Detection Of Green Fluorescent Protein Research Articles

Isoxaflutole treatment leads to reversible tissue bleaching and allows for more effective detection of GFP in transgenic soybean tissues

The effects of the herbicide isoxaflutole (IFT) on tissue growth and the detection of green fluorescent protein (GFP) in transgenic embryogenic soybean tissues were evaluated using image analysis. The inclusion of this “bleaching” herbicide at 3 or 10 mg l−1 in a standard soybean embryo proliferation medium resulted in a change in tissue color from green to non-pigmented over the course of a 4-wk experiment. Although the loss in pigmentation was observed in transgenic and non-transformed control tissues, tissue growth remained unaffected. GFP expression in three different transgenic soybean clones, representing low to moderate GFP expression levels, was easily detected and quantified using image analysis following culture of the tissues on an IFT-containing medium. Quantification of GFP in tissues from the same clones cultured in the absence of IFT, however, was difficult using image analysis. After transfer of transgenic embryogenic tissue from a medium containing IFT to a medium without IFT, the growth of pigment-containing tissue resumed. The bleaching effects from this herbicide appear to be reversible and make IFT and possibly other bleaching herbicides useful in the analysis of GFP expression in tissues, where interference from chlorophyll is problematic.

Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein (GFP) in Carrot ( Daucus carota L.) Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis (Southern and Western) of callus tissue (non-photosynthetic tissue) to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.

A rapid, high-throughput vaccinia virus neutralization assay for testing smallpox vaccine efficacy based on detection of green fluorescent protein

Virus neutralization remains a vital tool in assessment of vaccine efficacy for smallpox in the absence of animal smallpox models. In this regard, development of a rapid, sensitive, and high-throughput vaccinia neutralization assay has been sought for evaluating alternative smallpox vaccines, use in bridging studies, as well as understanding the effects of anti-viral immunotherapeutic regimes. The most frequently used method of measuring vaccinia virus neutralization by plaque reduction is time, labor, and material intensive, and therefore limiting in its utility for large scale, high-throughput analysis. Recent advances provide alternative methods that are less labor intensive and higher throughput but with limitations in reagents needed and ease of use. An innovative neutralization assay is described based on a modified Western Reserve vaccinia vector expressing green fluorescent protein (WR-GFP) and an adherent cell monolayer in multi-well plate format. The assay is quick, accurate, provides a large dynamic range and is well suited for large-scale vaccination studies using standard adherent cell lines.

Limitations of Green Fluorescent Protein as a Cell Lineage Marker

The enhanced green fluorescent protein (GFP) reporter has been widely adopted for tracking cell lineage. Here, we compare three transgenic mouse strains in which GFP is considered "ubiquitously expressed," with the GFP transgene under control of the chicken beta-actin (CBA) or human ubiquitin C (UBC) promoter. We compared the expression of GFP using flow cytometry, direct tissue fluorescence, and immunostaining with multiple commercially available anti-GFP antibodies. Mice of CBA-GFP strain 1Osb have strong but variegated expression of GFP in adult liver, kidney, small intestine, and blood. Mice of CBA-GFP strain Y01 have the highest proportion of GFP-positive peripheral blood cells yet limited GFP expression in liver, intestine, and kidney. UBC-GFP mice express GFP only weakly in solid organs and variably in blood. Direct fluorescent detection of GFP in formalin-fixed, paraffin-embedded tissue sections was the simplest approach, but it was useful only in high-expressing strains and potentially subject to artifact because of tissue autofluorescence. Immunofluorescence using either primary goat or primary rabbit antibodies was much more sensitive and allowed better discrimination of authentic signal from autofluorescence. Immunohistochemical staining was less sensitive than direct fluorescence or immunofluorescence and was subject to false-positive signal in the small intestine. In conclusion, there is considerable variability of expression within and between GFP transgenic strains. None of the tested strains gave truly ubiquitous GFP expression. A detailed analysis of GFP expression in one's tissues of interest must guide the choice of reporter mouse strain when GFP is used as a marker of cell lineage or donor origin. Disclosure of potential conflicts of interest is found at the end of this article.

Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung

Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h. Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone. These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression.

Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.

Detection of Green Fluorescent Protein in a Single Bacterium by Capillary Electrophoresis with Laser-Induced Fluorescence

Green fluorescence protein (GFP) is a common reporter used to monitor protein expression in single cells. However, autofluorescence from endogenous components can mask the signal from GFP, particularly at low expression levels in prokaryotes. We employ capillary electrophoresis with laser-induced fluorescence for the analysis of the expression of green fluorescent protein in a single bacterium. Capillary electrophoresis separates GFP from native cellular autofluorescent components, reducing the background signal and improving detection limits. Our system provides 100 ymol (60 copies) limits of detection for GFP. To demonstrate the performance of this instrument, we employ a model system of Deinococcus radiodurans that has been engineered to express GFP under the control of the recA promoter. We report resolution and detection of GFP and autofluorescent components in a single D. radiodurans bacterium. This paper presents the first example of expression of GFP in D. radiodurans and the first detection of GFP in a single bacterium by capillary electrophoresis.

A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs

The power and simplicity of genetically encoded fluorophores (fluorescent proteins, FPs) have drawn many molecular biologists to light microscopy. First generation FPs suffered from overlapping excitation and emission spectra, which limited their use together in pairs (Patterson et al., J Cell Sci 2001;114 (Part 5):837-838). Image acquisition and processing techniques, collectively known as linear unmixing, have been developed to separate overlapping fluorescence signals encountered in the imaging of FP pairs and also in FRET. These specialized techniques are not without their potential drawbacks, including limitations on sensitivity and time-resolution for live cell imaging, and the risk of artifact in the hands of nonspecialists. With the advent of a new generation of red-shifted FPs (Shaner et al., Nat Biotechnol 2004;22:1567-1572; Verkhusha and Lukyanov, Nat Biotechnol 2004;22:289-296) careful selection of excitation sources and emission filters obviate the need for linear unmixing when simple two channel imaging of FPs is required. Here we introduce a new configuration of the Zeiss LSM 510 laser scanning confocal microscope, optimized for live cell imaging of green fluorescent protein (GFP) together with spectral variants such as mRFP1 and mCherry using standard photo-multipliers. A 2 mW, 594 nm HeNe laser was chosen as the excitation source for the red FP. This wavelength efficiently excites the aforementioned red variants without limiting the detection range of GFP emission during simultaneous two-channel imaging. Compared to excitation of GFP and mCherry at 488 and 543 nm, excitation at 488 and 594 nm approximately doubles the sensitivity of GFP detection and eliminates bleed-through of GFP into the mCherry channel. However, sensitivity of mCherry detection is decreased by 30%, suggesting the need for red FPs having longer emission peaks. Practical advantages to the simultaneous optical separation of FPs with nonoverlapping emission spectra include simplicity, robustness, reduced risk of artifact, and increased sensitivity during live cell imaging.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector. Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured. The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMV-CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P < 0.001). Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Design and fabrication of a multilayered polymer microfluidic chip with nanofluidic interconnects via adhesive contact printing

