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Results for “Gene Targeting”

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At least 109 records · Page 6Linked to original sources
PubMed2001

Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo.

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Cre-dependent manner. Thus, the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo.

Animals↗
PubMed2003

Dissecting adrenal and behavioral responses to stress by targeted gene inactivation in mice.

To define the molecular pathways modulating adrenal and behavioral responses to stress, we have generated mice with inactivation of hypothalamic neuropeptides and signaling pathways. Studies in mice deficient in corticotropin-releasing hormone (CRH) have revealed the essential role for CRH in adrenal glucocorticoid production in response to many physiological and psychological stressors. Immune system activation in CRH-deficient mice provides a unique exception to the necessity for CRH in stimulating adrenal glucocorticoid production. By analyzing mice deficient in interleukin-6 (IL-6) and CRH, we find that restoration of glucocorticoid output with inflammation is largely mediated by dysregulated IL-6 production. Current studies focus on identifying cellular and gene targets by which glucocorticoids regulate immune system function. In contrast to impaired adrenocortical responses to stress, CRH-deficient mice exhibit normal behavioral responses to stress. To determine signaling pathways that may contribute to the behavioral responses to stress, we have generated and analyzed mice deficient in adenylyl cyclase type 8 (AC8). AC8 deficient mice have intact adrenocortical responses to stress, but an inability to undergo stress-induced alterations in behavior.

Adrenal Glands↗
PubMed2004

In vivo pattern of lipopolysaccharide and anti-CD3-induced NF-kappa B activation using a novel gene-targeted enhanced GFP reporter gene mouse.

NF-kappa B is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-kappa B has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-kappa B-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-kappa B cis elements (cis-NF-kappa B(EGFP)). A gene-targeting approach was used to insert a single copy of a NF-kappa B-dependent EGFP reporter gene 5' of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-kappa B(EGFP) mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-alpha that was blocked by the NF-kappa B inhibitors BAY 11-7082 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-kappa B(EGFP) promoter. Adenoviral delivery of NF-kappa B-inducing kinase strongly induced EGFP expression in the liver of cis-NF-kappa B(EGFP) mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-kappa B(EGFP) mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-kappa B during normal and diseased states.

Animals↗
PubMed2002

Direct production of gene-targeted mice from ES cells by nuclear transfer and gene transmission to their progeny.

In order to evaluate the usefulness of a cloning technique to produce gene-manipulated mice for the field of laboratory animal science, we produced mice cloned from gene-targeted embryonic stem (ES) cells and examined the vertical transmission of a targeted gene to their progeny. Of 1257 eggs constructed by nuclear transfer using M-phase ES donor cells targeted with an oviduct-specific glycoprotein (OGP) gene, 990 formed a pseudo-pronucleus and a polar body after activation. Of 504 cloned embryos transferred into recipients, 20 live cloned pups (2%) were recovered by Caesarean section at 19.5 days of gestation. Fourteen of these cloned mice were studied. Genotyping of the OGP locus and 20 microsatellite loci showed that they were genetically identical to the OGP gene-targeted TT2 cells. Eight cloned pups grew into adults, of which 7 were male and 1 was female (missing the Y chromosome). Mating experiments using the cloned mice were carried out. Of 89 F1 mice produced from the mating of cloned and C57BL/6J mice, 50 had the targeted OGP gene heterozygously. Thirty-seven F2 mice from 4 pairs of the OGP-/+ mice were composed of 9 OGP-/-, 18 OGP-/+, and 10 OGP+/+. Moreover, 26 offspring of one pair of the cloned mice were composed of 10 OGP-/-, 12 OGP-/+, and 4 OGP+/+. These offspring were fertile and transmitted the mutant OGP gene to the next generation. Comparison of these results with those of germline chimeric mice indicates that gene-targeted mice can be produced at least one generation earlier by nuclear transfer than by the conventional methods. In addition, the targeted OGP gene was constantly transmitted to the progeny of the gene-targeted mice. Cloning techniques are potentially a more efficient way to generate gene-manipulated mice for laboratory animal science, although such techniques include many unresolved problems, such as low production efficiency, and selection of a cell source for gene manipulation among others.

Animals↗
PubMed2006

miRNAMap: genomic maps of microRNA genes and their target genes in mammalian genomes.

