Astrocyte-to-neuron transportation of Enhanced Green Fluorescent Protein in cerebral cortex requires F-actin dependent Tunneling Nanotubes

Fluorescent Protein ◽

Cell Contact ◽

Neural Cells ◽

Tunneling Nanotubes ◽

Green Fluorescent ◽

And Function

Abstract Tunneling Nanotube (TNT) , a dynamic cell-cell contact, is dependent on actin polimerization. TNTs are efficient in transporting ions, proteins and organelles intercellularly, which are important mechanisms in physiological and phathological precesses. Reported studies on the existence and function of TNTs among neural cells focus on cultured cell for the convenience in detecting TNTs’ ultrastructure. In this study, the adeno-associated virus (AAV-GFAP-EGFP-p2A-cre) was injected in the cerebral cortex of knock-in mice ROSA26 GNZ. GFAP promoter initiated the expression of Enhanced Green Fluorescent Protein (EGFP) in infected astrocytes. At 10 days post injection (10 DPI), we found that EGFP could transfer from astrocytes in layerⅠ-Ⅲ to neurons in layer Ⅴ. The dissemination of EGFP was not through endocytosis or exosome. And the intercellular transportation of EGFP was F-actin dependent. Therefore, we concluded EGFP transported from astrocytes to neurons in coetex via F-actin dependent TNTs. Although it is hardly to detect the ultrastrucute of TNTs in brain for its transiency and the noisy background, we established an animal model and indirect experimental methods to explore TNTs in vivo.

Astrocyte-to-neuron transportation of enhanced green fluorescent protein in cerebral cortex requires F-actin dependent tunneling nanotubes

Scientific Reports ◽

10.1038/s41598-021-96332-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jing Chen ◽

Junyan Cao

Keyword(s):

Cerebral Cortex ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Cell Contact ◽

Neural Cells ◽

Tunneling Nanotubes ◽

Layer V ◽

AbstractTunneling nanotube (TNT), a dynamic cell–cell contact, is dependent on actin polymerization. TNTs are efficient in transporting ions, proteins and organelles intercellularly, which are important mechanisms in physiological and pathological processes. Reported studies on the existence and function of TNTs among neural cells focus on cultured cell for the convenience in detecting TNTs’ ultrastructure. In this study, the adeno-associated virus (AAV-GFAP-EGFP-p2A-cre) was injected into the cerebral cortex of knock-in mice ROSA26 GNZ. GFAP promoter initiated the expression of enhanced green fluorescent protein (EGFP) in infected astrocytes. At 10 days post injection (10 DPI), EGFP transferred from astrocytes in layer I–III to neurons in layer V. The dissemination of EGFP was not through endocytosis or exosome. Applying microscopes, we found that the intercellular transportation of EGFP through contact connection was F-actin dependent. Therefore, we concluded that EGFP transported from astrocytes to neurons in cortex via F-actin dependent TNTs. This study first proved that proteins transported intercellularly via TNTs in brain.

Effect of targeted ovarian cancer therapy using amniotic fluid mesenchymal stem cells transfected with enhanced green fluorescent protein-human interleukin-2 in vivo

Molecular Medicine Reports ◽

10.3892/mmr.2015.4076 ◽

2015 ◽

Vol 12 (4) ◽

pp. 4859-4866 ◽

Cited By ~ 9

Author(s):

QI YOU ◽

YUAN YAO ◽

YUANLONG ZHANG ◽

SONGBIN FU ◽

MEI DU ◽

...

Keyword(s):

Ovarian Cancer ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cancer Therapy ◽

Fluorescent Protein ◽

Interleukin 2 ◽

The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2015.08.013 ◽

2015 ◽

Vol 256 ◽

pp. 22-29 ◽

Cited By ~ 6

Author(s):

Jo E. Lewis ◽

John M. Brameld ◽

Phil Hill ◽

Perry Barrett ◽

Francis J.P. Ebling ◽

...

Keyword(s):

Fluorescent Protein ◽

Over Expression ◽

432 INHERITANCE OF LENTIVIRAL PHOSPHOGLYCERATE KINASE-ENHANCED GREEN FLUORESCENT PROTEIN (PGK-eGFP) INTEGRANTS OF TRANSGENIC CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab432 ◽

2010 ◽

Vol 22 (1) ◽

pp. 373

Author(s):

M. Reichenbach ◽

F. A. Habermann ◽

H. D. Reichenbach ◽

T. Guengoer ◽

F. Weber ◽

...

Keyword(s):

Fluorescent Protein ◽

Germ Line ◽

Phosphoglycerate Kinase ◽

Healthy Male ◽

Transgenic Founder ◽

An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.

Enhanced Green Fluorescent Protein Expression in Pleurotus ostreatus for In Vivo Analysis of Fungal Laccase Promoters

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-012-9816-3 ◽

2012 ◽

Vol 168 (4) ◽

pp. 761-769 ◽

Cited By ~ 6

Author(s):

Antonella Amore ◽

Yoichi Honda ◽

Vincenza Faraco

Keyword(s):

Protein Expression ◽

Pleurotus Ostreatus ◽

Green Fluorescent Protein Expression ◽

Fluorescent Protein ◽

Fungal Laccase ◽

In Vivo Analysis ◽

In Vitro and In Vivo Characterization of Recombinant Ebola Viruses Expressing Enhanced Green Fluorescent Protein

The Journal of Infectious Diseases ◽

10.1086/520590 ◽

2007 ◽

Vol 196 (s2) ◽

pp. S313-S322 ◽

Cited By ~ 59

Author(s):

Hideki Ebihara ◽

Steven Theriault ◽

Gabriele Neumann ◽

Judie B. Alimonti ◽

Joan B. Geisbert ◽

...

