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Results for “Gene Targeting”

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PubMed1998

Transgenic animals with inducible, targeted gene expression in brain.

Several inducible gene expression systems have been developed in vitro in recent years to overcome limitations with traditional transgenic mice. One of these, the tetracycline-regulated system, has been used successfully in vivo. Nevertheless, concerns remain about the ability of this system to direct high levels of transgene expression in vivo and to enable such expression to be turned on and off effectively. We report here the generation, using a modified tetracycline-regulated system under the control of the neuron-specific enolase promoter, of several lines of mice that direct transgene expression to specific brain regions, including the striatum, cerebellum, CA1 region of the hippocampus, or deep layers of cerebral neocortex. Transgene expression in these mice can be turned off completely with low doses of doxycycline (a tetracycline derivative) and driven to very high levels in the absence of doxycycline. We demonstrate this tissue-specific, inducible expression for three transgenes: those that encode luciferase (a reporter protein) or DeltaFosB or the cAMP-response element binding protein (CREB) (two transcription factors). The various lines of transgenic mice demonstrate an inducible system that generates high levels of transgene expression in specific brain regions and represent novel and powerful tools with which to study the functioning of these (or potentially any other) genes in the brain.

Animals↗
PubMed2003

Neuronal untranslated BC1 RNA: targeted gene elimination in mice.

Despite the potentially important roles of untranslated RNAs in cellular form or function, genes encoding such RNAs have until now received surprisingly little attention. One such gene encodes BC1 RNA, a small non-mRNA that is delivered to dendritic microdomains in neurons. We have now eliminated the BC1 RNA gene in mice. Three independent founder lines were established from separate embryonic stem cells. The mutant mice appeared to be healthy and showed no anatomical or neurological abnormalities. The gross brain morphology was unaltered in such mice, as were the subcellular distributions of two prototypical dendritic mRNAs (encoding MAP2 and CaMKIIalpha). Due to the relatively recent evolutionary origin of the gene, we expected molecular and behavioral consequences to be subtle. Behavioral analyses, to be reported separately, indicate that the lack of BC1 RNA appears to reduce exploratory activity.

Animals↗
PubMed2006

Oxygen treatment after experimental hypoxia-ischemia in neonatal rats alters the expression of HIF-1alpha and its downstream target genes.

Recently, mounting evidence has emerged to suggest that hyperbaric oxygenation (HBOT)-induced neuroprotection after experimental global ischemia and subarachnoid hemorrhage entails a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Therefore, the purpose of this study was to test the hypothesis that oxygen-induced neuroprotection after neonatal hypoxia-ischemia involves alterations in the expression of HIF-1alpha. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% O(2) at 37 degrees C). Pups were then treated with HBOT (2.5 ATA) or normobaric oxygenation treatment (NBOT) for 2 h. The expression and phosphorylation status of HIF-1alpha was evaluated at intervals up to 24 h after the insult, as was the expression of glucose transporter (GLUT)-1, GLUT-3, lactate dehydrogenase (LDH), aldolase (Ald), and p53. The protein-protein interaction of HIF-1alpha and p53 was also examined. An elevated expression of HIF-1alpha, GLUT-1, GLUT-3, Ald, and LDH was observed after the insult. An increase in the dephosphorylated form of HIF-1alpha was followed by an increase in the association of HIF-1alpha with p53 and an increase in p53 levels. Both HBOT and NBOT reduced the elevated expression of HIF-1alpha and decreased its dephosphorylated form. Furthermore, both treatments promoted a transient increase in the expression of GLUT-1, GLUT-3, LDH, and Ald, while decreasing the HIF-1alpha-p53 interaction and decreasing the expression of p53. Therefore, the alteration of the HIF-1alpha phenotype by a single oxygen treatment may be one of the underlying mechanisms for the observed oxygen-induced neuroprotection seen when oxygen is administered after a neonatal hypoxic-ischemic insult.

Adaptation, Physiological↗
PubMed2004

Identification of target genes regulated by FOXC1 using nickel agarose-based chromatin enrichment.

