Red Fluorescent Protein mCherry Research Articles

Pressure-Induced Changes in the Fluorescence Behavior of Red Fluorescent Proteins

We present an experimental study on the fluorescence behavior of the red fluorescent proteins TagRFP-S, TagRFP-T, mCherry, mOrange2, mStrawberry, and mKO as a function of pressure up to several GPa. TagRFP-S, TagRFP-T, mOrange2, and mStrawberry show an initial increase in fluorescence intensity upon application of pressure above ambient conditions. At higher pressures, the fluorescence intensity decreases dramatically for all proteins under study, probably due to denaturing of the proteins. Small blue shifts in the fluorescence spectra with increasing pressure were seen in all proteins under study, hinting at increased rigidity of the chromophore environment. In addition, mOrange2 and mStrawberry exhibit strong and abrupt changes in their fluorescence spectra at certain pressures. These changes are likely due to structural modifications of the hydrogen bonding environment of the chromophore. The strong differences in behavior between proteins with identical or very similar chromophores highlight how the chromophore environment contributes to pressure-induced behavior of the fluorescence performance.

One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus

The gene transfer agent (RcGTA) of Rhodobacter capsulatus is the model for a family of novel bacteriophage-related genetic elements that carry out lateral transfer of essentially random host DNA. Genuine and putative gene transfer agents have been discovered in diverse genera and are becoming recognized as potentially an important source of genetic exchange and microbial evolution in the oceans. Despite being discovered over 30 years ago, little is known about many essential aspects of RcGTA biology. Here, we validate the use of direct fluorescence reporter constructs, which express the red fluorescent protein mCherry in R. capsulatus. A construct containing the RcGTA promoter fused to mCherry was used to examine the single-cell expression profiles of wild type and RcGTA overproducer R. capsulatus populations, under different growth conditions and growth phases. The majority of RcGTA production clearly arises from a small, distinct sub-set of the population in the wild type strain and a larger sub-set in the overproducer. The most likely RcGTA release mechanism concomitant with this expression pattern is host cell lysis and we present direct evidence for the release of an intracellular enzyme accompanying RcGTA release. RcGTA ORF s is annotated as a ‘cell wall peptidase’ but we rule out a role in host lysis and propose an alternative function as a key contributor to RcGTA invasion of a target cell during infection.

A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination

BackgroundTargeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre.FindingsHere, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line.ConclusionsThe pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.

Abstract 2687: Imaging sub-cellular dynamics of proliferating intra- and extra-vascular cancer cells

Abstract In order to visualize nuclear-cytoplasmic dynamics during intravascular cancer cell-proliferation and extravasation, green fluorescent protein (GFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and red fluorescent protein (m-Cherry), linked to histone H2B, was expressed in the nucleus. Nuclear m-Cherry expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic GFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Intra- and extra-vascular mitotic cells were visualized by imaging after injection of the cancer cells in the epigastric cranials vein in an abdominal flap. After one hour, round and elongated cancer cells were observed in the vessel. Three hours after injection, invadopodia of the cancer cells was observed. Five hours after injection, dual-color cancer cells began to divide within the vessel. By 10 hours, some intra-vascular cancer cells underwent apoptosis. Deformed new blood vessels in the tumor were observed 10 days later. Extravascular cancer cells were dividing in the tumor at day 14. The subcellular in vivo imaging approach described here provides new visual targets for trafficking, extravasating and invading cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2687. doi:1538-7445.AM2012-2687

An siRNA Screen in Pancreatic Beta Cells Reveals a Role for Gpr27 in Insulin Production

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.

Probing S4 Length Changes during Gating with LRET

Voltage-activated proteins containing a Voltage Sensor Domain (VSD) respond to changes in the membrane potential by transferring across the electric field several positively charged residues (gating charges) located in the fourth transmembrane segment (S4). Even though the mechanistic details of gating charge translocation and S4 re-arrangement are presently unclear, a prevailing hypothesis suggests that the S4 segment adopts a 310 helix conformation during gating, thus aligning the gating charges and facilitating their transport across the membrane electric field. If a whole typical S4 segment were to change its conformation from an α-helix to a 310 helix, its length would stretch by about 8 Å. Here we tested the existence of such transition by measuring the length of the S4 segment during gating using the LRET technique. We used the VSD domain of the Ciona intestinalis Voltage-Sensitive Phosphatase (CiVSP), truncated from its phosphatase and phospholipid-binding domains, to genetically encode a lanthanide (Tb3+)-binding-tag at the extracellular end of S4 and the red fluorescent protein mCherry at its intracellular end. We expressed the protein in Xenopus oocytes from which we recorded gating currents using the cut-open and two-electrode voltage-clamp techniques. The distance-dependent efficiency of energy transfer between the two probes was measured at steady-state voltages and also during voltage pulse protocols of varying amplitudes and durations. Our technique could only detect distance changes larger than 2.5 Å. We found that in our CiVSP construct, the results are not consistent with the expected length change if the whole S4 were converted (and remained converted) from a 310 helix to an α-helix (or vice-versa) when the membrane potential is changed from −100 to +80 mV. Supported by NIH GM68044-07 and GM30376-30S1.

