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PubMed1999

Targeted gene disruption in the development of mouse models to elucidate the role of receptor guanylyl cyclase signaling pathways in physiological function.

The physiological role of receptor guanylyl cyclases (GCs), which transduce a signal via the generation of intracellular cyclic GMP, has been somewhat speculative since there are few specific inhibitors that discriminate among various receptor isoforms. Although the natriuretic peptide receptors have been thought to regulate cardiovascular and renal function, the exact contribution of the receptor subtypes has not been clarified. The normal role of the heat-stable enterotoxin receptor guanylyl cyclase remains undefined, and several orphan members of the family await the identification of ligands as well as function. Targeted gene disruption, familiarly known as gene knockout, has emerged during the past decade as a powerful technique for probing the function of gene products, and has been used to develop animal models of inherited human diseases. We are just beginning to apply gene targeting technology to the guanylyl cyclase receptor family. Reviewed here is the information gained to date from the targeted disruption of several members of the guanylyl cyclase receptor family, their ligands, or effector molecules.

Animals↗
PubMed1992

Modification of the apolipoprotein B gene in HepG2 cells by gene targeting.

The HepG2 cell line has been used extensively to study the synthesis and secretion of apolipoprotein (apo) B. In this study, we tested whether gene-targeting techniques can be used to inactivate one of the apo B alleles in HepG2 cells by homologous recombination using a transfected gene-targeting vector. Our vector contained exons 1-7 of the apo B gene, in which exon 2 was interrupted by a promoterless neomycin resistance (neo(r)) gene. The recombination of this vector with the cognate gene would inactivate an apo B allele and enable the apo B promoter to activate the transcription of the neo(r) gene. To detect the rare homologous recombinant clone, we developed a novel solid phase RIA that uses the apo B-specific monoclonal antibody MB19 to analyze the apo B secreted by G418-resistant (G418r) clones. Antibody MB19 detects a two-allele genetic polymorphism in apo B by binding to the apo B allotypes MB19(1) and MB19(2) with high and low affinity, respectively. HepG2 cells normally secrete both the apo B MB19 allotypes. Using the MB19 immunoassay, we identified a G418r HepG2 clone that had lost the ability to secrete the MB19(1) allotype. The inactivation of an apo B allele of this clone was confirmed by the polymerase chain reaction amplification of an 865-bp fragment unique to the targeted apo B allele and by Southern blotting of genomic DNA. This study demonstrates that gene-targeting techniques can be used to modify the apo B gene in HepG2 cells and demonstrates the usefulness of a novel solid phase RIA system for detecting apo B gene targeting events in this cell line.

Alleles↗
PubMed2000

Uracil incorporation into a gene targeting construct reduces the frequency of homologous and nonhomologous recombinants in human cells.

Gene targeting allows the introduction of specific modifications into the eukaryotic genome by homologous recombination, but its efficiency is low in many mammalian systems. We are exploring different ways to increase the efficiency of gene targeting and we report here the effect of uracil incorporation in the targeting construct. Plasmids containing uracil substituting for a fraction of thymine residues are hyperrecombinogenic in some bacterial systems. To test whether a similar stimulation of recombination occurs in mammalian cells, we have prepared a uracil-rich HPRT targeting construct and quantified its homologous and nonhomologous recombination frequencies compared to the same plasmid lacking uracil. The uracil-rich plasmid led to reductions in both homologous and nonhomologous recombination in human cells.

Analysis of Variance↗
PubMed1994

The Max transcription factor network: involvement of Mad in differentiation and an approach to identification of target genes.

The small bHLHZip protein, Max, was originally identified through its interaction with Myc family proteins and appears to be an obligate partner for Myc function. Max has now been found to interact with at least two other proteins, Mad and Mxi1. These also belong to the bHLHZip class but are otherwise unrelated to Myc. Mad has been shown to abrogate the positive transcriptional activity of Myc and to inhibit Myc in co-transformation assays. This suggests that Mad may antagonize Myc function. Mad is rapidly induced upon differentiation, a time when Myc is frequently down-regulated. We show here evidence for Mad expression upon differentiation of myeloblasts, monoblasts, and keratinocytes. Mad:Max complexes are detected during differentiation and appear to replace the Myc:Max complexes present in proliferating cell populations. Since these complexes appear to form even in the presence of Myc, there may exist mechanisms that act to inhibit Myc:Max, or to promote Mad:Max, complex formation. We speculate that Max complex switching causes a change in the transcriptional activity of groups of target genes. Mad is not induced in all differentiating cell types, suggesting that other, possibly tissue-restricted, proteins might act in similar switch mechanisms to effect changes in transcriptional programs. We have also developed an approach to identification of the gene targets for Myc:Max complexes. By employing an immunoisolation procedure, we have begun characterization of several clones whose expression levels correlate with those of c-myc. Further identification of Myc-regulated genes may allow us to determine the molecular mechanism by which Myc governs cell proliferation and differentiation.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2002