PURPOSE: To overcome the problem of antibody availability, often encountered during chromatin immunoprecipitation (ChIP) assays, nickel agarose-based chromatin enrichment (NACE) was developed. Based on the affinity of (His)-6-tagged proteins for the nickel ion, this modified form of ChIP allows the isolation of chromatin in the absence of specific antibodies. METHODS: Nonpigmented ciliary epithelium cells were transfected with (His)-6-tagged FOXC1. FOXC1-enriched chromatin complexes were isolated by using the tight electrostatic interaction between histidine residues of the recombinant FOXC1 protein and nickel. One hundred fifty NACE-enriched clones were sequenced and subjected to in silico and biochemical analyses. RESULTS: Twenty-six clones were detected near known genes: Eight were near predicted but uncharacterized genes, eight were within areas where neither known nor predicted genes have yet been mapped, four were chimeric, and the rest were either repetitive (n=81) or poor-quality (n=23) sequences. Twenty of the 26 known genes were expressed in the eye. Five of the NACE-enriched clones (BMP2K, DACH, FVT-1, SIX-1, and PGE-2 receptor), as well as nine clones selected from the literature, were validated by PCR amplification in two independent lots of NACE-enriched chromatin. All five NACE-selected genes were detected in two independent assays, as well as four (BMP7, SMAD2, TGF-B1, and WNT6) of the nine genes selected from the literature, consistent with these genes' being regulated by FOXC1. CONCLUSIONS: NACE is a useful technique allowing specific chromatin enrichment in cases where antibodies are unavailable. Specific recovery of PTGER, DACH1, WNT6, and FVT-1 implicates FOXC1 in a variety of cellular events including modulation of intraocular pressure, cell cycle, ocular development, and oncogenesis.

Chromatin↗
PubMed2005

Targeted gene delivery to cancer cells with nanometric DNA particles enveloped with folic acid using a polymerisable anchor.

Progress in the design of gene delivery system is vital for cancer gene therapy since many physiological and intracellular barriers remain. We have developed a technology for condensing genes into nanometric delivery systems. In this paper, we present a novel strategy for decorating 30 nm DNA particles with folic acid for cancer cell recognition. Physicochemical and biological experiments show that these DNA complexes selectively bind to cells expressing the corresponding folic acid receptor.

Carrier Proteins↗
PubMed2004

Down syndrome critical region protein 1 (DSCR1), a novel VEGF target gene that regulates expression of inflammatory markers on activated endothelial cells.

We conducted a genome-wide analysis of genes that are regulated by vascular endothelial growth factor (VEGF) in endothelial cells and identified DSCR1 to be most significantly induced. Consistent with an antagonistic function on calcineurin (CnA) signaling, expression of DSCR1 in endothelial cells blocked dephosphorylation, nuclear translocation, and activity of nuclear factor of activated T cell (NFAT), a transcription factor involved in mediating CnA signaling. DSCR1 was not only induced by VEGF, but also by other compounds activating CnA signaling, suggesting a more general role for DSCR1 in activated endothelial cells. Transient expression of DSCR1 attenuated inflammatory marker genes such as tissue factor (TF), E-selectin, and Cox-2, identifying a previously unknown regulatory role for DSCR1 in activated endothelial cells. In contrast, knock-down of endogenous DSCR1 increased NFAT activity and stimulated expression of inflammatory genes on activated endothelial cells. Thus, the negative regulatory feedback loop between DSCR1 and CnA signaling in endothelial cells identified may represent a potential molecular mechanism underlying the frequently transient expression of inflammatory genes following activation of endothelial cells.

Calcineurin↗
PubMed2006

Targeted gene deletion demonstrates that the cell adhesion molecule ICAM-4 is critical for erythroblastic island formation.

Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule 4 (ICAM-4), an immunoglobulin superfamily member, participates in island formation. Earlier, we identified alpha(V) integrins as ICAM-4 counterreceptors. Because macrophages express alpha(V), ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knock-out mice and developed quantitative, live cell techniques for harvesting intact islands and for re-forming islands in vitro. We observed a 47% decrease in islands reconstituted from ICAM-4 null marrow compared to wild-type marrow. We also found a striking decrease in islands formed in vivo in knock-out mice. Further, peptides that block ICAM-4/alpha(V) adhesion produced a 53% to 57% decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage alpha(V) functions in island integrity. Importantly, we documented that alpha(V) integrin is expressed in macrophages isolated from erythroblastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/alpha(V) adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.

