scholarly journals Three-Dimensional Model of Dorsal Root Ganglion Explant as a Method of Studying Neurotrophic Factors in Regenerative Medicine

Biomedicines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 49 ◽  
Author(s):  
Polina Klimovich ◽  
Kseniya Rubina ◽  
Veronika Sysoeva ◽  
Ekaterina Semina
Keyword(s):  
Cell Migration ◽  
Green Fluorescent Protein ◽  
Dorsal Root Ganglion ◽  
Neurotrophic Factors ◽  
Dorsal Root ◽  
Fluorescent Protein ◽  
Axon Regeneration ◽  
Ex Vivo ◽  
Axon Growth ◽  
Green Fluorescent

Neurotrophic factors play a key role in the development, differentiation, and survival of neurons and nerve regeneration. In the present study, we evaluated the effect of certain neurotrophic factors (NGF, BDNF, and GDNF) on axon growth and migration of Nestin-green fluorescent protein (GFP)-positive cells using a 3D model of dorsal root ganglion (DRG) explant culture in Matrigel. Our method generally represents a convenient model for assessing the effects of soluble factors and therapeutic agents on axon growth and nerve regeneration in R&D studies. By analyzing the DRG explants in ex vivo culture for 21 days, one can evaluate the parameters of neurite outgrowth and the rate of cell migration from the DRG explants into the Matrigel. For the current study, we used Nestin-GFP-expressing mice in which neural precursors express Nestin and the green fluorescent protein (GFP) under the same promoter. We revealed that GDNF significantly (two fold) stimulated axon outgrowth (p < 0.05), but not BDNF or NGF. It is well-known that axon growth can be stimulated by activated glial cells that fulfill a trophic function for regenerating nerves. For this reason, we evaluated the number of Nestin-GFP-positive cells that migrated from the DRG into the Matrigel in our 3D ex vivo explant model. We found that NGF and GDNF, but not BDNF, stimulated the migration of Nestin-GFP cells compared to the control (p < 0.05). On the basis of the aforementioned finding, we concluded that GDNF had the greatest stimulating potential for axon regeneration, as it stimulated not only the axon outgrowth, but also glial cell migration. Although NGF significantly stimulated glial cell migration, its effect on axon growth was insufficient for axon regeneration.

Electrophysiological Analysis of Dorsal Root Ganglion Neurons Pre- and Post-Coexpression of Green Fluorescent Protein and Functional 5-HT3Receptor

1997 ◽  
Vol 77 (6) ◽  
pp. 3115-3121 ◽  
Author(s):  
George M. Smith ◽  
Richard L. Berry ◽  
Jay Yang ◽  
Darrell Tanelian
Keyword(s):  
Green Fluorescent Protein ◽  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Fluorescent Protein ◽  
Dorsal Root Ganglion Neurons ◽  
Drg Neurons ◽  
Whole Cell ◽  
Electrophysiological Analysis ◽  
Green Fluorescent ◽  
Ganglion Neurons

Smith, George M., Richard L. Berry, Jay Yang, and Darrell Tanelian. Electrophysiological analysis of dorsal root ganglion neurons pre- and post-coexpression of green fluorescent protein and functional 5-HT3receptor. J. Neurophysiol. 77: 3115–3121, 1997. Aequorea green fluorescent protein (GFP) is an excellent marker to examine genetically altered live cells in whole animals or culture. Its potential use in identifying genetically modified neurons, however, has not been investigated extensively. To examine the usefulness, toxicity, and potential electrophyiological effects of GFP expression in neurons, we generated adenovirus containing the mGFP4 cDNA. One week after virus transfection of dorsal root ganglion neurons (DRG), 10% of postnatal DRG neurons appeared brightly fluorescent, labelling the soma and neurites. Temporal examination of these neurons demonstrated no toxicity to DRG neurons even after several weeks in culture with repeated daily epifluorescent exposure. Electrophysiological analysis and comparison of control and viral exposed (GFP− and GFP+) DRG neurons did not demonstrate any differences in whole cell resistance, resting potential, action potential (AP) threshold, AP duration, AP amplitude, or whole cell capacitance. To investigate the usefulness of GFP as a marker for identifying neurons genetically altered to express a novel neurotransmitter receptor, a second adenovirus construct was generated containing both GFP and serotonin type 3 (5-HT3) receptor cDNAs. Transfection of DRG neurons with this virus produced an inward current in the presence of serotonin only in DRG neurons that were GFP-positive. It is concluded that adenoviral transfection of neurons with GFP, for cellular labeling, and coexpression of GFP-neurotransmitter constructs are safe, nontoxic, methods for electrophysiologically investigating neurons over several weeks. The uniqueness of the vector used in these experiments is that it was constructed to express GFP in a second cassette so that it would label the transduced cells, but have no potential for interfering with the function of the foreign 5-HT3receptor.

scholarly journals Use of Green Fluorescent Protein To Tag Lactic Acid Bacterium Strains under Development as Live Vaccine Vectors