Keyword(s):

Fluorescent Protein ◽

Green Fluorescent ◽

In Vivo Characterization

Enhanced green fluorescent protein targeted to the Sca-1 (Ly-6A) locus in transgenic mice results in efficient marking of hematopoietic stem cells in vivo

Experimental Hematology ◽

10.1016/s0301-472x(02)01021-4 ◽

2003 ◽

Vol 31 (2) ◽

pp. 159-167 ◽

Cited By ~ 33

Author(s):

Piia Hanson ◽

Vikram Mathews ◽

Sarah H Marrus ◽

Timothy A Graubert

Keyword(s):

Stem Cells ◽

Transgenic Mice ◽

Hematopoietic Stem Cells ◽

Fluorescent Protein ◽

Hematopoietic Stem ◽

Electrotransfer of siRNA to Silence Enhanced Green Fluorescent Protein in Tumor Mediated by a High Intensity Pulsed Electromagnetic Field

Vaccines ◽

10.3390/vaccines8010049 ◽

2020 ◽

Vol 8 (1) ◽

pp. 49 ◽

Cited By ~ 3

Author(s):

Simona Kranjc Brezar ◽

Matej Kranjc ◽

Maja Čemažar ◽

Simon Buček ◽

Gregor Serša ◽

...

Keyword(s):

Electromagnetic Field ◽

High Intensity ◽

Fluorescent Protein ◽

Gene Electrotransfer ◽

Pulsed Electromagnetic Field ◽

Pulsed Electromagnetic ◽

The contactless high intensity pulsed electromagnetic field (HI-PEMF)-induced increase of cell membrane permeability is similar to conventional electroporation, with the important difference of inducing an electric field non-invasively by exposing a treated tissue to a time-varying magnetic field. Due to the limited number of studies in the field of electroporation induced by HI-PEMF, we designed experiments to explore the feasibility of such a contactless delivery technique for the gene electrotransfer of nucleic acids in tissues in vivo. By using HI-PEMF for gene electrotransfer, we silenced enhanced green fluorescent protein (EGFP) with siRNA molecules against EGFP in B16F10-EGFP tumors. Six days after the transfer, the fluorescent tumor area decreased by up to 39% as determined by fluorescence imaging in vivo. In addition, the silencing of EGFP to the same extent was confirmed at the mRNA and protein level. The results obtained in the in vivo mouse model demonstrate the potential use of HI-PEMF-induced cell permeabilization for gene therapy and DNA vaccination. Further studies are thus warranted to improve the equipment, optimize the protocols for gene transfer and the HI-PEMF parameters, and demonstrate the effects of HI-PEMF on a broader range of different normal and tumor tissues.

The EZC-Prostate Model: Noninvasive Prostate Imaging in Living Mice

Molecular Endocrinology ◽

10.1210/me.2003-0316 ◽

2004 ◽

Vol 18 (3) ◽

pp. 722-732 ◽

Cited By ~ 12

Author(s):

Xiaoming Xie ◽

Zheng Luo ◽

Kevin M. Slawin ◽

David M. Spencer

Keyword(s):

Fluorescent Protein ◽

Ex Vivo ◽

Firefly Luciferase ◽

In Vivo Models ◽

Charge Coupled Device ◽

Prostate Disease ◽

Abstract Recently, progress in the development of prostate-specific promoters and high resolution imaging techniques has made real-time monitoring of transgenic expression possible, opening a vista of potentially important in vivo models of prostate disease. Herein, we describe a novel prostate reporter model, called the EZC-prostate model that permits both ex vivo and in vivo imaging of the prostate using a sensitive charge-coupled device. Firefly luciferase and enhanced green fluorescent protein were targeted to the prostate epithelium using the composite human kallikrein 2 (hK2)-based promoter, hK2-E3/P. In EZC-prostate mice, the ventral and dorsal/lateral prostate lobes were brilliant green under fluorescence microscopy, with expression localized to the secretory epithelium. In contrast, enhanced green fluorescent protein was undetectable in the anterior lobes of prostate, seminal vesicles, testes, liver, lung, and brain. The kinetics of luciferase activity in intact and castrated living mice monitored with the IVIS charge-coupled device-based imaging system confirmed that firefly luciferase expression was largely prostate restricted, increased with age up to 24 wk, and was androgen dependent. Decreases in reporter expression after 24 wk may reflect well known, age-related decreases in androgen signaling with age in humans. Ex vivo imaging of microdissected animals further confirmed that the luminescence detected in living mice emanated predominately from the prostate, with minor signals originating from the testes and cecum. These data demonstrate that the hK2-E3/P promoter directs strong prostate-specific expression in a transgenic mouse model. Multigenic models, generated by crosses with various hyperplastic and neoplastic prostate disease models, could potentially provide powerful new tools in longitudinal monitoring of changes in prostate size, androgen signaling, metastases, or response to novel therapies without sacrificing large cohorts of animals.