scholarly journals Identification of HI-Like Loop in CELO Adenovirus Fiber for Incorporation of Receptor Binding Motifs

2007 ◽  
Vol 81 (18) ◽  
pp. 9641-9652 ◽  
Author(s):  
Denis Y. Logunov ◽  
Olga V. Zubkova ◽  
Anna S. Karyagina-Zhulina ◽  
Eugenia A. Shuvalova ◽  
Andrei P. Karpov ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Cell Types ◽  
Binding Motif ◽  
Recombinant Viruses ◽  
Rgd Motif ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The αv integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.

scholarly journals Retroviral-Mediated Transfer of the Green Fluorescent Protein Gene Into Murine Hematopoietic Cells Facilitates Scoring and Selection of Transduced Progenitors In Vitro and Identification of Genetically Modified Cells In Vivo

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1777-1786 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard J. Smeyne ◽  
Richard A. Ashmun ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
High Titer ◽  
Hematopoietic Cells ◽  
Green Fluorescent Protein Gene ◽  
Retroviral Gene Transfer ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.

scholarly journals Tropism Modification of Adenovirus Vectors by Peptide Ligand Insertion into Various Positions of the Adenovirus Serotype 41 Short-Fiber Knob Domain

2006 ◽  
Vol 81 (6) ◽  
pp. 2688-2699 ◽  
Author(s):  
Andrea Hesse ◽  
Daniela Kosmides ◽  
Roland E. Kontermann ◽  
Dirk M. Nettelbeck
Keyword(s):  
Gene Transfer ◽  
Cell Types ◽  
Short Fiber ◽  
Peptide Ligand ◽  
Cell Binding ◽  
Adenovirus Serotype ◽  
C Terminus ◽  
Genetic Targeting

ABSTRACT Recombinant adenoviruses have emerged as promising agents in therapeutic gene transfer, genetic vaccination, and viral oncolysis. Therapeutic applications of adenoviruses, however, would benefit substantially from targeted virus cell entry, for example, into cancer or immune cells, as opposed to the broad tropism that adenoviruses naturally possess. Such tropism modification of adenoviruses requires the deletion of their natural cell binding properties and the incorporation of cell binding ligands. The short fibers of subgroup F adenoviruses have recently been suggested as a tool for genetic adenovirus detargeting based on the reduced infectivity of corresponding adenovectors with chimeric fibers in vitro and in vivo. The goal of our study was to determine functional insertion sites for peptide ligands in the adenovirus serotype 41 (Ad41) short fiber knob. With a model peptide, CDCRGDCFC, we could demonstrate that ligand incorporation into three of five analyzed loops of the knob, namely, EG, HI, and IJ, is feasible without a loss of fiber trimerization. The resulting adenovectors showed enhanced infectivity for various cell types, which was superior to that of viruses with the same peptide fused to the fiber C terminus. Strategies to further augment gene transfer efficacy by extension of the fiber shaft, insertion of tandem copies of the ligand peptide, or extension of the ligand-flanking linkers failed, indicating that precise ligand positioning is pivotal. Our study establishes that internal ligand incorporation into a short-shafted adenovirus fiber is feasible and suggests the Ad41 short fiber with ligand insertion into the top (IJ loop) or side (EG and HI loops) of the knob domain as a novel platform for genetic targeting of therapeutic adenoviruses.

scholarly journals Live-Attenuated Measles Virus Vaccine Targets Dendritic Cells and Macrophages in Muscle of Nonhuman Primates

2014 ◽  
Vol 89 (4) ◽  
pp. 2192-2200 ◽  
Author(s):  
Linda J. Rennick ◽  
Rory D. de Vries ◽  
Thomas J. Carsillo ◽  
Ken Lemon ◽  
Geert van Amerongen ◽  
...  
Keyword(s):  
Measles Virus ◽  
Nonhuman Primates ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Vaccine Virus ◽  
Target Cells ◽  
Recombinant Viruses ◽  
Green Fluorescent