Activity-dependent coordinated mobility of hippocampal inhibitory synapses visualized with presynaptic and postsynaptic tagged-molecular markers

Axonal varicosities and dendritic spines at excitatory synapses are dynamic structures essential for synaptic plasticity, whereas the behavior of inhibitory synapses during development and plasticity remains largely unknown. To investigate the morphology and dynamics of inhibitory synapses, we used two distinct pre- and postsynaptic fluorescent probes: one is a yellow fluorescent protein, Venus, incorporated into vesicular GABA transporter (VGAT) gene as a specific marker of presynaptic inhibitory neurons and the other red fluorescent protein (mCherry)-tagged gephyrin, a postsynaptic scaffolding protein, as a postsynaptic marker. Using primary culture of mouse hippocampal neurons and confocal laser-scanning microscopy, we established a system by which close contacts of Venus-positive axonal varicosities with mCherry-labeled gephyrin clusters in the dendritic shafts of dissociated hippocampal pyramidal neurons could be clearly visualized. Time-lapse imaging revealed that: (1) the presynaptic varicosities actively moved with marked changes in their shapes, and the postsynaptic scaffolding protein gephyrin clusters underwent coordinated movements in a tight association with the presynaptic varicosities, (2) the extents of morphological changes and movements depended on the developmental stages, reaching a stable level as the inhibitory synaptic connections matured, and (3) the motility indexes of the varicosity and its counterpart gephyrin cluster were well correlated. Furthermore, action potential blockade with tetrodotoxin treatment reduced the varicosity size, gephyrin cluster mobility as well as the amplitude of GABAergic synaptic currents in pyramidal neurons. Such a neural activity-dependent dynamic change in GABAergic synaptic morphology is likely to play a critical role in the regulatory mechanism underlying the formation and plasticity of inhibitory synapses.

Gamma camera and optical imaging with a fusion reporter gene using human sodium/iodide symporter and monomeric red fluorescent protein in mouse model

Purpose: Multimodality imaging contributes to the activation of translational research by compensating for its weak points. Herein, we developed a noninvasive dual-reporter gene system for nuclear and optical imaging.Materials and methods: We constructed a fusion reporter vector concurrently expressing the human sodium/iodide symporter (hNIS) and monomeric red fluorescent protein (mCherry), and evaluated the function of this fusion reporter system under in vitro and in vivo conditions.Results: The expression of hNIS/mCherry fusion gene was confirmed in transfected cells using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. As the numbers of cells increased, the fluorescence and 125I uptake increased in the hNIS/mCherry-transfected cells, and a high correlation between fluorescence intensity and radioactivity was noted. The fluorescence intensities and radioactivity signals were also well-correlated in HT-29-hNIS/mCherry tumors (R2 = 0.9304) in in vivo fluorescence and gamma camera imaging.Conclusions: The dual-reporter imaging method using hNIS and mCherry genes reflected tumor extent as well as viable cell numbers, and correlated well with one another. This suggests that the hNIS/mCherry dual-reporter system can be a useful tool for multi-modal imaging.

Single cell detection of latent cytomegalovirus reactivation in host tissue

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor-GFP reporter mice, we never observed reactivation originating from CX3CR1(+) monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b(+) and CX3CR1(+) subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

Efficient phage display of intracellularly folded proteins mediated by the TAT pathway

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.

Modulation of Sarcoplasmic Reticulum Ca 2+ Release by Phosphatidylinositol-Phosphate Lipids in Isolated Mouse Skeletal Muscle Fibers

