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PubMed2002

Targeting gene expression in the preimplantation mouse embryo using morpholino antisense oligonucleotides.

Morpholino antisense oligonucleotides act by blocking translation of their target gene products and are effective tools for down-regulating gene expression. The current study was conducted to define treatment conditions for the use of morpholino oligonucleotides (MOs) in mammalian preimplantation embryos, and to employ MOs to target genes and study gene function in the early embryo. For the first time, ethoxylated polyethylenimine (EPEI), Lipofectin or Lysolecithin delivery agents were employed in combination with a fluorescent control MO and an alpha-catenin specific MO, to down-regulate gene expression during murine preimplantation development. Experiments applied to both two- and eight-cell stage murine preimplantation embryos contrasted the efficacy of MO concentrations of 1, 2, 5, 10, and 20 microM and treatment delivery times of 3, 6, 24, and 48 hr. Continuous treatment of two-cell embryos with Lipofectin and 20 microM alpha-catenin MO for 48 hr resulted in a significant (P < 0.05) reduction in development to the blastocyst stage and was accompanied by a marked reduction in alpha-catenin protein. These results indicate that morpholino antisense oligonucleotides are effective tools for down-regulating gene expression during mammalian preimplantation development.

Animals↗
PubMed2006

Kidney-specific upregulation of vitamin D3 target genes in ClC-5 KO mice.

Mutations in ClC-5 cause Dent's disease, a disorder associated with low molecular weight proteinuria, hyperphosphaturia, and kidney stones. ClC-5 is a Cl(-)/H(+)-exchanger predominantly expressed in the kidney, where it facilitates the acidification of proximal tubular endosomes. The reduction in proximal tubular endocytosis resulting from a lack of ClC-5 raises the luminal concentration of filtered proteins and peptides like parathyroid hormone (PTH). The increase in PTH may explain the hyperphosphaturia observed in Dent's disease. Expression profiling, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and hormone measurements were used to investigate whether the disruption of ClC-5 affects other signalling pathways. Although the upregulation of 25(OH)(2)-vitamin D(3) 1alpha-hydroxylase and downregulation of vitamin D(3) 24-hydroxylase suggested an increased formation of 1,25(OH)(2)-vitamin D(3), the concentration of this active metabolite was reduced in the serum of ClC-5 knockout (KO) mice. However, target genes of 1,25(OH)(2)-vitamin D(3) were upregulated in KO kidneys. Expression analysis of intestine and bone revealed that the upregulation of 1,25(OH)(2)-vitamin D(3) target genes was kidney intrinsic and not systemic. In spite of reduced serum levels of 1,25(OH)(2)-vitamin D(3) in ClC-5 KO mice, 1,25(OH)(2)-vitamin D(3) is increased in later nephron segments as a consequence of impaired proximal tubular endocytosis. This leads to a kidney-specific stimulation of 1,25(OH)(2)-vitamin D(3) target genes that may contribute to the pathogenesis of Dent's disease. The activation of genes in distal nephron segments by hormones that are normally endocytosed in the proximal tubule may extend to other pathways like those activated by retinoic acid.

Animals↗
PubMed2006

Prohibitin is a novel target gene of vitamin D involved in its antiproliferative action in breast cancer cells.

Previously, we showed that N-methyl-N-nitrosourea-transformed MCF12F breast epithelial cells exhibited differential expression of several genes, including up-regulation of prohibitin and elevated sensitivity to a relatively noncalcemic vitamin D analogue, 1alpha-hydroxyvitamin D5 [1alpha(OH)D5]. In this report, we evaluated the functional significance of prohibitin in relation to the cellular response to vitamin D. The in silico screening for putative transcription factor binding sites identified two vitamin D receptor (VDR)/retinoid X receptor binding sites in the 1-kb promoter region of prohibitin. Prohibitin up-regulation by 1alpha(OH)D5 treatment at both transcriptional and translational levels was confirmed by real-time reverse transcription-PCR and Western blot analysis in breast cancer cells, identifying prohibitin as a vitamin D target gene. Confocal microscopic analysis showed that prohibitin was localized in the nuclei of MCF-7 cells and a portion of prohibitin was colocalized with VDR, but direct physical interaction between VDR and prohibitin in cell lysates was not detectable. In MCF-7 cells expressing tetracycline-inducible prohibitin (Tet-On model), the overexpression of prohibitin inhibited cell proliferation and enhanced vitamin D-induced antiproliferative activity. Knockdown of prohibitin was accompanied by increased number of cells incorporating bromodeoxyuridine in the whole population and increased cell distribution in the S phase of cell cycle. In addition, prohibitin level had no significant effect on the vitamin D-induced transactivation of CYP24, a VDR target gene. This is the first report to suggest that prohibitin serves as a novel vitamin D target gene, which is involved in the antiproliferative action of vitamin D without affecting CYP24 transactivation in breast cancer cells.

