Amount Of Green Fluorescent Protein Research Articles

Non-destructive estimation of the amount of green fluorescent protein transiently expressing in <i>Nicotiana benthamiana</i> leaves with a viral vector

Non-destructive estimation of the amount of green fluorescent protein transiently expressing in <i>Nicotiana benthamiana</i> leaves with a viral vector

Tick Cell Culture Analysis of Growth Dynamics and Cellular Tropism of Rickettsia buchneri, an Endosymbiont of the Blacklegged Tick, Ixodes scapularis.

Simple SummaryThe blacklegged tick, Ixodes scapularis, a species of significant medical and veterinary importance, harbors an endosymbiont, Rickettsia buchneri. This bacterium is largely restricted to the ovaries, but all life stages can harbor different numbers or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. We characterized the cells using microscopy. The doubling time of wildtype R. buchneri and a transformant expressing green fluorescent protein was determined to be >7 days when measured by quantitative PCR. Quantification based on fluorescence indicated that 11 days were needed to double the amount of green fluorescent protein. Two rRNA probes were tested using rickettsiae grown in vitro and adapted to localize R. buchneri in different organs of field-collected female I. scapularis ticks. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. The sequestration of rickettsia in ticks and the slow growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.The blacklegged tick, Ixodes scapularis, a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. Rickettsia buchneri most readily grows and is maintained in the cell line IRE11 from the European tick, Ixodes ricinus. The line was characterized by light and electron microscopy and used to analyze the growth dynamics of wildtype and GFPuv-expressing R. buchneri. qPCR indicated that the genome copy doubling time in IRE11 was >7 days. Measurements of fluorescence using a plate reader indicated that the amount of green fluorescent protein doubled every 11 days. Two 23S rRNA probes were tested via RNA FISH on rickettsiae grown in vitro and adapted to evaluate the tissue tropism of R. buchneri in field-collected female I. scapularis. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. Tissue tropism in I. scapularis and in vitro growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.

Amount of Green Fluorescent Protein in the Anterior Chamber after Intravitreal Injection of Triple-Mutated Self-Complementary AAV2 Vectors is Not Affected by Previous Vitrectomy Surgery.

The adeno-associated virus (AAV) vector is a promising vector for ocular gene therapy. Surgical internal limiting membrane peeling before AAV vector administration is useful for efficient retinal transduction. However, no report has investigated localization of AAV vectors after administration into a post-vitrectomy eye. This study investigated the effects of vitrectomy surgery on intravitreal-injected AAV vector-mediated gene expression in the anterior segment and examined the presence of neutralizing antibodies (NAbs) in serum before and after AAV vector administration. Of six eyes from three female cynomolgus monkeys, four were vitrectomized (Group VIT) and two were non-vitrectomized (Group IV). All eyes were injected with 50 μL of triple-mutated self-complementary AAV2 vector (1.9 × 1013 v.g./mL) encoding green fluorescent protein (GFP). NAbs in the serum were examined before administration and at 2 and 6 weeks after administration. GFP expression was analyzed at 19 weeks after administration. Immunohistological analysis showed no GFP expression in the trabecular meshwork in any eye. The GFP genome copy in two slices of the anterior segment was 2.417 (vector genome copies/diploid genome) in Group VIT and 4.316 (vector genome copies/diploid genome) in group IV. The NAb titer was 1:15.9 (geometric mean) before administration, 1:310.7 at 2 weeks after administration, and 1:669.4 at 6 weeks after administration. Previous vitrectomy surgery did not affect gene expression in the anterior segment after intravitreal injection of AAV vectors.

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Standoff detection of explosives and buried landmines using fluorescent bacterial sensor cells

A standoff detection scheme for buried landmines and concealed explosive charges is presented. The detection procedure consists of the following: Live bacterial sensor strains, genetically engineered to produce a dose-dependent amount of green fluorescent protein (GFP) in the presence of explosives' vapors, are encapsulated and spread on the suspected area. The fluorescence produced by the bacteria in response to traces of the explosive material in their microenvironment is remotely detected by a phase-locked optoelectronic sampling system.This scheme enables fast direct access to a large minefield area, while obviating the need to endanger personnel and equipment. Moreover, the employment of phase locking detection efficiently isolates the bacterial sensors' fluorescent output from the background optical signals. This facilitates the application of bacterial sensors in an outdoor environment, where control of background illumination is not possible. Using this system, we demonstrate standoff detection of 2,4-DNT both in aqueous solution and when buried in soil, by sensor bacteria either in liquid culture or agar-immobilized, respectively, at a distance of 50m in a realistic optically noisy environment.

