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At least 487 records · Page 27Linked to original sources
PubMed2005

[Experimental study of dually targeting gene therapy system for pituitary adenomas].

OBJECTIVE: To construct a dually targeting gene therapy system for pituitary adenomas and investigate its effect. METHODS: Promoter hGHp containing human growth hormone gene was obtained from human genome and cloned into the plasmid pcDNA3.1/His A with the promoter cut to construct the recombinant plasmid pcDNA3.1/His A-hGHp. HSV-TK gene was obtained from the plasmid pcDNA3.1/His A-TK and integrated into the plasmid pcDNA3.1/His A-hGHp to construct the recombinant plasmid pcDNA3.1/His A-hGHp-TK. A GE7 gene delivery system-mediated human growth hormone promoter controlled gene therapy system was constructed by adding the mixture of GE7-polylysine and HA20-polylysine into the DNA solution. Human growth hormone-secreting pituitary adenoma cells of the GH3 line, human myeloma cells of the U-2OS line, and human oophoroma cells of the HO8910PM line were cultured and transfected with PBS or GE7 packaged pcDNA3.1/HisA-TK or pcDNA3.1/HisA-hGHp-TK. Western blotting was used to examine the expression of PBS or GE7 packaged pcDNA3.1/HisA-TK or pcDNA3.1/HisA-hGHp-TK protein, MTT method was used to detect the cell survival rate. Another GH3, U-2OS, and HO8910PM cells were cultured and transfected with PBS or GE7 packaged pcDNA3.1/HisA-TK or pcDNA3.1/HisA-hGHp-TK and then ganciclovir (GCV) was added. MTT method was used to examine the cell survival rates. GH3 cells were injected subcutaneously into the right axilla of 200 SD nude rats loaded with human pituitary adenoma. Three weeks after the rats were randomly divided into 5 equal groups: PBS group in which PBS was injected into the tumor and GCV was injected peritoneally; GE7 group in which GE7-polylysine and HA20-polylysine were injected into the tumor and GCV was injected peritoneally; without TK group in which GE7-packaged pcDNA3.1/His A-hGHp was injected into the tumor and GCV was injected peritoneally; without GCV group in which GE7-packaged pcDNA3.1/His A-hGHp-TK was injected into the tumor and PBS was injected peritoneally; and treatment group in which GE7-packaged pcDNA3.1/His A-hGHp-TK was injected into the tumor and GCV was injected peritoneally. Peritoneal injection lasted 21 days for all groups. On the days 3, 7, 14, and 21 eight rats from each group were killed to measure the volume of tumor. The survival rate of the rest 8 rats was observed. RESULTS: A dually targeting gene therapy system for pituitary adenoma was composed successfully. HSV-TK protein was expressed in the GH3 cells but not in the U-2OS and HO8910PM cells after transfection of GE7-packaged pcDNA3.1/HisA-hGHp-TK; and was expressed in the GH3 and HO8910PM cells but not in the U-2OS cells after transfection of GE7-packaged pcDNA3.1/HisA-TK. Transfection of GE7-packaged pcDNA3.1/HisA-hGHp-TK and addition of GCV significantly decreased the survival rate of the GH3 cells, but did not influence the survival rates of the U-2OS and HO8910PM cells. Transfection of GE7-packaged pcDNA3.1/HisA-TK and addition of GCV significantly decreased the survival rate of GH3 and HO8910PM cells but did not influence the survival of the U-2OS and HO8910PM cells. When the GH3 cells were transfected with GE7-packaged pcDNA3.1/HisA-hGHp-TK with the addition of GCV of the concentration of 4 mg/L the survival rate decreased to 10%, when the GCV concentration was raised to 8 mg/L the survival rate of the GH3 cells was < 5%. Three days after the beginning of treatment the tumor volume of different groups of rats increased at different degrees and the tumor was smallest in the treatment group in comparison with the other groups (all P < 0.05). Seven days after the beginning of treatment the tumor volume of the treatment group significantly decreased and the tumors of the other groups still increased (all P < 0.001). The survival time of the treatment group was over 120 days, significantly longer than those of the other groups (all about 40 days). CONCLUSION: GE7 system-mediated hGHp controlled gene therapy system is hopeful to be the targeted therapeutic strategy for pituitary adenomas.

Adenoma↗
PubMed2005

Hedgehog signaling in mouse ovary: Indian hedgehog and desert hedgehog from granulosa cells induce target gene expression in developing theca cells.