Base Sequence↗
PubMed2004

Williams-Beuren syndrome critical region-5/non-T-cell activation linker: a novel target gene of AML1/ETO.

The chromosomal translocation t(8;21) fuses the AML1 (RUNX1) gene on chromosome 21 and the ETO gene on chromosome 8 in human acute myeloid leukemias (AMLs), resulting in expression of the chimeric transcription factor AML1/ETO. AML1/ETO-mediated dysregulation of target genes critical for hematopoietic differentiation and proliferation is thought to contribute to the leukemic phenotype. Several mechanisms, including recruitment of histone deacetylases (HDACs) to AML1 target genes, may be responsible for altered gene expression. We used an ecdysone-inducible expression system in the human monoblastic U-937 cell line to isolate genes that were differentially expressed upon induction of AML1/ETO expression. By representational difference analysis (cDNA-RDA), we identified 26 genes whose expression levels were significantly modulated following AML1/ETO induction for 48 h. None of these genes has previously been described as a target of AML1, ETO or AML1/ETO. One gene downregulated by AML1/ETO in vitro, Williams Beuren syndrome critical region 5 (WBSCR5), was expressed in primary t(8;21)-negative AML blasts but not in primary t(8;21)-positive AML blasts, strongly implying a role of this gene in the phenotype of t(8;21)-positive AML. Four upregulated and four downregulated genes were further studied with all-trans-retinoic acid (ATRA), an inducer of differentiation of U-937 cells, and Trichostatin A (TSA), an HDAC inhibitor. Three out of eight genes including WBSCR5 were regulated during ATRA-induced monocytic differentiation of U-937 cells, however, none of them antagonistically, upon both ATRA treatment and AML1/ETO induction. AML1/ETO-associated dysregulation of gene expression was not mediated by a TSA-sensitive mechanism. The identified genes provide a useful model to study the mechanism by which the AML1/ETO fusion protein exerts its function in transcriptional dysregulation in AML. The possible role of WBSCR5 in normal and malignant hematopoiesis warrants further study.

Cell Differentiation↗
PubMedDate not supplied

Microarray analysis of p53 target gene expression patterns in the spleen and thymus in response to ionizing radiation.

The ability of p53 to induce apoptosis through transactivation of its target genes is critical for its function as a tumor suppressor. To identify the critical p53 target genes that mediate apoptosis through these pathways, we examined p53-dependent apoptosis in vivo where these apoptotic pathways are intact. p53 wild-type and null animals were irradiated and the global p53 gene expression patterns in response to ionizing radiation were examined in two tissues (spleen and thymus) which undergo p53-dependent apoptosis in response to ionizing radiation. We found that the vast majority of genes that increased or decreased in a p53-dependent manner did so in a tissue specific manner. Although the spleen and thymus had similar levels of p53-dependent apoptosis after ionizing radiation, there was very little overlap in the induced or repressed targets. This suggested that distinct targets may mediate apoptosis in these tissues. In addition, these microarray analyses also led to the identification of several novel p53 targets. Several of these targets appear to be involved in known p53 functions such as the induction of apoptosis (bim) or the initiation of DNA repair (DinB). However, some p53 targets (both confirmed and unconfirmed) link p53 to the anti-tumor immune response (MIP 1-alpha). Further characterization of the tissue specific mediators of p53-dependent responses will greatly increase our understanding of p53 function.

Animals↗
PubMed2000

Nuclear gene targeting using negatively charged liposomes.

Oligonucleotides are a very useful tool to control gene activity. Oligos work by complementary base-pairing with target sequences either in the nucleus or in the cytosol (Zelphati, O., Szoka, F.C., Jr., 1996. Liposomes as a carrier for intracellular delivery of antisense oligonucleotides: a real or magic bullet? J. Contr. Rel. 41, 99-119). In a new approach using chimeric oligonucleotides (Yoon, K., Cole Strauss, A., Kmiec, E.B., 1996. Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA-DNA oligonucleotide. Proc. Natl. Acad. Sci. USA 93, 2071-2076) conversion of single base mutations with help of intranuclear repair mechanisms maybe an advantageous method to cure genetic diseases which are based on single point mutations. These chimeric oligonucleotides are constructed in a way that they form an intramolecular double strand of DNA and modified RNA-bases. We used a fluorescent labelled pure 68-mer DNA-analogue of a chimeric oligonucleotides to follow the intracellular fate of these kind of genetic material. The oligos were complexed with protamine sulfate and coated with three different liposomal formulations. The AVE-3 formulation shows enhanced properties compared to a classical neutral and negatively charged formulation. Nuclear localisation of oligos could only be observed with the AVE-3 formulation. Furthermore only the negatively charged liposome formulations interact with the protamine-complexed oligonucleotides.