Previous results from ryanodine binding and single ryanodine receptor (RyR) channel measurements suggested that RyR1 activity may be modulated by phosphatidylinositol-phosphate lipids (PtdInsPs). Possibly related, recent data showed that skeletal muscle e-c coupling is altered in several models of PtdInsPs phosphatase deficiency. We measured intracellular Ca2+ in fibers from flexor digitorum brevis (fdb)muscles microinjected with a solution containing a given PtdInsP lipid (PtdIns(3,5)P2, PtdIns(3)P, PtdIns(5)P or PtdIns) together with the calcium dye indo-1. Following equilibration, fibers were stimulated by short voltage-clamp depolarizations of increasing amplitude from −80mV. Resting [Ca2+] did not differ between control fibers and any group of PtdInsPs-injected fibers; it was though ∼1.5 larger in fibers injected with PtdIns(3,5)P2 or PtdIns(3)P than in fibers injected with PtdIns(5)P or PtdIns. Peak SR Ca2+ release was specifically depressed in fibers injected with PtdIns(3,5)P2 or PtdIns(3)P with maximum values of 34.8 ± 3 µM.ms−1 in control fibers (n=15) and of 24.4 ± 2 and 17.4 ± 2 µM.ms−1 in fibers injected with PtdIns(3,5)P2 (n=8) and PtdIns(3)P (n=8), respectively. There was no concurrent effect on the membrane current measured during the pulses and the voltage dependence of Ca2+ release inactivation was also unaffected. These results highlight the possibility that SR Ca2+ release may be physiologically regulated by certain PtdInsPs. A plasmid coding for a wild-type form of the PtdIns phosphatase MTM1 tagged with the red fluorescent protein mCherry was transfected in fdb muscles. The protein was found distributed within the entire volume of the transfected fibers and to reproducibly yield a striated transversal expression pattern consistent with localization within the triadic area. Preliminary measurements of voltage-activated Ca2+ transients in these cells indicated that under these conditions over-expression of MTM1 had limited effects on SR Ca2+ release.

Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate

Fluorescence cross-correlation spectroscopy (FCCS) reveals information about the spatiotemporal coincidence of two spectrally well-defined fluorescent molecules in a small observation area at the level of single-molecule sensitivity. To simultaneously evaluate the activities of caspase-3 and caspase-9, we constructed a chimeral protein that consisted of tandemly fused enhanced cyan fluorescent protein (ECFP), monomeric red fluorescent protein (mCherry) and monomeric yellow fluorescent protein (Venus). In HeLa cell lysates, a combination of tumor necrosis factor-α (TNF-α)- and cycloheximide (CHX-)-induced apoptosis was monitored. In this, decreases of cross-correlation amplitudes were observed between ECFP and mCherry and between mCherry and Venus. Moreover, time-dependent monitoring of single cells revealed decreases in the cross-correlation amplitudes between ECFP and mCherry and between mCherry and Venus before morphologic changes were observed by laser scanning fluorescence microscopy (LSM). Thus, our method could predict the fate of the cell in the early apoptotic stage.

Ubiquitous expression of the monomeric red fluorescent protein mcherry in transgenic mice

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual-labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N-terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin-C promoter. mCherry is expressed in almost all tissues analyzed including pre- and post-implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals.

Genetically encoded dendritic marker sheds light on neuronal connectivity inDrosophila

In recent years, Drosophila melanogaster has emerged as a powerful model for neuronal circuit development, pathology, and function. A major impediment to these studies has been the lack of a genetically encoded, specific, universal, and phenotypically neutral marker of the somatodendritic compartment. We have developed such a marker and show that it is effective and specific in all neuronal populations tested in the peripheral and central nervous system. The marker, which we name DenMark (Dendritic Marker), is a hybrid protein of the mouse protein ICAM5/Telencephalin and the red fluorescent protein mCherry. We show that DenMark is a powerful tool for revealing novel aspects of the neuroanatomy of developing dendrites, identifying previously unknown dendritic arbors, and elucidating neuronal connectivity.

Identification of MOAG-4/SERF as a Regulator of Age-Related Proteotoxicity

Fibrillar protein aggregates are the major pathological hallmark of several incurable, age-related, neurodegenerative disorders. These aggregates typically contain aggregation-prone pathogenic proteins, such as amyloid-beta in Alzheimer's disease and alpha-synuclein in Parkinson's disease. It is, however, poorly understood how these aggregates are formed during cellular aging. Here we identify an evolutionarily highly conserved modifier of aggregation, MOAG-4, as a positive regulator of aggregate formation in C. elegans models for polyglutamine diseases. Inactivation of MOAG-4 suppresses the formation of compact polyglutamine aggregation intermediates that are required for aggregate formation. The role of MOAG-4 in driving aggregation extends to amyloid-beta and alpha-synuclein and is evolutionarily conserved in its human orthologs SERF1A and SERF2. MOAG-4/SERF appears to act independently from HSF-1-induced molecular chaperones, proteasomal degradation, and autophagy. Our results suggest that MOAG-4/SERF regulates age-related proteotoxicity through a previously unexplored pathway, which will open up new avenues for research on age-related, neurodegenerative diseases.

