Mechanism Study of Gene Delivery and Expression in PK-15 Cells Using Magnetic Iron Oxide Nanoparticles as Gene Carriers

Nano LIFE ◽  
2014 ◽  
Vol 04 (04) ◽  
pp. 1441018 ◽  
Author(s):  
Yan Wang ◽  
Haixin Cui ◽  
Yongguang Yang ◽  
Xiang Zhao ◽  
Changjiao Sun ◽  
...  
Keyword(s):  
Iron Oxide ◽  
Magnetic Nanoparticles ◽  
Gene Delivery ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mechanism Study ◽  
Magnetic Iron Oxide Nanoparticles ◽  
Egfp Gene ◽  
Gene Carriers ◽  
Green Fluorescent

The mechanism of gene delivery and expression is one of the most important concerns raised by the development of gene delivery methods. Limited investigation is performed on how magnetic nanoparticles combine with DNA and deliver gene into mammalian cells. In this context, polyethyleneimine (PEI) coated iron oxide magnetic nanoparticles (MNPs) were used as gene carriers for binding and condensing with plasmid DNA expressing enhanced green fluorescent protein (EGFP). The morphology and structure of MNP–DNA complexes were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). We evidenced that large amounts of DNA wrapped around the surface of MNPs and that the MNPs were physically entrapped by the DNA arranged both horizontally and vertically. EGFP gene was successfully expressed under mediation of an external magnetic field which is necessary to efficiently target EGFP gene to the cells. Fluorescence from EGFP was separately detected in the cell cytoplasm and cell nucleus.

scholarly journals Efficient and Genotype Independent Maize pollen Transfection Mediated by Magnetic Nanoparticles

2020 ◽  
Author(s):  
Zuo-Ping Wang ◽  
Zhong-Bao Zhang ◽  
Xiang-Long Li ◽  
Chun Zhang ◽  
Long-Fei Yin ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Magnetic Nanoparticles ◽  
Gene Delivery ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Exogenous Dna ◽  
Maize Pollen ◽  
Maize Varieties ◽  
Green Fluorescent

Abstract Background: Biotechnological engineering of maize to introduce favorable new traits relies on delivery of foreign DNA into its cells. Current gene delivery methods for maize is limited to specific genotypes, and depend on time-consuming and labor-intensive processes of tissue culture. Results: Here, we report a new method to transfect maize that is culture-free and genotype independent. Enhanced green fluorescent protein gene (EGFP) or bialaphos resistance gene (Bar) bound with magnetic nanoparticles (MNPs) was delivered into maize pollens, and female florets of five maize varieties were pollinated. Green fluorescence was detected in 92% transfected pollens and 70% immature embryos. EGFP gene detected by PCR ranged from 29 to 68% in T1 generation of these five transfected varieties, and 7-16% of the T1 seedlings showed immunologically active EGFP protein. Moreover, 1.41% of the Bar transfected T1 plants were glufosinate resistant, and heritable Bar gene was integrated into the maize genome effectively (verified by Southern blot), expressed normally and inherited stably in their progenies.Conclusion: These results demonstrate that exogenous DNA could be delivered into maize efficiently and expressed normally through our genotype-independent pollen transfection system, providing a reliable, fast and large-scale gene delivery choice for most elite maize varieties recalcitrant to tissue culture.

scholarly journals Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Danielle Weber-Adrian ◽  
Rikke Hahn Kofoed ◽  
Joseph Silburt ◽  
Zeinab Noroozian ◽  
Kairavi Shah ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Intravenous Injection ◽  
Viral Vectors ◽  
Focused Ultrasound ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Peripheral Organs ◽  
Neuronal Expression ◽  
Green Fluorescent ◽  
The Brain

AbstractNon-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

scholarly journals Dynamin-related Protein Drp1 Is Required for Mitochondrial Division in Mammalian Cells

2001 ◽  
Vol 12 (8) ◽  
pp. 2245-2256 ◽  
Author(s):  
Elena Smirnova ◽  
Lorena Griparic ◽  
Dixie-Lee Shurland ◽  
Alexander M. van der Bliek
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Subcellular Fractionation ◽  
Related Protein ◽  
Direct Role ◽  
Mitochondrial Division ◽  
Transfected Cells ◽  
Green Fluorescent ◽  
Time Lapse Photography

Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

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.

Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

1999 ◽  
Vol 112 (16) ◽  
pp. 2705-2714
Author(s):  
E.M. Burns ◽  
L. Christopoulou ◽  
P. Corish ◽  
C. Tyler-Smith
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Quantitative Measurement ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Chromosome Loss ◽  
Facs Analysis ◽  
Fluorescent Cells ◽  
Green Fluorescent ◽  
Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

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.

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 Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

1997 ◽  
Vol 136 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Erik A.C. Wiemer ◽  
Thibaut Wenzel ◽  
Thomas J. Deerinck ◽  
Mark H. Ellisman ◽  
Suresh Subramani
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Small Subset ◽  
Subcellular Compartment ◽  
Directional Movement ◽  
Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

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