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PubMed2003

Effects of additional sequences directly downstream from the AUG on the expression of GFP gene.

We have studied the expression of the green fluorescent protein (GFP) gene to gain more understanding of the effects of additional nucleotide triplets (codons) downstream from the initiation codon on the translation of the GFP mRNA in CHO and Cos1 cells. A leader sequence of six consecutive identical codons (GUG, CUC, AGU or UCA) was introduced into a humanized GFP (hm gfp) gene downstream from the AUG to produce four GFP gene variants. Northern blot and RT-PCR analysis indicated that mRNA transcription from the GFP gene was not significantly affected by any of the additional sequences. However, immunoblotting and FACS analysis revealed that AGU and UCA GFP variants produced GFP at a mean level per cell 3.5-fold higher than the other two GFP variants and the hm gfp gene. [35S]-Methionine labeling and immunoprecipitation demonstrate that GFP synthesis was very active in UCA variant transfected-cells, but not in GUG variant and hm gfp transfected-cells. Moreover, proteasome inhibitor MG-132 treatment indicated that the GFPs encoded by each of the GFP variants and the hm gfp were equally stable, and this together with the comparable mRNA levels observed for each construct suggested that the different steady-state GFP concentrations observed reflected different translation efficiencies of the various GFP genes. In addition, the CUC GFP variant, when transiently transfected into CHO or COS-1 cells, did not produce any GFP expressing cells (fully green cells), and the GUG variant produced GFP expressing cells less than 10%, while AGU and UCA GFP variants up to 30-35% in a time course study from 8 to 36 h posttransfection. Analysis of the potential secondary structure of the GFP variant mRNAs especially in the translation initiation region suggested that the secondary structure of the GFP mRNAs was unlikely to explain the different translation efficiencies of the GFP variants. The present findings indicate that a change of the initiation context of the GFP gene by addition of extra coding sequence can alter the translation efficiency of GFP mRNA, providing a means of more efficient expression of GFP in eukaryotic cells.

Animals↗

PubMed2001

The HDV large-delta antigen fused with GFP remains functional and provides for studying its dynamic distribution.

Hepatitis D virus (HDV) requires the isoprenylated large delta antigen (LDAg) for interaction with hepatitis B surface antigen (HBsAg) to allow packaging and secretion out of the host cell. Phosphorylated LDAg has been found but, as yet, neither localization of LDAg within the nucleus nor any other function has been correlated with modification. In this study, we transfected HuH-7 or HeLa cells with plasmids encoding various lengths of LDAg [designated GFP-LD and GFP-LD(31-214) for full length and a deletion, respectively] or non-isoprenylated mutants of these [designated GFP-LDM and GFP-LD(31-214)M] fused to the green fluorescent protein (GFP). These fusion proteins were then characterized and it was found that: (i) the addition of the GFP did not interfere with the functioning of the full-length or N-terminally deleted LDAgs when interacting with HBsAg for secretion; (ii) the HDV small antigen (SDAg) together with the GFP-LD, but not the GFP-LD(31-214), could be cosecreted by HBsAg; and (iii) the GFP-LD, but not the GFP-LD(31-214), exerted a dominant-negative role on HDV genome replication. Analyses of transiently transfected cells and postmitotic permanent cells revealed the sequential appearance of GFP-LD in the nucleoplasm, then in the nucleolus, and finally in nuclear speckles (NS). Isoprenylation of LDAg seems to be important for targeting to and accumulating in the NS, which was evident from the dynamic and static localization of the non-isoprenylation mutant (GFP-LDM) and the distribution of wild-type (GFP-LD) when treated with an isoprenylation inhibitor, lovastatin, for more than 48 h. Permanently expressing GFP-LD cells allowed us to show the dynamic redistribution of dephosphorylated GFP-LD from the nucleolus to the SC-35 containing NS in the presence of dichlororibofuranosyl benzimidazole (DRB) and then the translocation back of the GFP-LD to the nucleolus within 2 h after removal of DRB. Our studies thus suggest that the various versions of the GFP-LD fusion protein, having the same function as their nonfusion counterparts, can be a powerful tool for the study of the dynamic localization of LDAg when correlated with the functional modification of this protein.

