scholarly journals Progenitor Stage-Specific Activity of acis-Acting Double GATA Motif forGata1Gene Expression

2014 ◽  
Vol 35 (5) ◽  
pp. 805-815 ◽  
Author(s):  
Takashi Moriguchi ◽  
Mikiko Suzuki ◽  
Lei Yu ◽  
Jun Takai ◽  
Kinuko Ohneda ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Germ Line ◽  
Specific Activity ◽  
Artificial Chromosome ◽  
Mutant Mice ◽  
Hematopoietic Progenitors ◽  
Gfp Expression ◽  
Gfp Reporter ◽  
Green Fluorescent ◽  
Gata Motif

GATA1 is a master regulator of erythropoiesis, expression of which is regulated by multiple discretecis-acting elements. In this study, we examine the activity of a promoter-proximal double GATA (dbGATA) motif, using aGata1bacterial artificial chromosome (BAC)-transgenic green fluorescent protein (GFP) reporter (G1BAC-GFP) mouse system. Deletion of the dbGATA motif led to significant reductions in GFP expression in hematopoietic progenitors, while GFP expression was maintained in erythroblasts. Consistently, in mice with a germ line deletion of the dbGATA motif (Gata1ΔdbGATAmice), GATA1 expression in progenitors was significantly decreased. The suppressed GATA1 expression was associated with a compensatory increase in GATA2 levels in progenitors. When we crossedGata1ΔdbGATAmice withGata2hypomorphic mutant mice (Gata2fGN/fGNmice), theGata1ΔdbGATA::Gata2fGN/fGNcompound mutant mice succumbed to a significant decrease in the progenitor population, whereas both groups of single mutant mice maintained progenitors and survived to adulthood, indicating the functional redundancy between GATA1 and GATA2 in progenitors. Meanwhile, the effects of the dbGATA site deletion onGata1expression were subtle in erythroblasts, which showed increased GATA1 binding and enhanced accumulation of active histone marks around the 1st-intron GATA motif of the ΔdbGATAlocus. These results thus reveal a novel role of the dbGATA motif in the maintenance ofGata1expression in hematopoietic progenitors and a functional compensation between the dbGATA site and the 1st-intron GATA motif in erythroblasts.

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.

scholarly journals 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 ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cns Infection ◽  
Gfp Expression ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Differential Gene ◽  
Green Fluorescent

ABSTRACT Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungusCryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFα1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFα1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFα1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFα1 promoter is induced by nutrient deprivation and the MATα locus containing the MFα1 gene has been linked with virulence, yeast cells containing the pMFα1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFα1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFα1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.

scholarly journals Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Reproduction ◽  
2013 ◽  
Vol 145 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Shahin Eghbalsaied ◽  
Kamran Ghaedi ◽  
Götz Laible ◽  
Sayed Morteza Hosseini ◽  
Mohsen Forouzanfar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Gfp Expression ◽  
Exogenous Dna ◽  
Bovine Sperm ◽  
Gfp Fluorescence ◽  
Gfp Reporter ◽  
Green Fluorescent

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

scholarly journals Characterization of Sleeping Beauty Transposition and Its Application to Genetic Screening in Mice

2003 ◽  
Vol 23 (24) ◽  
pp. 9189-9207 ◽  
Author(s):  
Kyoji Horie ◽  
Kosuke Yusa ◽  
Kojiro Yae ◽  
Junko Odajima ◽  
Sylvia E. J. Fischer ◽  
...  
Keyword(s):  
Large Scale ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Donor Site ◽  
Insertion Site ◽  
Sleeping Beauty ◽  
Mutant Mice ◽  
Genome Wide ◽  
Green Fluorescent

ABSTRACT The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.

scholarly journals Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Danielle Weber-Adrian ◽  
Rikke Hahn Kofoed ◽  
Joseph Silburt ◽  
Zeinab Noroozian ◽  
Kairavi Shah ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Intravenous Injection ◽  
Viral Vectors ◽  
Focused Ultrasound ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Peripheral Organs ◽  
Neuronal Expression ◽  
Green Fluorescent ◽  
The Brain

AbstractNon-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

scholarly journals Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

2006 ◽  
Vol 17 (7) ◽  
pp. 3085-3094 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Anjon Audhya ◽  
Karen Oegema ◽  
Peter Schweinsberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Cortical Granule ◽  
Major Protein ◽  
Dynamic Regulation ◽  
Caveolin 1 ◽  
Green Fluorescent

