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A noncytotoxic DsRed variant for whole-cell labeling.

ABSTRACT: A common application of fluorescent proteins is to label whole cells, but many RFPs are cytotoxic when used with standard high-level expression systems. We engineered a rapidly maturing tetrameric fluorescent protein called DsRed-Express2 that has minimal cytotoxicity. DsRed-Express2 exhibits strong and stable expression in bacterial and mammalian cells, and it outperforms other available RFPs with regard to photostability and phototoxicity.

SUBMITTER: Strack RL

PROVIDER: S-EPMC4107390 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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A noncytotoxic DsRed variant for whole-cell labeling.

Strack Rita L RL Strongin Daniel E DE Bhattacharyya Dibyendu D Tao Wen W Berman Allison A Broxmeyer Hal E HE Keenan Robert J RJ Glick Benjamin S BS

Nature methods 20081026 11

A common application of fluorescent proteins is to label whole cells, but many RFPs are cytotoxic when used with standard high-level expression systems. We engineered a rapidly maturing tetrameric fluorescent protein called DsRed-Express2 that has minimal cytotoxicity. DsRed-Express2 exhibits strong and stable expression in bacterial and mammalian cells, and it outperforms other available RFPs with regard to photostability and phototoxicity. ...[more]

PMID: 18953349

Similar Datasets

Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling.

Project description:BackgroundWhole-cell labeling is a common application of fluorescent proteins (FPs), but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties.ResultsBuilding on previous studies of DsRed mutants, we created two DsRed-Express2 derivatives: E2-Orange, an orange FP, and E2-Red/Green, a dual-color FP with both red and green emission. We show that these new FPs retain the low cytotoxicity of DsRed-Express2. In addition, we show that these new FPs are useful as second or third colors for flow cytometry and fluorescence microscopy.ConclusionE2-Orange and E2-Red/Green will facilitate the production of healthy, stably fluorescent cell lines and transgenic organisms for multi-color labeling studies.

| S-EPMC2678115 | biostudies-literature

A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling.

Project description:Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy.

| S-EPMC2861903 | biostudies-literature

Noncytotoxic differentiation treatment of renal cell cancer.

Project description:Current drug therapy for metastatic renal cell cancer (RCC) results in temporary disease control but not cure, necessitating continued investigation into alternative mechanistic approaches. Drugs that inhibit chromatin-modifying enzymes involved in transcription repression (chromatin-relaxing drugs) could have a role, by inducing apoptosis and/or through differentiation pathways. At low doses, the cytosine analogue decitabine (DAC) can be used to deplete DNA methyl-transferase 1 (DNMT1), modify chromatin, and alter differentiation without causing apoptosis (cytotoxicity). Noncytotoxic regimens of DAC were evaluated for in vitro and in vivo efficacy against RCC cell lines, including a p53-mutated RCC cell line developed from a patient with treatment-refractory metastatic RCC. The cell division-permissive mechanism of action-absence of early apoptosis or DNA damage, increase in expression of HNF4? (hepatocyte nuclear factor 4?), a key driver associated with the mesenchymal to epithelial transition, decrease in mesenchymal marker expression, increase in epithelial marker expression, and late increase in cyclin-dependent kinase inhibitor CDKN1B (p27) protein-was consistent with differentiation-mediated cell-cycle exit. In vivo blood counts and animal weights were consistent with minimal toxicity of therapy. The distinctive mechanism of action of a dose and schedule of DAC designed for noncytotoxic depletion of DNMT1 suggests a potential role in treating RCC.

| S-EPMC3661214 | biostudies-literature

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

Project description:With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

| S-EPMC5882651 | biostudies-literature

Whole-mount immunofluorescent labeling of the adult fly retina.

Project description:The adult Drosophila compound eye is an ideal in vivo model for studying biological questions. However, light microscopy of this tissue requires cumbersome embedding and sectioning. Here, we document detailed whole-mount procedures for immunolabeling the adult retina, enabling high-quality studies of fluorescent-tagged targets with straightforward preparations. We describe the steps for visualizing the nuclear lamina, membrane-associated protein, and actin-rich rhabdomere, but this robust protocol can apply to other cellular structures and target proteins. For complete details on the use and execution of this protocol, please refer to Chang et al. (2021).

| S-EPMC9157552 | biostudies-literature

TGF-?-Induced Regulatory T Cells Directly Suppress B Cell Responses through a Noncytotoxic Mechanism.