The design and fabrication of a multilayered polymer micro-nanofluidic chip is described that consists of poly(methylmethacrylate) (PMMA) layers that contain microfluidic channels separated in the vertical direction by polycarbonate (PC) membranes that incorporate an array of nanometre diameter cylindrical pores. The materials are optically transparent to allow inspection of the fluids within the channels in the near UV and visible spectrum. The design architecture enables nanofluidic interconnections to be placed in the vertical direction between microfluidic channels. Such an architecture allows microchannel separations within the chip, as well as allowing unique operations that utilize nanocapillary interconnects: the separation of analytes based on molecular size, channel isolation, enhanced mixing, and sample concentration. Device fabrication is made possible by a transfer process of labile membranes and the development of a contact printing method for a thermally curable epoxy based adhesive. This adhesive is shown to have bond strengths that prevent leakage and delamination and channel rupture tests exceed 6 atm (0.6 MPa) under applied pressure. Channels 100 microm in width and 20 microm in depth are contact printed without the adhesive entering the microchannel. The chip is characterized in terms of resistivity measurements along the microfluidic channels, electroosmotic flow (EOF) measurements at different pH values and laser-induced-fluorescence (LIF) detection of green-fluorescent protein (GFP) plugs injected across the nanocapillary membrane and into a microfluidic channel. The results indicate that the mixed polymer micro-nanofluidic multilayer chip has electrical characteristics needed for use in microanalytical systems.

Donor hematopoietic cells from transgenic mice that express GFP are immunogenic in immunocompetent recipients

Objective: To study the immunogenicity of hematopoietic cells marked with green fluorescence protein (GFP) while avoiding the potentially confounding effects of viral gene transduction, marked cells from GFP + transgenic mice were tracked after transplantation into unconditioned immunocompetent recipients.Materials and Methods: Marrow was harvested from GFP + tran2sgenic mice that had been crossed onto a BALB/cByJ background. Unconditioned marrow transplantation involved infusion of sex-matched or sex-mismatched cells into female BALB/cByJ hosts. Engraftment and contribution to circulating nucleated blood cells were compared to recipients of donor cells that were not GFP-marked. Donor cells were detected by flow cytometry (GFP) and fluorescence in situ hybridization (FISH) for Y-chromosome sequences.Results: Donor cells from mice of the same genetic background that did not express GFP were detected for more than four-weeks in unconditioned recipients. In contrast, GFP-marked cells in the blood peaked at one-week, declined to undetectable levels by two-weeks and were not detected in the marrow at sacrifice. In sex-mismatched studies, detection of male GFP + donor cells by FISH yielded levels similar to those observed by flow cytometry, in contrast to the levels detected for many weeks in mice infused with male cells that did not express GFP. In immunocompetent recipients immunized with irradiated GFP-expressing cells, rechallenge with GFP + cells resulted in the accelerated loss of donor cells.Conclusion: Donor marrow cells from GFP + transgenic mice were lost after infusion into unconditioned immunocompetent mice and sensitization studies infer an immunologic mechanism. These results are similar to studies of virally transduced cells. Thus, infusion of cells with optimum engraftment potential could not compensate for the loss of donor cells due to immunogenicity.

Tandem screening of toxic compounds on GFP-labeled bacteria and cancer cells in microtiter plates

A 96-well fluorescence-based assay has been developed for the rapid screening of potential cytotoxic and bacteriocidal compounds. The assay is based on detection of green fluorescent protein (GFP) in HeLa human carcinoma cells as well as gram negative ( Escherichia coli) and gram positive bacteria ( Mycobacterium avium). Addition of a toxic compound to the GFP marked cells resulted in the loss of the GFP fluorescence which was readily detected by fluorometry. Thirty-nine distinct naphthoquinone derivatives were screened and several of these compounds were found to be toxic to all cell types. Apart from differences in overall toxicity, two general types of toxic compounds were detected, those that exhibited toxicity to two or all three of the cell types and those that were primarily toxic to the HeLa cells. Our results demonstrate that the parallel screening of both eukaryotic and prokaryotic cells is not only feasible and reproducible but also cost effective.

AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats

Leptin is a hormone secreted primarily by white adipocytes that regulates energy homeostasis and reproduction via CNS receptors. Koletsky ( f/ f) rats with a leptin receptor (OB-Rb) gene mutation are obese, diabetic and infertile. We employed recombinant adeno-associated viral (rAAV) vectors to transfer the human OB-Rb gene into the brains of female Koletsky rats to identify sites of leptin action in the brain. rAAV-OB-Rb was microinjected into the medial preoptic area (MPOA), the paraventricular nucleus (PVN), the ventromedial hypothalamus, the arcuate nucleus (ARC), or the dorsal vagal complex in the brainstem. Food intake and body weight were monitored bi-weekly for 55 days. Vaginal cytology was examined daily to assess estrous cyclicity. After sacrifice, uncoupling protein-1 (UCP-1) mRNA in brown adipose tissue and serum concentrations of leptin, insulin, glucose, estradiol and progesterone were measured. Expression of OB-Rb was documented by RT-PCR and site specificity of microinjection was verified by immunohistochemical detection of green fluorescent protein following a control microinjection of rAAV-GFP. OB-Rb installation in the ARC reduced food intake, however, energy expenditure, assessed by UCP-1 mRNA expression, was increased by OB-Rb installation in all sites except the PVN. When injected into the MPOA and ARC, rAAV-OB-Rb stimulated the reproductive axis as evidenced by normalization of estrous cycle length and increased luteinizing hormone releasing hormone concentrations in the hypothalamus. These studies show that long-term installation of a functional leptin receptor in the CNS is achievable using rAAV vectors and further show that leptin acts on specific sites in the brain to produce differential effects on food intake, energy expenditure and reproduction.

Paracrine and differentiation mechanisms underlying stem cell therapy for the damaged kidney

NATIVE KIDNEY ACUTE RENAL FAILURE (ARF) occurs in 2‐5% of hospitalized patients and is associated with high mortality (11). Allograft ARF occurs in 30‐50% of deceased donor kidney transplants and leads to longer hospital stays, a higher incidence of acute rejection, and reduced long-term graft survival (13). There is no specific therapy for ARF except for supportive care. A surge of evidence over the last decade supports an important role for inflammatory mediators, microvascular dysfunction, and apoptosis in the pathogenesis of the injury and extension phase of ARF (12). The mechanisms of recovery, less well understood, are felt to include a recapitulation of mechanisms originally involved in renal development. It is commonly believed that kidney progenitor cells, from either the kidney or an extrarenal source, repopulate the kidney during the recovery phase of ARF and drive the repair process (9). A popular model was that damaged/dead cells are removed and the kidney stem cells migrate to necrotic areas, differentiate, and repopulate the kidney. Indeed, injured human kidney transplants from female donors into male recipients were found to have Y chromosome staining of tubular epithelial cells, demonstrating that they were of recipient origin (5). In support of this model are studies in mice in which labeled bone marrow was tracked with -galactosidase (-gal) staining into postischemic kidney. -Gal staining was observed in 20% of the proximal tubular cells at 1 wk after ischemia, and also to some extent within 48 h of ischemia (7). Despite the focus on stem cell effects on repair, mice without functional bone marrow in that study had a worse early course of ARF, suggesting that bone marrow-derived cells had an early protective effect on the injury process. In another murine study, male-derived cells were found in female mouse kidney recipients after ischemia (8). However, recent work has shown that -gal staining to identify differentiated stem cells has considerable limitations, false positive results can result from the kidney’s endogenous -gal, and the staining validity is vulnerable to slight pH shifts (3). In a cisplatin model of ARF, administration of mesenchymal stem cells improved kidney function and structure (10). Given that Y chromosome-containing cells were found in recipient tubular epithelium of female mice and expressed specific lectin-binding proteins found in proximal tubules, transdifferentiation was concluded to be the mechanism of enhanced repair. Administration of skeletal muscle-derived stem cells that were differentiated into endothelial cells provided early protection from ischemic ARF in mice and were detected in glomeruli and peritubular capillaries using green fluorescent protein detection (1).