Cell Nucleolus↗

PubMed2006

In vivo and in vitro protein solubility assays using split GFP.

The rapid assessment of protein solubility is essential for evaluating expressed proteins and protein variants for use as reagents for downstream studies. Solubility screens based on antibody blots are complex and have limited screening capacity. Protein solubility screens using split beta-galactosidase in vivo and in vitro can perturb protein folding. Split GFP used for monitoring protein interactions folds poorly, and to overcome this limitation, we recently developed a protein-tagging system based on self-complementing split GFP derived from an exceptionally well folded variant of GFP termed 'superfolder GFP'. Here we present the step-by-step procedure of the solubility assay using split GFP. A 15-amino-acid GFP fragment, GFP 11, is fused to a test protein. The GFP 1-10 detector fragment is expressed separately. These fragments associate spontaneously to form fluorescent GFP. The fragments are soluble, and the GFP 11 tag has minimal effect on protein solubility and folding. We describe high-throughput protein solubility screens amenable both for in vivo and in vitro formats. The split-GFP system is composed of two vectors used in the same strain: pTET GFP 11 and pET GFP 1-10 (Fig. 1 and Supplementary Note online). The gene encoding the protein of interest is cloned into the pTET GFP 11 vector (resulting in an N-terminal fusion) and transformed into Escherichia coli BL21 (DE3) cells containing the pET GFP 1-10 plasmid. We also describe how this system can be used for selecting soluble proteins from a library of variants (Box 1). The large screening power of the in vivo assay combined with the high accuracy of the in vitro assay point to the efficiency of this two-step split-GFP tool for identifying soluble clones suitable for purification and downstream applications.

Base Sequence↗

PubMed2003

Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse.

Gamma-aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse. The distribution pattern of GFP-positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67-negative cells nor any apparent lack of GFP expression in GAD67-positive neurons in the two GAD67-GFP knock-in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock-in mouse brain. Moreover, we used the knock-in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was approximately 19.5%. All the CR-, PV-, and SS-positive cells appeared positive for GFP. The CR-, PV, and SS-positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR-, PV-, and SS-positive cells among GFP-positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP-positive cells, many medium-sized spherical somata emitting intense GFP fluorescence were observed in layer I.

Animals↗

PubMed2004

Neural stem and progenitor cells in nestin-GFP transgenic mice.

Neural stem cells generate a wide spectrum of cell types in developing and adult nervous systems. These cells are marked by expression of the intermediate filament nestin. We used the regulatory elements of the nestin gene to generate transgenic mice in which neural stem cells of the embryonic and adult brain are marked by the expression of green fluorescent protein (GFP). We used these animals as a reporter line for studying neural stem and progenitor cells in the developing and adult nervous systems. In these nestin-GFP animals, we found that GFP-positive cells reflect the distribution of nestin-positive cells and accurately mark the neurogenic areas of the adult brain. Nestin-GFP cells can be isolated with high purity by using fluorescent-activated cell sorting and can generate multipotential neurospheres. In the adult brain, nestin-GFP cells are approximately 1,400-fold more efficient in generating neurospheres than are GFP-negative cells and, despite their small number, give rise to 70 times more neurospheres than does the GFP-negative population. We characterized the expression of a panel of differentiation markers in GFP-positive cells in the nestin-GFP transgenics and found that these cells can be divided into two groups based on the strength of their GFP signal: GFP-bright cells express glial fibrillary acidic protein (GFAP) but not betaIII-tubulin, whereas GFP-dim cells express betaIII-tubulin but not GFAP. These two classes of cells represent distinct classes of neuronal precursors in the adult mammalian brain, and may reflect different stages of neuronal differentiation. We also found unusual features of nestin-GFP-positive cells in the subgranular cell layer of the dentate gyrus. Together, our results indicate that GFP-positive cells in our transgenic animals accurately represent neural stem and progenitor cells and suggest that these nestin-GFP-expressing cells encompass the majority of the neural stem cells in the adult brain.

Animals↗

PubMed1998

Functional characterization and visualization of a GABAA receptor-GFP chimera expressed in Xenopus oocytes.