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

2013 ◽  
Vol 95 (3) ◽  
pp. 319-329
Author(s):  
Atsushi Hirao ◽  
Tatsuo Kawarasaki ◽  
Kenjiro Konno ◽  
Satoko Enya ◽  
Masatoshi Shibata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Olfactory Epithelium ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Gfp Expression ◽  
Green Fluorescent

Abstract 38: Rbp-j Regulates the Genetic Program of the Myo-epithelioid JG Cell

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Ruth M Castellanos Rivera ◽  
Ellen S. Pentz ◽  
Kenneth W. Gross ◽  
Silvia Medrano ◽  
Jing Yu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Fluorescent Protein ◽  
Smooth Muscle Actin ◽  
Artificial Chromosome ◽  
Specific Protein ◽  
Genetic Program ◽  
Cell Phenotype ◽  
Gfp Expression ◽  
Renin Gene

RBP-J , the major downstream effector of Notch signaling, is necessary to maintain the number of juxtaglomerular (JG) cells. In addition, RBP-J regulates the plasticity of arteriolar smooth muscle cells to adopt the renin cell phenotype when homeostasis is threatened. We hypothesized that RBP-J acts as an on/off switch controlling the expression of genes that determine the renin phenotype. To determine whether RBP-J directly affects renin gene expression, we generated mice harboring a bacterial artificial chromosome (BAC) transgene with green fluorescent protein (GFP) under the control of the renin gene carrying a mutation in its RBP-J- binding site (Mut-BAC). Mut-BAC mice had markedly reduced GFP expression to 12.9 % ±0.01 (n=3) of the control (Wt-BAC) and a diminished response to homeostatic challenges: mut-BAC mice had a reduced number of GFP positive JG areas per total number of glomeruli (Wt-BAC: 25.1 % ±3.0, n=3; Mut-BAC: 9.3 % ±1.4, n=2, p<0.02) and no GFP expression along the arterioles. To determine whether the decrease in the number of JG cells in mice lacking RBP-J (cKO) was due to a diminished endowment of renin progenitor cells, we traced the fate of cells derived from the renin lineage by generating mice ( RBP-J fl/fl ; Ren1d +/cre ; R26R +/- ) in which cells lacking RBP-J simultaneously expressed β-galactosidase (β-gal). The pattern of β-gal in cKO and control kidneys was identical, indicating that cells derived from the renin lineage did not die but instead changed their phenotype. Next we investigated the phenotype adopted by the cells derived from the renin lineage. Expression of α-smooth muscle actin and smoothelin (a marker of mature smooth muscle) was significantly decreased to 41 % ±7.0 (n=2) and 44 % ±8.8 (n=2) respectively with respect to controls (p<0.01). In addition, mutant JG cells in vivo did not express genes characteristic of the renin phenotype such as renin, calponin1, Nfat and Akr1b7 expressing instead fibroblast-specific protein 1 indicating the adoption of a fibroblast-like phenotype. Results indicate that RBP-J directly governs a genetic program that controls the dual endocrine-contractile phenotype of the JG cell, which is crucial to maintain blood pressure and fluid-electrolyte homeostasis.

scholarly journals Regulation of RamA by RamR in Salmonella enterica Serovar Typhimurium: Isolation of a RamR Superrepressor

2012 ◽  
Vol 56 (11) ◽  
pp. 6037-6040 ◽  
Author(s):  
Vito Ricci ◽  
Stephen J. W. Busby ◽  
Laura J. V. Piddock
Keyword(s):  
Transcription Factor ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Promoter Sequence ◽  
Palindromic Sequence ◽  
Content Type ◽  
Gfp Reporter ◽  
Serovar Typhimurium ◽  
Green Fluorescent

ABSTRACTRamA is a transcription factor involved in regulating multidrug resistance inSalmonella entericaserovar Typhimurium SL1344. Green fluorescent protein (GFP) reporter fusions were exploited to investigate the regulation of RamA expression by RamR. We show that RamR represses theramApromoter by binding to a palindromic sequence and describe a superrepressor RamR mutant that binds to theramApromoter sequence more efficiently, thus exhibiting aramAinactivated phenotype.

scholarly journals Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

2018 ◽  
Vol 10 (4) ◽  
pp. 12
Author(s):  
Mahipal Singh ◽  
Xiaoling Ma
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Genetically Engineered ◽  
Dermal Fibroblasts ◽  
Biologically Active ◽  
Cell Type ◽  
Gfp Expression ◽  
Primary Fibroblasts ◽  
Species Specific ◽  
Green Fluorescent

Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression. 

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