Long-term cardiac gene expression using a coxsackieviral vector

Efficient myocardial gene transfer in the intact adult heart is difficult using conventional transfer vectors. Since coxsackievirus B3 (CVB3) is cardiotropic, it may be possible to exploit its cardiotropic characteristics to design a vector for gene transfer to the intact heart. We generated a recombinant CVB3 cDNA by inserting a green fluorescent protein (GFP) gene immediately upstream from the VP0 capsid protein of CVB3. The infectious virus (rCVB3-GFP) was recovered from the supernatants of the transfected Cos-7 cells, and was grown in HeLa cells to titers of 10 11 pfu/ml. In the rCVB3-GFP infected HeLa cells and neonatal rat cardiac myocytes, GFP protein expression was documented by immunoblot and by fluorescent microscopy. GFP expression was maintained after five passages in HeLa cells. To test in vivo expression of GFP, we infected 8-week-old inbred female Balb/C mice with 10 6 pfu of rCVB3-GFP, intraperitoneally. GFP was present in up to 30% of cardiac myocytes over the 8 weeks post infection (p.i.) and it was co-localized with CVB3 infection. Surprisingly, in spite of detection of GFP up to at least 8 weeks after infection, there was no mortality in the mice. It is possible to express exogenous proteins in the intact heart after an intraperitoneal (i.p.) injection of recombinant coxsackievirus. The duration of expression persisted for at least 8 weeks with little immune response nor mortality. These results demonstrated that the cardiac tropism of CVB3 could be used to design vectors for efficient gene expression in the intact heart.

Enhanced function with decreased internalization of carboxy-terminus truncated CXCR4 responsible for WHIM syndrome

WHIM (warts, hypogammaglobulinemia, recurrent bacterial infection, myelokathexis) syndrome is an autosomal dominant immune deficiency with severe chronic neutropenia and marrow neutrophil apoptosis. Carboxy-termini truncating mutations in the chemokine receptor CXCR4 have been identified in WHIM patients. We created a retrovirus encoding mutated CXCR4 (truncating point mutation 1000C-->T [R334X] inherited heterozygously in several WHIM patients) in order to transducer healthy human CD34 stem cells and K562 to overexpress mutated CXCR4 and determined its effect on receptor responses to stromal-derived factor-1 (SDF1). Retrovirus vector was engineered to coexpress WHIM-associated R334X mutated CXCR4 together with green fluorescent protein (GFP). Control vectors included similar constructs with wild-type CXCR4 (WT-CXCR4) or only GFP. CD34+ cells and K562 were transduced with these vectors. Populations of 100% transduced K562 were established by sorting GFP+ cells by flow cytometry. We performed migration and calcium flux assays of transduced CD34+ cells and transduced/sorted K562. We also examined receptor recycling in response to SDF1. Healthy human CD34+ cells and/or human erythroleukemia K562 cells transduced to express mutated CXCR4, WT-CXCR4, or GFP alone demonstrated that mutated CXCR4 was associated with enhanced calcium flux and enhanced migration. There was also decreased receptor internalization and enhanced recovery of surface mutated CXCR4 in response to SDF1 compared with WT-CXCR4. We propose that decreased internalization of WHIM-associated mutated CXCR4 leads to prolongation/enhancement of signaling in response to SDF1 and that this may provide the biochemical basis for the autosomal dominant abnormalities of cell trafficking and function associated with WHIM syndrome.

Identification of an N-terminal TRPC2 splice variant which inhibits calcium influx