The GABAA receptor is a ligand-gated chloride channel belonging to the superfamily of ligand-gated ion channels of which the nicotinic acetylcholine (nACh) receptor is prototypic. In the central nervous system the GABAA receptor mediates fast neuronal inhibition. To facilitate the study of this receptor, a GABAA receptor-green fluorescent protein (GABAAR-GFP) chimera was constructed by fusing green fluorescent protein (GFP) to the C-terminus region of the GABAA receptor alpha1 subunit. When expressed in Xenopus oocytes, this chimera responded in a manner indistinguishable from the wild-type GABAA receptor with respect to agonist potency, receptor desensitization, allosteric modulation, rectification, and ion selectivity of the channel. The addition of GFP to the GABAA receptor alpha1 subunit did not appear to alter the assembly or efficiency of expression of the GABAA receptor complex. The GABAAR-GFP chimera generated a strong fluorescent signal that was restricted to the animal pole of the oocyte plasma membrane. This signal was readily detectable using either epifluorescence or laser confocal microscopy. To confirm the extracellular location of the GFP portion of the chimera, non-permeabilized oocytes were immunolabeled with an anti-GFP antibody. Fluorescence microscopy showed that GFP was located extracellularly since it was accessible to the GFP antibody. These results confirm the predicted extracellular location of the C-terminus of the GABAA receptor alpha1 subunit and also demonstrate that GFP retains its fluorescent property when expressed extracellularly. The usefulness of the GABAAR-GFP chimera in receptor trafficking was investigated using non-hydrolyzable GTP analogues since GTP binding proteins participate in protein transport in oocytes. Microinjections of GTP-gamma-S but not GDP-beta-S reduced both GABA-gated chloride currents and cell surface GFP fluorescence in oocytes expressing the GABAAR-GFP chimera indicating that the chimera undergoes internalization upon stimulation of oocyte GTP-binding proteins. The results of the present study show that the GABAAR-GFP chimera is functionally similar to the wild-type GABAA receptor and can be used to study receptor trafficking in living cells. This is the first demonstration of a ligand-gated ion channel-GFP chimera for an ion channel belonging to this superfamily and also is the first example of the fusion of GFP to an extracellular domain of an integral membrane protein.

Anesthetics↗

PubMed2004

[The inhibitory effects on hepatitis B virus replication by stable expression of DN mutants of hepatitis B virus X gene pRev X-GFP].

OBJECTIVE: Persistent replication of hepatitis B virus (HBV) is one of the major obstacles in HBV infection treatment. Reduction or clearance of HBV propagation would be one of the aims of HBV therapy. The drugs approved in clinical used such as nucleotide analogs or interferon, were limited effects on HBV replication. The newly developing gene therapy method, dominant negative mutants, were be used as new promising HBV therapy strategy, and a dominant negative mutant of HBVX gene pRev X-GFP which we have reported in our previous study has some effects both on HBV replication and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG2 2.2.15 cells without transfection pRev X-GFP in the experiment. To make sure the effects of dominant negative mutant of pRev X-GFP, we established a HBX DN stable express cell clone, and evaluated the effects of HBX dominant negative mutant on HBV replication. METHODS: The X gene mutant, in which a specific point mutation of 3'-end ATG to AAG and fused with human green fluorescence protein (GFP) were cloned into pRev TRE vector, assigned to pRev HBX-GFP dominant mutant (pRev X-GFP). And the plasmid contains the wild type X gene or GFP gene was cloned into the same vector to construct pRev Xwt, pRev GFP constructs. All the constructs then transfected into HepG2 2.2.15 cells by liposome. After 7 days resistance selection of hygromycin (300 microg/ml), and cell clones which stable expression HBX-GFP, HBXwt, GFP were obtained. After reseeding of 106 cells of each clones in 12 wells with a 12 well cell plate and another 12 wells 2.2.15 cell were serve as blank control. The cells and media were harvested after cultured in DMEM with 10% FBS for 3 days. HBV-related DNA was assayed by dot blot and Southern blot. RESULTS: The 100% expression of pRev HBX-GFP, GFP and wild type X constructs were obtained. The stable expressed HBX-GFP can significantly reduce HBV DNA level both in cell media and cells by dot blot and Southern blot analysis, but not for pRev Xwt and pRev GFP. CONCLUSION: The dominant negative mutant pRev HBX-GFP can significantly inhibit the HBV gene expression. It also suggested that X gene might be one of promising target for HBV gene therapy.

