scholarly journals The Effect of Linear Versus Circular Vector on Enhanced Green Fluorescent Protein (EGFP) Expression in Transgenic Zebrafish (Danio rerio)

2010 ◽  
Vol 9 (8) ◽  
pp. 1232-1236
Author(s):  
Aygul Ekici ◽  
Digdem Aktoprakli ◽  
Metin Timur ◽  
Tolga Akkoc ◽  
Haydar Bagis
Keyword(s):  
Green Fluorescent Protein ◽  
Danio Rerio ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Green Fluorescent

128 MULTIPLICATION OF 8-CELL EMBRYOS BY AGGREGATION OF A SINGLE ENHANCED GREEN FLUORESCENT PROTEIN-LABELED BLASTOMERE WITH PUTATIVE TETRAPLOID EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 168
Author(s):  
M. I. Hiriart ◽  
R. J. Bevacqua ◽  
R. Fernandez-Martin ◽  
D. F. Salamone
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Egfp Expression ◽  
Simple Method ◽  
Transgenic Embryos ◽  
Green Fluorescent

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitro matured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50 ng mL–1 of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cell embryos were electrofused twice at 40V for 25 μs at 100-ms intervals to generate putative tetraploid embryos, visualised as a single blastomere 1 h after the fusion pulse (fused embryos, F). Two aggregation groups were included. A synchronic group (S): IVF for the production of both transgenic embryos and fused embryos was done on the same day; and an asynchronic group (AS): IVF for transgenic embryos took place 1 day before IVF for fused embryos production, so embryos from the A group were younger. Controls consisted of the same S and AS groups, but no fusion was included (NF). On day 3, the enhanced green fluorescent protein [EGFP(+)] blastomeres were selected. Using the well of well system, 1 or 2 embryos of each fusion group (S or AS and F or NF) were removed of their ZP and aggregated in a microwell with one EGFP(+) blastomere from a 5- to 8-cell stage embryo (day 3). In vitro development of the aggregates and green fluorescent protein expression localization of blastocysts were analysed. Blastocysts were obtained for all groups; however, the 2A-F and 2A-NF groups showed the highest rates (44%, P < 0.05) compared with one embryo aggregation. The highest aggregation rates of the EGFP(+) blastomere were observed for 2A-F (67%) and 2A-NF (44%) groups, too. A very poor integration was noted in the 2S-NF (100%), 2S-F (94%), 1A-NF (89%), and 1S-NF (80%) groups. Localised EGFP distribution was also high in the 2A-F group (42%). In all cases, EGFP expression seemed to localise by the inner cell mass. We demonstrated that it is possible to multiply 8-cell embryos of genetic value and also transgenic embryos, in theory reducing mosaicism rates in future offspring. Moreover, our results give rise to the possibility of using EGFP like a reporter gene that could be used to evaluate aggregation efficiency by a fluorescence microscope.

scholarly journals Transient Immunosuppression Stops Rejection of Virus-Transduced Enhanced Green Fluorescent Protein in Rabbit Retina

2004 ◽  
Vol 78 (20) ◽  
pp. 11327-11333 ◽  
Author(s):  
Kentaro Doi ◽  
Jian Kong ◽  
Janos Hargitai ◽  
Stephen P. Goff ◽  
Peter Gouras
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Retinal Pigment ◽  
Serum Antibody ◽  
Protein Detection ◽  
Foreign Protein ◽  
Egfp Expression ◽  
Systemic Immunosuppression ◽  
Green Fluorescent

ABSTRACT The expression of lentivirus-transduced enhanced green fluorescent protein (EGFP) was detectable in rabbit retinal pigment epithelium (RPE) within 3 to 5 days after subretinal injection of the vector. Within 2 to 3 weeks, EGFP-expressing cells were eliminated by rejection. In the current experiments, we monitor serum antibody titers for EGFP before and after transduction and determine whether systemic immunosuppression prevents recognition of EGFP by the immune system. While all control rabbits developed antibodies against EFGP and showed signs of rejection, no such evidence was observed with animals which received immunosuppression. One month of systemic immunosuppression permanently prevented rejection of RPE with EGFP expression. Fluorescence has been maintained for more than a year. If a control eye was injected with the same virus after terminating immunosuppression, both eyes showed signs of rejection. The lack of rejection is not due to tolerance but to a failure of the animals to detect the foreign protein. Detection must depend upon a brief window of time after surgery needed to introduce the vector, perhaps related to a concurrent but transient inflammation. This strategy may be useful in managing other types of rejection in the retina.