Animals↗
PubMed1998

Reconstitution of estrogen-dependent transcriptional activation of an adenoviral target gene in select regions of the rat mammary gland.

Estrogen regulates proliferation and morphogenesis of mammary ductal epithelium by interacting with a specific intracellular estrogen receptor (ER) that acts as a hormone-dependent transcriptional regulator of gene expression. The mechanisms by which ER regulates transcription in response to estrogen have been analyzed extensively in tissue culture and in cell-free systems. These studies have demonstrated that the transcriptional activity of ER is strongly influenced by cellular context and highlight the need to address ER transcriptional activity in an appropriate cellular background. Thus, to gain insight into the mechanistic role of ER in mammary epithelial morphogenesis, we have used an adenoviral gene delivery strategy to introduce an estrogen-responsive reporter gene into the mammary epithelium and to monitor the activity of endogenous ERs in their natural environment where cellular context including stromal-epithelial interactions can be taken into account. Using this approach, we first demonstrated highly efficient adenoviral delivery throughout the mammary epithelium using a beta-galactosidase (betagal) reporter gene under the control of the constitutively active cytomegalovirus (CMV) promoter. Next, we constructed an adenoviral vector by substituting the CMV promoter with an estrogen-dependent promoter fragment-linked betagal (Ad-ERE-tk-betagal). This adenoviral reporter system provides evidence that ER positive mammary epithelial cells display a differential sensitivity in a region-specific manner toward estrogen induction. Our data suggest that the availability of factor(s) other than ER is necessary for ER-mediated gene activation and may be important in modulating the differential responses of mammary epithelial cells to estrogen.

Adenoviridae↗
PubMed2001

Cluster analysis and promoter modelling as bioinformatics tools for the identification of target genes from expression array data.

Expression arrays yield enormous amounts of data linking genes, via their cDNA sequences, to gene expression patterns. This now allows the characterisation of gene expression in normal and diseased tissues, as well as the response of tissues to the application of therapeutic reagents. Expression array data can be analysed with respect to the underlying protein sequences, which facilitates the precise determination of when and where certain groups of genes are expressed. More recent developments of clustering algorithms take additional parameters of the experimental set-up into account, focusing more directly on co-regulated set of genes. However, the information concerning transcriptional regulatory networks responsible for the observed expression patterns is not contained within the cDNA sequences used to generate the arrays. Regulation of expression is determined to a large extent by the promoter sequences of the individual genes (and/or enhancers). The complete sequence of the human genome now provides the molecular basis for the identification of many regulatory regions. Promoter sequences for specific cDNAs can be obtained reliably from genomic sequences by exon mapping. In the many cases in which cDNAs are 5'-incomplete, high quality promoter prediction tools can be used to locate promoters directly in the genomic sequence. Once sufficient numbers of promoter sequences have been obtained, a comparative promoter analysis of the co-regulated genes and groups of genes can be applied in order to generate models describing the higher order levels of transcription factor binding site organisation within these promoter regions. Such modules represent the molecular mechanisms through which regulatory networks influence gene expression, and candidates can be determined solely by bioinformatics. This approach also provides a powerful alternative for elucidating the functional features of genes with no detectable sequence similarity, by linking them to other genes on the basis of their common promoter structures.

Algorithms↗
PubMed2003

Expression of the NF-kappa B target gene X-ray-inducible immediate early response factor-1 short enhances TNF-alpha-induced hepatocyte apoptosis by inhibiting Akt activation.

