Combination of gene targeting and gene transfer by adenoviral vectors in the analysis of neurotrophin-mediated neuronal plasticity.
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Adenoviridae↗
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Results for “Gene Targeting”
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Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene.
Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially CRE-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.
AMP-Activated Protein Kinases↗
Estrogen receptor target gene: an evolving concept.
Estrogen receptor (ER) functions as a transcription factor to induce gene expression events sufficient for cell division and breast cancer progression. A significant body of work exists on the identification of ER gene targets and the cofactors that contribute to these transcription events, yet surprisingly little is known of the cis-regulatory elements involved. In this review, we investigate the advances in technology that contribute to a comprehensive understanding of ER target genes and explore recent work identifying cis-regulatory domains that augment transcription of these targets. Specifically, we find that ER association with gene targets results from an association with the pioneer factor FoxA1, responsible for recruitment of ER to the genome. Recruitment of ER to the genome does not occur at promoter proximal regions, but instead involves distal enhancer elements that function to tether the ER complex to the target gene promoters. These advances in technology permit a more detailed investigation of ER activity and may aid in the development of superior drug interventions.
Animals↗
Target gene identification: target specific transcriptional activation by three murine homeodomain/VP16 hybrid proteins in Saccharomyces cerevisiae.
The mammalian homeodomain proteins encoded by Hox genes play an important role in embryonic development by providing positional queues which define developmental identities along the anteroposterior axis of developing organisms. These proteins bind DNA specifically through their homeodomain to sequences containing ATTA cores, and thereby are thought to exert their effect regulating downstream genes. Little is known about the specificity of binding of homeodomain proteins to their sequences and the identity of their target genes. We have developed a transcriptional activation assay in yeast which employs a homeobox/VP16 fusion gene as a transcriptional activator and a target construct in which test fragments of DNA are inserted upstream to a reporter gene. Using this assay, we compared transcriptional activation by three chimeric proteins containing the homeodomains of the mouse homeobox genes, Hoxa-5, Hoxb-6, and Hoxc-8. When tested on previously defined target sequences, strong differential specificities of activation were observed. In an effort to identify enhancers that normally respond to homeodomain transcriptional activators, random fragments of mouse genomic DNA were cloned upstream of the reporter gene. Genomic DNA fragments with distinct activation profiles were obtained and were found to share matches beyond the ATTA core with previously described enhancers. These results demonstrate that the transcriptional activation system in yeast can be used as a convenient system to detect DNA motifs which bind homeodomain proteins, and subsequently, to identify authentic target genes responsive to Hox gene proteins.
Amino Acid Sequence↗
Double targeted gene replacement for creating null mutants.
We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.
Alleles↗
Targeted gene deletion of Leishmania major genes encoding developmental stage-specific leishmanolysin (GP63).
The major surface glycoprotein of Leishmania major is a zinc metalloproteinase of 63 kDa referred to as leishmanolysin or GP63, which is encoded by a family of seven genes. Targeted gene replacement was used to delete gp63 genes 1-6 encoding the highly expressed promastigote and constitutively expressed GP63. In the L. major homozygous mutants deficient in gp63 genes 1-6, there was no expression of GP63 as detected by reverse transcription-polymerase chain reaction (RT-PCR) or fluorescent staining in promastigotes from the procyclic stage (logarithmic growth phase). The remaining L. major gP63 gene 7 was shown to be developmentally regulated, as it was expressed exclusively in infectious metacyclic stage (late stationary growth phase) promastigotes and in lesion amastigotes. The gp63 genes 1-6-deficient mutants showed increased sensitivity to complement-mediated lysis. The sensitivity to lysis was greater in procyclics than in metacyclics when compared with the equivalent wild-type stages. Increased resistance of the mutant metacyclic promastigotes correlated with the expression of gp63 gene 7 and was restored to the same levels as wild-type promastigotes by transfection with gp63 gene 1. Thus, expression of GP63 is clearly involved in