High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34+ cells

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 445-452 ◽  
Author(s):  
Robert E. Donahue ◽  
Robert P. Wersto ◽  
James A. Allay ◽  
Brian A. Agricola ◽  
Mark E. Metzger ◽  
...  
Keyword(s):  
Stem Cell ◽  
Green Fluorescent Protein ◽  
Peripheral Blood ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Rhesus Macaques ◽  
Peripheral Circulation ◽  
Egfp Expression ◽  
Cd34 Cells ◽  
Green Fluorescent

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34+ cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4+, CD8+, CD20+, and CD16/56+ lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP+ myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol.

scholarly journals Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 224 ◽  
Author(s):  
Lang Jin ◽  
Shahid Mehmood ◽  
Giikailang Zhang ◽  
Yuwei Song ◽  
Songkun Su ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Honey Bees ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Apis Cerana ◽  
Egfp Expression ◽  
Sacbrood Virus ◽  
Full Genomic Sequence ◽  
Green Fluorescent ◽  
Positive Strand Rna

Sacbrood virus (SBV) of honey bees is a picornavirus in the genus Iflavirus. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3′ untranslated region (UTR) and poly(A) can retain added sequences. We added enhanced green fluorescent protein (EGFP) expression at this site, creating a SBV clone with an expression tag that does not affect virus genes. An intergenic region internal ribosome entry site (IRES) from Black queen cell virus (BQCV) was inserted to initiate EGFP expression. The SBV-IRES-EGFP clone successfully infected Apis cerana and Apis mellifera, and in A. cerana larvae, it was isolated and passaged using oral inoculation. The inoculated larvae had higher mortality and the dead larvae showed sacbrood symptoms. The added IRES-EGFP remained in the clone through multiple passages and expressed the expected EGFP in all infected bees. We demonstrated the ability to add gene sequences in the site between 3′-UTR and poly(A) in SBV and the potential to do so in other bee iflaviruses; however, further investigations of the mechanisms are needed. A clone with a desired protein expression reporter will be a valuable tool in bee virus studies.

Recapitulating the Expression Pattern of Jak2 in Transgenic Zebrafish Using Jak2 Promoters and a GFP Reporter Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1156-1156
Author(s):  
Jing Zhang ◽  
Hui-Feng Lin ◽  
Robert I. Handin
Keyword(s):  
Green Fluorescent Protein ◽  
Expression Pattern ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Translation Initiation Site ◽  
Transgenic Zebrafish ◽  
Intermediate Cell ◽  
Green Fluorescent

Abstract The non-receptor tyrosine kinase Jak2 plays an important role in regulating erythro and thrombopoiesis. There has been intense interest in Jak2 since the observation that an activating mutation V617F is present in almost all patients with Polycythemia vera and many patients with Essential Thrombocytosis and Myelofibrosis. The analysis of Jak2 function in vivo has been limited as the murine jak2 knockout is lethal at day 10.5 of embryogenesis. Our laboratory has taken advantage of an ancestral partial duplication of the zebrafish genome, which has yielded two jak2 alleles --- jak2a and jak2b to study jak2 expression and function. Whole mount in situ hybridization studies confirm that the jak2a gene is only expressed in hematopoietic tissues, while jak2b is expressed in the developing lens and nephritic ducts. We have cloned and characterized the full-length jak2a and jak2b cDNAs and characterized the jak2a and 2b genomic loci. The jak2b gene has 24 exons spanning 79kb of genomic DNA. We amplified a 4kb zebrafish genomic fragment upstream of the first exon of the jak2b gene and linked it to the enhanced green fluorescent protein (EGFP) cDNA reporter and then microinjected the construct into single-cell zebrafish embryos. At 24 hours post fertilization (hpf), we observed fluorescence in the lens and nephritic ducts of developing embryos, with some expression in skin, muscle and notochord. The jak2a gene locus is complex as the jak2a gene is linked to a gene of unknown function, STARD4, in a head-to-tail manner with a small intergenic region of 1kb. As observed with jak2b, the first exon contains the jak2b 5′-UTR and the second exon contains the translation initiation site. We cloned a 1.9kb DNA fragment that included exons 1 and 2 the intervening first intron and an additional 800bp upstream of exon 1. This 1.9 kb promoter fragment was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that recapitulated the native expression pattern of jak2a. In injected embryos 24hpf, GFP+ cells were present in the anterior intermediate cell mass (ICM) and the lens. Fluorescent circulating blood cells, largely erythrocytes, were detected in 12 of 124 microinjected embryos 48 hours after fertilization. The jak2-EGFP transgenic zebrafish strains should be useful in the study of normal and pathologic hematopoiesis and in future studies of the pathogenesis of the MPDs.

scholarly journals Attenuation of Simian Varicella Virus Infection by Enhanced Green Fluorescent Protein in Rhesus Macaques

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Ravi Mahalingam ◽  
Benedikt B. Kaufer ◽  
Werner J. D. Ouwendijk ◽  
Georges M. G. M. Verjans ◽  
Colin Coleman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Varicella Zoster Virus ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Rhesus Macaques ◽  
Egfp Expression ◽  
Varicella Zoster ◽  
Varicella Virus ◽  
Green Fluorescent

ABSTRACT Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo . One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis. IMPORTANCE SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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