scholarly journals GFP transgenic animals in biomedical research: a review of potential disadvantages

2019 ◽  
pp. 525-530
Author(s):  
N. Lipták ◽  
Z. Bősze ◽  
L. Hiripi
Keyword(s):  
Transgenic Mice ◽  
Biomedical Research ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Transgenic Animals ◽  
In Vivo Studies ◽  
Reporter Protein ◽  
Physiological Processes ◽  
Green Fluorescent

Green Fluorescent protein (GFP) transgenic animals are accepted tools for studying various physiological processes, including organ development and cell migration. However, several in vivo studies claimed that GFP may impair transgenic animals’ health. Glomerulosclerosis was observed in transgenic mice and rabbits with ubiquitous reporter protein expression. Heart-specific GFP expression evoked dilated cardiomyopathy and altered cardiac function in transgenic mouse and zebrafish lines, respectively. Moreover, growth retardation and increased axon swelling were observed in GFP and yellow fluorescent protein (YFP) transgenic mice, respectively. This review will focus on the potential drawbacks of the applications of GFP transgenic animals in biomedical research.

scholarly journals Targeting Mucosal Sites by Polymeric Immunoglobulin Receptor-directed Peptides

2002 ◽  
Vol 196 (4) ◽  
pp. 551-555 ◽  
Author(s):  
Kendra D. White ◽  
J. Donald Capra
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Secretory Component ◽  
In Vivo Studies ◽  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor ◽  
Potential Binding Site ◽  
Green Fluorescent

Polymeric immunoglobulins provide first line humoral defense at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR) on mucosal and glandular epithelial cells. Previous studies from our laboratory suggested that amino acids 402–410 of the Cα3 domain of dimeric IgA (dIgA) represented a potential binding site for the pIgR. Here by binding human secretory component to overlapping decapeptides of Cα3, we confirm these residues and also uncover an additional site. Furthermore, we show that the Cα3 motif appears to be sufficient to direct transport of green fluorescent protein through the pIgR-specific cellular transcytosis system. An alternative approach identified phage peptides, selected from a library by the in vitro Madin Darby Canine Kidney transcytosis assay, for pIgR-mediated transport through epithelial cells. Some transcytosis-selected peptides map to the same 402–410 pIgR-binding Cα3 site. Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport. In addition to identifying small peptides that are both bound and transported by the pIgR, this study provides evidence that the pIgR-mediated mucosal secretion system may represent a means of targeting small molecule therapeutics and genes to mucosal epithelial cells.

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.

scholarly journals Clp and Lon Proteases Occupy Distinct Subcellular Positions in Bacillus subtilis

2008 ◽  
Vol 190 (20) ◽  
pp. 6758-6768 ◽  
Author(s):  
Lyle A. Simmons ◽  
Alan D. Grossman ◽  
Graham C. Walker
Keyword(s):  
Bacillus Subtilis ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Normal Growth ◽  
Temporal Organization ◽  
Hexameric Atpase ◽  
Fluorescent Moiety ◽  
Green Fluorescent

ABSTRACT Among other functions, ATP-dependent proteases degrade misfolded proteins and remove several key regulatory proteins necessary to activate stress responses. In Bacillus subtilis, ClpX, ClpE, and ClpC form homohexameric ATPases that couple to the ClpP peptidase. To understand where these peptidases and ATPases localize in living cells, each protein was fused to a fluorescent moiety. We found that ClpX-GFP (green fluorescent protein) and ClpP-GFP localized as focal assemblies in areas that were not occupied by the nucleoid. We found that the percentage of cells with ClpP-GFP foci increased following heat shock independently of protein synthesis. We determined that ClpE-YFP (yellow fluorescent protein) and ClpC-YFP formed foci coincident with nucleoid edges, usually near cell poles. Furthermore, we found that ClpQ-YFP (HslV) localized as small foci, usually positioned near the cell membrane. We found that ClpQ-YFP foci were dependent on the presence of the cognate hexameric ATPase ClpY (HslU). Moreover, we found that LonA-GFP is coincident with the nucleoid during normal growth and that LonA-GFP also localized to the forespore during development. We also investigated LonB-GFP and found that this protein localized to the forespore membrane early in development, followed by localization throughout the forespore later in development. Our comprehensive study has shown that in B. subtilis several ATP-fueled proteases occupy distinct subcellular locations. With these data, we suggest that substrate specificity could be determined, in part, by the spatial and temporal organization of proteases in vivo.

scholarly journals Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters

2010 ◽  
Vol 76 (17) ◽  
pp. 5990-5994 ◽  
Author(s):  
Thomas Drepper ◽  
Robert Huber ◽  
Achim Heck ◽  
Franco Circolone ◽  
Anne-Kathrin Hillmer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Flavin Mononucleotide ◽  
Fluorescence Signal ◽  
Reporter Protein ◽  
Reporter Proteins ◽  
Green Fluorescent

ABSTRACT Fluorescent proteins of the green fluorescent protein (GFP) family are commonly used as reporter proteins for quantitative analysis of complex biological processes in living microorganisms. Here we demonstrate that the fluorescence signal intensity of GFP-like proteins is affected under oxygen limitation and therefore does not reflect the amount of reporter protein in Escherichia coli batch cultures. Instead, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs) are suitable for quantitative real-time in vivo assays under these conditions.

Thick ascending limb-specific expression of Cre recombinase

2003 ◽  
Vol 285 (1) ◽  
pp. F33-F39 ◽  
Author(s):  
Peter K. Stricklett ◽  
Deborah Taylor ◽  
Raoul D. Nelson ◽  
Donald E. Kohan
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Yellow Fluorescent Protein ◽  
Cre Recombinase ◽  
Specific Gene ◽  
Thick Ascending Limb ◽  
Specific Expression ◽  
Enhanced Yellow Fluorescent Protein

Evaluation of thick ascending limb (TAL) function has been hindered by the limited ability to selectively examine the function of this nephron segment in vivo. To address this, a Cre/loxP strategy was employed whereby the Tamm-Horsfall (THP) promoter was used to drive Cre recombinase expression in transgenic mice. The THP gene was cloned from a mouse genomic library, and 3.7 kb of the mouse THP 5′-flanking region containing the first noncoding exon of the THP gene were inserted upstream of an epitope-tagged Cre recombinase (THP-CreTag). THP-CreTag transgenic mice were bred with ROSA26-enhanced yellow fluorescent protein (eYFP) mice (contain a loxP-flanked “STOP” sequence 5′ to eYFP), and doubly heterozygous offspring were analyzed. THP and eYFP were expressed in an identical pattern with predominant localization to the renal outer medulla without expression in nonrenal tissues. eYFP did not colocalize with thiazide-sensitive cotransporter (distal tubule) or neuronal nitric oxide synthase (macula densa) expression. THP mRNA expression was detected only in kidney, whereas CreTag mRNA was also present in testes. These data indicate that THP-CreTag transgenic mice can be used for TAL-specific gene recombination in the kidney.

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