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Results for “Gene Targeting”

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PubMed2000

The influence of antisense gene location on target gene suppression in the fission yeast Schizosaccharomyces pombe.

A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression. Fission yeast transformants were generated that contained the target lacZ gene at a fixed position and a single copy antisense lacZ gene integrated into various genomic locations, including the same locus as the target gene. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expression in vivo. Instead, increased lacZ downregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number.

Down-Regulation↗
PubMedDate not supplied

The Cre-loxP system: a versatile tool for targeting genes in a cell- and stage-specific manner.

Gene-targeted mice, derived from embryonic stem cells, are useful tools to study gene function during development. However, if the inactivation of the target gene results in embryonic lethality, the postdevelopmental function of the gene cannot be further studied. The Cre recombinase-loxP (Cre-loxP) system was developed to overcome this limitation as well as to confine the inactivation of the target gene in a cell- or tissue-specific manner. This system allows for the inactivation of the target gene in a single cell type, thereby allowing the analysis of physiological and pathophysiological consequences of the genetic alteration in mature animals. A unique property of the insulin gene to be expressed only in pancreatic beta cells has allowed using the beta-cell-specific rat insulin promoter (RIP) for Cre recombinase expression to inactivate genes in beta cells. The RIP has been used to inactivate genes in beta cells and analysis of these genetically altered mice has provided important information regarding the role of potential transcription factors and the receptors in vivo, for regulation of insulin gene transcription and in the development of beta cells. The Cre-loxP system is at a relatively early stage of development, and the ability of this technique to virtually target any gene in any tissue at any stage of development makes the study of gene function in a single cell type in vivo an attainable goal. It is anticipated that the continued experience with this system will provide an important tool to determine the role of the transcription factors involved in insulin gene regulation and islet cell differentiation and ultimately provide the basis for novel therapy to treat diabetes.

Animals↗
PubMed2006

Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines.

Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human pre-B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest can be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian--particularly human--genes.

Base Sequence↗
PubMed1991

Gene targeting and gene trap screens using embryonic stem cells: new approaches to mammalian development.

Mouse embryonic stem cell lines offer an attractive route for introducing rare genetic alternations into the gene pool since the cells can be pre-screened in culture and the mutations then transmitted into the germline through chimera production. Two applications of this technique that seem ideally suited for a genetic analysis of development are enhancer and gene trap screens for loci expressed during gastrulation and production of targeted mutations using homologous recombination. These approaches should greatly increase the number of mouse developmental mutants available and help to elucidate the genetic hierarchy controlling embryogenesis.

Animals↗
PubMed2000

Efficient gene targeted random mutagenesis in genetically stable Escherichia coli strains.

We describe a method to generate in vivo collections of mutants orders of magnitude larger than previously possible. The method favors accumulation of mutations in the target gene, rather than in the host chromosome. This is achieved by propagating the target gene on a plasmid, in Escherichia coli cells, within the region preferentially replicated by DNA polymerase I (Pol I), which replicates only a minor fraction of the chromosome. Mutagenesis is enhanced by a conjunction of a Pol I variant that has a low replication fidelity and the absence of the mutHLS system that corrects replication errors. The method was tested with two reporter genes, encoding lactose repressor or lipase. The proportion of mutants in the collection was estimated to reach 1% after one cycle of growth and 10% upon prolonged cell cultivation, resulting in collections of 10(12)-10(13) mutants per liter of cell culture. The extended cultivation did not affect growth properties of the cells. We suggest that our method is well suited for generating protein variants too rare to be present in the collections established by methods used previously and for isolating the genes that encode such variants by submitting the cells of the collections to appropriate selection protocols.

Bacterial Proteins↗
PubMed2004

Somatic gene targeting with RNA/DNA chimeric oligonucleotides: an analysis with a sensitive reporter mouse system.

BACKGROUND: Targeted gene correction provides a potentially powerful method for gene therapy. RNA/DNA chimeric oligonucleotides were reported to be able to correct a point mutation with a high efficiency in cultured rodent cells, in the body of mice and rats, and in plants. The efficiency of correction in the liver of rats was claimed to be as high as 20% after tail-vein injection. However, several laboratories have failed to reproduce the high efficiency. METHODS: In order to sensitively detect and measure sequence changes by the chimeric oligonucleotides, we used Muta Mouse, a transgenic mouse system for mutation detection in vivo. It carries, on its chromosome, multiple copies of the lambda phage genome with the lacZ(+) gene. Two chimeric oligonucleotides were designed to make a point mutation at the active site of the LacZ gene product. They were injected into the liver with HVJ liposomes, which were demonstrated to allow reliable gene delivery. One week later, DNA was extracted from the liver, and lambda::lacZ particles were recovered by in vitro packaging. The lacZ-negative phage was detected by selection with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the injected mice was at the same level as the control mouse (approximately 1/10000). Our further restriction analysis and sequencing did not detect the designed mutations. CONCLUSIONS: Gene correction frequency in mouse liver by these oligonucleotides was shown to be less than 1/20000 in our assay with the Muta Mouse system.

