Design and Synthesis of a Multifunctional Probe for Bio-Imaging and Therapeutics

2006 ◽  
Vol 943 ◽  
Author(s):  
Yu-San Liu ◽  
Chi Hui Liang ◽  
P. Thomas Vernier ◽  
Yinghua Sun ◽  
Martin A. Gundersen
Keyword(s):  
Quantum Dots ◽  
Fluorescent Protein ◽  
Fluorescence Probe ◽  
Cancer Therapeutics ◽  
Medical Diagnostics ◽  
Egfp Gene ◽  
Design And Synthesis ◽  
Potential Applications ◽  
Bio Imaging ◽  
Green Fluorescent

ABSTRACTWe report the synthesis of CdSe/ZnSe/ZnS (core/shell/shell) quantum dots (QDs) as a fluorescence probe for biological cells, and their functionalization with carbon nanotube (CNT)-retrovirus complexes (labeled with the QDs) to form multi-functional tool with potential applications for cancer therapeutics. A mild method for preparing this QD-CNT-retrovirus complex, which preserves viral function, is reported. Observations and imaging under fluorescence confocal microscope show the expression of enhanced green fluorescent protein (EGFP) gene and QD uptake in viable cells, indicating that this novel probe is able to enter cells, crossing the natural barrier of the cell membranes. Such multi-functional probes have potential as medical diagnostics and for therapeutics for diseases including cancer. The multifunctional probe is of interest for potentially broad applications – with the quantum dots as the visualization aid, carbon nanotubes as an efficient opto-thermo converter for selective extermination of cells with the help of light irradiation at near-IR range, and the retrovirus as a vehicle for genetic therapy.

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.

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 Inhibitory receptor FcγRIIb mediates the effects of IgG on a phagosome acidification and a sequential dephosphorylation system comprising SHIPs and Inpp4a

2017 ◽  
Vol 23 (4) ◽  
pp. 401-409 ◽  
Author(s):  
Tomohiro Segawa ◽  
Kaoru Hazeki ◽  
Kiyomi Nigorikawa ◽  
Atsuko Nukuda ◽  
Tomoki Tanizawa ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescence Probe ◽  
Pleckstrin Homology Domain ◽  
Raw 264.7 Macrophages ◽  
Lipid Phosphatases ◽  
Src Homology 2 ◽  
Lipid Phosphatase ◽  
Src Homology ◽  
Src Homology 2 Domain ◽  
Green Fluorescent

The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in Raw 264.7 macrophages. The clearance of PIs was much faster when the phagocytosed particles were coated with IgG. The effect of IgG was not observed in the macrophages deficient in FcγRIIb, an inhibitory IgG receptor. To identify the lipid phosphatases responsible for the FcγRIIb-accelerated PI clearance, we prepared a panel of lipid phosphatase-deficient cells. The lack of a PI 5-phosphatase Src homology 2 domain-containing inositol-5-phosphatase (SHIP)1 or SHIP2 impaired the FcγRIIb-accelerated clearance of PIs. The lack of a PI 4-phosphatase Inpp4a also impaired the accelerated PIs clearance. In the FcγRIIb- and Inpp4a-deficient cells, acidification of the formed phagosome was slowed. These results suggested that FcγRIIb drives the sequential dephosphorylation system comprising SHIPs and Inpp4a, and accelerates phagosome acidification.

Cellular Uptake of Carbon Nanotubes Conjugated to Semiconductor Quantum Dots for Breast Cancer Imaging and Treatment Applications

2010 ◽  
Author(s):  
Kristen A. Zimmermann ◽  
Jianfei Zhang ◽  
Harry Dorn ◽  
Christopher Rylander ◽  
Marissa Nichole Rylander
Keyword(s):  
Carbon Nanotubes ◽  
Quantum Dots ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Properties ◽  
Breast Cancer Imaging ◽  
Fluorescent Markers ◽  
Green Fluorescent

Carbon nanotubes (CNTs) are attractive materials for early detection, treatment, and imaging of cancer malignancies; however, they are limited by their inability to be monitored in vitro and in vivo [1]. Unlabeled CNTs are difficult to distinguish using elemental analysis because they are composed entirely of carbon, which is also characteristic of cellular membranes. Although some single walled nanotubes (SWNT) have been found to exhibit fluorescent properties, not all particles in a single batch fluoresce [2]. Additionally, these emissions may be too weak to be detected using conventional imaging modalities [3]. Incorporating fluorescent markers, such as fluorescent proteins or quantum dots, allows the non-fluorescent particles to be visualized. Previously, fluorophores, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), have been used to visualize and track cells or other particles in biological environments, but their low quantum yield and tendency to photobleach generate limitations for their use in such applications.

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

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.

A Facile Method To Encapsulate Proteins in Silica Nanoparticles: Encapsulated Green Fluorescent Protein as a Robust Fluorescence Probe

2010 ◽  
Vol 122 (17) ◽  
pp. 3086-3089 ◽  
Author(s):  
Aoneng Cao ◽  
Zhangmei Ye ◽  
Zhengwei Cai ◽  
Erya Dong ◽  
Xiaowei Yang ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Silica Nanoparticles ◽  
Fluorescent Protein ◽  
Fluorescence Probe ◽  
Green Fluorescent

scholarly journals A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1512
Author(s):  
Gang Chen ◽  
Yu Kong ◽  
You Li ◽  
Ailing Huang ◽  
Chunyu Wang ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Intracellular Protein ◽  
Full Size ◽  
Intracellular Protein Degradation ◽  
Potential Applications ◽  
Ubiquitin Proteasome ◽  
Green Fluorescent

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin–proteasome and autophagy–lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

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