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Results for “Gene Targeting”

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At least 145 records · Page 8Linked to original sources
PubMed2004

Tor signaling and nutrient-based signals converge on Mks1p phosphorylation to regulate expression of Rtg1.Rtg3p-dependent target genes.

The heterodimeric bZip/HLH transcription factors Rtg1p and Rtg3p regulate the expression of a concise set of metabolic genes (termed RTG target genes) required for de novo biosynthesis of glutamate and glutamine. Several components have now been identified that control both the intracellular localization as well as activity of the Rtg1p.Rtg3p complex, yet the precise upstream regulatory signals involved remain unclear. For example, it has been proposed that Rtg1p.Rtg3p activity is repressed by glutamate, acting through the mitochondrial retrograde response pathway or, alternatively, by glutamine, acting through the Tor kinase pathway. Here we demonstrate that RTG target gene regulation is remarkably complex, with glutamate and glutamine as well as ammonia collaborating as potentially distinct signals to regulate RTG target gene expression. We show that both Tor and these nutrient-based signals converge on Mks1p, the immediate upstream inhibitor of Rtg1p.Rtg3p, and that a direct correlation exists between the degree of Mks1p phosphorylation and the extent of RTG target gene repression. Finally, we find that Tor- and glutamine-mediated RTG-target gene repression can be experimentally uncoupled, indicating that glutamine and Tor act, at least in part, independently to inhibit this pathway.

Ammonia↗
PubMed2003

Effect of the Pro12Ala polymorphism in the peroxisome proliferator-activated receptor (PPAR) gamma2 gene on the expression of PPARgamma target genes in adipose tissue of massively obese subjects.

The aim was to study the effect of the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor (PPAR) gamma2 gene on the expression of PPARgamma target genes in adipose tissue. Adipose tissue samples were collected from 30 massively obese subjects (10 men and 20 women) from omental, sc abdominal, and femoral depots. The mRNA expression of PPARgamma1, PPARgamma2, lipoprotein lipase, p85alpha phosphatidylinositol 3-kinase, and uncoupling protein 2 were quantified by reverse transcription-competitive PCR. The genotypes of Pro12Ala polymorphism were determined by single-strand conformation polymorphism analysis. The frequency of the Ala12 allele was 13.3% (8 Pro12Ala and 22 Pro12Pro). There were no differences in body weight, fat mass, and fasting serum leptin between the genotypes. The mRNA expression of p85alpha phosphatidylinositol 3-kinase was significantly lower in the omental fat of the Pro12Ala carriers than the Pro12Pro carriers (P < 0.01). It also appeared that PPARgamma2 expression was higher in men with Ala12 allele (P < 0.01). Interestingly, particularly in women, the expression of both PPARgamma splice variants was lower in omental than sc fat independently of the genotype (P < 0.05-0.01). The common Pro12Ala polymorphism of the PPARgamma2 gene has minor influence on mRNA expression of PPARgamma target genes in adipose tissue of obese subjects. Expression of both PPARgamma splice variants is dependent on fat depot: omental fat shows lower mRNA levels, compared with sc fat depots.

Adipose Tissue↗
PubMed2003

1,5-isoquinolinediol increases the frequency of gene targeting by homologous recombination in mouse fibroblasts.

Gene targeting is a technique that allows the introduction of predefined alterations into chromosomal DNA. It involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In theory, gene targeting constitutes the ideal method of gene therapy for single gene disorders. In practice, gene targeting remains extremely inefficient for at least two reasons: very low frequency of homologous recombination in mammalian cells and high proficiency of the mammalian cells to randomly integrate the targeting vector by illegitimate recombination. One known method to improve the efficiency of gene targeting is inhibition of poly(ADP-ribose)polymerase (PARP). It has been shown that PARP inhibitors, such as 3-methoxybenzamide, could lower illegitimate recombination, thus increasing the ratio of gene targeting to random integration. However, the above inhibitors were reported to decrease the absolute frequency of gene targeting. Here we show that treatment of mouse Ltk cells with 1,5-isoquinolinediol, a recent generation PARP inhibitor, leads to an increase up to 8-fold in the absolute frequency of gene targeting in the correction of the mutation at the stable integrated HSV tk gene.