Remote Ischemic Preconditioning Improves the Viability of Donor Lipoaspirate during Murine Fat Transfer

Variable results associated with fat grafting have been attributed to local trauma, inconsistencies in transfer, and ischemia before the development of recipient circulation. Remote ischemic preconditioning is an inexpensive noninvasive technique that has been used in animal models and multicenter clinical trials to protect organ systems. In this work, the authors describe a novel animal model for analyzing the efficacy of fat grafting, and investigate the effect of remote ischemic preconditioning on volume retention. Subcutaneous adipose tissue samples from green fluorescent protein/luciferase-expressing FVB mice were obtained with or without pretreatment with a temporary hind-limb tourniquet. The samples were injected into the dorsal skin folds of wild-type FVB mice. The viability of the transferred tissue was examined over a 28-day period with quantitative bioluminescence after luciferin injection. Transferred tissue was also explanted for histologic analysis. The remote ischemic preconditioning group had significantly increased bioluminescence at days 0, 1, and 28. Histologic analysis at day 28 confirmed the presence of vascularized adipose in both groups. However, significant amounts of interstitial fibrosis were found in the control group, whereas substantially less was found in the remote ischemic preconditioning group. The remote ischemic preconditioning group retained a substantially greater amount of green fluorescent protein, suggesting increased survival of donor adipocytes. In this work, the authors describe a novel animal model for quantitative evaluation of fat grafting using in vivo bioluminescence of adipocytes from luciferase-expressing mice. The authors also demonstrate that remote ischemic preconditioning increases the viability of fat transfer and decreases interstitial fibrosis.

Dynamics of vacuoles and H+-pyrophosphatase visualized by monomeric green fluorescent protein in Arabidopsis: artifactual bulbs and native intravacuolar spherical structures.

We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H(+)-pyrophosphatase (H(+)-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H(+)-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H(+)-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H(+)-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H(+)-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.

336 QUANTITATIVE ANALYSIS OF TETRACYCLINE-INDUCIBLE EXPRESSION OF THE GREEN FLUORESCENT PROTEIN GENE IN TRANSGENIC CHICKENS

The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognised limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet (Kwon et al. 2011 Biochem. Biophys. Res. Commun. 410, 890–894). As a proof of principle study, however, quantitative assessment of expression was not possible, as only 1 G0 and 1 G1 transgenic chicken was obtained. In the current study, with 7 G2 transgenic chickens obtained from 1 G1 hen, we confirmed stable genomic integration of a single copy number of the transgene by Southern blot analysis. As we have observed in G1 transgenic chicken previously, all of the G2 transgenic chickens emitted a green fluorescence upon doxycycline feeding (50 mg kg–1 of formula feed). Fluorescence became detectable 4 days after starting doxycycline feeding, and maximum GFP expression was detected after 2 weeks. Removal of doxycycline from the diet after 14 days of induction feeding resulted in the return of external fluorescence to pre-induction levels after 39 days. Quantitative analysis of gene induction was done using protein and mRNA extracted from primary cultured cells derived from 6-day transgenic chicken embryos. The eggs were obtained by mating a nontransgenic wild-type hen with 1 of G2 transgenic roosters. Protein levels of GFP were analysed by immunoblot and quantified using a densitometer. In the absence of doxycycline, the amount of GFP per 1 µg of total protein was 0.2 ng. However, when the cells were treated with doxycycline for 6 days, the amount of GFP increased to 3.1 ng per 1 µg of total protein, which was 16-fold higher than that of the cells pre-treated with doxycycline. Switching to doxycycline-free medium after doxycycline induction resulted in significant abrogation of GFP expression in 6 days; the amount of GFP reduced from 3.1 to 0.5 ng, a 6.2-fold reduction. Transcription of the GFP gene was also assessed by Northern blot. The amount of GFP mRNA measured by band density increased as much as 20-fold (3.9/0.2) with 6 days of doxycycline induction and declined to 1/8 (3.9/0.5) when doxycycline was removed from the cell culture media for 6 days. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene.

