scholarly journals Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing

2017 ◽  
Author(s):  
Birgit Koch ◽  
Bianca Nijmeijer ◽  
Moritz Kueblbeck ◽  
Yin Cai ◽  
Nike Walther ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Lines ◽  
Blot Analysis ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cell Clone ◽  
Dynamic Properties ◽  
Functional Expression ◽  
Fluorescent Markers ◽  
Target Effects

AbstractGene tagging with fluorescent proteins is essential to investigate the dynamic properties of cellular proteins. CRISPR/Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and permits functionality and physiological expression of the fusion protein. It is essential to evaluate such genome-edited cell lines carefully in order to preclude off-target effects caused by either (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) non-specific genomic DNA damage by CRISPR/Cas9. In this protocol1, we provide a step-by-step description of our systematic pipeline to generate and validate homozygous fluorescent knock-in cell lines.We have used the paired Cas9D10A nickase approach to efficiently insert tags into specific genomic loci via homology-directed repair with minimal off-target effects. It is time- and cost-consuming to perform whole genome sequencing of each cell clone. Therefore, we have developed an efficient validation pipeline of the generated cell lines consisting of junction PCR, Southern Blot analysis, Sanger sequencing, microscopy, Western blot analysis and live cell imaging for cell cycle dynamics. This protocol takes between 6-9 weeks. Using this protocol, up to 70% of the targeted genes can be tagged homozygously with fluorescent proteins and result in physiological levels and phenotypically functional expression of the fusion proteins.Editorial SummaryThis protocol provides a detailed workflow describing how to insert fluorescent markers into all alleles of a gene of interest using CRISPR/Cas 9 technology and how to generate and validate homozygous fluorescent knock-in cell lines.

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.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

scholarly journals Properties of Wild-Type and Fluorescent Protein-Tagged Mouse Tetrodotoxin-Resistant Sodium Channel (NaV1.8) Heterologously Expressed in Rat Sympathetic Neurons

2008 ◽  
Vol 99 (4) ◽  
pp. 1917-1927 ◽  
Author(s):  
Geoffrey G. Schofield ◽  
Henry L. Puhl ◽  
Stephen R. Ikeda
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Sympathetic Neurons ◽  
Functional Expression ◽  
Nodose Ganglion Neurons ◽  
Similar Expression Pattern ◽  
Cervical Ganglion ◽  
Clonal Cell Lines ◽  
Mouse Dorsal Root Ganglion ◽  
Ganglion Neurons

The tetrodotoxin (TTX)-resistant Na+ current arising from NaV1.8-containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. Here, we show that injection of cDNA encoding mouse NaV1.8 into the nuclei of rat superior cervical ganglion (SCG) neurons results in TTX-resistant Na+ currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed NaV1.8 Na+ channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed NaV1.8 channels was shifted about 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of NaV1.8 did not substantially affect functional expression in SCG neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged NaV1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum, thus providing a basis for poor functional expression in clonal cell lines. Our results establish SCG neurons as a favorable surrogate for the expression and study of molecularly defined NaV1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of NaV1.8 with a biophysical phenotype identical to that found in sensory neurons.

scholarly journals Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5104-5110 ◽  
Author(s):  
Robert E. Throm ◽  
Annastasia A. Ouma ◽  
Sheng Zhou ◽  
Anantharaman Chandrasekaran ◽  
Timothy Lockey ◽  
...  
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Cell Clone ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Scale Production ◽  
Producer Cell

AbstractRetroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

scholarly journals Fluorescence Recovery Allows the Implementation of a Fluorescence Reporter Gene Platform Applicable for the Detection and Quantification of Horizontal Gene Transfer in Anoxic Environments

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Rafael Pinilla-Redondo ◽  
Leise Riber ◽  
Søren J. Sørensen
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Microbial Communities ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescence Recovery ◽  
Fluorescent Markers ◽  
Anoxic Environments ◽  
Labeling System

ABSTRACTThe study of horizontal gene transfer (HGT) in microbial communities has been revolutionized by significant advances in cultivation-independent methods based on fluorescence reporter gene technologies. Recently, the combination of these novel approaches with flow cytometry has presented itself as one of the most powerful tools to study the spread of mobile genetic elements (MGEs) in the environment. However, the use of fluorescent markers, like green fluorescent protein (GFP) and mCherry, is limited by environmental constraints, such as oxygen availability and pH levels, that affect the correct maturation of their fluorophores. Few studies have characterized the effects of such environmental conditions in a systematic way, and the sheer amount of distinct protein variants requires each system to be examined in an individual fashion. The lack of efficient and reliable markers to monitor HGT in anaerobic environments, coupled to the abundance of ecologically and clinically relevant oxygen-deprived niches in which bacteria thrive, calls for the urgent development of suitable tools that permit its study. In an attempt to devise a process that allows the implementation of the mentioned dual-labeling system to anoxic milieus, the aerobic fluorescence recovery of mCherry and GFPmut3, as well as the effect of pH on their fluorescence intensities, was studied. The findings present a solution to an intrinsic problem that has long hampered the utilization of this system, highlight its pH limitations, and provide experimental tools that will help broaden its horizon of application to other fields.IMPORTANCEMany anaerobic environments, like the gastrointestinal tract, anaerobic digesters, and the interiors of dense biofilms, have been shown to be hotspots for horizontal gene transfer (HGT). Despite the increasing wealth of reports warning about the alarming spread of antibiotic resistance determinants, to date, HGT studies mainly rely on cultivation-based methods. Unfortunately, the relevance of these studies is often questionable, as only a minor fraction of bacteria can be cultivated. A recently developed approach to monitoring the fate of plasmids in microbial communities is based on a fluorescence dual-labeling system and allows the bypassing of cultivation. However, the fluorescent proteins on which it is founded are constrained by pH levels and by their strict dependence on oxygen for the maturation of their fluorophores. This study focused on the development and validation of an appropriate aerobic fluorescence recovery (AFR) method for this platform, as this embodies the missing technical link impeding its implementation in anoxic environments.