Carcinoma, Hepatocellular↗

PubMed2005

C-terminal region of Bfl-1 induces cell death that accompanies caspase activation when fused with GFP.

Previously, we reported that anti-apoptotic Bfl-1 is converted to a pro-apoptotic protein following fusion at its N-terminus with green fluorescent protein (GFP) (GFP-Bfl-1). In this study, we performed a Bfl-1 deletion study in order to elucidate the underlying mechanism of GFP-Bfl-1-induced cell death. We found that the Bcl-2 homology (BH) domains in Bfl-1 are dispensable with respect to cell death and that GFP fusion with the 29 amino acids of the C-terminal region of Bfl-1 (GFP-BC) is sufficient to induce cell death. Moreover, when BC was fused with other tagging partners like GST or MBP, little cell death was observed, implying that the GFP region is as important as the BC region for GFP-BC-induced cell death. Further deletion analysis defined a region of GFP as a determinant of GFP-BC-induced cell death. Confocal microscopic analysis showed that GFP-chimeras containing the BC region of Bfl-1 are located mainly in mitochondria. The GFP-BC-induced cell death accompanied cellular caspase activation, and treatment with the pan-caspase inhibitor, Boc-D-FMK, partially inhibited GFP-BC-induced cell death. However, the over-expression of anti-apoptotic molecules, such as Bcl-x(L) and CrmA, did not block GFP-BC-induced cell death. In summary, GFP-BC induces cell death with caspase activation through mitochondria dependent process.

Apoptosis↗

PubMed2002

Functional expression and cellular localization of cercosporin-resistance proteins fused with the GFP in Cercospora nicotianae.

The Cercospora nicotianae pdx1 and crg1 genes were previously identified as genes required for resistance to the singlet oxygen ((1)O(2))-generating toxin cercosporin. The pdx1 gene has subsequently been shown to be required for pyridoxine biosynthesis, but both the precise biochemical function of the PDX1 protein and the function of the CRG1 protein remain undefined, as both sequences lack defined enzymatic domains or cofactor-binding sites. The gfp gene encoding green fluorescent protein was translationally fused with pdx1 and crg1. Transformation of these constructs into strains mutant in these respective genes resulted in green-fluorescent transformants complemented for the mutant phenotype. Microscopic studies revealed that in transformants transformed with gfp alone, fluorescence was distributed evenly throughout the cytoplasm and excluded from the vacuoles. Expression of PDX1::GFP either under the constitutive Aspergillus nidulans gpdA promoter or its own native promoter was visualized as distinct fluorescent circular structures in the cytoplasm, suggesting that PDX1::GFP was probably localized in the intracellular vesicles. Expression of CRG1 fused with GFP at either its N- or C-terminus resulted in low green fluorescence, compared with that of GFP alone or PDX1::GFP. The green fluorescence of either of the CRG1::GFP fusion proteins was barely observable in transformants and was generally seen as a few scattered regions of fluorescence in the hyphae. Southern blot analysis indicated multiple copies of the constructs were integrated into the fungal genome. Northern analysis revealed that pdx1:: gfp and crg1:: gfp were each expressed as an intact transcriptional unit. Cell fractionation followed by immunoblotting against a GFP antibody showed that GFP alone and PDX1::GFP were detected exclusively in the cytoplasmic fraction. The two CRG1::GFP proteins were barely detected in the cytoplasmic fraction and not at all from the membrane fraction, a result inconsistent with microscopic observation and computer sequence analysis, which suggests that CRG1 is a membrane protein.

Ascomycota↗

PubMed2004

Novel adenoviral vectors coding for GFP-tagged wtCFTR and deltaF508-CFTR: characterization of expression and electrophysiological properties in A549 cells.