Recent work has demonstrated that microRNAs (miRNAs) are involved in critical biological processes by suppressing the translation of coding genes. This work develops an integrated database, miRNAMap, to store the known miRNA genes, the putative miRNA genes, the known miRNA targets and the putative miRNA targets. The known miRNA genes in four mammalian genomes such as human, mouse, rat and dog are obtained from miRBase, and experimentally validated miRNA targets are identified in a survey of the literature. Putative miRNA precursors were identified by RNAz, which is a non-coding RNA prediction tool based on comparative sequence analysis. The mature miRNA of the putative miRNA genes is accurately determined using a machine learning approach, mmiRNA. Then, miRanda was applied to predict the miRNA targets within the conserved regions in 3'-UTR of the genes in the four mammalian genomes. The miRNAMap also provides the expression profiles of the known miRNAs, cross-species comparisons, gene annotations and cross-links to other biological databases. Both textual and graphical web interface are provided to facilitate the retrieval of data from the miRNAMap. The database is freely available at http://mirnamap.mbc.nctu.edu.tw/.

Animals↗
PubMed2002

The 'biased' evolution of targeted gene repair.

Targeted gene repair is a powerful yet controversial technique invented to direct single-base changes in chromosomal genes. The reaction mechanism has only recently come to light, and parameters that confer a level of control on the frequency of repair are still being defined in model genetic systems. Regulatory factors, such as strand bias, vector length, and the phase of the cell cycle in which targeting events take place, are now appreciated. This knowledge will lead to development of this technique for everyday genomics applications and, hopefully, gene therapeutics.

Animals↗
PubMed2002

Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO.

TAGKO ( transposon- arrayed gene knock out) is a highly efficient method for gene discovery and gene function assignment in the rice blast fungus Magnaporthe grisea. Here, we report the application of genome-wide TAGKO to the wheat blotch fungus Mycosphaerella graminicola, including the successful development of electroporation-based transformation for this fungus. A M. graminicola genomic cosmid library was constructed and a pool of 250 cosmid clones was mutagenized by in vitro transposition. Sequence analysis identified 5,110 unique insertion events in the M. graminicola genome. Eleven transposon-tagged cosmid clones (TAGKO clones) were chosen and transformed into the wild-type strain by electroporation. Ten TAGKO clones out of 11 produced gene-specific mutants at a targeting frequency of 15-28%, significantly higher than that of conventional gene-disruption constructs. The remaining clone failed to produce viable mutants, thereby providing indirect evidence for the identification of an essential gene.

Ascomycota↗
PubMed1997

Human immunodeficiency virus-1 proviral gene disruption by targeted gene therapy: a hypothetical technique for the elimination of provirus from the infected cells.

A hypothetical technique is proposed for the elimination of all the integrated human immunodeficiency virus-1 provirus from infected cells, based on the developing technology of selective gene excision through homologous recombination. In this technique, a recombinant retroviral packaging cell-line which would produce integrase-Rep78 chimeric protein would be constructed. Replication defective viral stocks would be made from this system which would have recombinant integrase-Rep78 protein packaged along with human immunodeficiency virus-1 long terminal repeat DNA. Since the Rep78 protein, which is a major regulatory protein of adeno-associated virus, has high affinity for human immunodeficiency virus-1 long terminal repeat, it would tether the newly synthesized human immunodeficiency virus-1 long terminal repeat (therapeutic DNA) to the human immunodeficiency virus-1 proviral site in the infected cell. This newly reverse transcribed human immunodeficiency virus-1 long terminal repeat would undergo homologous recombination with the provirus in the infected cells, facilitated by the nicking of the integrase part of the integrase-Rep78 recombinant protein. This selective gene knockout would be accomplished by the combined action of the chimeric integrase-Rep78 protein, where the Rep78 part would help docking of the therapeutic DNA to the proviral integration site and the integrase would provide nicking activity after homologous recombination, resulting in the replacement of human immunodeficiency virus-1 proviral genome with therapeutic DNA.

DNA, Recombinant↗
PubMed2001

Conditional gene targeting for cancer gene therapy.

Current treatment of solid tumors is limited by severe adverse effects, resulting in a narrow therapeutic index. Therefore, cancer gene therapy has emerged as a targeted approach that would significantly reduce undesired side effects in normal tissues. This approach requires a clear understanding of the molecular biology of both the malignant clone and the biological vectors that serve as vehicles to target cancer cells. In this review we discuss novel approaches for conditional gene expression in cancer cells. Targeting transgene expression to malignant tissues requires the use of specific regulatory elements including promoters based on tumor biology, tissue-specific promoters and inducible regulatory elements. We also discuss the regulation of both replication and transgene expression by conditionally-replicative viruses. These approaches have the potential to restrict the expression of transgenes exclusively to tissues of interest and thereby to increase the therapeutic index of future vectors for cancer gene therapy.

Animals↗
PubMed2003

GATA-4 and GATA-5 transcription factor genes and potential downstream antitumor target genes are epigenetically silenced in colorectal and gastric cancer.

