An Avian Sarcoma/Leukosis Virus-Based Gene Trap Vector for Mammalian Cells

ABSTRACT RCASBP-M2C is a retroviral vector derived from an avian sarcoma/leukosis virus which has been modified so that it uses the envelope gene from an amphotropic murine leukemia virus (E. V. Barsov and S. H. Hughes, J. Virol. 70:3922–3929, 1996). The vector replicates efficiently in avian cells and infects, but does not replicate in, mammalian cells. This makes the vector useful for gene delivery, mutagenesis, and other applications in mammalian systems. Here we describe the development of a derivative of RCASBP-M2C, pGT-GFP, that can be used in gene trap experiments in mammalian cells. The gene trap vector pGT-GFP contains a green fluorescent protein (GFP) reporter gene. Appropriate insertion of the vector into genes causes GFP expression; this facilitates the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of GFP. With this vector, we have generated about 90 gene-trapped lines using D17 and NIH 3T3 cells. Five trapped NIH 3T3 lines were selected based on the distribution of GFP in cells. The cellular genes disrupted by viral integration have been identified in four of these lines by using a 5′ rapid amplification of cDNA ends protocol.

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A Monoclonal Antibody to Visualize PtdIns(3,4,5)P3 in Cells

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540205000511 ◽

2002 ◽

Vol 50 (5) ◽

pp. 697-708 ◽

Cited By ~ 55

Author(s):

Riyan Chen ◽

Veronica H. Kang ◽

Jian Chen ◽

Joseph C. Shope ◽

Javad Torabinejad ◽

...

Keyword(s):

Mammalian Cells ◽

Cell Function ◽

Fluorescent Protein ◽

Human Neutrophils ◽

Ph Domain ◽

Extraction And Separation ◽

3T3 Fibroblasts ◽

Green Fluorescent ◽

Nih 3T3

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsPn) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsPn quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsPns. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P3 immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P3 IgM, and immunodetection of PtdIns(3,4,5)P3 in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P3 relative to its precursor, phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P2), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was bench-marked against HPLC analysis of [3H]-myo-inositol-labeled cellular PtdInsPns. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P3 and PtdIns(4,5)P2 in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P3 levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P2 levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.

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Rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells

Enzyme and Microbial Technology ◽

10.1016/j.enzmictec.2010.08.004 ◽

2011 ◽

Vol 48 (1) ◽

pp. 13-18 ◽

Cited By ~ 1

Author(s):

Wen-Hsin Lo ◽

Chi-Yuan Chen ◽

Chia-Ni Yeh ◽

Chin-Yu Lin ◽

Yu-Chen Hu

Keyword(s):

Green Fluorescent Protein ◽

Protein Expression ◽

Mammalian Cells ◽

Green Fluorescent Protein Expression ◽

Fluorescent Protein ◽

Green Fluorescent ◽

Expression In Mammalian Cells

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Dynamin-related Protein Drp1 Is Required for Mitochondrial Division in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.12.8.2245 ◽

2001 ◽

Vol 12 (8) ◽

pp. 2245-2256 ◽

Cited By ~ 1052

Author(s):

Elena Smirnova ◽

Lorena Griparic ◽

Dixie-Lee Shurland ◽

Alexander M. van der Bliek

Keyword(s):

Mammalian Cells ◽

Fluorescent Protein ◽

Time Lapse ◽

Subcellular Fractionation ◽

Related Protein ◽

Direct Role ◽

Mitochondrial Division ◽

Transfected Cells ◽

Green Fluorescent ◽

Time Lapse Photography

Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

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Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

Journal of Cell Science ◽

10.1242/jcs.112.16.2705 ◽

1999 ◽

Vol 112 (16) ◽

pp. 2705-2714

Author(s):

E.M. Burns ◽

L. Christopoulou ◽

P. Corish ◽

C. Tyler-Smith

Keyword(s):

Green Fluorescent Protein ◽

Mammalian Cells ◽

Quantitative Measurement ◽

Fluorescent Protein ◽

Cultured Cells ◽

Chromosome Loss ◽

Facs Analysis ◽

Fluorescent Cells ◽

Green Fluorescent ◽

Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

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The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study

The Journal of Cell Biology ◽

10.1083/jcb.137.7.1525 ◽

1997 ◽

Vol 137 (7) ◽

pp. 1525-1535 ◽

Cited By ~ 134

Author(s):

Jianwei Liu ◽

Thomas E. Hughes ◽

William C. Sessa

Keyword(s):

Nitric Oxide ◽

Amino Acids ◽

Fluorescent Protein ◽

3T3 Cells ◽

Fatty Acylation ◽

Golgi Region ◽

Green Fluorescent ◽

Oxide Synthase ◽

Nih 3T3 ◽

Endothelial Nitric Oxide

Catalytically active endothelial nitric oxide synthase (eNOS) is located on the Golgi complex and in the caveolae of endothelial cells (EC). Mislocalization of eNOS caused by mutation of the N-myristoylation or cysteine palmitoylation sites impairs production of stimulated nitric oxide (NO), suggesting that intracellular targeting is critical for optimal NO production. To investigate the molecular determinants of eNOS targeting in EC, we constructed eNOS–green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The full-length eNOS–GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, suggesting that the GFP tag does not interfere with eNOS localization or function. Experiments with different size amino-terminal fusion partners coupled to GFP demonstrated that the first 35 amino acids of eNOS are sufficient to target GFP into the Golgi region of NIH 3T3 cells. Additionally, the unique (Gly-Leu)5 repeat located between the palmitoylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoylation and thus localization, but not for N-myristoylation, membrane association, and NOS activity. The palmitoylation-deficient mutants displayed a more diffuse fluorescence pattern than did WT eNOS–GFP, but still were associated with intracellular membranes. Biochemical studies also showed that the palmitoylation-deficient mutants are associated with membranes as tightly as WT eNOS. Mutation of the N-myristoylation site Gly-2 (abolishing both N-myristoylation and palmitoylation) caused the GFP fusion protein to distribute throughout the cell as GFP alone, consistent with its primarily cytosolic nature in biochemical studies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells via the first 35 amino acids, including N-myristoylation and palmitoylation sites, and its overall membrane association requires N-myristoylation but not cysteine palmitoylation. These results suggest a novel role for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex.