E1/E3-deleted adenoviral vectors expressing an N-terminal green fluorescent protein (GFP) reporter gene fused to either wtCFTR (H5.040CMVEGFP-wtCFTR) or deltaF508-CFTR (H5.040CMVEGFP-deltaF508CFTR) were generated. To characterize the expression and activity, A549 cells were infected with vectors expressing GFP-tagged and non-tagged forms of CFTR and deltaF508CFTR. CFTR activity was assayed in cell-attached and excised patches. For H5.040CMVEGFP-wtCFTR, forskolin-dependent outward current was observed in cell-attached patches from 56 of 67 GFP-positive cells. Single-channel conductances, open probability, mean open and mean closed time values for GFP-CFTR and CFTR were not significantly different. After excision, GFP-CFTR activity required ATP and exhibited a linear I-V relationship. For H5.040CMVEGFP-deltaF508CFTR, media were supplemented with 5 mM butyrate 16 h after infection. Forskolin-dependent outward current was observed in cell-attached patches from 21 of 30 butyrate-treated GFP-positive cells and 0 of 8 GFP-positive cells without butyrate. Single-channel conductances, open probability, mean open and mean closed time values for GFP-deltaF508CFTR and deltaF508CFTR were not significantly different. However, the increase in open probability with genistein was significantly smaller for GFP-deltaF508CFTR than for deltaF508CFTR. In excised patches, GFP-deltaF508CFTR activity required ATP and exhibited a linear I-V relationship. Despite the consistent detection of GFP-CFTR and GFP-deltaF508CFTR channels in the plasma membrane by patch clamping, GFP fluorescence was observed only in intracellular regions and was not altered by butyrate. The data show that high levels of functional GFP-tagged CFTR channels can be expressed with these adenoviral vector constructs.

Adenocarcinoma↗

PubMed2000

Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames.

As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins. Ynl080c-GFP and Ynl081c-GFP fluorescence was visible in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C-terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5'-GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP-Ynl020c and GFP-Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases.

Gene Deletion↗

PubMed2006

By-stander activation in autoimmune thyroiditis: studies on experimental autoimmune thyroiditis in the GFP+ fluorescent mouse.

We have taken advantage of GFP+ fluorescent protein (GFP) tagged lymphocytes to examine by-stander activity in experimental autoimmune thyroiditis in the mouse. To generate GFP-positive EAT-susceptible CBA/J mice (H-2k) (GFP-CBA/J mice), we backcrossed CBA/J (H-2k) with heterozygous GFP+ transgenic mice (C57Bl/6; H-2b). I-Ak and GFP expression on peripheral lymphocytes was used to select the resulting progeny up to the N7 generation. Mixed lymphocyte reactions using spleen cells from N7 GFP-CBA/J mice showed negative responses to spleen cells from CBA/J confirming the inbreeding and with marked reactivity to cells from C57BL/6. Immunization with human thyroglobulin (hTg) in GFP-CBA/J mice induced thyroiditis in 50% of the animals and high titers of Tg antibodies in all the animals. In addition, priming of GFP+ spleen cells in vitro with hTg induced a marked proliferative response (mean stimulation index = 24.7), These proliferating spleen cells were then transferred to CBA/J recipients. Fourteen days after transferring 30 x 10(6) Tg-primed GFP+ spleen cells into irradiated (500 rad) normal syngeneic hosts, a GFP+ lymphocytic infiltration was seen within their thyroid glands along with a GFP- lymphocytic infiltration arising from the host. This suggested that the hTg-specific transferred cells had initiated by-stander activation of naive host lymphocytes. This model of bystander cell detection confirmed that such an effect occurs in EAT and adds weight to the importance of this phenomenon in the initiation of autoimmune thyroid disease.

Adoptive Transfer↗

PubMed2004

Stem/progenitor cells in the postnatal inner ear of the GFP-nestin transgenic mouse.