TRPC2 is a member of the transient receptor potential (TRP) superfamily of Ca 2+-permeable channels expressed in nonexcitable cells. TRPC2 is involved in a number of physiological processes including sensory activation of the vomeronasal organ, sustained Ca 2+ entry in sperm, and regulation of calcium influx by erythropoietin. Here, a new splice variant of TRPC2, called “Similar to mouse TRPC2” (smTRPC2), was identified consisting of 213 amino acids, largely coincident with the N-terminus of TRPC2 clone 17. This splice variant lacks all six TRPC2 transmembrane domains and the calcium pore. Expression of smTRPC2 was found in all tissues examined by RT–PCR and in primary erythroid cells by RT–PCR and Western blotting. Confocal microscopy of CHO-S cells transfected with TRPC2 clone 14 and smTRPC2 demonstrated that TRPC2 clone 14 and smTRPC2 both localize at or near the plasma membrane and in the perinuclear region. Cell surface localization of TRPC2 was confirmed with biotinylation, and was not substantially affected by smTRPC2 expression. Coassociation of TRPC2 c14 and α with smTRPC2 was confirmed by immunoprecipitation. To examine the functional significance of smTRPC2 expression, a CHO-S model was used to study its effect on calcium influx stimulated by Epo through TRPC2. Single CHO-S cells which express transfected Epo-R were identified by detection of green fluorescent protein (GFP). Cells that express transfected TRPC2 c14 or α were identified by detection of blue fluorescent protein (BFP). [Ca] i was quantitiated with Fura Red fluorescence using digital video imaging. Epo stimulated calcium influx through TRPC2 isoforms c14 and α, which was inhibited by coexpression of smTRPC2. These data demonstrate that a short splice variant of TRPC2 exists in many cell types, which associates with and modifies the activity of functional TRPC2 splice variants.

Laser Scanning Cytometry for selection of green fluorescent protein transgenic mice using small number of blood cells

A Laser Scanning Cytometry-based method was developed for identification of transgenic mice expressing green fluorescent protein (GFP) using minute amounts of peripheral blood. The difference between the autofluorescence of cells not expressing GFP and the fluorescence of GFP expressing cells after excitation with Ar-ion laser (wavelength 488 nm) and detection of emitted fluorescent light in the green channel was high enough for unambiguous identification of the GFP expressing mice. The sensitivity of this method was estimated 1:10 4 for detection of rare GFP expressing cells under the conditions used. This sensitivity should be sufficient for many studies on microchimerism. Because of the possibility for relocation of the cells, this method will be particularly useful for characterizing the cells with high GFP expression using other markers of cell phenotype or conventional morphological analysis.

Efficient Intracellular Delivery of a Protein and a Low Molecular Weight Substance via Recombinant Polyomavirus-like Particles

Efficient encapsulation of foreign molecules like proteins and low molecular weight drugs into polyoma virus-like particles (capsoids) was achieved by the development of an anchoring technique based upon the specific interaction of the inner core protein VP2 with VP1 pentamers. A stretch of 49 amino acids of VP2 served as an anchor molecule, either expressed as a fusion protein with green fluorescent protein (GFP) or covalently linked to methotrexate (MTX). The loaded capsoids showed regular morphology and stability for several months. GFP and MTX were internalized into cells in vitro, as was demonstrated by the detection of GFP and VP1 fluorescence in mouse fibroblasts and the cytostatic effect of intracellularly released MTX on leukemia T cells.

Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes

Embryonic stem cells (ES) can self-replicate and differentiate into all cell types including insulin-producing, beta-like cells and could, therefore, be used to treat diabetes mellitus. To date, results of stem cell differentiation into beta cells have been debated, largely due to difficulties in defining the identity of a beta cell. We have recently differentiated non-human primate (rhesus) embryonic stem (rES) cell lines into insulin producing, beta-like cells with the beta cell growth factor, Exendin-4 and using C-peptide as a phenotype marker. Cell development was characterized at each stage by gene and protein expression. Insulin, NKX6.1 and glucagon mRNA were expressed in stage 4 cells but not in early undifferentiated cells. We concluded that rES cells could be differentiated ex vivo to insulin producing cells. These differentiated rES cells could be used to develop a non-human primate model for evaluating cell therapy to treat diabetes. To facilitate the identification of beta-like cells and to track the cells post-transplantation, we have developed a marker gene construct: fusing the human insulin promoter (HIP) to the green fluorescent protein (GFP) gene. This construct was transfected into stage 3 rES derived cells and subsequent GFP expression was identified in C-peptide positive cells, thereby substantiating endogenous insulin production by rES derived cells. Using this GFP detection system, we will enrich our population of insulin producing rES derived cells and track these cells post-transplantation in the non-human primate model.