Project description:Foxp3(+) regulatory T cells (Treg) playing a crucial role in the maintenance of immune tolerance and prevention of autoimmune diseases consist of thymus-derived naturally occurring CD4(+)Foxp3(+) Treg cells (nTreg) and those that can be induced ex vivo with TGF-? (iTreg). Although both Treg subsets share similar phenotypes and functional characteristics, they also have potential biologic differences on their biology. The role of iTreg in regulating B cells remains unclear so far. The suppression assays of Treg subsets on activation, proliferation, and Abs production of B cells were measured using a Treg and B cell coculture system in vitro. Transwell and Ab blockade experiments were performed to assess the roles of cell contact and soluble cytokines. Treg were adoptively transferred to lupus mice to assess in vivo effects on B cells. Like nTreg, iTreg subset also directly suppressed activation and proliferation of B cells. nTreg subset suppressed B cell responses through cytotoxic manner related to expression of granzyme A, granzyme B, and perforin, whereas the role of iTreg subset on B cells did not involve in cytotoxic action but depending on TGF-? signaling. Furthermore, iTreg subset can significantly suppress Ab produced by lupus B cells in vitro. Comparison experiments using autoantibodies microarrays demonstrated that adoptive transfer of iTreg had a superior effect than nTreg subset on suppressing lupus B cell responses in vivo. Our data implicate a role and advantage of iTreg subset in treating B cell-mediated autoimmune diseases, boosting the translational potential of these findings.

| S-EPMC4868785 | biostudies-literature

Noncytotoxic lytic granule-mediated CD8+ T cell inhibition of HSV-1 reactivation from neuronal latency.

Project description:Reactivation of herpes simplex virus type 1 (HSV-1) from neuronal latency is a common and potentially devastating cause of disease worldwide. CD8+ T cells can completely inhibit HSV reactivation in mice, with interferon-gamma affording a portion of this protection. We found that CD8+ T cell lytic granules are also required for the maintenance of neuronal latency both in vivo and in ex vivo ganglia cultures and that their directed release to the junction with neurons in latently infected ganglia did not induce neuronal apoptosis. Here, we describe a nonlethal mechanism of viral inactivation in which the lytic granule component, granzyme B, degrades the HSV-1 immediate early protein, ICP4, which is essential for further viral gene expression.

| S-EPMC2680315 | biostudies-literature

One Injection of DsRed Followed by Bites from Transgenic Mosquitoes Producing DsRed in the Saliva Elicits a High Titer of Antibody in Mice.

Project description:It has been proposed that transgenic mosquitoes can be used as a "flying syringe" for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes. We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect.

| S-EPMC3473083 | biostudies-literature

Chromophore formation in DsRed occurs by a branched pathway.

Project description:Like GFP, the fluorescent protein DsRed has a chromophore that forms autocatalytically within the folded protein, but the mechanism of DsRed chromophore formation has been unclear. It was proposed that an initial oxidation generates a green chromophore, and that a final oxidation yields the red chromophore. However, this model does not adequately explain why a mature DsRed sample contains a mixture of green and red chromophores. We present evidence that the maturation pathway for DsRed branches upstream of chromophore formation. After an initial oxidation step, a final oxidation to form the acylimine of the red chromophore is in kinetic competition with a dehydration to form the green chromophore. This scheme explains why green and red chromophores are alternative end points of the maturation pathway.

| S-EPMC2891888 | biostudies-literature

Single-cell RNAseq of Neurog1-Neurog2-DsRed transduced DRG cells

Project description:Cultured adult mouse dorsal root ganglia (DRG) cells exhibit glial sensory progenitor properties in vitro. Therefore, they might be a good starter cell for reprogramming into sensory neurons. Here, we infected them with the retroviral vector Neurog2-Neurog1-DsRed to induce sensory neuron development and analyzed by scRNAseq at 14 days post infection whether infected cells show properties of sensory neurons such as nociceptors. After 10x Genomics, data analysis of 4,549 individual cells indicated the generation of neurons, but at an immature cell state.

2024-06-20 | GSE261529 | GEO

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