Animals↗
PubMed2006

Carbon monoxide rescues ischemic lungs by interrupting MAPK-driven expression of early growth response 1 gene and its downstream target genes.

Carbon monoxide (CO), an endogenous cytoprotective product of heme oxygenase type-1 regulates target thrombotic and inflammatory genes in ischemic stress. Regulation of the gene encoding early growth response 1 (Egr-1), a potent transcriptional activator of deleterious thrombotic and inflammatory cascades, may govern CO-mediated ischemic lung protection. The exact signaling mechanisms underlying CO-mediated cytoprotection are not well understood. In this study we tested the hypothesis that inhibition of mitogen-activated protein kinase-dependent Egr-1 expression may be pivotal in CO-mediated ischemic protection. In an in vivo isogeneic rat lung ischemic injury model, inhaled CO not only diminished fibrin accumulation and leukostasis and improved gas exchange and survival but also suppressed extracellular signal-regulated kinase (ERK) activation, Egr-1 expression, and Erg DNA-binding activity in lung tissue. Additionally, CO-mediated inhibition of Egr-1 reduced expression of target genes, such as tissue factor, serpine-1, interleukin-1, and TNF-alpha. However, CO failed to inhibit serpine-1 expression after unilateral lung ischemia in mice null for the Egr-1 gene. In RAW macrophages in vitro, hypoxia-induced Egr-1 mRNA expression was ERK-dependent, and CO-mediated suppression of ERK activation resulted in Egr-1 inhibition. Furthermore, CO suppression of ERK phosphorylation was reversed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but was insensitive to cAMP-dependent protein kinase A inhibition with H89 and NO synthase inhibition with l-nitroarginine methyl ester. This finding indicates that CO suppresses ERK in a cGMP-dependent but cAMP/protein kinase A- and NO-independent manner. Together, these data identify a unifying molecular mechanism by which CO interrupts proinflammatory and prothrombotic mediators of ischemic injury.

Animals↗
PubMed1996

An animal model for Norrie disease (ND): gene targeting of the mouse ND gene.

In order to elucidate the cellular and molecular processes which are involved in Norrie disease (ND), we have used gene targeting technology to generate ND mutant mice. The murine homologue of the ND gene was cloned and shown to encode a polypeptide that shares 94% of the amino acid sequence with its human counterpart. RNA in situ hybridization revealed expression in retina, brain and the olfactory bulb and epithelium of 2 week old mice. Hemizygous mice carrying a replacement mutation in exon 2 of the ND gene developed retrolental structures in the vitreous body and showed an overall disorganization of the retinal ganglion cell layer. The outer plexiform layer disappears occasionally, resulting in a juxtaposed inner and outer nuclear layer. At the same regions, the outer segments of the photoreceptor cell layer are no longer present. These ocular findings are consistent with observations in ND patients and the generated mouse line provides a faithful model for study of early pathogenic events in this severe X-linked recessive neurological disorder.

Amino Acid Sequence↗
PubMed2006

Homologous recombination is required for AAV-mediated gene targeting.

High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325-330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612-4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735-738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting approximately 5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR.

Cell Line, Tumor↗
PubMed1994

Targeted gene duplication and disruption for analyzing quantitative genetic traits in mice.

Experimental analysis of complex quantitative genetic traits, such as essential hypertension, should be greatly facilitated by being able to manipulate the expression of a gene in living animals without altering the nucleotide sequence, chromosomal location, or regulatory elements of the gene. To explore this possibility, we have used targeted gene disruption and duplication to generate mice that are genetically identical [(129 x C57BL6)F1] except for having one, two, or three functional copies of the gene coding for angiotensinogen. The two-copy animals have two normal copies of the angiotensinogen gene; the one-copy and three-copy animals have one normal copy with the other either disrupted or duplicated by gene targeting. The duplicated pair of genes was generated by a special form of gap-repair gene targeting that tandemly duplicates the whole of a gene together with 5' and 3' flanking regions. We find progressively and significantly higher levels of the gene product in the animals having increasing numbers of gene copies: the one-copy animals have steady-state plasma angiotensinogen levels approximately 35% of normal (P < 0.0001), and the three-copy animals have levels approximately 124% of normal (P < 0.004). Detailed information about regulatory sequences is not required for this type of experiment; nor is it necessary to have DNA clones or targeting constructs that cover the whole of the target gene. Varying gene copy numbers by targeting consequently offers a promising approach to quantitative genetics.