conferring resistance to complement-mediated lysis. The L. major GP63 1-6 mutants were capable of infecting mouse macrophages and differentiating into amastigotes. Similar levels of infection and subsequent intracellular survival were observed when mouse macrophages were infected in vitro with wild type, GP63 1-6 mutants and mutants transfected with gp63 gene 1. The GP63 1-6 mutants were capable of lesion formation in BALB/c mice and, thus, gp63 genes 1-6 do not play a role in the survival of the parasite within mouse macrophages. The role of gp63 genes 1-6 in parasite development within the sandfly vector was studied. GP63 1-6 mutants grew normally in the blood-engorged midgut of both Phlebotomus argentipes and P. papatasi However, both wild-type and mutant promastigotes were lost after 2 days' growth in P. papatasi. The complete developmental pathway in P. argentipes was observed for wild-type promastigotes, GP63 1-6 mutants and mutants transfected with gp63 gene 1. Normal stage differentiation from amastigotes to procyclics, to nectomonads, to haptomonads and to infectious metacyclics was observed. Thus, the highly expressed promastigote forms of GP63, encoded by gp63 genes 1-6, do not appear to be required for nutrient utilization in the bloodmeal during the early stages of development in the sandfly or for midgut attachment and further development. gp63 1-6 genes do, however, play a major protective role against complement-mediated lysis when promastigotes are introduced into the mammalian host.
Animals↗
Gene-targeting technologies for the study of neurological disorders.
Studies using genetic manipulations have proven invaluable in the research of neurological disorders. In the forefront of these approaches is the knockout technology that engineers a targeted gene mutation in mice resulting in inactivation of gene expression. In many cases, important roles of a particular gene in embryonic development have precluded the in vivo study of its function in the adult brain, which is usually the most relevant experimental context for the study of neurological disorders. The conditional knockout technology has provided a tool to overcome this restriction and has been used successfully to generate viable mouse models with gene inactivation patterns in certain regions or cell types of the postnatal brain. This review first describes the methodology of gene targeting in mice, detailing the aspects of designing a targeting vector, introducing it into embryonic stem cells in culture and screening for correct recombination events, and generating chimeric and null mutant mice from the positive clones. It then discusses the special issues and considerations for the generation of conditional knockout mice, including a section about approaches for inducible gene inactivation in the brain and some of their applications. An overview of gene-targeted mouse models that have been used in the study of several neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, seizure disorders, and schizophrenia, is also presented. The importance of the results obtained by these models for the understanding of the pathogenic mechanism underlying the disorders is discussed.
Animals↗
Target genes and target cells in carcinogenesis.
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Animals↗
Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice.
BACKGROUND: The role of plasminogen system components in focal cerebral ischemic infarction (FCI) was studied in mice deficient in plasminogen (Plg-/-), in tissue or urokinase plasminogen activator (tPA-/- or uPA-/-), or in plasminogen activator inhibitor-1 or alpha2-antiplasmin (PAI-1(-/-) or alpha2-AP-/-). METHODS AND RESULTS: FCI was produced by ligation of the left middle cerebral artery and measured after 24 hours by planimetry of stained brain slices. In control (wild-type) mice, infarct size was 7.6+/-1.1 mm3 (mean+/-SEM), uPA-/- mice had similar infarcts (7.8+/-1.0 mm3, P=NS), tPA-/- mice smaller (2.6+/-0.80 mm3, P<0.0001), PAI-1(-/-) mice larger (16+/-0.52 mm3, P<0.0001), and Plg-/- mice larger (12+/-1.2 mm3, P=0.037) infarcts. alpha2-AP-/- mice had smaller infarcts (2. 2+/-1.1 mm3, P<0.0001 versus wild-type), which increased to 13+/-2.5 mm3 (P<0.005 versus alpha2-AP-/-) after intravenous injection of human alpha2-AP. Injection into alpha2-AP-/- mice of Fab fragments of affinospecific rabbit IgG against human alpha2-AP, after injection of 200 microg human alpha2-AP, reduced FCI from 11+/-1.5 to 5.1+/-1.1 mm3 (P=0.004). CONCLUSIONS: Plg system components affect FCI at 2 different levels: (1) reduction of tPA activity (tPA gene inactivation) reduces whereas its augmentation (PAI-1 gene inactivation) increases infarct size, and (2) reduction of Plg activity (Plg gene inactivation or alpha2-AP injection) increases whereas its augmentation (alpha2-AP gene inactivation or alpha2-AP neutralization) reduces infarct size. Inhibition of alpha2-AP may constitute a potential avenue to treatment of FCI.
