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PubMed2000

Characterization of organelles in the vacuolar-sorting pathway by visualization with GFP in tobacco BY-2 cells.

We have shown the localization and mobilization of modified green fluorescent proteins (GFPs) with various signals in different compartments in a vacuolar-sorting system of tobacco BY-2 cells. In contrast to the efficient secretion of GFP from the transformed cells expressing SP-GFP composed of a signal peptide and GFP, accumulation of GFP in the vacuoles was observed in the cells expressing SP-GFP fused with the C-terminal peptide of pumpkin 2S albumin. This indicated that this peptide is sufficient for vacuolar targeting. Interestingly, the fluorescence in the vacuoles disappeared sharply at 7 d after inoculation of the cells, but it appeared again after re-inoculation into a new culture medium. When SP-GFP was fused with the region, termed PV72C, including a transmembrane domain and a cytosolic tail of a vacuolar-sorting receptor PV72, GFP-PV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that reached the prevacuolar compartments was cleaved off the PV72C region to produce GFP, that arrived at the vacuoles to be diffused. These findings suggested that the vacuolar-sorting receptor might be recycled between the Golgi complex and prevacuolar compartments.

Base Sequence↗
PubMed2006

Analysis of tumor-associated stromal cells using SCID GFP transgenic mice: contribution of local and bone marrow-derived host cells.

The green fluorescence protein (GFP) from the UBI-GFP/BL6 transgenic line was bred into C57BL/6J-scid and C.B-17-scid mice for investigating host-tumor cell interactions. These mice express high levels of GFP under the control of the ubiquitin promoter in virtually all cells examined. In tumor tissue generated by implanting tumor cells in the GFP transgenic SCID mice, the tumor cells and tumor-associated murine host cells were clearly distinguished by GFP expression. A population of cells expressing the endothelial cell marker VEGFR-2/Flk-1, and the progenitor markers c-Kit and Sca-1, were incorporated into tumor tissue. The majority of the Flk-1-positive cells were hematopoietic-derived cells that coexpressed CD45. To investigate the contribution of bone marrow-derived cells to the formation of tumor vessels and stroma, tumor cells were implanted in nontransgenic SCID mice that received a bone marrow transplant from GFP-expressing SCID mice. Although GFP-positive cells were readily detected by histology in tumors taken from bone marrow transplanted animals, they were spatially isolated and lacked organization. In contrast, if tumors were implanted in nontransgenic SCID mice adjacent to a patch of transplanted GFP-expressing skin, these tumors recruited GFP-positive cells that organized into tumor vessels. The results demonstrate that hematopoietic-derived cells, including Flk-1+/CD45+ cells, readily colonized the tumor stroma but were minimally incorporated in the tumor vasculature. The majority of the tumor vessels were instead recruited from tissue adjacent to the tumor. The expression of Flk-1 on nonendothelial, tumor-associated host cells raises the possibility that VEGF antagonists, such as Avastin, could inhibit tumor growth by a mechanism involving hematopoietic-derived CD45+/Flk-1+ cells, in addition to direct suppression of endothelial cell function.

Animals↗
PubMed2001

Remote delivery of rAAV-GFP to the rat brainstem through the recurrent laryngeal nerve.

