Red Fluorescent Protein Expression in Transgenic Founder of Angelfish (Pterophyllum sp) Driven by Zebrafish Myosin Light Chain 2 Promoter

Angelfish (Pterophyllum sp.) are attractive fish popular with aquarists. The introduction of fluorescent protein genes into angelfish has been reported, but specific techniques have not been revealed. This study aimed to develop a strategy to produce red fluorescent protein (RFP) transgenic angelfish driven by the myosin light chain 2 (mylz2) promoter from zebrafish. A 1999 bp Mylz2 promoter fragment was isolated from zebrafish muscle genomic DNA. This promoter fragment was then cloned into the plasmid pDsred2-1 open-loop at restriction enzyme SacI and AgeI sites to create the final transgene construct pMylz2-RFP. Angelfish embryos at one cell stage were microinjected with approximately 100 pg of the plasmid pMylz2-RFP. From 524 microinjected embryos, 16 successfully hatched, while 12 showed red fluorescence signals. Two larvae survived to 2 months of age. They showed significant red fluorescence expression in the muscles, suggesting that the angelfish could be used as potential transgenic founders to evaluate the next generation of stable red fluorescence expression transgenic fish.

mBeRFP, a versatile fluorescent tool to enhance multichannel live imaging and its applications

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and the development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins, such as mBeRFP, offers interesting new perspectives to bypass this limitation. Here, we constructed a set of mBeRFP-expressing vectors and provided a detailed characterization of this fluorescent protein for in vivo live imaging and its applications in Drosophila. Briefly, we showed that a single illumination source is sufficient to simultaneously stimulate mBeRFP and GFP. We demonstrated that mBeRFP can be easily combined with classical green and red fluorescent protein without any crosstalk. We also showed that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano biologists in their current research.

mCherry contains a fluorescent protein isoform that interferes with its reporter function

Fluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

Advances in the Genetically Engineered KillerRed for Photodynamic Therapy Applications

Photodynamic therapy (PDT) is a clinical treatment for cancer or non-neoplastic diseases, and the photosensitizers (PSs) are crucial for PDT efficiency. The commonly used chemical PSs, generally produce ROS through the type II reaction that highly relies on the local oxygen concentration. However, the hypoxic tumor microenvironment and unavoidable dark toxicity of PSs greatly restrain the wide application of PDT. The genetically encoded PSs, unlike chemical PSs, can be modified using genetic engineering techniques and targeted to unique cellular compartments, even within a single cell. KillerRed, as a dimeric red fluorescent protein, can be activated by visible light or upconversion luminescence to execute the Type I reaction of PDT, which does not need too much oxygen and surely attract the researchers’ focus. In particular, nanotechnology provides new opportunities for various modifications of KillerRed and versatile delivery strategies. This review more comprehensively outlines the applications of KillerRed, highlighting the fascinating features of KillerRed genes and proteins in the photodynamic systems. Furthermore, the advantages and defects of KillerRed are also discussed, either alone or in combination with other therapies. These overviews may facilitate understanding KillerRed progress in PDT and suggest some emerging potentials to circumvent challenges to improve the efficiency and accuracy of PDT.

Construction of an Integrated mCherry Red Fluorescent Protein Expression System for Labeling and Tracing in Lactiplantibacillus plantarum WCFS1

Thorough intestinal adhesion and colonization greatly promote the probiotic properties of lactic acid bacteria (LAB). Labeling and tracing with fluorescent proteins are effective and reliable for studying the in vivo physiological activities of LAB including localization, adhesion, and colonization. Lactiplantibacillus plantarum WCFS1 was successfully traced with a red fluorescent protein (RFP), which was expressed by the bacteria-carrying recombinant plasmids. In this study, we aimed to construct a stable RFP mCherry expression system, whose encoding gene was integrated into the bacterial chromosome via double-crossed homologous recombination, and use it for labeling WCFS1 with the goal of avoiding the potential loss of non-chromosomal plasmids along with intestinal growth. First, the constitutive expression of the mCherry protein was improved after adjusting the length of the spacer between the promoter and the gene start codon. Then, the optimized mCherry gene expression cassette was integrated into the chromosome of WCFS1. The resulting strain had normal unimpaired growth and strong fluorescent signals, even after 100 generations, indicating its stability. Furthermore, quantitative polymerase chain reaction (PCR) results revealed a strong positive correlation between the fluorescence intensity of the strain and the number of viable cells, demonstrating its potential usage for the quantification of in vivo WCFS1 cells. Finally, the increased adhesion ability of WCFS1 due to the recombinant expression of the bsh gene was visualized and evaluated using fluorescence intensity, the results of which were consistent with those obtained using the previously established quantification methods. These results suggest that the chromosomal-integrated mCherry labeling system can be extensively used to examine the distribution, colonization, and survival of LAB in vivo in order to determine the mechanism of its probiotic function.

Platelets release mitochondrial antigens in systemic lupus erythematosus

The accumulation of DNA and nuclear components in blood and their recognition by autoantibodies play a central role in the pathophysiology of systemic lupus erythematosus (SLE). Despite the efforts, the sources of circulating autoantigens in SLE are still unclear. Here, we show that in SLE, platelets release mitochondrial DNA, the majority of which is associated with the extracellular mitochondrial organelle. Mitochondrial release in patients with SLE correlates with platelet degranulation. This process requires the stimulation of platelet FcγRIIA, a receptor for immune complexes. Because mice lack FcγRIIA and murine platelets are completely devoid of receptor capable of binding IgG-containing immune complexes, we used transgenic mice expressing FcγRIIA for our in vivo investigations. FcγRIIA expression in lupus-prone mice led to the recruitment of platelets in kidneys and to the release of mitochondria in vivo. Using a reporter mouse with red fluorescent protein targeted to the mitochondrion, we confirmed platelets as a source of extracellular mitochondria driven by FcγRIIA and its cosignaling by the fibrinogen receptor α2bβ3 in vivo. These findings suggest that platelets might be a key source of mitochondrial antigens in SLE and might be a therapeutic target for treating SLE.

Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

Reporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we compared the characteristics of five reporter genes (Firefly luciferase, stable- and unstable Nano luciferase, secretable Gaussia luciferase and Red Fluorescent Protein) to study complex body fluids. For this comparison, the NFκB Response Element (NFκB-RE) and Smad Binding Element (SBE) were identically cloned into the five different reporter vectors. Reporter characteristics were evaluated by kinetic and concentration–response measurements in SW1353 and HeLa cell lines. Finally, reporter compatibility with complex body fluids (fetal calf serum, knee joint synovial fluid and human serum) and inter-donor variation were evaluated. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases. Intracellularly measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. Secreted Gaussia luciferase appeared to be incompatible with complex body fluids, due to variability in inter-donor signal interference. Unstable Nano luciferase demonstrated clear inducibility, high sensitivity and compatibility with complex body fluids and therefore can be recommended for cellular signaling studies using complex body fluids.