Mechanisms underlying targeted gene correction using chimeric RNA/DNA and single-stranded DNA oligonucleotides.

Chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides have been developed for site-specific correction of episomal and chromosomal target genes. The gene repair approach relies on specific hybridization of the oligonucleotides to the target gene generating a mismatch with the targeted point mutation. Restored gene function is anticipated to occur through activation of endogenous repair systems that recognize the created mismatch. We present an overview of the gene correction results obtained in several target genes by employing various oligonucleotide designs and a discussion of the possible mechanisms underlying the gene correction techniques. Experimental data suggest that modified single-stranded oligonucleotides form intermediate three-stranded heteroduplexes involving the human RecA homologue, hRad51, whereas chimeric RNA/DNA oligonucleotides may participate in three or four-stranded intermediate structures. Protein factors such as hRad52, hRad54, hRPA, and p53 may modulate the heteroduplex formation and participate in the activation of the endogenous mismatch repair and/or nucleotide excision repair pathway(s). The efficiency of the gene correction process may furthermore be influenced by the differential recognition of mismatches by repair enzymes and possible sequence context effects.

Adenosine Triphosphatases↗
PubMed2004

Kupffer cells and reactive oxygen species partially mediate lipopolysaccharide-induced downregulation of nuclear receptor pregnane x receptor and its target gene CYP3a in mouse liver.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates target gene transcription in a ligand-dependent manner. The in vivo effects of lipopolysaccharide (LPS) on expression of PXR and its target gene cytochrome P450 3A (CYP3A) in mouse liver were investigated in this study. Mice were injected intraperitoneally with different doses of LPS (0.1-5.0 mg/kg). PXR and CYP3A11 mRNA levels were measured using reverse transcription polymerase chain reaction. Results indicate that LPS significantly inhibits the expression of PXR mRNA in a dose-dependent manner, followed by suppression of CYP3A11 mRNA in mouse liver. LPS also represses the upregulation of CYP3A11 mRNA levels and erythromycin N-demethylase (ERND) catalytic activity in mice pretreated with PXR ligands dexamethasone, rifampicin, mifepristone, and phenobarbital. LPS-induced downregulation of PXR and CYP3A11 mRNA in liver was significantly attenuated in mice pretreated with gadolinium chloride, a selective Kupffer cell toxicant. Pretreatment with a single dose of gadolinium chloride (10 mg/kg) also significantly attenuated LPS-induced downregulation of dexamethasone-, rifampicim-, mifepristone-, and phenobarbital-inducible, CYP3A11 mRNA expression and ERND activity in mouse liver. Furthermore, LPS-induced downregulation of PXR and CYP3A11 mRNA was significantly attenuated in mice pretreated with allopurinol, an inhibitor of xanthine oxidase, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. Allopurinol and diphenyleneiodonium chloride pretreatment also attenuated the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. However, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, has no effect on LPS-induced downregulation of PXR and CYP3A11 mRNA. Finally, LPS-induced downregulation of PXR and CYP3A11 mRNA was prevented in mice pretreated with either N-acetylcysteine or ascorbic acid. These antioxidants also prevented the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. These results indicate that Kupffer cells contribute to LPS-induced downregulation of PXR and CYP3A in mouse liver. Reactive oxygen species, produced possibly by NADPH oxidase and perhaps by xanthine oxidase, are involved in LPS-induced downregulation of nuclear receptor PXR and its target gene CYP3A in mouse liver.

Animals↗
PubMed2004

Neuroblastoma and pre-B lymphoma cells share expression of key transcription factors but display tissue restricted target gene expression.