OBJECTIVE: To demonstrate that a recombinant adeno-associated viral vector (rAAV) carrying the gene for green fluorescent protein (GFP) could be delivered to the rat brainstem by remote injection into the recurrent laryngeal nerve. STUDY DESIGN/METHODS: rAAV-GFP is a serotype 2 adeno-associated vector containing the cDNA of GFP and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under the control of the CAG promoter (obtained from Matthew During, PhD, Thomas Jefferson Medical College). Five microliters or 10 microL of 1.4 x 109 particles/microL of rAAV-GFP were injected into the right recurrent laryngeal nerve of adult Sprague-Dawley rats. Rats were killed and perfused at 3 (n = 3) and 11 weeks (n = 3). Brainstems were removed and cryosectioned. Fluorescent in-situ hybridization (FISH) was performed on cryosections from animals killed at 3 weeks using a cDNA probe for woodchuck polyribosomal enzyme within the rAAV vector. In a third group (n = 2), Fluoro-Gold (Fluorochrome, Inc., Denver, CO) was injected into the right thyroarytenoid muscle for comparison of neuronal uptake distribution. These rats were killed and perfused at 3 weeks. RESULTS: The presence of GFP was noted in neurons throughout the medulla of all rat brainstems after unilateral rAAV-GFP injection at both 3 and 11 weeks. In contrast to the Fluoro-Gold, GFP was noted bilaterally and outside of the nucleus ambiguus. FISH confirmed the presence of virus within neurons expressing GFP at 3 weeks. CONCLUSIONS: Remote delivery of rAAV-GFP to the rat brainstem is possible through injection into the recurrent laryngeal nerve. This has important therapeutic implications for the future treatment of recurrent laryngeal nerve injury and neurodegenerative diseases.

Animals↗
PubMed2005

Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots.

Transgenic Arabidopsis plants were constructed to express a range of GFP-fusion proteins (36-67 kDa) under the companion cell (CC)-specific AtSUC2 promoter. These plants were used to monitor the trafficking of these GFP-fusion proteins from the CCs into the sieve elements (SEs) and their subsequent translocation within and out of the phloem. The results revealed a large size exclusion limit (SEL) (>67 kDa) for the plasmodesmata connecting SEs and CCs in the loading phloem. Membrane-anchored GFP-fusions and a GFP variant targeted to the endoplasmic reticulum (ER) remained inside the CCs and were used as 'zero trafficking' controls. In contrast, free GFP and all soluble GFP-fusions, moved from the CCs into the SEs and were subsequently translocated through the phloem. Phloem unloading and post-phloem transport of these mobile GFP-fusions were studied in root tips, where post-phloem transport occurred only for the free form of GFP. All of the other soluble GFP-fusion variants were unloaded and restricted to a narrow zone of cells immediately adjacent to the mature protophloem. It appears that this domain of cells, which has a peripheral SEL of about 27-36 kDa, allows protein exchange between protophloem SEs and surrounding cells, but restricts general access of large proteins into the root tip. The presented data provide additional information on phloem development in Arabidopsis in relation to the formation of symplasmic domains.

Arabidopsis↗
PubMed2002

Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells.

AIMS: To study the regulation of type 1 insulin like growth factor receptor (IGF-1R) tyrosine kinase activity using the fission yeast Schizosaccharomyces pombe and a green fluorescent protein (GFP) tagged, full length IGF-1R. METHODS: The beta chain of the IGF-1R (betawt) was expressed under inducible conditions in the fission yeast S. pombe. Western blot analysis with antiphosphotyrosine antibodies was used to assess the kinase activity of betawt. A GFP tagged IGF-1R (GFP-IGF-1R) was constructed to study the tyrosine kinase activity of the full length IGF-1R. The signalling capabilities of GFP-IGF-1R in response to IGF-1 stimulation were investigated in transiently transfected fibroblasts. Immunofluorescent staining for cellular phosphotyrosine content was used to assess the localisation and tyrosine kinase activity of GFP-IGF-1R. RESULTS: The betawt protein displayed functional tyrosine kinase activity in S pombe and phosphorylated endogenous yeast proteins. In response to IGF-1 stimulation, the GFP-IGF-1R became autophosphorylated and also activated the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways. Tyrosine phosphorylation and kinase activity of the GFP-IGF-1R could be visualised by immunofluorescence with antiphosphotyrosine antibodies. Coexpression of a mammalian tyrosine phosphatase PTP1B with betawt completely inhibited this tyrosine kinase activity in yeast and also reduced the tyrosine phosphorylation in COS cells transfected with the GFP-IGF-1R. CONCLUSIONS: Schizosaccharomyces pombe can be used to analyse the tyrosine kinase activity of the IGF-1R beta chain and its regulation by tyrosine phosphatases. In addition, the regulation of IGF-1R tyrosine kinase activity can be studied using a GFP tagged IGF-1R. Using both of these methods, IGF-1R kinase activity was shown to be inhibited by the protein tyrosine phosphatase, PTP1B.