Combined pluronic P85- and ultrasound contrast agents-mediated gene transfection to HepG2 cells

This study examined the effect of P85 (a pluronic block copolymer) and microbubble (MB) ultrasound contrast agents under ultrasound irradiation on gene transfection and expression. The pEGFP plasmids that can encode enhanced green fluorescent protein (pEGFP) served as a report gene and were mixed with different concentrations of MB/0.05% (w/v) P85. Then the plasmids were transfected into human hepatoma G2 (HepG2) cells. The HepG2 cells treated with MB/P85 or without treatment were exposed to ultrasound (US parameters: 1 MHz, 1.0 W/cm(2), 20 s, 20% duty cycle). Twenty-four hours later, the transfection efficiency was assessed by fluorescence microscopy and fluorescence activated cell sorting (FACS) analysis. The cell viability was evaluated by Trypan blue exclusion test. The results showed that the gene transfection efficiency in HepG2 cells under ultrasound irradiation was significantly higher than that without ultrasound irradiation. HepG2 cells in the MB or P85 group in the absence of ultrasound expressed less amount of green fluorescent protein. The expression efficiency reached (22.14 ± 3.06)% and the survival rate was as high as (55.73 ± 3.32)% in the 30% MB plus P85 group. It was concluded that MB and P85 in the presence of ultrasound can enhance gene transfection and expression.

Comparison of AAV Serotypes for Gene Delivery to Dorsal Root Ganglion Neurons

For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP(+) at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4(+) neurons, and CGRP(+) neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.

Quantitation of green fluorescent protein using a gel-based imaging method

Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.

Bone marrow-derived progenitor cells contribute to lung remodelling after myocardial infarction

Congestive heart failure (CHF) causes structural modifications of the lungs that contribute to the functional limitations of affected subjects. We hypothesized that bone marrow-derived progenitor cells could contribute to lung structural remodelling after myocardial infarction (MI). Wistar rats were irradiated and received a bone marrow transplant (BMT) from green fluorescent protein (GFP) transgenic rats, followed 5 weeks later by coronary artery ligation or sham operation. Five weeks after MI, lung immunofluorescence studies were performed and GFP expression evaluated by Western immunoblotting. After MI, rats developed lung structural remodelling characterized by myofibroblast (MF) proliferation in the alveolar septa. After BMT, some GFP+ cells were found in the lungs of sham animals. The amount of GFP+ cells in the lungs of MI rats was greatly increased with evidence of differentiation into MFs, as evaluated by co-localization correlation analysis with smooth muscle alpha-actin (P<.01). These cells were particularly abundant in the perivenular regions where they incorporated into the wall of blood vessels. There was a threefold increase in lung GFP protein expression after MI (P=.01). After MI, bone marrow-derived progenitor differentiates into lung MFs. This novel pathophysiologic process may contribute to the pulmonary manifestations of CHF and could have significant therapeutic implications.

The Hematopoietic Isoform of Cas-Hef1-Associated Signal Transducer Regulates Chemokine-Induced Inside-Out Signaling and T Cell Trafficking

Leukocyte migration and trafficking is dynamically regulated by various chemokine and adhesion molecules and is vital to the proper function of the immune system. We describe a role for the Cas and Hef-1-associated signal transducer in hematopoietic cells (Chat-H) as a critical regulator of T lymphocyte migration, by using lentivirus-mediated RNA interference (RNAi). Impaired migration of Chat-H-depleted cells coincided with defective inside-out signaling shown by diminished chemokine-induced activation of the Rap-1 GTPase and integrin-mediated adhesion. Localization of Chat-H to the plasma membrane, association with its binding partner Crk-associated substrate in lymphocytes (CasL), and Chat-H-mediated CasL serine-threonine phosphorylation were required for T cell migration. These results identify Chat-H as a critical signaling intermediate acting upstream of Rap1 to regulate chemokine-induced adhesion and migration.