Using a cDNA microarray analysis, we identified x-ray-inducible immediate early response factor-1 (IEX-1) as a proapoptotic gene which was induced by TNF-alpha and also depend on NF-kappaB activation in Hc human hepatocytes. In these cells only the original form of IEX-1, termed IEX-1S, but not its longer transcript IEX-1L, was expressed. Overexpression of IEX-1S resulted in promotion of TNF-alpha-induced apoptosis in Hc cells expressing a mutant form of IkappaB. This proapoptotic action can be explained by its inhibitory findings on survival signals; inhibition of TNF-alpha-induced activation and expression of phosphatidylinositol 3-kinase (PI3K)/Akt, and also blockage of expression of Mcl-1, an antiapoptotic Bcl-2 family member which is located downstream of Akt, was inhibited by IEX-1S. LY 294002, an inhibitor of PI3K, increased IEX-1S expression induced by TNF-alpha and accelerated TNF-alpha-induced apoptosis in IkappaB-treated Hc cells. Overexpression of the dominant-negative Akt enhanced, but the constitutively active Akt suppressed, TNF-alpha-induced IEX-1S expression, suggesting that PI3K/Akt negatively regulated IEX-1S expression. These results demonstrate that NF-kappaB-dependent recruitment of IEX-1S may play a proapoptotic role in TNF-alpha-stimulated hepatocytes through blockage of the PI3K/Akt pathway. Moreover, the reciprocal cross-talk between IEX-1S and PI3K/Akt may closely be involved in the regulation of TNF-alpha-induced hepatocyte apoptosis.

Adjuvants, Immunologic↗
PubMed2002

Tumor-targeted gene therapy for nasopharyngeal carcinoma.

The unique feature of human nasopharyngeal carcinoma (NPC) is its almost universal association with the EBV, which is expressed in a latent form exclusively in cancer cells, and not in the surrounding tissues. We have exploited this differential by constructing a novel replication-deficient adenovirus vector (ad5.oriP) in which transgene expression is under the transcriptional regulation of the family of repeats domain of the origin of replication (oriP) of EBV. When EBNA1, one of the latent gene products of EBV, binds to the family of repeats sequence, this activates transcription of downstream genes. Vector constructs were made using the beta-galactosidase and luciferase reporter genes (ad5oriP.betagal and ad5oriP.luc) or the p53 tumor suppressor gene (ad5oriP.p53). 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining demonstrated extensive expression only in EBV-positive NPC cells, specifically in response to the presence of EBNA1. The relative difference in expression between EBV-positive and -negative cell lines is approximately 1000-fold. This selective expression was corroborated in EBV-positive and -negative tumor models, along with an absence of transgene expression in the host liver. Significant cytotoxicity was achieved using the adv.oriP.p53 therapeutic gene only in EBV-positive NPC cells, which was enhanced with the addition of ionizing radiation. Cytotoxicity was mediated primarily by induction of apoptosis. These results demonstrate that the oriP sequence can achieve high levels of gene expression targeted specifically to EBV-positive NPC cells in the context of the adv vector. This has now provided the tumor-specific expression system from which additional interventions can be evaluated in future treatment strategies for patients with nasopharyngeal cancers.

Adenoviridae↗
PubMed2004

Targeted gene expression in the chicken eye by in ovo electroporation.

PURPOSE: The chicken embryo lens is a classical model system for developmental and cell biology studies. To understand the molecular mechanisms that underlie the morphological changes that occur during lens development, it is important to develop an effective gene transfer method that permits the analysis of gene functions in vivo. In ovo electroporation has been successfully used for introducing DNA into neural and mesenchymal tissues of chicken embryos. In this study, we explored the possibility of using this technique to manipulate gene expression in lens epithelial and fiber cells, as well as in other cells of the chicken eye. METHODS: Two DNA constructs were used in this study. pCAX contains a chicken beta-actin promoter fused to the CMV IE enhancer to drive enhanced green fluorescent protein (EGFP) expression. pMES-cNf2 uses the same chimeric promoter to drive the expression of the chicken neurofibromatosis 2 (cNf2) and EGFP proteins in the same cell. Plasmid DNA was injected into the lumen of the lens vesicle in chicken embryos at stage 15. For corneal epithelial and retinal cell electroporation, DNA was placed near the surface ectoderm in the eye region or injected into the vitreous cavity, respectively. Electroporation was performed with one electrode above the eye and the other underneath the head of the embryo. Chicken embryos were harvested at different time points for EGFP expression analysis by immunohistochemistry. 5-bromo-2'-deoxyuridine (BrdU) incorporation assays were used to evaluate the effects of cNf2 on lens epithelial cell proliferation. RESULTS: A strong EGFP signal can be detected in lens cells 4 h after electroporation. The transfected cells maintain high levels of EGFP expression for at least 5 days. Overexpressing cNf2 in lens epithelial cells significantly inhibits cell proliferation. Ectopic expression of EGFP in corneal epithelial and retinal cells was also achieved by in ovo electroporation. CONCLUSIONS: We have demonstrated that exogenous DNA can be effectively introduced into lens, corneal and retinal cells in the living embryo by in ovo electroporation. In comparison to viral infection and transgenic mouse approaches, in ovo electroporation offers an easier and quicker way to manipulate gene expression during embryonic development. This technique will be a useful tool for exploring the molecular mechanisms of lens and eye development.