Animals↗
PubMed2002

Analysis of Chlamydomonas SF-assemblin by GFP tagging and expression of antisense constructs.

Striated fiber assemblin (SF-assemblin or SFA) is the major component of the striated microtubule-associated fibers (SMAFs) in the flagellar basal apparatus of green flagellates. We generated nuclear transformants of Chlamydomonas expressing green fluorescent protein (GFP) fused to the C-terminus of SFA. SFA-GFP assembled into striated fibers that exceeded those of wild-type cells in size by several fold. At elevated temperatures (>or=32 degrees C) SFA-GFP was mostly soluble and heat shock depolymerized the SMAFs. C-terminal deletions of 18 or only six residues disturbed the ability of SFA-GFP to polymerize, indicating an important role of the C-terminal domain for fiber formation. The exchange of the penultimate Ser275 with alanine made SFA-GFP highly insoluble, causing aberrant fiber formation and conferring heat stability to the fibers. By contrast, a replacement with glutamic acid increased the solubilty of the molecule, indicating that phosphorylation on Ser275 might control solubility of SFA. In vivo observation of GFP fluorescence showed that SFA-GFP fibers were disassembled during mitosis. In cells overexpressing full-length or truncated SFA-GFP, the amount of wild-type protein was reduced. Elevated temperatures dissolved SFA-GFP fibers and induced the synthesis of SFA, suggesting that cells control both the amount of soluble and polymeric SFA. By expressing constructs consisting of cDNA and genomic DNA for parts of SFA in antiparallel configuration, the amount of SFA was severely reduced. In these strains we observed defects in flagellar assembly, indicating an important role for noncontractile striated roots in the flagella apparatus.

Amino Acid Sequence↗
PubMed1999

GFP chimeras of E-MAP-115 (ensconsin) domains mimic behavior of the endogenous protein in vitro and in vivo.

E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters of ensconsin distribution and dynamics; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells.

Animals↗
PubMed2005

Histological analysis of GFP expression in murine bone.

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a beta-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.

Animals↗
PubMed2005

Trafficking pathways of Cx49-GFP in living mammalian cells.

In the present study we examined the trafficking pathways of connexin49 (Cx49) fused to green fluorescent protein (GFP) in polar and non-polar cell lines. The Cx49 gene was isolated from ovine lens by RT-PCR. Cx49 cDNA was fused to GFP and the hybrid cDNA was transfected into several cell lines. After transfection of Cx49-GFP cDNA into HeLa cells, it was shown using the double whole-cell patch-clamp technique that the expressed fusion protein was still able to form conducting gap junction channels. Synthesis, assembly, and turnover of the Cx49-GFP hybrid protein were investigated using a pulse-chase protocol. A major 78-kDa protein band corresponding to Cx49-GFP could be detected with a turnover of 16-20 h and a half-life time of 10 h. The trafficking pathways of Cx49-GFP were monitored by confocal laser microscopy. Fusion proteins were localized in subcellular compartments, including the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment, the Golgi apparatus, and the trans-Golgi network, as well as vesicles traveling towards the plasma membrane. Time-dependent sequential localization of Cx49-GFP in the ER and then the Golgi apparatus supports the notion of a slow turnover of Cx49-GFP compared to other connexins analyzed so far. Gap junction plaques resembling the usual punctuate distribution pattern could be demonstrated for COS-1 and MDCK cells. Basolateral distribution of Cx49-GFP was observed in polar MDCK cells, indicating specific sorting behavior of Cx49 in polarized cells. Together, this report describes the first characterization of biosynthesis and trafficking of lens Cx49.