BACKGROUND: Transcription factors are frequently involved in the process of cellular transformation, and many malignancies are characterized by a distinct genetic event affecting a specific transcription factor. This probably reflects a tissue specific ability of transcription factors to contribute to the generation of cancer but very little is known about the precise mechanisms that governs these restricted effects. METHODS: To investigate this selectivity in target gene activation we compared the overall gene expression patterns by micro-array analysis and expression of target genes for the transcription factor EBF in lymphoma and neuroblastoma cells by RT-PCR. The presence of transcription factors in the different model cell lines was further investigated by EMSA analysis. RESULTS: In pre-B cells mb-1 and CD19 are regulate by EBF-1 in collaboration with Pax-5 and E-proteins. We here show that neuroblastoma cells express these three, for B cell development crucial transcription factors, but nevertheless fail to express detectable levels of their known target genes. Expression of mb-1 could, however, be induced in neuroblastoma cells after disruption of the chromatin structure by treatment with 5-azacytidine and Trichostatin A. CONCLUSION: These data suggest that transcription factors are able to selectively activate target genes in different tissues and that chromatin structure plays a key role in the regulation of this activity.

Antigens, CD↗
PubMed2006

TC1(C8orf4) correlates with Wnt/beta-catenin target genes and aggressive biological behavior in gastric cancer.

PURPOSE: We have recently reported that TC1(C8orf4), a small protein present in vertebrates, functions as a novel regulator of the Wnt/beta-catenin pathway. TC1 up-regulates beta-catenin target genes that are implicated in the aggressive behavior of cancers. Our aim was to investigate the clinical and pathobiological relevance of TC1 in gastric cancer. EXPERIMENTAL DESIGN: The expression of TC1 was analyzed using tissue microarray in correlation with clinicopathologic variables and beta-catenin target genes in 299 gastric cancers. The biological effects of TC1 on Matrigel invasiveness and the proliferation of cancer cells were analyzed. TC1 expression was analyzed in gastric cancer cells after serial peritoneal implantation in nude mice. RESULTS: TC1 expression was present in 111 carcinomas (37.1%), correlating with tumor stage (P < 0.002), poor differentiation (P < 0.001), lymphatic infiltration (P < 0.005), and lymph node metastasis (P < 0.006). TC1 also correlated with poor survival in diffuse type carcinomas (P < 0.0001), and even in patients with lymph node metastasis (P < 0.0014). TC1 also correlated with the expression of beta-catenin target genes including laminin gamma2, metalloproteinase-7 and metalloproteinase-14, cyclin D1, c-Met, and CD44. TC1 enhanced Matrigel invasiveness and proliferation, supporting its role in the aggressive biological behavior of cancers. The expression of TC1 increased in MKN45 cells after serial peritoneal seeding in nude mice. CONCLUSIONS: Our data suggests that TC1 coordinates the up-regulation of Wnt/beta-catenin target genes that are implicated in the aggressive biological behavior of cancers. The strong clinical relevance, even in patients with lymph node metastasis, suggested that TC1 could be a potential therapeutic target of advanced gastric cancers.

Animals↗
PubMed2004

Targeted gene expression profiling in the rainbow trout (Oncorhynchus mykiss) ovary during maturational competence acquisition and oocyte maturation.

A real-time polymerase chain reaction-based gene expression survey was performed using 37 target genes and 22 female rainbow trout sampled during follicular maturational competence (FMC) acquisition or during oocyte maturation. In females sampled before meiosis resumption, FMC was estimated using an in vitro assay. Several growth factors, bone morphogenetic proteins, steroidogenic enzymes, cathepsins, genes known to play a role in the fish preovulatory ovary, as well as previously unstudied genes, were analyzed in this survey. Gene expression profiling was performed using a supervised clustering analysis in order to identify groups of genes exhibiting similar expression profiles in the ovary during FMC acquisition and follicular maturation. From the clustering analysis, three clusters exhibiting a specific expression during FMC acquisition or at the time of oocyte maturation were identified. Cluster 1 was characterized by a progressive increase in gene expression during FMC acquisition, whereas cluster 2 exhibited an increased expression at the time of oocyte maturation. In contrast, cluster 3 was characterized by a decreased mRNA expression at the time of oocyte maturation. Among the 37 target genes used in this survey, 18 were significantly regulated during maturational competence acquisition or at the time of oocyte maturation. Among these 18 genes, 16 belonged to one of the three clusters identified. Although the results allowed a global description of gene expression profiles, they also suggest an important role for several factors, including some previously unstudied bone morphogenetic proteins, in the paracrine control of FMC acquisition and meiosis resumption.