The GATA family of transcription factors participates in gastrointestinal (GI) development. Increases in GATA-4 and -5 expression occur in differentiation and GATA-6 expression in proliferation in embryonic and adult settings. We now show that in colorectal cancer (CRC) and gastric cancer promoter hypermethylation and transcriptional silencing are frequent for GATA-4 and -5 but are never seen for GATA-6. Potential antitumor target genes upregulated by GATA-4 and -5, the trefoil factors, inhibinalpha, and disabled-2 (Dab2) are also silenced, in GI cancers, with associated methylation of the promoters. Drug or genetically induced demethylation simultaneously leads to expression, in CRC cells, of all of the GATA-4, -5, and downstream genes. Expression of exogenous GATA-5 overrides methylation at the downstream promoters to activate the target genes. Selection for silencing of both upstream transcription factors and their target genes in GI cancers could indicate that epigenetic silencing of the involved genes provides a summated contribution to tumor progression.

Cell Line, Tumor↗
PubMed2002

The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD.

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C(2)C(12) cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially up-regulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G(1) phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G(1)-to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.

Animals↗
PubMed2006

The antiapoptotic gene A1/BFL1 is a WT1 target gene that mediates granulocytic differentiation and resistance to chemotherapy.

Previous work has demonstrated that WT1 (-Ex5/-KTS) potentiates granulocyte colony-stimulating factor (G-CSF)-mediated granulocytic differentiation. This WT1 isoform suppresses cyclin E, which may contribute to the prodifferentiation effect by slowing proliferation, but WT1 target genes that affect survival might also be involved. We screened a cDNA array and identified the bCL2 family member A1/BFL1 as a new WT1 target gene in 32D cl3 murine myeloblast cells. Induction of WT1 (-Ex5/-KTS) expression is accompanied by up-regulation of A1 on the cDNA array, and this up-regulation was confirmed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Moreover, both promoter-reporter assays and chromatin immunoprecipitation assays suggest that this isoform of WT1 activates the promoter directly. Constitutive expression of A1 in 32D cl3 cells induces spontaneous granulocytic differentiation, with both morphologic and cell-surface antigen changes, as well as resistance both to chemotherapy and to withdrawal of interleukin-3 (IL-3). Finally, we note an association between WT1 expression and A1 expression in primary acute myeloid leukemia samples. Taken together, these results demonstrate that A1 is a new WT1 target gene involved in both granulocytic differentiation and resistance to cell death, and suggests that these genes might play an important role in the biology of high-risk leukemias.

Animals↗
PubMed2006

Molecular characterization of true and ectopic gene targeting events at the acetolactate synthase gene in Arabidopsis.

Precise modification of plant genomes via gene targeting (GT) is important for the study of gene function in vivo. A reliable GT system using the protoporphyrinogen oxidase (PPO) gene in Arabidopsis was reported 4 years ago; however, there are no subsequent successful reports of GT in Arabidopsis. A previous study showed ectopic gene targeting (EGT) of the endogenous gene in two-thirds of GT plants, which was an obstacle to efficient true gene targeting (TGT). The endogenous acetolactate synthase (ALS) gene is involved in the biosynthesis of branched chain amino acids in plants and is the site of action of several herbicides. To confirm the generality of the GT system in Arabidopsis, and to characterize the EGT event in plants in detail, we converted ALS from a herbicide (imazapyr)-susceptible to a -resistant form by GT. We obtained two imazapyr-resistant plants following GT. One of the targeting events was TGT while the other was EGT. After detailed Southern blotting, PCR and nucleotide sequence analysis of the EGT plant, we determined the genomic position and structure of the ectopically targeted site. Based on our findings, we discuss the possible mechanisms of EGT in plants.

Acetolactate Synthase↗
PubMed1997

Endothelial cell adhesion molecule expression in gene-targeted mice.

Gene-targeted mice are now routinely employed as tools for defining the contribution of different leukocyte and endothelial cell adhesion molecules to the leukocyte recruitment and tissue injury associated with acute and chronic inflammation. The objective of this study was to determine whether gene-targeted mice that are deficient in CD11/CD18, intracellular adhesion molecule-1 (ICAM-1), or P-selectin exhibit an altered constitutive or induced expression of the endothelial cell adhesion molecules E- and P-selectin. The gene-targeted mice were all developed in the 129Sv mouse strain and backcrossed into C57B1/6J mice. The number of backcrosses ranged between 8 (P-selectin) and 10 (CD18 and ICAM-1) generations. The dual-radiolabeled monoclonal antibody technique was used to quantify E- and P-selectin expression in different vascular beds. In the unstimulated state, E-selectin expression was significantly elevated (relative to wild-type mice) in the stomach, large intestine, and brain of mutants deficient in ICAM-1. In general, constitutive expression of P-selectin did not differ between wild-type, ICAM-1-deficient, and CD11/CD18-deficient mutants. In CD11/CD18-deficient mice, tumor necrosis factor-alpha (TNF-alpha) administration elicited a more profound upregulation of P-selectin in several vascular beds, compared with wild-type and ICAM-1-deficient mice. E-selectin expression in brain of TNF-alpha-stimulated, ICAM-1-deficient, and P-selectin-deficient mice was attenuated compared with wild-type mice. These findings indicate that chronic deficiency of some of the adhesion glycoproteins that mediate leukocyte recruitment alters basal and induced surface expression of other adhesion molecules on endothelial cells.