Follicle development in the mammalian ovary requires interactions among the oocyte, granulosa cells, and theca cells, coordinating gametogenesis and steroidogenesis. Here we show that granulosa cells of growing follicles in mouse ovary act as a source of hedgehog signaling. Expression of Indian hedgehog and desert hedgehog mRNAs initiates in granulosa cells at the primary follicle stage, and we find induced expression of the hedgehog target genes Ptch1 and Gli1, in the surrounding pre-theca cell compartment. Cyclopamine, a highly specific hedgehog signaling antagonist, inhibits this induced expression of target genes in cultured neonatal mouse ovaries. The theca cell compartment remains a target of hedgehog signaling throughout follicle development, showing induced expression of the hedgehog target genes Ptch1, Ptch2, Hip1, and Gli1. In periovulatory follicles, a dynamic synchrony between loss of hedgehog expression and loss of induced target gene expression is observed. Oocytes are unable to respond to hedgehog because they lack expression of the essential signal transducer Smo (smoothened). The present results point to a prominent role of hedgehog signaling in the communication between granulosa cells and developing theca cells.

Animals↗
PubMed2005

Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target genes.

In higher eukaryotes, the Retinoblastoma and E2F families of proteins control the transcription of a large number of target genes. Here, we have mutated the second Drosophila Retinoblastoma family gene (Rbf2), and contrasted the in vivo molecular functions of RBF2 with dE2F2, the only E2F partner of RBF2. Previous studies failed to uncover a unique role for RBF2 in E2F regulation. Here we find that RBF2 functions in concert with dE2F2 in vivo to repress the expression of differentiation markers in ovaries and embryos where RBF2 is highly expressed. We have compared the profiles of transcripts that are mis-expressed in ovaries, embryos and S2 cells where RBF2 function has been ablated and find that RBF2 and dE2F2 control strikingly different transcriptional programs in each situation. In vivo promoter occupancy studies point to the redistribution of dE2F/RBF complexes to different promoters in different cell types as one mechanism governing the tissue-specific regulation of dE2F/RBF target genes. These results demonstrate that RBF2 has a unique function in repressing E2F-regulated differentiation markers and that dE2F2 and RBF2 are required to regulate different sets of target genes in different tissues.

Alleles↗
PubMed1999

What have we learned from gene targeting studies for the renin angiotensin system of the kidney?

Over the last decade, gene targeting technologies have provided investigators with powerful new tools to study the physiology and pathophysiology of the kidney. In that, the renin-angiotensin system (RAS) has been a subject of intense investigation. Detailed analyses of mutant mice have not only confirmed notions already suggested by other studies, but also shed a new light on previously unrecognized functions of RAS. In this review, we will focus on what we have learned from these gene targeted animals in particular relevance to nephrology.

Animals↗
PubMed2004

The proneural proteins Atonal and Scute regulate neural target genes through different E-box binding sites.

For a particular functional family of basic helix-loop-helix (bHLH) transcription factors, there is ample evidence that different factors regulate different target genes but little idea of how these different target genes are distinguished. We investigated the contribution of DNA binding site differences to the specificities of two functionally related proneural bHLH transcription factors required for the genesis of Drosophila sense organ precursors (Atonal and Scute). We show that the proneural target gene, Bearded, is regulated by both Scute and Atonal via distinct E-box consensus binding sites. By comparing with other Ato-dependent enhancer sequences, we define an Ato-specific binding consensus that differs from the previously defined Scute-specific E-box consensus, thereby defining distinct E(Ato) and E(Sc) sites. These E-box variants are crucial for function. First, tandem repeats of 20-bp sequences containing E(Ato) and E(Sc) sites are sufficient to confer Atonal- and Scute-specific expression patterns, respectively, on a reporter gene in vivo. Second, interchanging E(Ato) and E(Sc) sites within enhancers almost abolishes enhancer activity. While the latter finding shows that enhancer context is also important in defining how proneural proteins interact with these sites, it is clear that differential utilization of DNA binding sites underlies proneural protein specificity.

3' Flanking Region↗
PubMed2000

Peroxisome proliferator-activated receptor gamma target gene encoding a novel angiopoietin-related protein associated with adipose differentiation.

The nuclear receptor peroxisome proliferator-activated receptor gamma regulates adipose differentiation and systemic insulin signaling via ligand-dependent transcriptional activation of target genes. However, the identities of the biologically relevant target genes are largely unknown. Here we describe the isolation and characterization of a novel target gene induced by PPARgamma ligands, termed PGAR (for PPARgamma angiopoietin related), which encodes a novel member of the angiopoietin family of secreted proteins. The transcriptional induction of PGAR follows a rapid time course typical of immediate-early genes and occurs in the absence of protein synthesis. The expression of PGAR is predominantly localized to adipose tissues and placenta and is consistently elevated in genetic models of obesity. Hormone-dependent adipocyte differentiation coincides with a dramatic early induction of the PGAR transcript. Alterations in nutrition and leptin administration are found to modulate the PGAR expression in vivo. Taken together, these data suggest a possible role for PGAR in the regulation of systemic lipid metabolism or glucose homeostasis.