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Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

The Journal of Cell Biology ◽

10.1083/jcb.136.1.71 ◽

1997 ◽

Vol 136 (1) ◽

pp. 71-80 ◽

Cited By ~ 112

Author(s):

Erik A.C. Wiemer ◽

Thibaut Wenzel ◽

Thomas J. Deerinck ◽

Mark H. Ellisman ◽

Suresh Subramani

Keyword(s):

Mammalian Cells ◽

Laser Scanning ◽

Fluorescent Protein ◽

Time Lapse ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Small Subset ◽

Subcellular Compartment ◽

Directional Movement ◽

Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

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310 PRODUCTION OF TRANSGENIC CLONED PORCINE EMBRYOS EXPRESSING EGFP OR LacZ GENE THROUGH REPLICATION DEFECTIVE RETROVIRAL VECTOR

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab310 ◽

2008 ◽

Vol 20 (1) ◽

pp. 235

Author(s):

S. J. Uhm ◽

M. K. Gupta ◽

T. Kim ◽

H. T. Lee

Keyword(s):

Fluorescent Protein ◽

Leukemia Virus ◽

Fluorescein Isothiocyanate ◽

Retroviral Vectors ◽

Retroviral Vector ◽

Lacz Gene ◽

Cell Stage ◽

Uv Illumination ◽

Green Fluorescent

We have demonstrated previously that retroviral-mediated gene transfer is a promising method to produce transgenic avian, porcine, and bovine embryos. This study was designed to evaluate the development potential of transgenic porcine embryos produced by somatic cell nuclear transfer (SCNT) of fetal fibroblast (pFF) cells transfected by a robust replication-defective retroviral vector harboring enhanced green fluorescent protein (EGFP) or β-galactosidase (LacZ) gene. Moloney murine leukemia virus (MoMLV)-based retroviral vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein and harboring EGFP or LacZ under the control of β-actin promoter were produced and used to transfect primary pFF cells that were subsequently used for SCNT of enucleated porcine oocytes matured in vitro. Our results showed that all surviving cells after transfection and antibiotic selection expressed the genes without any evidence of replication-competent retrovirus. The fusion, cleavage, and blastocyst rates were 85.6 � 6.5, 53.6 � 6.4, and 12.0 � 5.7% for EGFP; 83.5 � 8.2, 57.5 � 6.3 and 10.1 � 4.1% for LacZ; and 80.5 � 4.2, 60.9 � 8.2 and 12.3 � 4.0% for controls, respectively. Mosaicism was not observed in any of the group as evidenced by the expression of LacZ or EGFP in individual blastomeres of all embryos upon staining with β-galactosidase (for LacZ) or when visualized under UV illumination of an epifluorescent microscope using the fluorescein isothiocyanate (FITC) filter set (for EGFP). Further recloning of EGFP-expressing blastomeres, obtained from 4-cell-stage cloned embryos produced by SCNT of pFF cells infected with EGFP harboring vector, into enucleated metaphase II (MII) oocytes resulted in consistent expression of EGFP in recloned blastocysts. Interspecies SCNT (iSCNT) of transfected pFF into enucleated bovine oocytes could also result in consistent gene expression without any adverse effect on blastocyst rate (5.5 v. 4.9%) compared with non-transfected pFF. These data indicate that the replication-defective retroviral vector used in the present study is robust and independent of the genes inserted. Furthermore, introduction of transgenes by this method does not influence the in vitro development rate of cloned embryos. This work was supported by a grant from Biogreen 21 Program, RDA, Republic of Korea.

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Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo

Immunology Letters ◽

10.1016/j.imlet.2017.12.005 ◽

2018 ◽

Vol 194 ◽

pp. 29-39 ◽

Cited By ~ 10

Author(s):

Fatemeh Motevalli ◽

Azam Bolhassani ◽

Shilan Hesami ◽

Sepideh Shahbazi

Keyword(s):

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Green Fluorescent

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Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells

The Journal of Cell Biology ◽

10.1083/jcb.200101089 ◽

2001 ◽

Vol 154 (1) ◽

pp. 71-84 ◽

Cited By ~ 276

Author(s):

Nathalie Daigle ◽

Joël Beaudouin ◽

Lisa Hartnell ◽

Gabriela Imreh ◽

Einar Hallberg ◽

...

Keyword(s):

Mammalian Cells ◽

Fluorescent Protein ◽

Nuclear Lamina ◽

Nuclear Pore ◽

Global Changes ◽

Nuclear Pore Complexes ◽

Annulate Lamellae ◽

Lamin B1 ◽

Green Fluorescent ◽

Pore Complexes

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121–green fluorescent protein (GFP) and GFP-Nup153, and GFP–lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

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