Angiotensinogen↗
PubMed2001

Contemporary gene targeting strategies for the novice.

Gene targeting in mouse embryonic stem (ES) cells is a fundamental methodology for generating mice with precise genetic modifications. Although there are many complex gene targeting strategies for creating a variety of diverse mutations in mice, most investigators initially choose to generate a null allele. Here we provide a guide for the novice to generate a null allele for a protein coding gene using a fundamental gene targeting strategy. Ultimately, a well considered gene targeting strategy saves significant amounts of time, money, and research animal lives. The straightforward strategy presented here bypasses many of the pitfalls associated with gene knockouts generated by novices. This guide also serves as a foundation for subsequently designing more complex gene targeting strategies.

Animals↗
PubMed1999

Gene targeting of retinoid receptors.

Gene targeting in embryonic stem (ES) cells has been employed to investigate the role of the retinoid receptors and binding proteins both in the mouse as well as in embryocarcinoma cells. It is a powerful technique for the modification of the mouse genome. With more recent refinements in gene targeting technology, it is now possible to introduce more subtle mutations in the murine genome, as well as to investigate gene function in a tissue and temporally-restricted manner. It should also be possible to modify genes in diverse diploid cell lines, to generate diverse model systems for analysis of retinoid receptor function. In this article, some of the basic principles for gene targeting are described.

Animals↗
PubMed2002

A chromosomal position effect on gene targeting in human cells.

We describe gene targeting experiments involving a human cell line (RAN10) containing, in addition to its endogenous alleles, two ectopic alleles of the interferon-inducible gene 6-16. The frequency of gene targeting at one of the ectopic 6-16 alleles (H3.7) was 34-fold greater than the combined frequency of gene targeting involving endogenous 6-16 alleles in RAN10. Preference for H3.7 was maintained when the target loci in RAN10 were transcriptionally activated by interferon. Despite the 34-fold preference for H3.7, the absolute gene targeting efficiency in RAN10 was only 3-fold higher than in the parental HT1080 cell line. These data suggest that different alleles can compete with each other, and perhaps with non-homologous loci, in a step which is necessary, but not normally rate-limiting, for gene targeting. The efficiency of this step can therefore be more sensitive to chromosomal position effects than the rate-determining steps for gene targeting. The nature of the position effects involved remains unknown but does not correlate with transcription status, which in our system has a very modest influence on the frequency of gene targeting. In summary, our work unequivocally identifies a position effect on gene targeting in human cells.

Alleles↗
PubMed1994

Gene-targeting and the p53 tumor-suppressor gene.

Gene-targeting techniques are now frequently applied to embryonic stem (ES) cells to introduce mutations of endogenous genes in mice. Modifications introduced into tumor-suppressor genes by this technology have produced mice and cell lines with unique tumorigenic and growth characteristics, respectively. A number of strategies have been developed to enhance the efficiency of homologous recombination between targeting vectors and endogenous genes. This review describes recent advances in the techniques used to construct mice with a variety of genetic alterations. In addition, an application of gene-targeting is illustrated in the study of a class of genes with tumor-suppressor function. Recent findings from experiments using gene targeted mice to study the p53 tumor-suppressor gene are discussed and the potential of gene-targeting for the discovery and study of novel tumor-suppressor genes are explored.

Animals↗
PubMed1997

Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria.

We describe several plasmids that are designed to create fusions between chromosomal or plasmid-encoded genes and the lacZ, phoA or gfp reporter genes. These plasmids all contain the vegetative origin of R6K, but lack the R6K pir gene, and therefore fail to replicate in strains lacking pir. Fragments of target genes are introduced into these plasmids, and fusions are created in a single step as a consequence of (Campbell-type) integration of the entire plasmid by homologous recombination. Cloned fragments containing either an intact 5'-end of the target gene including its promoter or an intact 3'-end of the gene preserve a functional copy of that gene, while fragments lacking both 5'- and 3'-ends of the target gene cause a gene disruption. In addition to facilitating measurements of gene expression, some plasmids create translational fusions to beta-galactosidase or alkaline phosphatase and are therefore useful in studying the membrane topology of a target protein. We demonstrate the utility of these plasmids by constructing and testing two operon fusions and two protein fusions between the virG gene of Agrobacterium tumefaciens and lacZ.