Animals↗
PubMed1995

The effect of target site transcription on gene targeting in human cells in vitro.

We evaluate the effect of target site transcription on gene targeting in cultured human fibrosarcoma cells. A number of cell lines that harbored a plasmid recombination substrate within their chromosomal DNA were created. Gene targeting frequency was then measured at these different loci in the presence and absence of an agent that stimulated target site transcription. We observed that gene targeting was significantly enhanced by RNA transcription. The magnitude of transcription-stimulated gene targeting varied from 3-fold to > 20-fold. No increase in gene targeting was observed, however, when transcription proceeded away from, rather than through, the recombination site. Transcription-stimulated gene targeting was also observed when single-stranded plasmid vectors complementary to either the coding or template strand were used as recombination substrates. Our results indicate that gene targeting, like other forms of DNA recombination, can be stimulated by target site transcription. The implications of our observations on current models of transcription-stimulated recombination are discussed.

Binding Sites↗
PubMed2000

Gene-targeting strategies.

Gene targeting in mouse ES cells is a powerful method for studying gene function in vivo. For the novice, this combination of molecular biology, specialized tissue-culture cell lines, and mouse reproductive biology can be daunting. We present a straightforward, one might say constrained, guide for novices of gene targeting to generate a null allele in large or small protein-coding genes. The method we outline has evolved from years of experience of training and advising beginners on this powerful technology. We believe that a good design for a gene-targeting strategy ultimately saves time, money, and research animal lives. Once you feel comfortable with a fundamental knockout, we suggest you consider the new and exciting gene-targeting variations that can be used to address important biological questions (2).

Animals↗
PubMed2004

Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations.

The stability of standard gene expression is an elementary prerequisite for internal standardisation of target gene expression data and many so called housekeeping genes with assumed stable expression can exhibit either up- or down-regulation under some experimental conditions. The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points. The BestKeeper software tool was validated on four housekeeping genes and 10 members of the somatotropic axis differentially expressed in bovine corpora lutea total RNA. The BestKeeper application and necessary information about data processing and handling can be downloaded on http://www.wzw.tum.de/gene-quantification/bestkeeper.html.

Animals↗
PubMed2000

[Construction of gene targeting vector for duplicating p35Nck5a gene and its application in the gene targeting of ES cells].

To duplicating the regulatory subunit p35Nck5a gene of mouse neuronal cdc2-like kinase in embryonic stem (ES) cells, about 12.2 kb of pGDTV vector for p35Nck5a gene duplication was constructed. The linearized pGDTV vectors were transfected into ES cells by electroporation. 245 drug-resistant cell clones were obtained in both G418 and GANC medium and the frequency of surviving cells was 6.22 x 10(-5). The ES cell clones were identified to have duplicated the p35Nck5a gene by use of both PCR and genomic Southern blotting, and the frequency of homologous recombination is 5.08 x 10(-7). The use of negative selection gene (HSV-tk) results in 7-fold increase at selection efficiency. This study lays the foundations of preparing mouse models of Alzheimer's disease.

Alzheimer Disease↗
PubMed2006

Targeted gene silencing in the model mushroom Coprinopsis cinerea (Coprinus cinereus) by expression of homologous hairpin RNAs.

The ink cap Coprinopsis cinerea is a model organism for studying fruiting body (mushroom) formation in homobasidiomycetes. Mutant screens and expression studies have implicated a number of genes in this developmental process. Functional analysis of these genes, however, is hampered by the lack of reliable reverse genetics tools for C. cinerea. Here, we report the applicability of gene targeting by RNA silencing for this organism. Efficient silencing of both an introduced GFP expression cassette and the endogenous cgl1 and cgl2 isogenes was achieved by expression of homologous hairpin RNAs. In latter case, silencing was the result of a hairpin construct containing solely cgl2 sequences, demonstrating the possibility of simultaneous silencing of whole gene families by a single construct. Expression of the hairpin RNAs reduced the mRNA levels of the target genes by at least 90%, as determined by quantitative real-time PCR. The reduced mRNA levels were accompanied by cytosine methylation of transcribed and nontranscribed DNA at both silencing and target loci in the case of constitutive high-level expression of the hairpin RNA but not in the case of transient expression. These results suggest the presence of both posttranscriptional and transcriptional gene silencing mechanisms in C. cinerea and demonstrate the applicability of targeted gene silencing as a powerful reverse genetics approach in this organism.

Coprinus↗
PubMed1995

Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.