Animals↗
PubMed2001

The Expression of Adenoviral Mediated gfp Reporter Gene in Tumor Cells Induced and Regulated by Irradiation.

In order to establish a radiation inducible gene expression system for cancer gene therapy, the promoter sequence of radiation-inducible Egr-1 gene was amplified from genomic DNA of BALB/c mouse with PCR method, and linked to gfp reporter gene. Then the pEgr-gfp expression cassette was subcloned into an adenoviral shuttle plasmid to generate recombinant adenovirus of AdEgr-GFP by using a novel, high efficient method of homologous recombination in bacteria. After infection with AdEgr-GFP, MM45T.Li tumor cells were exposed to different doses of gamma-irradiation from 0 Gy to 15 Gy in vitro. The percentage of GFP expression positive cells increased greatly in a dose-dependent manner as detected by FACS and Western blot analysis. For in vivo study, AdEgr-GFP were injected intratumorally, and tumor site received different doses of local gamma-irradiation 48 h after injection, and after 8 h the tumor samples were biopsed for investigating the GFP expression. Tumor tissue image analysis revealed that gamma-irradiation could markedly increase GFP expression in a dose-dependent manner as compared with that of non-irradiated control group. Our results indicate that the irradiation can effectively control adenoviral-mediated GFP expression in tumor cells via Egr-1 promoter, and these data laid basis for further gene radiotherapy study.

Journal Article↗
PubMed2003

Chromosomal location and expression of green fluorescent protein (gfp) gene in microspore derived transgenic barley (Hordeum vulgare L.).

Four doubled haploid barley lines (A, C, D, E) derived from gfp (green fluorescent protein) transformation and selection following particle bombardment of microspores were studied for gene expression pattern and the location of genome inserts. The integration sites were detected by fluorescence in situ hybridization (FISH) using the gfp plasmid DNA as a probe. Plants from events A, C, D and E all have a single insert site on chromosome 7L(5HL) at different locations while line E has a second insert site on chromosome 5S(7HS). All original transgenic plants were hemizygous for the transgenes and segregated in the T1 and T2 generations. Although line D had no GFP expression, FISH and PCR could detect gfp gene on its chromosome in transformed plants. Expression levels of GFP varied with lines and tissues examined. Plants from line C showed good expression in pollen and an intermediate level in root tips. Plants from A have intermediate expression of GFP in the pollen and light expression in the root tips. Line E showed strong expression in the root-tips and an intermediate level of GFP in the pollen. Lines A and C segregated as a single Mendelian locus while E segregated in a duplicate loci ratio (15:1) on seedling root tips but had low expression frequency in the pollen. PCR results were consistent with GFP expression on root tips in the three segregating lines. The expression of GFP for lines D and E was abnormal and may be related to the physical location of the transgene or the gene construct used.

Chromosome Mapping↗
PubMedDate not supplied

Quantification of VP22-GFP spread by direct fluorescence in 15 commonly used cell lines.

BACKGROUND: The intercellular transport property of VP22 chimeric proteins offers the opportunity for the improvement of gene therapy delivery systems. Since enhanced therapeutic effects of transduced genes already have been exemplified for chimeric proteins VP22-p53 and VP22-tk, we were interested in examining whether spread of VP22 chimeric proteins is a general biological phenomenon not restricted to distinct tissues or species. METHODS: To study intercellular spread of VP22-GFP fusion proteins, 15 different mammalian cell lines were transfected with 200-2000 ng of VP22-GFP or GFP expression plasmids. Expression of VP22-GFP or GFP was monitored by fluorescence microscopy of live GFP fluorescence and direct FACS analysis. For selected cell lines, antibody detection of VP22-GFP spread was analysed by confocal microscopy as a control. RESULTS: Spread of VP22-GFP fusion proteins was detected in all 15 cell lines tested, and quantified by FACS analysis. Experimental conditions were found to be critical in the investigation of VP22-mediated intercellular spread. CONCLUSION: Results of our study indicate that spread of VP22 chimeric proteins is a general biological phenomenon not restricted to distinct tissues or species. Therefore, further evidence is provided that VP22-enhanced gene therapeutic effects may be obtained irrespective of the target organ/tissue to be addressed.