Biological Transport↗
PubMed2002

Roles for cytosolic phospholipase A2alpha as revealed by gene-targeted mice.

Cytosolic phospholipase A2alpha (cPLA2alpha) has unique characteristics among phospholipase A2 (PLA2) family members. Under regulation by intracellular signaling system, cytosolic phospholipase A2alpha liberates arachidonic acid that can be metabolized by downstream enzymes to generate prostaglandins (PGs) and leukotrienes (LTs). Mice deficient in this enzyme have been generated by gene-targeting techniques. Cytosolic phospholipase A2alpha-deficient mice have a normal appearance and grow normally. Close examinations have revealed a renal concentration defect and intestinal ulcerative lesions. There may also be other disadvantages that are not manifested in well-regulated housing conditions. Although female mice are fertile, they become pregnant less frequently and have small litter sizes; moreover, impaired parturition results in few surviving pups. Primary cultured cells prepared from cytosolic phospholipase A2alpha-deficient mice produce significantly smaller amounts of prostaglandins and leukotrienes. Various disease models such as anaphylaxis, acute lung injury, brain injury induced by ischemia/reperfusion and neurotoxin, and polyposis have been investigated. In all these settings, cytosolic phospholipase A2alpha-deficient mice show significantly milder phenotypes. The mechanisms by which deficiencies of this enzyme exert protective effects may differ, but, a cytosolic phospholipase A2alpha inhibitor could have a wide spectrum of clinical targets. Specific functions of cytosolic phospholipase A2alpha have been clearly demonstrated using the gene-targeted mice. Also, comparisons with mice in which related enzymes and receptors have been manipulated using genetic technologies provide further insights into roles of lipid mediators in physiology and pathology.

Anaphylaxis↗
PubMed2025

In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene.

Homology-directed repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested Repair Drive, a platform technology for selectively expanding HDR-corrected hepatocytes in adult mice in vivo. Repair Drive involves transient conditioning of the liver by knocking down an essential gene, fumarylacetoacetate hydrolase (Fah), and delivering an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive increased the percentage of correctly targeted hepatocytes in healthy wild-type mice up to 25%, which resulted in a fivefold increased expression of a therapeutic transgene, human factor IX (FIX). Repair Drive was well tolerated and did not induce toxicity or tumorigenesis during a 1-year follow-up. This approach may broaden the range of liver diseases that can be treated with somatic genome editing.

Animals↗
PubMed2006

Simplified primer design for PCR-based gene targeting and microarray primer database: two web tools for fission yeast.

PCR-based gene targeting is a popular method for manipulating yeast genes in their normal chromosomal locations. The manual design of primers, however, can be cumbersome and error-prone. We have developed a straightforward web-based tool that applies user-specified inputs to automate and simplify the task of primer selection for deletion, tagging and/or regulated expression of genes in Schizosaccharomyces pombe. This tool, named PPPP (for Pombe PCR Primer Programs), is available at http://www.sanger.ac.uk/PostGenomics/S_pombe/software/. We also present a searchable Microarray Primer Database to retrieve the sequences and accompanying information for primers and PCR products used to build our in-house Sz. pombe microarrays. This database contains information on both coding and intergenic regions to provide context for the microarray data, and it should be useful also for other applications, such as quantitative PCR. The database can be accessed at http://www.sanger.ac.uk/PostGenomics/S_pombe/microarray/.

DNA Primers↗
PubMed2004

A nonviral carrier for targeted gene delivery to tumor cells.

