Bright Green Fluorescent Protein Research Articles

A highly photostable and bright green fluorescent protein

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

A Genetically Encoded Bioluminescent System for Fast and Highly Sensitive Detection of Antibodies with a Bright Green Fluorescent Protein.

A method for fast and highly sensitive detection of antibodies in serum would greatly facilitate the early diagnosis of disease and infection and dose optimization of therapeutic antibody. Bioluminescence detection with LUMABS (renamed mNeonG-LUMABS, where mNeonG is short for mNeonGreen) sensors based on bioluminescence resonance energy transfer (BRET) between blue-emitting luciferase Nluc and green fluorescent protein (FP) mNeonGreen has been demonstrated to enable fast detection of antibodies directly in serum with reasonable sensitivity. However, some mNeonG-LUMABS sensors exhibit low sensitivity, and thus, sensitivity improvement remains imperative. Here, we report a bright green FP, Clover4, obtained by structure-guided mutagenesis of green FP Clover. Despite similar brightness and fluorescence spectra of Clover and mNeonGreen, Clover4-LUMABS sensors exhibit a largely increased dynamic range (maximum 20-fold) and much lower limit of detection (LOD) (maximum 5.6-fold), most likely because Clover4 is positioned in a more parallel orientation to Nluc in LUMABS. Due to modular design, Clover4-LUMABS offers a general BRET system for fast and highly sensitive antibody detection in serum.

Developing Bright Green Fluorescent Protein (GFP)-like Fluorogens for Live-Cell Imaging with Nonpolar Protein-Chromophore Interactions.

Fluorescence-activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence-activating and absorption-shifting tag (FAST) is a light-weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super-resolution imaging. However, the rational design of FAST-specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M-1 cm-1 , is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein-fluorogen interactions. Our deep insights into structure-function relationships could guide the rational design of bright fluorogens for live-cell imaging with extended spectral properties such as redder emissions.

Bright and High-Performance Genetically Encoded Ca2+ Indicator Based on mNeonGreen Fluorescent Protein.

Genetically encodable calcium ion (Ca2+) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the Aequorea victoria GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

Effect of endometriosis on the fecal bacteriota composition of mice during the acute phase of lesion formation.

Accumulating evidence indicates that there is an interaction between the gut microbiota and endometriotic lesions. The new formation of these lesions is associated with stem cell recruitment, angiogenesis and inflammation, which may affect the composition of the gut microbiota. To test this hypothesis, we herein induced endometriotic lesions by transplantation of uterine tissue fragments from green fluorescent protein (GFP)+ donor mice into the peritoneal cavity of GFP- C57BL/6 wild-type mice. Sham-transplanted animals served as controls. Fecal pellets of the animals were collected 3 days before as well as 7 and 21 days after the induction of endometriosis to analyze the composition of the gut microbiota by means of 16S ribosomal RNA gene sequencing. The transplantation of uterine tissue fragments resulted in the establishment of endometriotic lesions in all analyzed mice. These lesions exhibited a typical histomorphology with endometrial glands surrounded by a vascularized stroma. Due to their bright GFP signal, they could be easily differentiated from the surrounding GFP- host tissue. Bacterial 16S rRNA genes were successfully PCR-amplified from the DNA extracts of all obtained mice fecal samples. However, no significant effect of endometriosis induction on the composition of the bacterial microbiota was detected with our experimental setup. Our findings allow careful speculation that endometriosis in mice does not induce pronounced dysbiosis during the acute phase of lesion formation.

Live-cell nanoscopy with spontaneous blinking of conventional green fluorescent proteins

Single-molecule localization microscopy with spontaneously blinking fluorescent tags holds a promise of simplified imaging setup for live-cell application. However, robust blinking has been reported for just a few fluorescent proteins. Here we report on a comparison of spontaneous blinking for three bright green fluorescent proteins, mAvicFP1, AausFP1, and mNeonGreen. mAvicFP1 outperforms other fluorescent proteins in this list in a wide range of camera exposure times and illumination intensities. We establish imaging conditions for live-cell nanoscopy and single-particle tracking with mAvicFP1.