Double-strand breaks (DSBs) are recombinogenic lesions in chromosomal DNA in yeast, Drosophila and Caenorhabditis elegans. Recent studies in mammalian cells utilizing the I-Scel endonuclease have demonstrated that in some immortalized cell lines DSBs in chromosomal DNA are also recombinogenic. We have now tested embryonic stem (ES) cells, a non-transformed mouse cell line frequently used in gene targeting studies. We find that a DSB introduced by I-Scel stimulates gene targeting at a selectable neo locus at least 50-fold. The enhanced level of targeting is achieved by transient expression of the I-Scel endonuclease. In 97% of targeted clones a single base pair polymorphism in the transfected homologous fragment was incorporated into the target locus. Analysis of the targeted locus demonstrated that most of the homologous recombination events were 'two-sided', in contrast to previous studies in 3T3 cells in which 'one-sided' homologous events predominated. Thus ES cells may be more faithful in incorporating homologous fragments into their genome than other cells in culture.

Animals↗
PubMed1997

Advances in gene targeting methods.

Gene targeting in embryonic stem cells is commonly used for gene inactivation and the generation of mouse mutants. The combined use of methods for site-specific and homologous DNA recombination expands the potential of gene targeting in embryonic stem cells considerably and offers the opportunity of conditional gene targeting in mice.

Animals↗
PubMed2005

Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting.

Targeted gene replacement (TGR) in yeast and mammalian cells is initiated by the two free ends of the linear targeting molecule, which invade their respective homologous sequences in the chromosome, leading to replacement of the targeted locus with a selectable gene from the targeting DNA. To study the postinvasion steps in recombination, we examined the effects of DNA structure-specific proteins on TGR frequency and heteroduplex DNA formation. In strains deleted of RAD1, MSH2, or MSH3, we find that the frequency of TGR is reduced and the mechanism of TGR is altered while the reverse is true for deletion of SGS1, suggesting that Rad1 and Msh2:Msh3 facilitate TGR while Sgs1 opposes it. The altered mechanism of TGR in the absence of Msh2:Msh3 and Rad1 reveals a separate role for these proteins in suppressing an alternate gene replacement pathway in which incorporation of both homology regions from a single strand of targeting DNA into heteroduplex with the targeted locus creates a mismatch between the selectable gene on the targeting DNA and the targeted gene in the chromosome.

DNA↗
PubMed2001

Identification of novel TGF-beta /Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach.

Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-beta (TGF-beta) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation, (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-beta/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smad-dependent TGF-beta target genes in fibroblasts.

Animals↗
PubMed1995

Scavenger receptor A gene regulatory elements target gene expression to macrophages and to foam cells of atherosclerotic lesions.

Transcription of the macrophage scavenger receptor A gene is markedly upregulated during monocyte to macrophage differentiation. In these studies, we demonstrate that 291 bp of the proximal scavenger receptor promoter, in concert with a 400-bp upstream enhancer element, is sufficient to direct macrophage-specific expression of a human growth hormone reporter in transgenic mice. These regulatory elements, which contain binding sites for PU.1, AP-1, and cooperating ets-domain transcription factors, are also sufficient to mediate regulation of transgene expression during the in vitro differentiation of bone marrow progenitor cells in response to macrophage colony-stimulating factor. Mutation of the PU.1 binding site within the scavenger receptor promoter severely impairs transgene expression, consistent with a crucial role of PU.1 in regulating the expression of the scavenger receptor gene. The ability of the scavenger receptor promoter and enhancer to target gene expression to macrophages in vivo, including foam cells of atherosclerotic lesions, suggests that these regulatory elements will be of general utility in the study of macrophage differentiation and function by permitting specific modifications of macrophage gene expression.

Animals↗
PubMed1996

Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci.

P element-induced gene conversion has been previously used to modify the white gene of Drosophila melanogaster in a directed fashion. The applicability of this approach of gene targeting in Drosophila melanogaster, however, has not been analyzed quantitatively for other genes. We took advantage of the P element-induced forked allele, f(hd), which was used as a target, and we constructed a vector containing a modified forked fragment for converting f(hd). Conversion frequencies were analyzed for this locus as well as for an alternative white allele, w(eh812). Combination of both P element-induced mutant genes allowed the simultaneous analysis of conversion frequencies under identical genetic, developmental, and environmental conditions. This paper demonstrates that gene conversion through P element-induced gap repair can be applied with similar success rates at the forked locus and in the white gene. The average conversion frequency at forked was 0.29%, and that at white was 0.17%. These frequencies indicate that in vivo gene targeting in Drosophila melanogaster should be applicable for other genes in this species at manageable rates. We also confirmed the homolog dependence of reversions at the forked locus, indicating that P elements transpose via a cut-and-paste mechanism. In a different experiment, we attempted conversion with a modified forked allele containing the su(Hw) binding site. Despite an increased sample size, there were no conversion events with this template. One interpretation (under investigation) is that the binding of the su(Hw) product prevents double-strand break repair.

ATP-Binding Cassette Transporters↗
PubMed2003

New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region.

The use of PCR-based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR-amplified functional analysis (FA)-cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3'-ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C-terminal GFP-tagging were generated by adding GFP-sequences flanked at the 5'-end by a (Gly-Ala)3-linker and at the 3'-end by the S. cerevisiae URA3-terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3'-ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene-specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future.

Candida albicans↗