCT + Ad5-LO (7–10 days) compared with those treated with control vector, MCT + adenovirus expressing green fluorescent protein (AdGFP), or MCT alone (15–18 days). The weight ratio of the right ventricle to left ventricle plus septum was higher in the MCT + Ad5-LO group than that of the MCT + AdGFP or MCT group (0.45 ± 0.08 vs. 0.35 ± 0.03 or 0.33 ± 0.06). Lung tissue histological sections from MCT + Ad5-LO rats exhibited more severe inflammatory cell infiltration and pulmonary vascular muscularization than those from MCT + AdGFP- or MCT-treated rats. Administration of 5-LO inhibitors, zileuton or MK-886, to either MCT- or MCT + Ad5-LO-treated rats prevented the development of pulmonary hypertension. These data suggest that 5-LO plays a critical role in the progression of pulmonary hypertension in rats and that the detrimental effect of 5-LO is manifest only in the setting of pulmonary vascular endothelial cell dysfunction.

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