In this study, we developed a nonviral, cationic, targeted DNA-carrier system by coupling SAINT/DOPE lipids to monoclonal antibodies. The two monoclonal antibodies used were both tumor specific, that is, MOC31 recognizes the epithelial glycoprotein EGP-2 present in carcinomas and Herceptin recognizes the HER-2/neu protein in breast and ovarian cancers. Coupling was performed under nonreducing conditions by covalent attachment. The coupling procedure appeared to be reproducible and the binding capacity of the antibody was not affected by linking them to the cationic lipid. Binding and transfection efficiency was assayed with target cells and nontarget cells. SAINT/DOPE lipoplexes as such appeared to be an effective transfection reagent for various cell lines. After coupling SAINT/DOPE to the monoclonal antibodies or F(ab)2 fragments, it was shown that the targeted MoAb-SAINT/DOPE lipoplexes preferably bound to target cells, compared to binding to the nontarget cells, especially for the Herceptin-SAINT/DOPE lipoplexes. More importantly, transfection of the target cells could also be improved with these targeted lipoplexes. In conclusion, we have shown that by using monoclonal antibody-coupled SAINT/DOPE lipoplexes cells targeted gene delivery can be achieved, and also a higher number of transfected target cells was seen.

Animals↗
PubMed1997

Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.

Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways. The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells. Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs. The addition of Cre recombinase activity promotes deletions of three types that can be scored. We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele. However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed. Therefore, Mre11 is required for normal cell proliferation. This methodology introduces a means to study important regions of essential genes in cell culture models.

Amino Acid Sequence↗
PubMed2004

Targeted gene alteration in Caenorhabditis elegans by gene conversion.

Now that some genomes have been completely sequenced, the ability to direct specific mutations into genomes is particularly desirable. Here we present a method to create mutations in the Caenorhabditis elegans genome efficiently through transgene-directed, transposon-mediated gene conversion. Engineered deletions targeted into two genes show that the frequency of obtaining the desired mutation was higher using this approach than using standard transposon insertion-deletion approaches. We also targeted an engineered green fluorescent protein insertion-replacement cassette to one of these genes, thereby confirming that custom alleles of different types can be created in vitro to make the corresponding mutations in vivo. This approach should also be applicable to heterologous transposons in C. elegans and other organisms, including vertebrates.

Animals↗
PubMed2003

pRB2/p130 target genes in non-small lung cancer cells identified by microarray analysis.

The retinoblastoma gene family consisting of RB/p105, p107, and RB2/p130 cooperate to regulate cell-cycle progression through the G1 phase of the cell cycle. Previous data demonstrated an independent role for the reduction or loss of pRb2/p130 expression in the formation and/or progression of lung carcinoma. Rb2/p130 is mutated in a human cell line of lung small cell carcinoma as well as in primary lung tumors. To identify potential pRb2/p130 target genes in an unbiased manner, we have utilized an adenovirus-mediated expression system of pRb2/p130 in a non-small lung cancer cell line to identify specific genes that are regulated by pRb2/p130. Using oligonucleotide arrays, a number of Rb2/p130 downregulated genes were identified and their regulation was confirmed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. As a result, 40 genes showed greater than 2.0-fold modification in their expression level after the RB2/p130 viral transduction. In conclusion, coupling adenoviral overexpression with microarray and semiquantitative RT-PCR analyses proved to be a versatile strategy for identifying pRb2/p130 target genes and for better understanding the expression profiles of these genes. Our results may also contribute to identifying novel therapeutic biomarkers in lung carcinoma.

Adenoviridae↗
PubMed2004

Real-time PCR for Chlamydia pneumoniae utilizing the Roche Lightcycler and a 16S rRNA gene target.

Chlamydia pneumoniae (CPN) causes pneumonia in humans, and has emerged as an important respiratory pathogen. There are also established links between CPN infection and coronary artery disease. Traditional culture methods for CPN detection can be time consuming and difficult. There are a variety of molecular-based amplification methods for CPN detection. These methods are more sensitive than culture, but have the disadvantage of being inconsistent and non-comparable across studies. In this paper, we describe the adaptation of the existing primer set CPN 90/CPN91 for use in a real-time PCR assay using the Roche Lightcycler and a Taqman probe. This assay had an analytical sensitivity of between 4 and 0.4 infection-forming units (IFUs)/PCR reaction. A total of 355 samples were tested for validation of the assay. Tested samples included two standardized panels of blinded samples from culture (N = 70), archived specimens consisting of a CPN dilution series, CPN-spiked porcine aortal tissue and endarterectomy specimens (N = 87). The third group consisted of prospectively collected PBMCs from clinical samples (N = 198). Results were compared to nested PCR, which targets the ompA gene of CPN; TETR PCR, which targets the 16S rRNA gene of CPN; or the known result for the sample. Overall, the assay had a sensitivity of 88.5% (69 of 78) and a specificity of 99.3% (275 of 277). This method should prove useful for accurate, high throughput detection of CPN.

