scholarly journals Analysis of Green Fluorescent Protein Expression in Transgenic Rats for Tracking Transplanted Neural Stem/Progenitor Cells

2005 ◽  
Vol 53 (10) ◽  
pp. 1215-1226 ◽  
Author(s):  
Andrea J. Mothe ◽  
Iris Kulbatski ◽  
Rita L. van Bendegem ◽  
Linda Lee ◽  
Eiji Kobayashi ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Green Fluorescent Protein ◽  
Progenitor Cells ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Rat Strain ◽  
Direct Fluorescence ◽  
Green Fluorescent

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents—Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]—by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wildtype spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo.

scholarly journals A Replication-Competent Feline Leukemia Virus, Subgroup A (FeLV-A), Tagged with Green Fluorescent Protein Reporter Exhibits In Vitro Biological Properties Similar to Those of the Parental FeLV-A

2001 ◽  
Vol 75 (18) ◽  
pp. 8837-8841 ◽  
Author(s):  
Zongli Chang ◽  
Judong Pan ◽  
Christopher Logg ◽  
Noriyuki Kasahara ◽  
Pradip Roy-Burman
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Intradermal Injection ◽  
Feline Leukemia Virus ◽  
Gfp Expression ◽  
Entry Site ◽  
Green Fluorescent

ABSTRACT We previously established that lymphoid tumors could be induced in cats by intradermal injection of ecotropic feline leukemia virus (FeLV), subgroup A, plasmid DNA. In preparation for in vivo experiments to study the cell-to-cell pathway for the spread of the virus from the site of inoculation, the green fluorescent protein (GFP) transgene fused to an internal ribosome entry site (IRES) was inserted after the last nucleotide of theenv gene in the ecotropic FeLV-A Rickard (FRA) provirus. The engineered plasmid was transfected into feline fibroblast cells for production of viruses and determination of GFP expression. The virions produced were highly infectious, and the infected cells could continue to mediate strong expression of GFP after long-term propagation in culture. Similar to parental virus, the transgene-containing ecotropic virus demonstrated recombinogenic activity with endogenous FeLV sequences in feline cells to produce polytropic recombinant FeLV subgroup B-like viruses which also contained the IRES-GFP transgene in the majority of recombinants. To date, the engineered virus has been propagated in cell culture for up to 8 months without diminished GFP expression. This is the first report of a replication-competent FeLV vector with high-level and stable expression of a transgene.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

In vitro and in vivo evaluation of a green fluorescent protein-HSV1-TK dual-function pet reporter gene

2001 ◽  
Vol 44 (S1) ◽  
pp. S339-S341
Author(s):  
K. E. Luker ◽  
G. D. Luker ◽  
C. M. Pica ◽  
J. L. Dahlheimer ◽  
T. J. Fahrner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Dual Function ◽  
In Vivo Evaluation ◽  
Green Fluorescent

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

2010 ◽  
Vol 22 (1) ◽  
pp. 373
Author(s):  
M. Reichenbach ◽  
F. A. Habermann ◽  
H. D. Reichenbach ◽  
T. Guengoer ◽  
F. Weber ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Phosphoglycerate Kinase ◽  
Healthy Male ◽  
Transgenic Founder ◽  
Green Fluorescent

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.

Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cryptococcus Neoformans ◽  
Differential Gene Expression ◽  
Fluorescent Protein ◽  
Differential Gene ◽  
Green Fluorescent

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

2018 ◽  
Vol 194 ◽  
pp. 29-39 ◽  
Author(s):  
Fatemeh Motevalli ◽  
Azam Bolhassani ◽  
Shilan Hesami ◽  
Sepideh Shahbazi
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals In vivo analysis of key elements within the renin regulatory region

2008 ◽  
Vol 35 (3) ◽  
pp. 243-253 ◽  
Author(s):  
Sean T. Glenn ◽  
Craig A. Jones ◽  
Li Pan ◽  
Kenneth W. Gross
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Region ◽  
Renin Angiotensin System ◽  
Artificial Chromosome ◽  
Proximal Promoter ◽  
Gfp Expression ◽  
Renin Gene ◽  
Green Fluorescent

Renin is responsible for initiating the enzymatic cascade that results in the production of angiotensin II, the major effector molecule of the renin-angiotensin system (RAS). Extensive information on the regulatory region of the renin gene has been derived by transient transfection studies in vitro, particularly using the As4.1 cell line. To verify key factors within the regulatory region of renin in vivo, homologous recombination was used to introduce a green fluorescent protein (GFP) cassette into exon one of the renin gene contained within a 240 kb bacterial artificial chromosome (BAC) to create a construct that has GFP expression controlled by the renin regulatory region (RenGFP BAC). Within the regulatory region of the RenGFP BAC construct we independently deleted the enhancer, as well as mutated the HOX-PBX site within the proximal promoter element. Transgenic lines were generated for each of these BAC constructs and GFP expression was analyzed throughout a spectrum of tissues positive for renin expression including the kidney, adrenal gland, gonadal artery, and submandibular gland. The results described within this manuscript support the interpretation that the renin enhancer is critical for regulating baseline expression where as the Hox/Pbx site is important for the tissue specificity of renin expression.

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

2007 ◽  
Vol 196 (s2) ◽  
pp. S313-S322 ◽  
Author(s):  
Hideki Ebihara ◽  
Steven Theriault ◽  
Gabriele Neumann ◽  
Judie B. Alimonti ◽  
Joan B. Geisbert ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
In Vivo Characterization

scholarly journals Illuminating the Formation of Lumens

2007 ◽  
Vol 15 (3) ◽  
pp. 3-5
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Animal Model ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Solid Core ◽  
Red Fluorescent Protein ◽  
Neuronal Process ◽  
Two Photon ◽  
Green Fluorescent

How do lumens form? Two mechanisms that come readily to mind are a wrapping model, similar to the wrapping of the myelin sheath around a neuronal process, and a solid core of cells followed by apoptosis of the central cells. Another obvious mechanism that was suggested over 100 years ago is the fusion of intracellular vacuoles. Whereas several recent studies have supported this latter mechanism, it has not yet been proven. Now, the appropriate animal model (zebrafish), the modern techniques (transgenic chimeras), dyes (green fluorescent protein and monomeric red fluorescent protein) that can be linked to proteins to label vacuoles, and two-photon imaging in real time finally have provided the strongest support yet. In an article by Makoto Kamei, Brian Saunders, Kayla Bayless, Louis Dye, George Davis, and Brant Weinstein the assembly of endothelial tubes from intracellular vacuoles was observed in vitro and in vivo.

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