Adenoviridae↗
Gene radiotherapy; gene targeted versus targeted by gene product.
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Animals↗
Site-specific gene targeting for gene expression in eukaryotes.
Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase-DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration. In addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.
Animals↗
Targeting genes and proteins in the analysis of learning and memory: caveats and future directions.
Gene targeting using homologous recombination in embryonic stem (ES) cells and transgenic approaches in general allow one to precisely manipulate single genes and investigate their in vivo function in the mouse. Geneticists argue that these techniques are superior to pharmacological approaches as they obviate the lack of highly specific pharmacological agents in the study of brain function and behavior. However, by now it has become clear that transgenic approaches also have some limitations. One problem is spatial and temporal specificity of the genetic manipulation. The other is the possibility that the introduced genetic alteration gives rise to complex, secondary phenotypic changes. This may be a disadvantage in the functional analysis of genes associated with learning and memory especially if the gene of interest plays roles in embryonic development of the brain as well as in adult neural function. Examples of such genes include, but are not limited to, those encoding neurotrophins, cell adhesion molecules, and protein kinases. Second generation gene targeting with inducible and cell type restricted knock-out, or transgenic approaches with inducible gene expression systems, will solve some problems. However, at present these strategies also suffer from difficulties inherent to the traditional knock-out. Several strategies alternative to transgenic approaches are also available. Antisense oligonucleotides, antibodies, or pharmacological agents may be used to manipulate molecular events at the transcription, translation, or protein function levels. I review these strategies briefly and suggest yet another approach: protein targeting with the use of recombinant immunoadhesins. I suggest that this latter approach has the specificity of gene targeting but lacks some of its disadvantages.
Animals↗
Stable transformation of Trypanosoma cruzi: inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption.
Analysis of gene expression in Trypanosoma cruzi has been impeded by the lack of efficient, stable, DNA-mediated transfection systems. We describe here the establishment of such a system for T. cruzi. Stable transformants were isolated following integration of the circular transforming plasmid into the chromosome by homologous recombination. Mutants with a disrupted PUB12.5 polyubiquitin gene, resulting from targeted integration of the plasmid vector, have been isolated. A mutant harboring the disrupted PUB12.5 gene lacks the intact PUB12.5 mRNA as well as transcripts corresponding to the truncated gene. Genomic Southern-blot analysis indicates that the inserted plasmid is tandemly repeated in each of the clones analyzed. A secondary recombination event in one clone resulted in a deletion within the 2.65 calmodulin-ubiquitin locus, encompassing the sequence from the CalA2 calmodulin gene to the PUB12.5 polyubiquitin gene.
Animals↗
Oligonucleotide-directed mutagenesis and targeted gene correction: a mechanistic point of view.
Within the last decade, a number of nucleic acid-based gene targeting strategies have been developed with the ultimate goal to cure human genetic disorders caused by mutations. Thus far, site-directed gene targeting is the only procedure that can make predefined alterations in the genome. The advantage of this approach is that expression of the corrected gene is regulated in the same way as a normal gene. In addition, targeted specific mutations can be made in the genome for functional analysis of proteins. Several approaches, including chimeric RNA-DNA oligonucleotides, short single-stranded oligonucleotides, small fragment homologous replacements, and triple-helix-forming oligonucleotides have been used for targeted modification of the genome. Due to the absence of standardized assays and mechanistic studies in the early developmental stages of oligonucleotide-directed gene alteration, it has been difficult to explain the large variations and discrepancies reported. Here, we evaluate the progress in the field, summarize the achievements in understanding the molecular mechanism, and outline the perspective for the future development. This review will emphasize the importance of reliable, sensitive and standardized assays to measure frequencies of gene repair and the use of these assays in mechanistic studies. Such studies have become critical for understanding the gene repair process and setting realistic expectations on the capability of this technology. The conventionally accepted but unproven dogmas of the mechanism of gene repair are challenged and alternative points of view are presented. Another important focus of this review is the development of general selection procedures that are required for practical application of this technology.