Animals↗
PubMed2003

Low-dose bleomycin induces targeted gene repair frequency in cultured melan-c cells using chimeric RNA/DNA oligonucleotide transfection.

We attempted to raise the frequency of targeted gene repair attained by RNA/DNA chimeraplasty through activation of endogenous DNA recombination/repair system in cultured melan-c cells. For elevating the repair activity, either gamma-ray irradiation at 2 Gy or bleomycin addition at 5 nM to the culture medium was done. Subsequently, the tyrosinase-targeted chimeric RNA/DNA oligonucleotides were transfected. The frequency of targeted gene repair was assessed by three means, i.e., changes in cell color from transparent to dark black, restriction fragment band patterns, and DNA sequencing analysis. The results indicated that the gamma-ray irradiation did not improve the efficiency at all. In contrast, the low-dose bleomycin addition induced the frequency of targeted base conversion, compared with transfection with chimeric RDOs alone, although the frequency was still insufficient and varied from 0.1 to 10% of the total cultured cells.

Amino Acid Sequence↗
PubMed2000

The role of p53-target genes in human cancer.

The tumor suppressor gene p53 is mutated in a large proportion of human cancers. In some cellular conditions like DNA damage, the p53 gene is induced and its gene product is posttranscriptionally activated. p53 works as a transcriptional activator and induces the expression of its downstream target genes. This review will explain why expression of the normal p53 gene leads to tumor growth suppression. The p53 has several biological effects involving cell-cycle arrest, DNA replication and repair, proliferation, apoptosis, angiogenesis inhibition, and cellular stress response. These effects of the p53 result mainly from the activation of expression of a large number of p53-target genes. Here we have focused on the biological functions of the transcriptional targets of p53.

Animals↗
PubMed1997

Light and electron microscopic analysis of the central and peripheral nervous systems of acid sphingomyelinase-deficient mice resulting from gene targeting.

The acid sphingomyelinase (aSmase)-deficient mouse line recently generated by gene targeting (Otterbach and Stoffel, 1995) develops a lethal storage disease which is phenotypically comparable to the neurovisceral form of the human sphingomyelinosis, Niemann-Pick disease type A (NPA). This report describes the progressive accumulation of uncatabolized lipid substrates at the cellular and ultrastructural level in different regions of the nervous system of homozygous aSmase-/- mice, including cerebrum, cerebellum, spinal cord, optic nerve and peripheral nerves. We saw a cytoplasmic accumulation of pleomorphic lysosomal structures in cells of all regions under study, most extensively in macrophages, vascular endothelial cells, and also in neuronal perikarya. The complete and early degeneration of Purkinje cells was particularly striking. Moreover, we found a storage material in the cytoplasm of Schwann cells and to a minor extent in oligodendrocytes. In most advanced stages of the disorder, we detected an axonal dystrophy in both the central nervous system (CNS) and peripheral nervous system (PNS), without signs of dysmyelination or demyelination. The morphological changes of the central and peripheral nervous systems in the homozygous aSmase-/- mouse line closely resemble those in human NPA.

Animals↗
PubMed2000

B-cell lymphoma idiotypes chimerized by gene targeting can induce tumor immunity.

Immunization with modified immunoglobulin (Ig) idiotypes (Ids) of B-cell lymphomas is an attractive approach of experimental tumor immunotherapy. We show here that B-lymphoma cells can be gene-modified by homologous recombination at the Ig heavy chain locus. Although it has been demonstrated previously that a protein vaccine containing a mouse/human chimeric Ig had no immunostimulatory effect, we show that a xenogeneic Fc segment attached to the Id by gene targeting in autologous murine tumor cells can serve as an immunogenic carrier and is capable of inducing tumor protection. A prerequisite for successful vaccination is the delivery of tumor cells that have been engineered to express the Id in the chimeric form rather than administration of the soluble chimeric protein. Also DNA vaccination with plasmids encoding chimeric Ids was reported to induce an anti-idiotypic response, suggesting that there might be related mechanisms such as enhanced antigen presentation. Immunization with engineered lymphoma cells is a very potent protocol: in the cell-based setting, minute levels of expression in the gene-targeted cells are sufficient to confer tumor immunity. Because the titers of anti-Id antibodies induced do not reflect the degree of tumor protection, the immune mechanisms responsible for tumor rejection cannot be ascribed exclusively to a humoral response.

Adoptive Transfer↗