A Genetically‐Encoded Fluorescent Assay to Detect Drug Toxicity and Genetic Stress in Living Cells

We have created a genetically‐encoded fluorescent biosensor to detect chemically and genetically induced stress in living cells. Identifying stressed cells long before they enter the apoptotic pathway opens a therapeutic window between cell stress and death. Further, the biosensor can also be used to identify toxic side effects of drugs or lead compounds.The sensor co‐opts one pathway of the unfolded protein response as the driving mechanism to detect cell stress. Non‐canonical splicing of an intron derived from the XBP1 protein shifts into frame a bright green fluorescent protein, which indicates the onset of cell stress. Importantly, this intron is only spliced upon activation of endoplasmic reticulum stress or the unfolded protein response. Ratioing this signal with a constitutively expressed red fluorescent protein signal allows real‐time monitoring of cells stress and protein production. We have also engineered this biosensor to be reversible, allowing detection of the onset of stress as well as the amelioration of stress. This positions the biosensor to be used for testing compounds that may relieve cell stress.We applied this assay to examine the toxic side effects of cardiac glycosides and doxorubicin on cardiomyocytes derived from human induced pluripotent stem cells. Drug toxicity was detected within 24 hours of treatment in a dose dependent manner. The cardiac glycosides induced cell stress mediated through either the unfolded protein response or endoplasmic reticulum stress while doxorubicin, which inhibits the XBP1 pathway, mainly displayed cytotoxic effects. We next used this assay to detect cellular stress induced by genetic mutations. Many neurological diseases, such as Retinitis Pigmentosa, Parkinson's and Amyotrophic lateral Sclerosis (ALS), are associated with protein folding or aggregation defects which induce high levels of cellular stress. This live‐cell stress assay is capable of monitoring cellular stress induced by mutations causing Retinitis Pigmentosa (Rhodopsin P23H mutation) and ALS (SOD G86R mutation) in less than 24 hours after mutant gene expression.Lastly, multiplexing this cell stress assay with other live‐cell assays detecting second messenger signaling revealed cells experiencing either chemically or genetically induced stress had altered basal levels of cytoplasmic Ca2+ and cAMP as well as a blunted Ca2+ signaling response. Together these studies demonstrate the broad utility of this newly developed live‐cell assay for cell stress and toxicity for use in automated screening of drugs and compounds, determination of adverse drug toxicity, and analysis of disease mechanisms.Support or Funding InformationSupported by ASEE/NSF Small Business Postdoctoral Research Diversity Fellowship Program NSF Award IIP‐1552305This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNAi-Mediated Loss of Function of Xenopus Immune Genes by Transgenesis.

Generation of transgenic frogs through the stable integration of foreign DNA into the genome is well established in Xenopus This protocol describes the combination of transgenesis with stable RNA interference as an efficient reverse genetic approach to study gene function in Xenopus Initially developed in the fish medaka and later adapted to Xenopus, this transgenic method uses the I-SceI meganuclease, a "rare-cutter" endonuclease with an 18 bp recognition sequence. In this protocol, transgenic X. laevis with knocked down expression of a specific gene are generated using a double promoter expression cassette. This cassette, which is flanked by I-SceI recognition sites, contains the shRNA of choice under the control of the human U6 promoter and a green fluorescent protein (GFP) reporter gene under the control of the human EF-1α promoter. Prior to microinjection the plasmid is linearized by digestion with I-SceI and the entire reaction is then microinjected into one-cell stage eggs. The highly stringent recognition sequence of I-SceI is thought to maintain the linearized plasmid in a nonconcatamerized state, which promotes random integration of the plasmid transgene in the genome. The injected embryos are reared until larval stage 56 and then screened for GFP expression by fluorescence microscopy and assessed for effective knockdown by quantitative RT-PCR using a tail biopsy. Typically, the I-SceI meganuclease transgenesis technique results in 35%-50% transgenesis efficiency, a high survival rate (>35%) and bright nonmosaic GFP expression. A key advantage of this technique is that the high efficiency and nonmosaic transgene expression permit the direct use of F0 animals.