Cryo-Fixation and Gene Product Localization in Fungi

2001 ◽  
Vol 7 (S2) ◽  
pp. 1202-1203
Author(s):  
T.M. Bourett ◽  
K.E. Duncan ◽  
K.J. Czymmek ◽  
J.A. Sweigard ◽  
T.M. Dezwaan ◽  
...  
Keyword(s):  
Magnaporthe Grisea ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Sequence Data ◽  
Fluorescent Proteins ◽  
Simultaneous Recording ◽  
Temporal Information ◽  
Cell Fractionation ◽  
Plant Host ◽  
Fluorescent Markers

There are many strategies for documenting the distribution of gene products or other molecules within cells or tissues. Depending on the required information, techniques can range from cell fractionation to methods that employ electron microscopy: some preclude examination of living cells, and thus can not provide dynamic temporal information. Time sequence data are paramount when gene expression is transient, as is often the case during fungal-pathogen plant-host interactions. Fluorescent dyes and protein tags (e.g., green fluorescent protein, GFP) allow monitoring of specific molecules, organelles or cells over time, for simultaneous recording of both spatial and temporal information. in addition, fluorescent markers are amenable to powerful 3-D analysis using confocal or multi-photon imaging methods. We have achieved good expression of fluorescent proteins in the fungal blast pathogen Magnaporthe grisea, and have generated a functional fusion protein between GFP and P-tubulin (Figs. 1,2).

Synthetic Fluorophores for Visualizing Biomolecules in Living Systems

Acta Naturae ◽  
2016 ◽  
Vol 8 (4) ◽  
pp. 33-46 ◽  
Author(s):  
V. I. Martynov ◽  
A. A. Pakhomov ◽  
N. V. Popova ◽  
I. E. Deyev ◽  
A. G. Petrenko
Keyword(s):  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Infrared Region ◽  
Living Systems ◽  
Significant Advance ◽  
Low Molecular Weight ◽  
Fluorescent Markers ◽  
Wide Range

The last decade has witnessed significant advance in the imaging of living systems using fluorescent markers. This progress has been primarily associated with the discovery of different spectral variants of fluorescent proteins. However, the fluorescent protein technology has its own limitations and, in some cases, the use of low-molecular-weight fluorophores is preferable. In this review, we describe the arsenal of synthetic fluorescent tools that are currently in researchers hands and span virtually the entire spectrum, from the UV to visible and, further, to the near-infrared region. An overview of recent advances in site-directed introduction of synthetic fluorophores into target cellular objects is provided. Application of these fluorescent probes to the solution of a wide range of biological problems, in particular, to the determination of local ion concentrations and pH in living systems, is discussed.

scholarly journals Functional Expression of Aquaporin-2 Tagged with Photoconvertible Fluorescent Protein in mpkCCD Cells

2015 ◽  
Vol 36 (2) ◽  
pp. 670-682 ◽  
Author(s):  
Kay-Pong Yip ◽  
Byeong J. Cha ◽  
Chung-Ming Tse ◽  
Mateus E. Amin ◽  
Jahanshah Amin
Keyword(s):  
High Speed ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Chimeric Protein ◽  
Functional Expression ◽  
Apical Plasma Membrane ◽  
Aquaporin 2 ◽  
Osmotic Water ◽  
Apical Trafficking ◽  
Acidic Organelles

Background: Vasopressin induced trafficking of aquaporin-2 (AQP2) containing vesicles has been studied in kidney cell lines using conventional fluorescent proteins as tags. However, trafficking of fluorescent tagged AQP2, which resembles the vectorial translocation of native AQP2 from cytoplasm to apical membrane has not been demonstrated at real time. Using a photoconvertible fluorescent protein tag on AQP2 might allow the simultaneous tracking of two separate populations of AQP2 vesicle after subcellular local photoconversion. Methods: A spacer was used to link a photoconvertible fluorescent protein (mEos2) to the amino-terminus of AQP2. The DNA constructs were expressed in mpkCCD cells. The trafficking of chimeric protein was visualized with high speed confocal microscopy in 4 dimensions. Results: Chimeric AQP2 expressed in mpkCCD cell conferred osmotic water permeability to the cells. Subcellular photoconversion with a 405 nm laser pulse converted green chimeras to red chimeras locally. Forskolin stimulation triggered chimeric AQP2 to translocate from acidic organelles to apical plasma membrane. By serendipity, the rate of apical accumulation was found to increase when mEos2 was tagged to the carboxyl-terminus in at least one of the AQP2 molecules within the tetramer. Conclusion: Functional photoconvertible chimeric AQP2 was successfully expressed in mpkCCD cells, in which forskolin induced apical trafficking and accumulation of chimeric AQP2. The proof-of-concept to monitor two populations of AQP2 vesicle simultaneously was demonstrated.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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