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.

Smooth muscle expression of Cre recombinase and eGFP in transgenic mice

2002 ◽  
Vol 10 (3) ◽  
pp. 211-215 ◽  
Author(s):  
H.-B. Xin ◽  
K.-Y. Deng ◽  
M. Rishniw ◽  
G. Ji ◽  
M. I. Kotlikoff
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Transgenic Mice ◽  
Gene Function ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Smooth Muscles ◽  
Cre Recombinase ◽  
Specific Expression

We report the generation of transgenic mice designed to facilitate the study of vascular and nonvascular smooth muscle biology in vivo. The smooth muscle myosin heavy chain (smMHC) promoter was used to direct expression of a bicistronic transgene consisting of Cre recombinase and enhanced green fluorescent protein (eGFP) coding sequences. Animals expressing the transgene display strong fluorescence confined to vascular and nonvascular smooth muscle. Enzymatic dissociation of smooth muscle yields viable, fluorescent cells that can be studied as single cells or sorted by FACS for gene expression studies. smMHC/Cre/eGFP mice were crossed with ROSA26/lacZ reporter mice to determine Cre recombinase activity; Cre recombinase was expressed in all smooth muscles in adult mice, and there was an excellent overlap between expression of the recombinase and eGFP. Initial smooth muscle-specific expression of fluorescence and Cre recombinase was detected on embryonic day 12.5. These mice will be useful to define smooth muscle gene function in vivo in mice, for the study of gene function in single, live cells, and for the determination of gene expression in vascular and nonvascular smooth muscle.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

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 In vivo visualization of uterine mast cells by two-photon microscopy

Reproduction ◽  
2014 ◽  
Vol 147 (6) ◽  
pp. 781-788 ◽  
Author(s):  
Franziska Schmerse ◽  
Katja Woidacki ◽  
Monika Riek-Burchardt ◽  
Peter Reichardt ◽  
Axel Roers ◽  
...  
Keyword(s):  
Mast Cells ◽  
Intravital Microscopy ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Specific Cell ◽  
Enhanced Yellow Fluorescent Protein ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Custom Made

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for the study ofin vivobehavior of these cells. We have recently reported that uterine mast cells (uMCs) are important for implantation and placentation. However, theirin vivolocalization in uterus before and during pregnancy is unknown. Herein, we report the direct observation of uMCsin vivousing double-transgenic C57BL/6JMcpt5-Cre ROSA26-EYFPmice with high expression of enhanced yellow fluorescent protein in MC protease 5 (Cma1(Mcpt5))-expressing cells by intravital two-photon microscopy. We were able to monitor MCs livein uteroduring the murine estrous cycle and at different days of pregnancy. We demonstrated that uMCs accumulated during the receptive phase of the female (estrus) and persisted in large numbers at early pregnancy stages and around mid-gestation and declined in number in non-pregnant animals at diestrus. This intravital microscopy technique, including a custom-made microscope stage and the adaption of the surgical procedure, allowed the access of the uterus and implantations for imaging. The introduced application of intravital microscopy to C57BL/6J-Mcpt5-Cre ROSA26-EYFPmice offers a novel and powerfulin vivoapproach to further address the evident relevance of uMCs to reproductive processes with obvious clinical implications.

scholarly journals In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles

2002 ◽  
Vol 157 (4) ◽  
pp. 615-629 ◽  
Author(s):  
Anthony K.L. Leung ◽  
Angus I. Lamond
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nucleolar Localization ◽  
Target Sequence ◽  
Enhanced Yellow Fluorescent Protein ◽  
Splicing Speckles

The NHPX protein is a nucleolar factor that binds directly to a conserved RNA target sequence found in nucleolar box C/D snoRNAs and in U4 snRNA. Using enhanced yellow fluorescent protein (EYFP)– and enhanced cyan fluorescent protein–NHPX fusions, we show here that NHPX is specifically accumulated in both nucleoli and Cajal bodies (CBs) in vivo. The fusion proteins display identical localization patterns and RNA binding specificities to the endogenous NHPX. Analysis of a HeLa cell line stably expressing EYFP–NHPX showed that the nucleolar accumulation of NHPX was preceded by its transient accumulation in splicing speckles. Only newly expressed NHPX accumulated in speckles, and the nucleolar pool of NHPX did not interchange with the pool in speckles, consistent with a unidirectional pathway. The transient accumulation of NHPX in speckles prior to nucleoli was observed in multiple cell lines, including primary cells that lack CBs. Inhibitor studies indicated that progression of newly expressed NHPX from speckles to nucleoli was dependent on RNA polymerase II transcription, but not on RNA polymerase I activity. The data show a specific temporal pathway involving the sequential and directed accumulation of NHPX in distinct subnuclear compartments, and define a novel mechanism for nucleolar localization.