New tracer to estimate community predation rates of phagotrophic protists

Predation of eukaryotic microbes on prokaryotes is one of the most important trophic interactions on Earth, representing a major mortality term and shaping morphology and composi- tion of prokaryotic communities. Here we introduce and validate a new tracer to determine pre- dation rates on prokaryotes. Minicells of Escherichia coli marked with a bright green fluorescent protein (GFP) vector have many operational advantages over previously used prey analogs such as fluorescently labeled bacteria. GFP-minicells are similar in size to naturally occurring bacteria from a variety of environments including the oligotrophic open ocean and the deep sea. They are relatively stable against microbial and light degradation, are easy to grow and process, and can be produced inexpensively in large numbers. No chemical alteration of the particle surface due to heat killing and staining is involved. Grazing coefficients were compared between GFP-minicells and other GFP-modified bacteria, as well as 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF)- stained cells. The grazing coefficients obtained from the removal of GFP-minicells compared favorably with estimates from tracer-independent estimates of grazing in the same experiments. Experiments with GFP-minicells resulted in community grazing coefficients similar to those reported for many different marine environments and those derived using various methods.

Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system.

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100β-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve.

A Dual-Color Genetically Engineered Mouse Model for Multispectral Imaging of the Pancreatic Microenvironment

To develop a mouse model for multispectral fluorescence imaging of the pancreas and pancreatic microenvironment. Cre/loxP technology was used to develop this model. We crossed mT/mG indicator mice, engineered to constitutively express a conditional tdTomato transgene that converts to green fluorescent protein (GFP) expression after exposure to Cre recombinase, with Pdx1-Cre transgenic mice. To characterize this model for studies of pancreas biology, we performed bright light and fluorescence imaging of body cavities and intact organs and confocal microscopy of pancreata from offspring of Pdx1-Cre and mT/mG crosses. Pdx1-Cre-mT/mG mice demonstrated bright GFP expression within the pancreas and duodenum and intense tdTomato expression in all other organs. Green fluorescent protein expression was mosaic in Pdx1-Cre-mT/mG pancreata, with most showing extensive conversion from tdTomato to GFP expression within the epithelial-derived elements of the pancreatic parenchyma. Because both GFP and tdTomato are membrane targeted, individual cell borders were clearly outlined in confocal images of mT/mG pancreata. This mouse model enables multispectral fluorescence imaging of individual cells and cell processes at the microscopic level of the pancreatic microenvironment; it should prove valuable for a variety of fluorescence imaging studies, ranging from pancreatic development to pancreatic cancer biology.

Muscarinic activation of Ca2+‐activated Cl− current in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity and functional bridges between enteric motor nerve terminals and gastrointestinal smooth muscle cells. The ionic conductance(s) in ICC that are activated by excitatory neural inputs are unknown. Transgenic mice (Kit(copGFP/+)) with constitutive expression of a bright green fluorescent protein were used to investigate cellular responses of ICC to cholinergic stimulation. ICC displayed spontaneous transient inward currents (STICs) under voltage clamp that corresponded to spontaneous transient depolarizations (STDs) under current clamp. STICs reversed at 0 mV when E(Cl) = 0 mV and at -40 mV when E(Cl) was -40 mV, suggesting the STICs were due to a chloride conductance. Carbachol (CCh, 100 nm and 1 μm) induced a sustained inward current (depolarization in current clamp) and increased the amplitude and frequency of STICs and STDs. CCh responses were blocked by atropine (10 μm) or 4-DAMP (100 nm), an M(3) receptor antagonist. STDs were blocked by niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (both 100 μm), and CCh had no effect in the presence of these drugs. The responses of intact circular muscles to CCh and stimulation of intrinsic excitatory nerves by electrical field stimulation (EFS) were also compared. CCh (1 μm) caused atropine-sensitive depolarization and increased the maximum depolarization of slow waves. Similar atropine-sensitive responses were elicited by stimulation of intrinsic excitatory neurons. Niflumic acid (100 μm) blocked responses to EFS but had minor effect on responses to exogenous CCh. These data suggest that different ionic conductances are responsible for electrical responses elicited by bath-applied CCh and cholinergic nerve stimulation.

A functional role for the ‘fibroblast‐like cells’ in gastrointestinal smooth muscles

Smooth muscles, as in the gastrointestinal tract, are composed of several types of cells. Gastrointestinal muscles contain smooth muscle cells, enteric neurons, glial cells, immune cells, and various classes of interstitial cells. One type of interstitial cell, referred to as 'fibroblast-like cells' by morphologists, are common, but their function is unknown. These cells are found near the terminals of enteric motor neurons, suggesting they could have a role in generating neural responses that help control gastrointestinal movements. We used a novel mouse with bright green fluorescent protein expressed specifically in the fibroblast-like cells to help us identify these cells in the mixture of cells obtained when whole muscles are dispersed with enzymes. We isolated these cells and found they respond to a major class of inhibitory neurotransmitters - purines. We characterized these responses, and our results provide a new hypothesis about the role of fibroblast-like cells in smooth muscle tissues.