Animals↗
PubMed2005

Identification of target genes of the transcription factor HNF1beta and HNF1alpha in a human embryonic kidney cell line.

Hepatocyte nuclear factor 1beta (HNF1beta, TCF2) is a tissue-specific transcription factor whose mutation in humans leads to renal cysts, genital malformations, pancreas atrophy and maturity onset diabetes of the young (MODY5). Furthermore, HNF1beta overexpression has been observed in clear cell cancer of the ovary. To identify potential HNF1beta target genes whose activity may be deregulated in human patients, we established a human embryonic kidney cell line (HEK293) expressing HNF1beta conditionally. Using Flp recombinase, we introduced wild type or mutated HNF1beta at a defined chromosomal position allowing a most reproducible induction of the HNF1beta derivatives upon tetracycline addition. By oligonucleotide microarrays we identified 25 HNF1beta-regulated genes. By an identical approach, we identified that the related transcription factor HNF1alpha (TCF1) affects only nine genes in HEK293 cells and thus is a less efficient factor in these kidney cells. The HNF1beta target genes dipeptidyl peptidase 4 (DPP4), angiotensin converting enzyme 2 (ACE2) and osteopontin (SPP1) are most likely direct target genes, as they contain functional HNF1 binding sites in their promoter region. Since nine of the potential HNF1beta target genes are deregulated in clear cell carcinoma of the ovary, we propose that HNF1beta overexpression in the ovarian cancer participates in the altered expression pattern.

Angiotensin-Converting Enzyme 2↗
PubMed1999

Identification of target genes of oncogenic transcription factors.

Disregulation of many transcription factors is associated with the development of human neoplasia. Transcription factors regulate cell growth, differentiation, and apoptosis by binding to specific DNA sequences within the promoter regions of growth-regulatory genes and modulating expression of these genes. This simple model is complicated by the fact that mammalian transcription factors are often members of large protein families that bind to similar DNA sequences. This raises the question as to whether members of a particular family regulate expression of overlapping or unique sets of genes. This review is focused on addressing this question using the Ets, Myc, and E2F transcription factor families as examples. Deregulated activity of some, but not all, members of these families is observed in cancer. Here, we summarize the data illustrating the concept that binding of individual members of these families of factors can result in promoter-specific responses and review the studies that have provided some insight into how target gene specificity is achieved. Since, for all of these oncogenic transcription factors, it remains unclear exactly which target genes are important in neoplasia, we have also reviewed the many approaches researchers are using to identify target genes of the various Ets, Myc, and E2F family members.

Carrier Proteins↗
PubMed2001

Gene targeting via triple-helix formation.

A report on a recent workshop entitled "Gene-Targeted Drugs: Function and Delivery" conveys a justified optimism for the eventual feasibility and therapeutic benefit of gene-targeting strategies. Although multiple approaches are being explored, this chapter focuses primarily on the uses of triplex-forming oligonucleotides (TFOs). TFOs are molecules that bind in the major groove of duplex DNA and by so doing can produce triplex structures. They bind to the purine-rich strand of the duplex through Hoogsteen or reverse Hoogsteen hydrogen bonding. They exist in two sequence motifs, either pyrimidine or purine. Improvements in delivery of these TFOs are reducing the quantities required for an effective intracellular concentration. New TFO chemistries are increasing the half-life of these oligos and expanding the range of sequences that can be targeted. Alone or conjugated to active molecules, TFOs have proven to be versatile agents both in vitro and in vivo. Foremost, TFOs have been employed in antigene strategies as an alternative to antisense technology. Conversely, they are also being investigated as possible upregulators of transcription. TFOs have also been shown to produce mutagenic events, even in the absence of tethered mutagens. TFOs can increase rates of recombination between homologous sequences in close proximity. Directed sequence changes leading to gene correction have been achieved through the use of TFOs. Because it is theorized that these modifications are due to the instigation of DNA repair mechanisms, an important area of TFO research is the study of triple-helix recognition and repair.

Chromatin↗
PubMed2003

New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells.