3T3 Cells↗
PubMed2007

Genome instability and DNA damage accumulation in gene-targeted mice.

Six major pathways for DNA repair have been identified. These include (1) DNA repair by direct reversal, (2) base excision repair, (3) mismatch repair, (4) nucleotide excision repair, (5) homologous recombination, and (6) non-homologous end-joining. In addition, several other cellular processes influence the response to DNA damage. The generation of gene-targeted organisms is crucial for assessing the relative contribution of single DNA repair proteins and DNA repair pathways in maintaining genome stability. In particular, the accumulation of DNA damage, mutations and cancer in unexposed gene-targeted animals illuminates the spontaneous load of a particular lesion and the relative significance of a single gene in a specific pathway. Strategies for the generation of gene-targeted mice have been available for 15 years and more than 100 different genes relevant to DNA repair have been targeted. This review describes some important progress made toward understanding spontaneous DNA damage and its repair, exemplified through one, or a few, gene-targeted mice from each major DNA repair pathway.

Animals↗
PubMed2002

Brachyury proteins regulate target genes through modular binding sites in a cooperative fashion.

Brachyury proteins, a conserved subgroup of the T domain transcription factors, specify gut and posterior mesoderm derivatives throughout the animal kingdom. The T domain confers DNA-binding properties to Brachyury proteins, but little is known how these proteins regulate their target genes. We characterized a direct target gene of the Drosophila Brachyury-homolog Brachyenteron. Brachyenteron activates the homeobox gene orthopedia in a dose-dependent manner via multiple binding sites with the consensus (A/G)(A/T)(A/T)NTN(A/G)CAC(C/T)T. The sites and their A/T-rich flanking regions are conserved between D. melanogaster and Drosophila virilis. Reporter assays and site-directed mutagenesis demonstrate that Brachyenteron binding sites confer in part additive, in part synergistic effects on otp transcription levels. This suggests an interaction of Brachyenteron proteins on the DNA, which we could map to a conserved motif within the T domain. Mouse Brachyury also interacts with Brachyenteron through this motif. We further show that the Xenopus and mouse Brachyury homologs activate orthopedia expression when expressed in Drosophila embryonic cells. We propose that the mechanisms to achieve target gene expression through variable binding sites and through defined protein-protein interactions might be conserved for Brachyury relatives.

Amino Acid Motifs↗
PubMed2005

Identification of the human ApoAV gene as a novel RORalpha target gene.

Retinoic acid receptor-related orphan receptor-alpha (RORalpha) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that RORalpha regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that RORalpha also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of RORalpha increased the endogenous expression of ApoAV in HepG2 cells and RORalpha also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate RORalpha transactivation, one of which overlaps with a previously identified binding site for PPARalpha. Together, these results suggest a novel mechanism whereby RORalpha modulates lipid metabolism and implies RORalpha as a potential target for the treatment of dyslipidemia and atherosclerosis.

Apolipoprotein A-V↗
PubMed2004

[A strategy for targeting gene therapy against cancer mediated by epidermal growth factor receptor].

OBJECTIVE: To establish a protocol for the targeting gene therapy against cancer with rich epidermal growth factor receptor (EGFR). METHODS: A recombinant pcDNA3.1-PE III mut was constructed and combined with a non-viral vector, a fusion protein histone H1, epidermal growth factor C-loop previously expressed by us, to be a protein-DNA complex in vitro. Using the complex to treat BT-325 and Hela cancer cells with EGFR and JK cells without EGFR. The killing rates of the cells was calculated after 48 h of incubation at 37 degrees C. RESULTS: To BT-325 and Hela cells, the killing rates were 46.03% and 48.12% respectively. To JK cells, the complex had no killing function. CONCLUSION: The protocol for targeting gene therapy against cancer with EGFR has been established successfully.

ADP Ribose Transferases↗
PubMed1995

Gene-targeting and transgenic approaches to IGF and IGF binding protein function.