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

BackgroundFluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development.ResultsHere we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M−1 cm−) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness.ConclusionsThe spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.

A Ca2+‐activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity

Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized 'slow wave' current. Reversal of tail current analysis showed this current was due to a Cl(-) selective conductance. ICC express ANO1, a Ca(2+)-activated Cl(-) channel. Slow wave currents are not voltage dependent, but a secondary voltage-dependent process underlies activation of these currents. Removal of extracellular Ca(2+), replacement of Ca(2+) with Ba(2+), or extracellular Ni(2+) (30 microm) blocked the slow wave current. Single Ca(2+)-activated Cl() channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration-dependent manner by niflumic acid (IC(50) = 4.8 microm). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca(2+)-activated Cl(-) conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.

Green fluorescence induced by EF‐hand assembly in a split GFP system

The affinity between the 1-157 and 158-238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D(9k) to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1-157 of GFP are fused to residues 1-43 of calbindin, and in EF2-GFPC, residues 44-75 of calbindin are fused to residues 158-238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca(2+)-dependent.

Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells

The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells

The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon’s anti-HCV action and for screening and testing new anti-HCV drugs.

Correction

The Plant JournalVolume 54, Issue 5 p. 964-964 Free Access Correction This article corrects the following: Transient transformation and RNA silencing in Zinnia tracheary element differentiating cell cultures Satoshi Endo, Edouard Pesquet, Gen Tashiro, Hideo Kuriyama, Deborah Goffner, Hiroo Fukuda, Taku Demura, Volume 53Issue 5The Plant Journal pages: 864-875 First Published online: November 23, 2007 First published: 12 May 2008 https://doi.org/10.1111/j.1365-313X.2008.03536.xCitations: 2AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Endo et al. (2007), there was an error in the labelling of Figure 1(d), (e), (f) and (g). The correct figure and figure legend are given below. (1) [ Time course of green fluorescent protein (GFP) expression and tracheary element (TE) differentiation after electroporation. (a) Rates of TEs in total cells: , native culture conditions; , unpulsed cultures; , pulsed cultures. (b) Rate of cell division, estimated by the number of cells with septa, in pulsed () and unpulsed ( cultures. (c) Rates of GFP-expressing cells in pulsed () and unpulsed () cultures, shown with the scale on the left y-axis; , with the scale on the right y-axis shows rates of GFP-expressing TEs in total GFP-expressing cells. Each data point presents the mean of three independent cultures ± SD. (d, e) Zinnia cells electroporated with P35S::GFP plasmid and cultured for 24 h, which were observed in bright field (d) and GFP fluorescence (e). (f, g) Differentiating TEs electroporated with P35S::GFP plasmid and cultured for 84 h, which were observed in bright field (f) and GFP fluorescence (g). Bars = 25 μm. ] We apologize for this error. Reference Endo, S., Pesquet, E., Tashiro, G., Kuriyama, H., Goffner, D., Fukuda, H. and Demura, T. (2007) Transient transformation and RNA silencing in Zinnia tracheary element differentiating cell cultures. Plant J. 53, 864– 875. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Citing Literature Volume54, Issue5June 2008Pages 964-964 ReferencesRelatedInformation

A vector for purification-free cloning of polymerase chain reaction products

Conventional cloning requires the purification of restriction-enzyme-digested vectors prior to the ligation reaction. The purification often involves the separation of restriction fragments via electrophoresis, the cutting out of a piece of gel, and the gel extraction of the linearized vector. In addition to the loss of significant amounts of DNA, reduced cloning efficiency, time, and cost, these steps are also mutagenic to DNA and hazardous to humans. We developed a purification-free cloning vector pGT3 with a bright green fluorescent protein indicator that is suitable for TA cloning of polymerase chain reaction (PCR) products. PCR products were cloned into pGT3 efficiently without the gel purification steps.