Until recently, precise modification of the animal genome by gene targeting was restricted to the mouse because germline competent embryonic stem cells are not available in any other mammalian species. Nuclear transfer (NT) technology now provides an alternative route for cell-based transgenesis in domestic species, offering new opportunities in genetic modification. Livestock that produce human therapeutic proteins in their milk, have organs suitable for xenotransplantation, or that could provide resistance to diseases such as spongiform encephalopathies have been produced by NT from engineered, cultured somatic cells. However, improvements in the efficiency of somatic cell gene targeting and a greater understanding of the reprogramming events that occur during NT are required for the routine application of what is currently an inefficient process. The ability to reprogramme and genetically manipulate cells will also be crucial for full exploitation of human embryonic stem (hES) cells, which offer unparalleled opportunities in human health and biotechnology. Particularly pertinent are directed differentiation of hES lines to specific cell lineages, production of cells that evade the patient's immune system and ensuring the safety of ensuing transplants. This review will discuss some of the successes, applications and challenges facing gene targeting in livestock and hES cells.

Animals↗
PubMed2002

Study of diverse mechanisms of cell-mediated cytotoxicity in gene-targeted mice using flow cytometric cytotoxicity assay.

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms-(1) release of lytic granules (containing perforin and granzymes), and (2) Fas ligand (FasL)/Fas or TNF-initiated apoptosis. We have examined mechanisms of target cell lysis by effector T cells from gene-targeted and mutant mice using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH67 dye. Cell death was estimated by 7-AAD inclusion and annexin V-PE binding. A direct correlation has been found between the percentage of dead target cells in FC Assay and the results of 111In release cytotoxicity assay when effector T cells from either Pfp -/- (perforin knockout) or gld (non-functional Fas Ligand) mice were used. As shown by the 4 h FC assay, the granule-mediated mechanism was utilized by T cells from gld mice. In contrast, T cells from Pfp -/- mice used death receptor-mediated lysis. Therefore, cytotoxic cells from gene-targeted and mutant mice can serve as valuable tools for studying different mechanisms of cell-mediated cytotoxicity, and the FC assay could be applied irrespective of which cytotoxic effector pathway is involved.

Animals↗
PubMed1998

Rare homologous gene targeting in Histoplasma capsulatum: disruption of the URA5Hc gene by allelic replacement.

URA5 genes encode orotidine-5'-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the Histoplasma capsulatum URA5 gene (URA5Hc) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized ura5 strains of H. capsulatum. We attempted to target the genomic URA5 locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in H. capsulatum and has been challenging in some other pathogenic fungi. Several strategies commonly used in Saccharomyces cerevisiae and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for URA5 gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (hph, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Deltaura5Hc::hph plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in H. capsulatum, or in any dimorphic systemic fungal pathogen of humans.

Alleles↗
PubMed2005

Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs.

Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Here, we demonstrate that three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or "seed" regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and we show all three classes of motif to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, we find that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, we demonstrate that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila.

3' Untranslated Regions↗
PubMed2003

Gene-targeted mice lacking the Ung uracil-DNA glycosylase develop B-cell lymphomas.

Mice deficient in the Ung uracil-DNA glycosylase have an increased level of uracil in their genome, consistent with a major role of Ung counteracting U:A base pairs arising by misincorporation of dUMP during DNA replication. A complementary uracil-excising activity apparently acts on premutagenic U:G lesions resulting from deamination of cytosine throughout the genome. However, Ung specifically processes U:G lesions targeted to immunoglobulin variable (V) genes during somatic hypermutation and class-switch recombination. Gene-targeted Ung(-/-) null mice remained tumour-free and showed no overt pathological phenotype up to approximately 12 months of age. We have monitored a large cohort of ageing Ung(-/-) mice and, beyond 18 months of age, they had a higher morbidity than Ung(+/+) controls. Post-mortem analyses revealed pathological changes in lymphoid organs, abnormal lymphoproliferation, and a greatly increased incidence of B-cell lymphomas in older Ung-deficient mice. These are the first data reporting the development of spontaneous malignancies in mice due to deficiency in a DNA glycosylase. Furthermore, they support a specific role for Ung in the immune system, with lymphomagenesis being related to perturbed processing of antibody genes in germinal centre B cells.

Animals↗