Animals↗
PubMed2004

Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent.

BACKGROUND: Serum Response Factor (SRF) is a transcription factor that is required for the expression of many genes including immediate early genes, cytoskeletal genes, and muscle-specific genes. SRF is activated in response to extra-cellular signals by its association with a diverse set of co-activators in different cell types. In the case of the ubiquitously expressed immediate early genes, the two sets of SRF binding proteins that regulate its activity are the TCF family of proteins that include Elk1, SAP1 and SAP2 and the myocardin-related MKL family of proteins that include MKL1 and MKL2 (also known as MAL, MRTF-A and -B and BSAC). In response to serum or growth factors these two classes of co-activators are activated by different upstream signal transduction pathways. However, it is not clear how they differentially activate SRF target genes. RESULTS: In order to identify the serum-inducible SRF target genes that are specifically dependent on the MKL pathway, we have performed microarray experiments using a cell line that expresses dominant negative MKL1. This approach was used to identify SRF target genes whose activation is MKL-dependent. Twenty-eight of 150 serum-inducible genes were found to be MKL-dependent. The promoters of the serum-inducible genes were analyzed for SRF binding sites and other common regulatory elements. Putative SRF binding sites were found at a higher rate than in a mouse promoter database but were only identified in 12% of the serum-inducible promoters analyzed. Additional partial matches to the consensus SRF binding site were found at a higher than expected rate in the MKL-dependent gene promoters. The analysis for other common regulatory elements is discussed. CONCLUSIONS: These results suggest that a subset of immediate early and SRF target genes are activated by the Rho-MKL pathway. MKL may also contribute to the induction of other SRF target genes however its role is not essential, possibly due to other activation mechanisms such as MAPK phosphorylation of TCFs.

Animals↗
PubMed2003

Homologous recombination and gene targeting in plant cells.

Gene targeting has become an indispensable tool for functional genomics in yeast and mouse; however, this tool is still missing in plants. This review discusses the gene targeting problem in plants in the context of general knowledge on recombination and gene targeting. An overview on the history of gene targeting is followed by a general introduction to genetic recombination of bacteria, yeast, and vertebrates. This abridged discussion serves as a guide to the following sections, which cover plant-specific aspects of recombination assay systems, the mechanism of recombination, plant recombination genes, the relationship of recombination to the environment, approaches to stimulate homologous recombination and gene targeting, and a description of two plant systems, the moss Physcomitrella patens and the chloroplast, that naturally have high efficiencies of gene targeting. The review concludes with a discussion of alternatives to gene targeting.

Bryopsida↗
PubMed1994

Lymphocyte ontogeny and activation in gene targeted mutant mice.

Gene targeting by homologous recombination in pluripotent embryonic stem cells enabled the systematic creation of mouse strains with defined genetic alterations. During the past few years a rapidly growing number of gene targeted mice has provided new insights into development, selection, activation, and signaling of T and B cells, as well as into the functions of cytokines. Here we discuss the present state of targeting technology and summarize the phenotypic changes observed in gene targeted mouse strains of immunological interest. These data allow us for the first time to define genetic checkpoints in lymphocyte development that are crucial for the orderly progression of T and B cells through ontogeny and for the generation of an immune response.

Animals↗
PubMed2006

The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination.

Gene targeting provides a powerful means for analyzing gene function, as exemplified by knockout mouse studies and recent work with the highly recombinogenic chicken DT40 B-lymphocyte line. In human cultured cells, however, the low frequency of gene targeting is a serious barrier to efficiently generate knockout clones. Moreover, commonly used human cell lines are karyotypically abnormal or unstable. Here, we show using promoterless targeting constructs that Nalm-6, a human pre-B ALL cell line, is highly proficient for gene targeting by homologous recombination. Indeed, the efficiency of TP53 gene targeting in Nalm-6 appears nearly two orders of magnitude higher than that in HCT116, a colon cancer cell line popularly used for gene targeting. Expression analysis revealed a lack of MSH2 expression in this cell line. As Nalm-6 has a stable neardiploid karyotype with normal p53 status, our results underscore the usefulness of Nalm-6 for gene knockout studies in humans.

B-Lymphocytes↗