Animals↗
Triplex forming oligonucleotides: sequence-specific tools for gene targeting.
Sequence-specificity is the key to effective genetic targeting. With specificity, targeted genes can be manipulated in multiple ways; without it, gene therapy agents become loose canons within cells. Triplex forming oligonucleotides (TFOs) bind in the major groove of duplex DNA with high specificity and affinity. Because of these characteristics, TFOs have been proposed as homing devices for genetic manipulation in vivo. Here we review work demonstrating the ability of TFOs and related molecules to alter gene expression and mediate genome modification in mammalian cells. Recent studies have established that TFOs can mediate targeted gene knock out in mice, laying the foundation for the potential application of these molecules in human gene therapy.
DNA↗
Identification of PLTP as an LXR target gene and apoE as an FXR target gene reveals overlapping targets for the two nuclear receptors.
Affymetrix microarray data and Northern blot assays demonstrated that phospholipid transfer protein (PLTP) was induced 6-fold when either murine or human macrophages were incubated in the presence of ligands for the liver X receptor (LXR) and the retinoid X receptor. Two functional LXR response elements (LXREs) were identified and characterized in the proximal promoter of the human PLTP gene. One LXRE corresponds to a traditional direct repeat separated by 4 bp. However, the second LXRE is novel in that it corresponds to an inverted repeat separated by 1 bp, and is identical to the farnesoid X receptor response element. These studies demonstrate that PLTP is a direct target for activated LXR and farnesoid X receptor (FXR). In addition, apolipoprotein E (apoE), a known LXR target gene in macrophages, was shown to be activated in liver cells by FXR ligands. Taken together, the current data suggest that a small number of genes that currently include PLTP, apoE, and apoC-II, are induced in macrophages by activated LXR and in liver by activated FXR.
Animals↗
[Structure-function relationship of thyroid hormone receptor and analysis of its physiological role by gene targeting].
Thyroid hormone plays important role in growth, development and maintenance of homeostasis through regulation of the expression of its target genes. The receptor of thyroid hormone belongs to the superfamily of nuclear hormone receptors, and there are two isotype receptors, alpha and beta, encoded by two distinct homologous genes. Molecular dissection and crystalographical analysis clarified the detailed structure-function relationship of the receptor such as DNA binding, dimerization, hormone binding, and interaction with transcription factors, leading to the understanding of the mechanism of regulation of transcription by thyroid hormone receptor. Furthermore, gene targeting revealed the physiological functions of the alpha and beta thyroid hormone receptor isotypes.
Animals↗
[Role of Cre-loxP-mediated conditional gene targeting in understanding the function of Notch receptors].
The Notch family of cell surface receptors and their ligands constitute an evolutionarily conserved signaling pathway that is used by invertebrates and vertebrates to regulate a broad spectrum of cell specification events through local cell interactions. After ligand binding Notch receptor undergoes proteolytic processing ultimately liberating the cytoplasmic domain of the Notch receptor which translocates to the nucleus and activates target genes. In all animal models tested, mutations in Notch genes invariably resulted in developmental abnormalities. In mammals, Notch signaling controls key stages of lymphocyte differentiation as well as activation and several abnormalities in Notch pathway have been suggested to cause human leukemias. Cre-loxP mediated conditional gene targeting significantly contributed to our current understanding of the physiological roles of different Notch family members in hematopoietic compartment. This technique helped to overcome embryonic lethality of Notch mutants providing the opportunity to inactivate specific Notch gene in adulthood.
Animals↗