Animals↗
PubMed1998

Mice with STAT6-targeted gene disruption develop a Th1 response and control cutaneous leishmaniasis.

The cutaneous growth of Leishmania mexicana was measured in STAT6-deficient mice (STAT6-/-) and compared with that in similarly infected wild-type (STAT6+/+) mice. Following s.c. inoculation with 5 x 10(6) amastigotes of L. mexicana into the shaven rump, STAT6+/+ mice developed large, nonhealing cutaneous lesions, while STAT6-/- mice failed to develop detectable lesions during most of the course of study. As infection progressed, STAT6+/+ mice infected with L. mexicana displayed significantly higher titers of Leishmania-specific IgG1 and IgE compared with STAT6-/- mice, which conversely produced significantly higher titers of Leishmania-specific IgG2a, indicating development of a Th1-like response in the latter group. At 12 wk postinfection, Leishmania Ag-stimulated lymph node cells from STAT6-/- mice produced significantly higher amounts of IL-12 and IFN-gamma than those from STAT6+/+ mice as measured by ELISA. However, there was no significant difference in IL-4 production between the two groups. Semiquantitative RT-PCR of transcript levels in intact draining lymph nodes and skin from inoculation sites confirmed a similar pattern of cytokines in vivo as that observed in stimulated lymph node cells in vitro. These results indicate that STAT6-mediated IL-4 signaling is critical for progression of L. mexicana infection in genetically susceptible mice and demonstrate that in the absence of STAT6, susceptible mice default toward a Th1-like response and control cutaneous L. mexicana infection.

Animals↗
PubMed1994

Nonenzymatic glycosylation of poly-L-lysine: a new tool for targeted gene delivery.

The basic approach in targeted gene delivery relies on the formation of a complex between a vector and a molecule that will be selectively internalized by the target cells. In the case of hepatocytes, asialoglycoproteins are convenient targeting molecules because of the high affinity and avidity of the hepatocyte galactose receptor. In this system, poly-L-lysine is cross-linked to an asialoglycoprotein, and the resulting conjugate is complexed with the expression vector (DNA). The electrostatic binding between DNA and poly-L-lysine-asialoglycoprotein ensures delivery of the intravenously injected complex to the liver, where it is subjected to endocytosis by hepatocytes. However, the poly-L-lysine-asialoglycoprotein complexes tend to be unstable, of limited solubility and of fixed carbohydrate content. For these reasons we searched for a simpler alternative. We exploited the known capacity of reducing sugars to be reductively coupled to the epsilon-amino groups in proteins and used lactose to obtain poly-L-lysine with "exposed" galactose. Glycosylation with sodium cyanoborohydride at high pH in borate buffer is a simple, reproducible procedure. The "lactosylated" poly-L-lysine has proved very stable, highly soluble and easily bound to plasmids. In a set of experiments we compared the asialofetuin-poly-L-lysine vector complexes with lactosylated poly-L-lysine vector complexes by transfecting hepatoma cells (HepG2) in culture. For these experiments we used a pRc/cytomegalovirus eukaryotic expression vector containing a mutant TGF-beta 1 complementary DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