2000 ◽  
Vol 66 (1) ◽  
pp. 383-391 ◽  
Author(s):  
Marie-Claude Geoffroy ◽  
Cyril Guyard ◽  
Brigitte Quatannens ◽  
Sonia Pavan ◽  
Marc Lange ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Live Vaccine ◽  
Expression Vectors ◽  
Fermented Foods ◽  
Vaccine Vectors ◽  
Green Fluorescent

ABSTRACT The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e.,Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying thegfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescentL. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.

scholarly journals Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

2000 ◽  
Vol 84 (09) ◽  
pp. 460-467 ◽  
Author(s):  
M. L. M. Lamfers ◽  
M. J. Wijnberg ◽  
J. M. Grimbergen ◽  
L. G. M. Huisman ◽  
M. C. Aalders ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Receptor Binding ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Adenoviral Gene Transfer ◽  
Amino Terminal ◽  
Green Fluorescent

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

Μελέτη παραγόντων βελτιστοποίησης των τεχνικών μεταφοράς και διατήρησης του αυτόλογου λίπους (fat transer)

2016 ◽  
Author(s):  
Μαργαρίτα Μουστάκη
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Adipose Derived Stem Cells ◽  
Green Fluorescent

Εισαγωγή:Ο αυτόλογος λιπώδης ιστός έχει αποδείχθηκε ένα εξαιρετικό υλικό αύξησης μαλακών μορίων. Η απομόνωση των βλαστικών κυττάρων από τον λιπώδη ιστό (Adipose Derived Stem Cells, ADSCs) μοιραία οδήγησε την έρευνα να στραφεί στη μελέτη της συνδυασμένης μεταμόσχευσης αυτόλογου λίπους και ADSCs (cell assisted lipotransfer-CAL).Σκοπός:Η συγκεκριμένη μελέτη είναι μια in vivo μελέτη σε ζωικό πρότυπο της υποβοηθούμενης με ADSCs λιπομεταφοράς. Σκοπός της μελέτης είναι να αποδείξει με ποσοτικές μετρήσεις τη δυνατότητα των ADSCs να βελτιώνουν την ποιότητα και την μακροπρόθεσμη διατήρηση του μοσχεύματος λίπους κατά τη λιπομεταφορά, συγκρίνοντας την με την παραδοσιακή λιπομεταφορά.Μέθοδοι:Για τους σκοπούς της μελέτης, βλαστικά κύτταρα απομονώθηκαν από διαγονιδιακούς μύες C57BL / 6J-GFΡ που εκφράζουν την πράσινη φθορίζουσα πρωτεΐνη (Green Fluorescent Protein, GFP) με αποτέλεσμα τα ίδια τα βλαστικά κύτταρα να φθορίζουν, γεγονός που μας επέτρεψε την παρακόλουθηση τους απεικονιστικά. Τα GFP-θετικά βλαστικά κύτταρα αφού απομονώθηκαν από το λίπος αναμείχθηκαν με τεμαχισμένο λιπώδη ιστό που συλλέχθηκε από τις βουβωνικές χώρες των C57BL / 6JolaHsd μυών και στη συνέχεια συν-εμφυτεύτηκαν σε Hsd:Athymic Nude-Foxn1nu μύες. Σε όλους τους μύες η έγχυση του ADSC-εμπλουτισμένου μείγματος έγινε στη μία πλευρά της ράχης ενώ στην αντίθετη πλευρά της ράχης πραγματοποιήθηκε η έγχυση ελέγχου (control) με ίδια ποσότητα τεμαχισμένου λιπώδους ιστού (μη εμπλουτισμένου) από τις βουβωνικές χώρες των C57BL / 6JolaHsd μυών .Η επιβίωση των εμφυτευμένων GFP-θετικών βλαστικών κυττάρων παρακολουθήθηκε με in νίνο μοριακή απεικόνιση φθορισμού 56 ημερών. Για τη συγκριτική μελέτη του ποσοστού επιβίωσης το μοσχεύματος λίπους τα μοσχεύματα αφαιρέθηκαν χειρουργικά στις 7 και στις 56 ημέρες μετά τη μεταμόσχευση, ζυγίστηκαν και υποβλήθηκαν σε ιστολογική εξέταση και ανοσοϊστοχημικό έλεγχο για τους δείκτες CD34 και Ki67.Αποτελέσματα:Τα αποτελέσματα έδειξαν καλύτερη επιβίωση του ADSC-εμπλουτισμένου λίπους σε σύγκριση με το μη εμπλουτισμένο (63% του αρχικής μάζας έναντι 33% μεγαλύτερη σε σχέση με την παραδοσιακή λιπομεταφορά,p<0.05). Η παρουσία των φθοριζόντων ADSCs μέχρι και την 56η ημέρα παρακολούθησης επιβεβαιώθηκε με την in vivo απεικόνιση φθορισμού. Η νεο-αγγειακή πυκνότητα ήταν αυξημένη στα εμπλουτισμένα με ADSCs λιπομοσχεύματα με διαφορά στατιστικά σημαντική (p<0.05)Συμπεράσματα: Ο εμπλουτισμός των λιπο-μοσχευμάτων με ADSCs είναι μια αξιόπιστη και αποτελεσματική μέθοδος βελτιστοποίησης της τεχνικής μεταφοράς αυτόλογου λίπους. Τα ex-vivo καλλιεργημένα ADSCs συμβάλλουν στη νεο-αγγείωση και αναγέννηση του ισχαιμικού ισχαιμικού λιπομοσχεύματος .