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry

Some root-associated pseudomonads sustain plant growth by suppressing root diseases caused by pathogenic fungi. We investigated to which extent select cereal cultivars influence expression of relevant biocontrol traits (i.e., root colonization efficacy and antifungal activity) in Pseudomonas fluorescens CHA0. In this representative plant-beneficial bacterium, the antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN), pyoluteorin (PLT), and hydrogen cyanide (HCN) are required for biocontrol. To monitor host plant effects on the expression of biosynthetic genes for these compounds on roots, we developed fluorescent dual-color reporters suited for flow cytometric analysis using fluorescence-activated cell sorting (FACS). In the dual-label strains, the constitutively expressed red fluorescent protein mCherry served as a cell tag and marker for root colonization, whereas reporter fusions based on the green fluorescent protein allowed simultaneous recording of antifungal gene expression within the same cell. FACS analysis revealed that expression of DAPG and PRN biosynthetic genes was promoted in a cereal rhizosphere, whereas expression of PLT and HCN biosynthetic genes was markedly less sustained. When analyzing the response of the bacterial reporters on roots of a selection of wheat, spelt, and triticale cultivars, we were able to detect subtle species- and cultivar-dependent differences in colonization and DAPG and HCN gene expression levels. The expression of these biocontrol traits was particularly favored on roots of one spelt cultivar, suggesting that a careful choice of pseudomonad-cereal combinations might be beneficial to biocontrol. Our approach may be useful for selective single-cell level analysis of plant effects in other bacteria-root interactions.

Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle α‐actin gene

Smooth muscle alpha actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full-length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1-3 copies of the mCherry-substituted BAC vector. Furthermore, we characterized the expression of SMA-mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence-activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA-expressing cells via FACS and for studying the emergence, behavior, and regulation of SMA-expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development.

Abstract 5503: Dual fluorescence high throughput screen detects MUC16 selective therapeutic candidates

Abstract BACKGROUND: The MUC16 gene encodes the CA125 ovarian cancer antigen. This glycoprotein is expressed in the majority of Epithelial Ovarian Cancers (EOC) but has limited expression elsewhere in the adult human. Expression of the carboxy-portion of MUC16 results in increased invasion, soft agar growth and in vivo growth. OBJECTIVES: We sought to develop a suitable microscopy based assay system to screen for and identify small molecules which have a differential effects against MUC16+ cells compared to the isogenic MUC16 negative ovarian cancer cell lines. Using this assay we seek to identify MUC16 specific small molecules for development of potential chemotherapy drug candidates against ovarian cancer. METHODS: Carboxy-terminus portions of the Human MUC16 gene were introduced into SK-Ov-3 and A2780 EOC cell lines and labeled with the red fluorescent protein (mCherry) vector. Control lines were labeled with green fluorescent protein (GFP) vector. Relative toxicity of tested compounds on MUC16+ (mCherry) and MUC16- (GFP) cell populations was assessed by cell viability using ratiometric fluorescence intensities of both co-cultured isogenic cells. Compounds with differential activity were selected, resupplied and tested in a 96 well plate viability assay for confirmation. RESULTS: Both A2780 and SK-Ov-3 isogenic pairs had similar in vitro growth characteristics. A small scale pilot screen of 3,119 compounds was completed for each pair of isogenic cells. Six compounds, including Quinacrine, Ellipticine and Acriflavinium were relatively selective for MUC16+ A2780 cells and these three compounds were among the hits identified in the secondary SK-Ov-3 dual fluorescence screen. Caspase3 apoptosis assay was used to confirm Quinacrine as the most selective inhibitor in the MUC16+ cells. CONCLUSIONS: MUC16 +/− dual fluorescence screen is an example of a high throughput screening strategy to identify potentially specific modulators of key oncogenic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5503.

Intracellular Bioconjugation of Targeted Proteins with Semiconductor Quantum Dots

We demonstrate controlled in vivo bioconjugation of a targeted intracellular protein to semiconductor quantum dots (QDs). Metal-affinity driven coordination of oligohistidine-appended proteins for chelated divalent cations was exploited to facilitate this interaction. Monomeric mCherry red fluorescent protein recombinantly engineered to express an N-terminal hexahistidine sequence was expressed from a eukaryotic plasmid vector following transfection into COS-1 cells. QDs solubilized with a carboxylated polymeric ligand and pretreated with Ni(2+) were then microinjected into the mCherry-expressing COS-1 cells. Förster resonance energy transfer (FRET) between the central QD donors and mCherry acceptors specifically coordinated to their surface was utilized to probe and confirm intracellular conjugate formation. We unexpectedly found that mCherry attachment to the QDs also substantially improves its resistance to photobleaching. This proof-of-concept, highlighting targeted intracellular bioconjugation to QDs, suggests that many cytoplasmic proteins expressing the ubiquitous hexahistidine affinity handle can be specifically attached to QDs in vivo. This approach can facilitate long-term monitoring of their spatio-temporal activity or, alternatively, allow engineering and in situ assembly of designer chimeric QD-fluorescent protein sensors.