ABSTRACTAlthough live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replicationin vivoare still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MVEZ. Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMVEZEGFP(1), rMVEZEGFP(3), and rMVEZEGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were comparedin vitroby growth curves, which indicated that rMVEZEGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMVEZEGFP(1) and rMVEZEGFP(6) did not induce satisfactory serum antibody responses, whereas bothin vitroandin vivorMVEZEGFP(3) was functionally equivalent to the commercial MVEZ-containing vaccine. Intramuscular vaccination of macaques with rMVEZEGFP(3) resulted in the identification of EGFP+cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV.IMPORTANCEEven though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicatesin vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

scholarly journals Foreign Gene Expression in the Mouse Cauda Epididymis is Regulated by Androgens

2018 ◽  
Vol 2 (4) ◽  
pp. 671-678
Author(s):  
Pedro Esponda
Keyword(s):  
Gene Transfer ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Female Reproductive Tract ◽  
Foreign Gene ◽  
Total Period ◽  
Green Fluorescent ◽  
In Vivo Gene Transfer

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

Partial Reprogramming As An Emerging Strategy for Safe Induced Cell Generation and Rejuvenation

2019 ◽  
Vol 19 (4) ◽  
pp. 248-254
Author(s):  
Marianne Lehmann ◽  
Martina Canatelli-Mallat ◽  
Priscila Chiavellini ◽  
Gloria M. Cónsole ◽  
Maria D. Gallardo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Somatic Cell ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Cell Types ◽  
Cell Reprogramming ◽  
Green Fluorescent ◽  
Original Cell

Background: Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research. Objective: Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation. Methods: In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM). Results: Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter. Conclusion: Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.

A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane

2001 ◽  
Vol 114 (4) ◽  
pp. 719-726 ◽  
Author(s):  
M.I. Milewski ◽  
J.E. Mickle ◽  
J.K. Forrest ◽  
D.M. Stafford ◽  
B.D. Moyer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Binding Motif ◽  
C Terminus ◽  
Polarized Epithelia ◽  
Selective Retention ◽  
Green Fluorescent ◽  
Apical Localization

Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370–1394 and 1404–1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.

scholarly journals The C-terminal Domain Is the Primary Determinant of Histone H1 Binding to Chromatinin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 20028-20034 ◽  
Author(s):  
Michael J. Hendzel ◽  
Melody A. Lever ◽  
Ellen Crawford ◽  
John P. H. Th'ng
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Histone H1 ◽  
Single Amino Acid ◽  
Kinetic Measurements ◽  
Terminal Domain ◽  
C Terminus ◽  
Green Fluorescent

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 bindingin vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.

scholarly journals Genetically modified adenoviral vector with the protein transduction domain of Tat improves gene transfer to CAR-deficient cells

2009 ◽  
Vol 29 (2) ◽  
pp. 103-109 ◽  
Author(s):  
Shihai Liu ◽  
Qinwen Mao ◽  
Weifeng Zhang ◽  
Xiaojing Zheng ◽  
Ye Bian ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Function Analysis ◽  
Cell Types ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Protein Transduction ◽  
Protein Transduction Domain ◽  
Green Fluorescent

The transduction efficiency of Ad (adenovirus) depends, to some extent, on the expression level of CAR (coxsackievirus and Ad receptor) of a target cell. The low level of CAR on the cell surface is a potential barrier to efficient gene transfer. To overcome this problem, PTD.AdeGFP (where eGFP is enhanced green fluorescent protein) was constructed by modifying the HI loop of Ad5 (Ad type 5) fibre with the Tat (trans-activating) PTD (protein transduction domain) derived from HIV. The present study showed that PTD.AdeGFP significantly improved gene transfer to multiple cell types deficient in expression of CAR. The improvement in gene transfer was not the result of charge-directed binding between the virus and the cell surface. Although PTD.AdeGFP formed aggregates, it infected target cells in a manner different from AdeGFP aggregates precipitated by calcium phosphate. In addition, PTD.AdeGFP was able to transduce target cells in a dynamin-independent pathway. The results provide some new clues as to how PTD.AdeGFP infects target cells. This new vector would be valuable in gene-function analysis and for gene therapy in cancer.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

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