Ca2+ mobilization through dorsal root ganglion Ca2+-sensing receptor stably expressed in HEK293 cells

2007 ◽  
Vol 292 (5) ◽  
pp. C1895-C1905 ◽  
Author(s):  
Emmanuel M. Awumey ◽  
Allyn C. Howlett ◽  
James W. Putney ◽  
Debra I. Diz ◽  
Richard D. Bukoski
Keyword(s):  
Dorsal Root Ganglion ◽  
Fusion Protein ◽  
Dorsal Root ◽  
Fluorescent Protein ◽  
Hek293 Cells ◽  
Pcr Analysis ◽  
Hek 293 ◽  
Intracellular Ca ◽  
Egfp Fusion Protein ◽  
Green Fluorescent

The rat dorsal root ganglion (DRG) Ca2+-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca2+ concentration ([Ca2+]i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca2+] ([Ca2+]e) from 0.5 to 1 mM resulted in increases in [Ca2+]i levels, which were blocked by 30 μM 2-aminoethyldiphenyl borate. [Ca2+]e-response studies indicate a Ca2+ sensitivity with an EC50 of 1.75 ± 0.10 mM. NPS R-467 and Gd3+ activated the CaR. When [Ca2+]e was successively raised from 0.25 to 4 mM, peak [Ca2+]i, attained with 0.5 mM, was reduced by ∼50%. Similar reductions were observed with repeated applications of 10 mM Ca2+, 1 and 10 μM NPS R-467, or 50 and 100 μM Gd3+, indicating desensitization of the response. Furthermore, Ca2+ mobilization increased phosphorylated protein kinase C (PKC)α levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca2+ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Cae2+. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca2+]i transients by 49.9 ± 5.2% (at 1 mM Ca2+) and 40.5 ± 6.5% (at 2 mM Ca2+), compared with controls. The findings suggest involvement of PKC in the pathway for Ca2+ mobilization following CaR activation.

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

2004 ◽  
Vol 18 (3) ◽  
pp. 722-732 ◽  
Author(s):  
Xiaoming Xie ◽  
Zheng Luo ◽  
Kevin M. Slawin ◽  
David M. Spencer
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Firefly Luciferase ◽  
In Vivo Models ◽  
Charge Coupled Device ◽  
Prostate Disease ◽  
Green Fluorescent

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.

scholarly journals Coculture system of keratinocytes and dorsal-root-ganglion-derived cells for screening neurotrophic factors involved in guidance of neuronal axon growth in the skin

2013 ◽  
Vol 23 (1) ◽  
pp. 58-60 ◽  
Author(s):  
Jun-ichi Kumamoto ◽  
Masashi Nakatani ◽  
Moe Tsutsumi ◽  
Makiko Goto ◽  
Sumiko Denda ◽  
...  
Keyword(s):  
Dorsal Root Ganglion ◽  
Neurotrophic Factors ◽  
Dorsal Root ◽  
Axon Growth ◽  
Coculture System

In vitro and ex vivo green fluorescent protein expression in alveolar mammary epithelial cells and mammary glands driven by the distal 5´-regulative sequence and intron 1 of the goat β-casein gene

2003 ◽  
Vol 15 (4) ◽  
pp. 231 ◽  
Author(s):  
Hsi-Tien Wu ◽  
Chich-Sheng Lin ◽  
Mu-Chiou Huang
Keyword(s):  
Green Fluorescent Protein ◽  
Green Fluorescent Protein Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Mammary Epithelial ◽  
Casein Gene ◽  
Intron 1 ◽  
Lactogenic Hormones ◽  
Green Fluorescent

The 5′-regulative sequence and intron 1 of the goat β-casein gene from −4044 to +2123 bp was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragments of the mammary gland was observed 24 h after gene transfer. The novel strategy of ex vivo gene transfer into mammary tissue using GFP as a reporter gene to detect the function of a tissue-specific promoter is efficient and convenient. The data obtained herein reveal that the 5′-regulative sequence and intron 1 of the 6.2 kb goat β-casein gene can enhance the efficiency of transgene expression. Thus, the GB562 sequence may act as a good promoter and effectively elevate the production of exogenous protein in mammary glands.

Sign in / Sign up

Export Citation Format

Share Document