Single‐Cell Analysis by Chemical Cytometry Combined with Fluorescence Microscopy

Chemical cytometry uses capillary microseparation with highly sensitive detection for detailed chemical analyses of single cells. Here, we studied microscopy and chemical cytometry in measurements of total fluorescence from single cells. An inverted fluorescence microscope was modified so that the fluorescence intensity of a sample could be automatically measured for different vertical positions of the sample with respect to the objective lens. The capillary of a custom‐made chemical cytometer was mounted in a vertical position over the microscope stage. A diluted suspension of 4T1 (mouse mammary gland tumor) cells stably expressing green fluorescent protein (GFP) was placed on a microscope slide. A single cell was positioned in the center of the field of view of the microscope. The intensity of its total GFP fluorescence was measured with the microscope detector first. The cell was then analyzed in the chemical cytometer as follows. The cell was injected into a capillary and lysed to form a homogeneous cellular lysate. The lysate was driven through the capillary by pressure and its GFP fluorescence was quantified at the output of the capillary with a laser‐induced fluorescence (LIF) detector. We demonstrated for the first time that in microscopy, the maximum fluorescence signal, as well as maximum signal to noise ratio, could be obtained when the cell was in one of two extremely out‐of‐focus positions. We proved that in chemical cytometry, the intensity of fluorescence had no memory of cell geometry and depended solely on the amount of GFP. This feature of chemical cytometry allowed us to use this technique as a reference method in the study and optimization of quantitative fluorescence microscopy. Using chemical cytometry as a reference method and 4T1 cells as a biological model, we proved that microscopy can be employed for reliable measurements of total fluorescence intensity from single cells in a heterogeneous population of non‐synchronized cells. This finding opens the opportunity for a wide use of tandem microscopy/chemical cytometry in analytical cytology.

Combinatorial Genetic Technology for the Development of New Anti-infectives

We previously developed a novel technology known as instant evolution for high-throughput analysis of mutations in Escherichia coli ribosomal RNA. To develop a genetic platform for the isolation of new classes of anti-infectives that are not susceptible to drug resistance based on the instant evolution system. Mutation libraries were constructed in the 16S rRNA gene of E coli and analyzed. In addition, the rRNA genes from a number of pathogenic bacteria were cloned and expressed in E coli. The 16S rRNA genes were incorporated into the instant-evolution system in E coli. The Department of Biological Sciences, Wayne State University, Detroit, Mich. Ribosome function was assayed by measuring the amount of green fluorescent protein produced by ribosomes containing mutant or foreign RNA in vivo. We have developed a new combinatorial genetic technology (CGT) platform that allows high-throughput in vivo isolation and analysis of rRNA mutations that might lead to drug resistance. This information is being used to develop anti-infectives that recognize the wild type and all viable mutants of the drug target. CGT also provides a novel mechanism for identifying new drug targets. Antimicrobials produced using CGT will provide new therapies for the treatment of infections caused by human pathogens that are resistant to current antibiotics. The new therapeutics will be less susceptible to de novo resistance because CGT identifies all mutations of the target that might lead to resistance during the earliest stages of the drug discovery process.

Oxygen consumption profiles of Sf-9 insect cells and their culture at low temperature to circumvent oxygen starvation

The oxygen consumption profile of Sf-9 insect cells was determined under various conditions using a 250 ml spinner flask. The specific oxygen consumption rate, q, of Sf-9 was scarcely changed with cell density and growth phases except for stationary phase. However, the q was varied with dissolved oxygen (DO) concentration and described by a Monod-form equation in the range of 20–34 °C. For uninfected Sf-9, the maximum specific oxygen consumption rate, q max, in the equation decreased with decreasing temperature, whereas the saturation constant, K S, remained constant independent of temperature up to 28 °C, at which Sf-9 cultures were usually performed. This indicated that the cellular physiology of Sf-9 was not altered in the temperature region. On a viral infection, the q max increased by 30–40% irrespective of temperature, and the K S increased more largely at higher temperature. The virus-infected Sf-9 culture was performed at 20 and 28 °C using a 2 l spinner flask with a working volume of 1 l, and DO behavior and recombinant protein production were determined. The oxygenation was done by supplying only air to mimic a large-scale culture of oxygenation-limited conditions. As a result, only the 20 °C culture was successful to keep DO concentration above the critical DO concentration (3 K S) and produce a large amount of green fluorescent protein, a recombinant model protein, but not the 28 °C culture. The DO behavior in both the 20 and 28 °C cultures was well simulated by model calculation using the oxygen consumption parameters obtained above. These results indicated that a low temperature culture was useful to circumvent oxygen starvation of virus-infected Sf-9 cells and to successfully produce recombinant proteins.