Agrobacterium tumefaciens↗
PubMedDate not supplied

Suppression of gene expression by tethering KRAB domain to promoter of ER target genes.

Estrogens play an important role in the development and progression of breast cancer. Although estrogen antagonist treatment often results in the arrest or remission of breast cancer growth, most breast cancers recur and become resistant to estrogen ablative therapy. The molecular mechanisms underlying these actions remain largely undefined. It is hypothesized that tumor cells of an advanced stage may develop compensatory pathways to stimulate the expression of estrogen receptor (ER) target genes or downstream events, independent of estrogen action. In this study, we developed a chimeric repressor to turn off ER target genes with the aim of directly investigating the role of ER target genes in tumor progression. The chimeric repressor contains the ER DNA-binding domain that recognizes estrogen response elements (EREs), a Krupple-associated box (KRAB) repressor domain which silences target genes when tethered to their promoter regions and a truncated progesterone ligand-binding domain which responds only to the exogenous synthetic ligand, RU486. The ability of the chimeric repressor to block ER mediated transcription was assessed in transient transfection assays. ER-induced reporter activity was inhibited by the repressor in a dose-dependent manner, with the maximum effect of more than 80% reduction. The inhibitory activity of the chimeric repressor was tightly under the control of RU486. Effective suppression by the repressor on the natural promoter of ER target gene, complement factor 3 (C3), was also observed. The inhibitory activity was specific to ER, since the repressor has no effect on other nuclear receptor systems tested. Furthermore, the repressor could inhibit the 4-hydroxy-tamoxifen (4OH-T)-induced ER activity. Taken together, our results demonstrate that the inducible repressor we have designed could specifically inhibit ER target gene expression in response to an exogenous synthetic ligand. This repressor will provide a useful tool to study the role of ER target genes in breast cancer progression and it may be potentially useful for gene therapy of breast cancer.

Base Sequence↗
PubMed1995

Inducible gene targeting in mice.

A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an interferon-responsive promoter to control the expression of Cre recombinase. Here, Cre was used to delete a segment of the DNA polymerase beta gene flanked by IoxP recombinase recognition sites. Deletion was complete in liver and nearly complete in lymphocytes within a few days, whereas partial deletion was obtained in other tissues. This method can be used for the inducible inactivation of any other gene in vivo.

Animals↗
PubMed2006

TIS11D is a candidate pro-apoptotic p53 target gene.

A number of target genes for the tumor suppressor, p53, have been identified, however, the mechanisms that contribute to p53-dependent apoptosis remain to be fully elucidated. In a comprehensive screen for p53 target genes by differential display, we have identified TIS11D as a p53-inducible gene. Induction of TIS11D mRNA was confirmed by Northern Blot in response to p53 expression. Inducible expression of TIS11D resulted in inhibition of cell proliferation and apoptosis. These data suggest TIS11D as a candidate p53 target gene that may be part of the network of genes responsible for p53-dependent apoptosis.

Animals↗
PubMed1989

Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology.

We have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418r cell per 3 x 10(6) infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

Animals↗
PubMed2006

A gene-targeting approach for functional characterization of KIAA genes encoding extremely large proteins.

Given that thousands of genes exist in the mammalian genome, criteria are needed to prioritize their functional analysis and to decrease the likelihood of producing gene-targeted mice that lack overt phenotypes. Initial analysis efforts are likely to be fruitful if focused on genes encoding large proteins, since at least some large proteins serve as frameworks for intricate assembly of protein complexes, and their inactivation would render definitive, observable phenotypes. Here, we describe the functional characterization of the murine homologues of five human KIAA genes (KIAA1409, KIAA1440, KIAA1447, KIAA1768, and KIAA1276) that encode large proteins. Gene-targeted mice had phenotypic and developmental defects resulting from the functional deletion of three of these five genes. Mice with targeted disruption of KIAA1409 lacked the ability to drink, and those with targeted disruption of KIAA1447 displayed hind leg motor dysfunction. Disruption of KIAA1440 led to embryonic lethality at the blastocyst stage. The high success rate of our approach demonstrates the rationale for the genome-wide functional examination of large proteins in mice using reverse genetics.

Animals↗