The ability to manipulate genetic information in the germ line of mice has provided powerful approaches to study gene function in vivo. These approaches have included the establishment of mouse lines in which a specified gene or genes are overexpressed, ectopically expressed, or deleted. Transgenic and gene-targeted mouse lines have been used extensively to study the function of the insulin-like growth factors (IGF), IGF-I and IGF-II, and their receptors and binding proteins. In the IGF system, these technologies have elucidated the roles of the IGFs in fetal and somatic growth and have demonstrated a critical role for this system in transformation and tumorigenesis. Analysis of combinatorial crosses of gene-targeted mouse lines also has suggested the existence of an as yet unidentified IGF receptor that regulates fetal growth. Similar approaches using transgenic and gene-targeted mouse models have been initiated to study the in vivo functions of the IGF binding proteins. These mouse models provide important tools to test specific functional questions in vivo as well as to study the long-term physiological consequences of chronic gene alterations.

Animals↗
PubMed2001

Construction of gene-targeting vectors: a rapid Mu in vitro DNA transposition-based strategy generating null, potentially hypomorphic, and conditional alleles.

Gene targeting into mammalian genomes by means of homologous recombination is a powerful technique for analyzing gene function through generation of transgenic animals. Hundreds of mouse strains carrying targeted alleles have already been created and recent modifications of the technology, in particular generation of conditional alleles, have extended the usefulness of the methodology for a variety of special purposes. Even though the standard protocols, including the construction of gene-targeting vector plasmids, are relatively straightforward, they typically involve time-consuming and laborious gene mapping and/or sequencing steps. To produce various types of gene-targeting constructions rapidly and with minimum effort, we developed a strategy, that utilizes a highly efficient in vitro transposition reaction of phage Mu, and tested it in a targeting of the mouse Kcc2 gene locus. A vast number and different types of targeting constructions can be generated simultaneously with little or no prior sequence knowledge of the gene locus of interest. This quick and efficient general strategy will facilitate easy generation of null, potentially hypomorphic, and conditional alleles. Especially useful it will be in the cases when effects of several exons within a given gene are to be studied, a task that necessarily will involve generation of multiple constructions. The strategy extends the use of diverse recombination reactions for advanced genome engineering and complements existing recombination-based approaches for generation of gene-targeting constructions.

Alleles↗
PubMed2005

EWS-FLI1 target genes recovered from Ewing's sarcoma chromatin.

In all, 85% of Ewing's sarcoma family tumors (ESFT), a neoplasm of unknown histogenesis, express EWS-FLI1 transcription factor gene fusions. To characterize direct target genes avoiding artificial model systems, we cloned genomic DNA from ESFT chromatin precipitating with EWS-FLI1. We now present a comprehensive list of 99 putative transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction. Gene-derived chromatin fragments co-precipitating with EWS-FLI1 were nonrandomly distributed over the human genome and localized predominantly to the upstream region and the first two introns of the genes. At least 20% of putative direct EWS-FLI1 targets were neural genes. One-third of genes recovered showed a significant ESFT-specific expression pattern and were found to be altered upon RNAi-mediated knockdown of EWS-FLI1. Among them, MK-STYX, encoding a MAP kinase phosphatase-like protein, was consistently expressed in ESFT. EWS-FLI1 was found to drive MK-STYX expression by binding to a single ETS binding motif within the first gene intron. MK-STYX serves as precedence for successful recovery of direct EWS-FLI1 targets from the authentic ESFT cellular context, the most relevant system to study oncogenic mechanisms for the discovery of new therapeutic targets in this disease.

Bone Neoplasms↗
PubMed2005

Role of the NF-kappaB signaling cascade and NF-kappaB-targeted genes in failing human hearts.

Nuclear factor-kappaB (NF-kappaB) is a ubiquitous transcription factor that has been indicated to play a causal role for many pathological states. Heart failure is a major cause of morbidity all over the world. In this study, we examined the role of NF-kappaB in failing (F) human hearts and nonfailing (NF) controls. Our data showed that an enhanced activation of this nuclear factor occurs in the F hearts along with its components, like I-kappaB kinase (IKK)beta and IkappaBalpha, both at transcript and translational levels. To obtain a profile of NF-kappaB-targeted gene expression in F hearts, we profiled, for the first time, the expression analysis of NF-kappaB-linked gene using a TranSignal human NF-kappaB-targeted gene array. Our data suggest that more than 50 genes were consistently upregulated in F hearts more than 1.5-fold (vs NF hearts, p<0.001). Our studies demonstrated that NF-kappaB is specifically and significantly activated via IKKbeta in F hearts. The most intriguing aspects of our studies are molecular profiles of NF-kappaB-linked or targeted gene expression in F hearts. Our data suggest that the regulation and control of NF-kappaB activation is, therefore, a powerful therapeutic strategy for delaying or attenuating such deadly disease.

Adult↗
PubMed2004

Identification of Tbr-1/CASK complex target genes in neurons.