Animals↗
PubMed2002

Simultaneous detection of bacteria expressing GFP and DsRed genes with a flow cytometer.

BACKGROUND: In this study, Escherichia coli cells producing red fluorescent protein of Discosoma sp. (drFP583 DsRed) were investigated with flow cytometry by using 488 nm excitation. We also studied whether green fluorescent protein (GFP) and drFP583 could be detected simultaneously from a single bacterial cell. METHODS: Plasmids pDsRed and pEGFP were used for the production of drFP583 and enhanced GFP, respectively, in E. coli MC1061 cells. To produce enhanced GFP and drFP583 simultaneously, plasmids pG9R and pG19R were constructed. These encode tandem fusions of enhanced GFP and drFP583 to ensure similar production levels for both proteins. RESULTS: Bacteria producing enhanced GFP and drFP583 were found to be brightly green and red fluorescent, respectively. Production of enhanced GFP and drFP583 fusion proteins resulted in bacteria that emitted both green and red fluorescence, which was detected easily by a flow cytometer using single laser excitation. Previously reported tetramerization of drFP583 did not restrict its use as a reporter gene, although it maturated significantly slower than enhanced GFP. CONCLUSIONS: The results show that enhanced GFP and drFP583 proteins can be detected simultaneously from single bacteria with a standard flow cytometer with simple optical configuration.

Cells, Cultured↗
PubMed2003

Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter.

A 1,934-bp muscle-specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5' promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis-elements in the promoter and that a minimal -77-bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2-gfp constructs. GFP expression in the 1,934-bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871-bp promoter. Our observation indicates that there is no distinct cis-element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP-dependent protein kinase A was injected into mylz2-gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP-expressing fast muscle. The mylz2-gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered.

Animals↗
PubMed2005

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos.

Animals↗
PubMed2001

Characterization of GFP-MAL expression and incorporation in rafts.

During myelin formation, membrane-associated proteins have to be sorted and transported in specified membrane regions such as compact and non-compact myelin membranes. One protein that may be involved in such a process is the Myelin and Lymphocyte protein MAL (VIP17/ MVP17). MAL was identified as a novel myelin membrane component expressed by oligodendrocytes and Schwann cells. Since MAL has been shown to be important in the apical sorting machinery of polarized cells, we have started to investigate the possible functional role of MAL in sorting myelin membrane-associated molecules. In this study, we have generated cDNA constructs with green fluorescent protein (GFP) either at the N- or C-terminus of MAL. Transfection experiments showed that GFP-MAL expression resembles that of normal MAL, whereas the MAL-GFP fusion construct was not properly transported within the cell. Furthermore, we could demonstrate that GFP-MAL is enriched in detergent insoluble glycolipid-enriched microdomains as already seen for untagged MAL. As a prerequisite for the generation of transgenic mice expressing GFP-MAL under the control of its own regulatory elements, we have generated a cDNA construct with an 8-kb MAL promotor fragment fused to GFP-MAL. Transfection experiments of the Oli-neu oligodendrocyte cell line showed that GFP-MAL was expressed, but only in cells, which were stimulated for differentiation with cAMP. In summary, the results confirm that the fusion protein GFP-MAL is incorporated into detergent-insoluble complexes and the 8-kb MAL promotor fragment is sufficient to be activated in oligodendrocytes.

Animals↗
PubMed2003

Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants.

The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.

Enzyme-Linked Immunosorbent Assay↗
PubMed2001

A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.

We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.

Animals↗