Characterization of the protein crystal growth apparatus for microgravity aboard the space station

We have conducted experiments to determine the equilibration rates of some major precipitants used in protein crystallography aboard the International Space Station (ISS). The solutions were placed in the Protein Crystallization Apparatus for Microgravity (PCAM) which mimic Cryschem sitting drop trays. The trays were placed in cylinders. These cylinders were placed inside a Single locker Thermal Enclosure System (STES), and were activated for different durations during the flight. Bumpers pressed against elastomers seal drops in a deactivated state during pre-flight and prior to transfer to the ISS. Activation occurs while in flight on the ISS by releasing the bumpers allowing the drops to be exposed to the reservoir. PCAM was flown to the ISS on STS 100, Flight 6A, on April 19, 2001. Six series of equilibration experiments were tested for each precipitant with a small amount of Green Fluorescent Protein (GFP). Cylinder 10 was never activated, 7 was activated for 40 days, 8 was activated for 20 days, 9 was activated for 10 days, 11 was activated for 4 days and 12 was activated for 2 days. Upon the return to Earth by STS 104 on July 24,2001 the samples were transferred to Marshall Space Flight Center. The samples were then brought to the lab and the volumes of each sample were measured.

Genetic Analysis of the Twin Arginine Translocator Secretion Pathway in Bacteria

The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tat-specific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wild-type TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.

A Deviant Immune Response to Viral Proteins and Transgene Product Is Generated on Subretinal Administration of Adenovirus and Adeno-associated Virus

The immune response after ocular exposure to foreign antigens varies substantially from that of a typical systemic response. Anterior chamber associated immune deviation (ACAID) has been well documented. The immune response of the subretinal space has not been studied in as much detail. Here, we characterized the immune response of the subretinal space when it encounters the antigens AdV-GFP and AAV-GFP (recombinant adenovirus or adeno-associated virus, respectively), each delivering the reporter gene encoding green fluorescent protein (GFP). Results indicate that the subretinal space possesses an immune-deviant property similar to ACAID. AdV-elicited immune responses following subretinal injections are significantly reduced compared with systemic responses elicited by intradermal injections of the same virus. Furthermore, subretinal AdV administration results in transduction of retinal pigment epithelial cells (RPE), which are the potential antigen presenting cells of the retina. This subsequently generates a population of immunosuppressive Th2-type, cytokine-secreting, splenic T cells. This response may be advantageous to the development of ocular gene therapy.

Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted much attention as a tool to study a number of biological processes. This study describes the use of GFP as a vital reporter molecule for localization and expression studies in Saccharomyces cerevisiae. Construction of GFP expression vectors which allow N- or C-terminal fusion of the gfp gene to a gene of interest allowed the generation of fusion proteins whose subcellular localization was followed by fluorescence microscopy in living yeast cells. Analysis of three unknown open reading frames obtained from the budding yeast chromosome XIV resulted in distinct staining patterns, allowing prediction of the cellular localization of these unknown proteins. Furthermore, GFP was used to construct a gene replacement cassette which, after homologous integration into the genomic locus, placed the gfp gene behind a promoter of interest. The amount of GFP produced from this promoter was then quantified in living yeast cells by flow cytometry. With this novel replacement cassette a gene of interest can be deleted and at the same time its expression level studied under various growth conditions. The experiments presented here suggest that GFP represents a convenient fluorescent marker for localization studies as well as gene expression studies in budding yeast. Systematic studies of a large number of genes should benefit from such assays.