Tbr-1, a neuron-specific T-box transcription factor, plays a critical role in brain development. Here, we performed a computational search using the non-palindromic T-box binding sequence, namely the non-palindromic T-element, to determine the putative downstream target genes of Tbr-1. More than 20 identified genes containing the non-palindromic T-element in the 5' regulatory region were found expressed in brain. Luciferase reporter assays using cultured hippocampal neurons showed that overexpression of Tbr-1 and CASK-enhanced promoter activities of some of these putative target genes, including NMDAR subunit 2b (NR2b), glycine transporter, interleukin 7 receptor (IL-7R) and OX-2. Among these genes, NR2b promoter responded strongest to overexpression of Tbr-1 and CASK. Deletion of the non-palindromic T-elements from NR2b promoter impaired the induction by Tbr-1 and CASK. We also examined expression of these target genes in Tbr-1 knockout mice, it was found that NR2b expression was consistently downregulated. Similarly, both RNA and protein expression levels of NMDAR subunit 1 (NR1), which also contains the non-palindromic T-elements in its 5' regulatory region, were reduced in Tbr-1 knockout mice. We suggest that Tbr-1/CASK protein complex regulates expression of these downstream target genes and thus modulates neuronal activity and function.

Amino Acid Transport Systems, Neutral↗
PubMed2006

Peroxisome proliferator-activated receptor gamma is a Zac target gene mediating Zac antiproliferation.

Zac is a C2H2 zinc finger protein, which regulates apoptosis and cell cycle arrest through DNA binding and transactivation. During tumorigenesis and in response to mitogenic activation, Zac gene expression is down-regulated in a methylation-sensitive manner. As yet, no target genes have been identified that could explain the potent antiproliferative function of Zac. Here, applying genome-wide expression analysis, we identify peroxisome proliferator-activated receptor gamma (PPARgamma) as a new bona fide Zac target gene, which is induced by direct Zac binding to the proximal PPARgamma1 promoter. We show that in human colon carcinoma cells, ZAC activates expression of PPARgamma target genes in a PPARgamma-dependent manner. Moreover, we show that treatment of pituitary tumor cells with octreotide, a somatostatin analogue, leads to Zac induction and subsequent Zac-dependent up-regulation of PPARgamma, which thereupon mediates part of the antiproliferative activity of Zac. Our work provides a first step toward elucidating a functional relationship between Zac and PPARgamma that could be relevant to the understanding of tumorigenesis and diabetes as well.

Cell Cycle Proteins↗
PubMed1997

Drosophila cubitus interruptus forms a negative feedback loop with patched and regulates expression of Hedgehog target genes.

The Drosophila segment polarity gene cubitus interruptus (ci) encodes a zinc finger protein that is required for the proper patterning of segments and imaginal discs. Epistasis analysis indicates that ci functions in the Hedgehog (Hh) signal transduction pathway and is required to maintain wingless expression in the embryo. In this paper, the role of the Ci protein in the Hh signaling pathway is examined in more detail. Our results show that ectopic expression of ci in imaginal discs and the embryo activates the expression of Hh target genes. One of these target genes, patched, forms a negative feedback loop with ci that is regulated by Hh signal transduction. Activation is also achieved using the Ci zinc finger domain fused to a heterologous transactivation domain. Conversely, repression of Hh target genes occurs in animals expressing the Ci zinc finger domain fused to a repression domain. To examine Ci function in more detail, regions of the Ci protein that are responsible for its ability to transactivate and its subcellular distribution have been identified.

Animals↗
PubMed1993

The effects of terminal heterologies on gene targeting by insertion vectors in embryonic stem cells.

We have examined the effects of placing nonhomologous DNA on the ends of an insertion-type gene targeting vector. The presence of terminal heterologies was found to be compatible with insertion targeting, and the terminal heterologies were efficiently removed. Terminal heterologies reduced the frequency of gene targeting to variable extents. The degree of inhibition of targeting was dependent on the length and the position of the heterology: 2.1kb heterologous sequences were more inhibitory than shorter regions of heterology, and heterology placed on the end of the long (4.8kb) arm of homology was more inhibitory than heterology positioned on the end of the short (0.8kb) arm. When heterology was placed on both arms of the targeting vector the targeting efficiencies were similar to or higher than when heterology was present on the long arm only. These results suggest that terminal sequences are removed simultaneously from both ends of targeting vectors. The removal of terminal sequences probably occurs by exonucleolytic degradation of both strands at each end, and removal of at least one of the strands is intimately coupled with the process of homologous recombination. These findings have implications for the design of gene targeting vectors.

Animals↗