Nestin promoter-GFP (green fluorescent protein) transgenic mice were used to determine the presence of stem/progenitor cells in the mouse inner ear. We examined the inner ear of mice at the following postnatal days (P): P0, P4, P5, P15 and P60. Hair cells stereocilia were identified with the use of the histochemical marker phalloidin. Whole endorgans or cryosections were analyzed under epi-fluorescent or confocal microscopy. From P0 to P5, GFP expressing cells were found in the vestibular sensory epithelia of the macula utricle, but not in the crista ampullaris. Cells within the stroma (tissue underneath the sensory epithelia), utricle, and crista were also GFP-positive. Satellite cells in the vestibular ganglia were GFP-positive, while vestibular ganglia neurons were not. In the organ of Corti, GFP signal was found in inner border and inner phalangeal cells that surround the inner hair cells (GFP-negative), Dieters cells and cells in the great epithelial ridge. Outer hair cells were mildly positive for GFP. Satellite cells in the spiral ganglia were GFP-positive, while spiral ganglia neurons were not. Similar GFP expression was found in the vestibule and cochlea of animals at P15, however, outer hair cells showed no GFP expression. The inner ear of P60 animals contained moderate GFP expression in the stroma of the crista ampullaris and utricle, but not within the sensory epithelia. In the organ of Corti, moderate GFP expression was found in a few Deiters cells. The present data indicates that the expression of nestin in the mouse inner ear is developmentally regulated; yet in the adult inner ear there are some nestin expressing cells, suggesting an intrinsic repair potential, although to a more limited extent than during early post-natal life.

Age Factors↗

PubMed1997

Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion.

The green fluorescent protein (GFP) was used as a noninvasive probe to quantify the rheological properties of cell cytoplasm. GFP mutant S65T was purified from recombinant bacteria for solution studies, and expressed in CHO cell cytoplasm. GFP-S65T was brightly fluorescent in solution (lambda ex 492 nm, lambda em 509 nm) with a lifetime of 2.9 ns and a rotational correlation time (tc) of 20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 +/- 2 ms (5-micron diameter spot), giving a diffusion coefficient of 8.7 x 10(-7) cm2/s. The t1/2 was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein. GFP photobleaching efficiency was not affected by solution O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), was independent of spot diameter, and was unaffected by O2 or quenchers. In cell cytoplasm, time-resolved microfluorimetry indicated a GFP lifetime of 2.6 ns and a tc of 36 +/- 3 ns, giving a relative viscosity (cytoplasm versus water) of 1.5. Photobleaching recovery of GFP in cytoplasm was 82 +/- 2% complete with a t1/2 of 83 +/- 6 ms, giving a relative viscosity of 3.2. GFP translational diffusion increased 4.7-fold as cells swelled from a relative volume of 0.5 to 2. Taken together with measurements of GFP translation and rotation in aqueous dextran solutions, the data in cytoplasm support the view that the primary barrier to GFP diffusion is collisional interactions between GFP and macromolecular solutes.

Animals↗

PubMed2005

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+ transgenic 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.

Animals↗

PubMed2003

Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells.

BACKGROUND: Plant phloem consists of an interdependent cell pair, the sieve element/companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants. RESULTS: In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs. CONCLUSION: A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

3' Untranslated Regions↗

PubMed2001

[Impact of AFP promoter on the expressions of GFP in tumor cells].

OBJECTIVE: To study the impact of AFP 5'flanking promoter (enhancer) on the expression of GFP in hepatocarcinoma cell. METHODS: Green Fluorescent Protein (GFP) reporter gene expression plasmid pcDNA3-GFP-AFP-w under the direction of AFP 5' flanking promoter (enhancer) was constructed by recombinant DNA technology and confirmed by restriction analyses. pcDNA3-GFP-AFP-w, pcDNA3-GFP and pcDNA3 were transfected into Hela and Bel7402 cells by lipofectin and selected by G418 respectively, after amplification of the positive cell clones, expression of GFP was detected by Western blotting and quantitatively analysed by GEL Doc 2000 digital image systems. RESULTS: The expression of GFP was lower in Bel-GFP-AFP-w than in Bel-GFP but was significantly higher than in Hela-GFP-AFP-w. CONCLUSION: GFP reporter gene plasmid pcDNA3-GFP-AFP-w under the direction of the 3.1 kb AFP 5'flanking promoter (enhancer) can be expressed in HCC Bel7402 cell definitely and specifically.

Carcinoma, Hepatocellular↗

PubMed2005

Observer-based online compensation of inner filter effect in monitoring fluorescence of GFP-expressing plant cell cultures.

The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.

Algorithms↗