scholarly journals Epidermolysis Bullosa Simplex-Type Mutations Alter the Dynamics of the Keratin Cytoskeleton and Reveal a Contribution of Actin to the Transport of Keratin Subunits

2004 ◽  
Vol 15 (3) ◽  
pp. 990-1002 ◽  
Author(s):  
Nicola Susann Werner ◽  
Reinhard Windoffer ◽  
Pavel Strnad ◽  
Christine Grund ◽  
Rudolf Eberhard Leube ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Fluorescent Protein ◽  
Dynamic Equilibrium ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Epidermolysis Bullosa Simplex ◽  
Enhanced Yellow Fluorescent Protein ◽  
Dominant Negative Mutation

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.

scholarly journals Constitutive Somatostatin Receptor Subtype 2 Activity Attenuates GH Synthesis

Endocrinology ◽  
2013 ◽  
Vol 154 (7) ◽  
pp. 2399-2409 ◽  
Author(s):  
Anat Ben-Shlomo ◽  
Oxana Pichurin ◽  
Ramtin Khalafi ◽  
Cuiqi Zhou ◽  
Vera Chesnokova ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Fluorescent Protein ◽  
Somatostatin Receptor ◽  
Yellow Fluorescent Protein ◽  
Histone Deacetylation ◽  
Receptor Subtype ◽  
Mrna Levels ◽  
Enhanced Yellow Fluorescent Protein ◽  
Stable Transfectants

Abstract Somatostatin signals predominantly through somatostatin receptor (SSTR) subtype 2 to attenuate GH release. However, the independent role of the receptor in regulating GH synthesis is unclear. Because we had previously demonstrated constitutive SSTR2 activity in mouse corticotrophs, we now analyzed GH regulation in rat pituitary somatotroph (GC) tumor cells, which express SSTR2 exclusively and are devoid of endogenous somatostatin ligand. We demonstrate that moderately stable SSTR2 overexpression (GpSSTR2WT cells) was associated with decreased GH promoter activity, GH mRNA, and hormone levels compared with those of control transfectants (GpCon cells). In contrast, levels of GH mRNA and peptide and GH promoter activity were unchanged in GpSSTR2DRY stable transfectants moderately expressing DRY motif mutated SSTR2 (R140A). GpSSTR2DRY did not exhibit an enhanced octreotide response as did GpSSTR2WT cells; however, both SSTR2WT-enhanced yellow fluorescent protein (eYFP) and SSTR2DRY-eYFP internalized on octreotide treatment. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, increased GH synthesis in wild-type GC cells and primary pituitary cultures. GpSSTR2WT cells induced GH synthesis more strongly on SAHA treatment, evident by both higher GH peptide and mRNA levels compared with the moderate but similar GH increase observed in GpCon and GpSSTR2DRY cells. In vivo SAHA also increased GH release from GpSSTR2WT but not from control xenografts. Endogenous rat GH promoter chromatin immunoprecipitation showed decreased baseline acetylation of the GH promoter with exacerbated acetylation after SAHA treatment in GpSSTR2WT compared with that of either GpSSTR2DRY or control cells, the latter 2 transfectants exhibiting similar GH promoter acetylation levels. In conclusion, modestly increased SSTR2 expression constitutively decreases GH synthesis, an effect partially mediated by GH promoter histone deacetylation.

scholarly journals Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

2015 ◽  
Vol 81 (24) ◽  
pp. 8277-8293 ◽  
Author(s):  
Tony Tumlirsch ◽  
Anna Sznajder ◽  
Dieter Jendrossek
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Proteome Analysis ◽  
Enhanced Yellow Fluorescent Protein ◽  
Granule Formation ◽  
Content Type ◽  
Cell Extracts ◽  
C And N

ABSTRACTA protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion ofphaXresulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs.In vivocolocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of theRalstonia eutropha ppk(ppkReu) genes in anEscherichia coliΔppkbackground and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds.

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