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PubMed2003

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.

Amino Acid Sequence↗
PubMed1999

Adenovirus-mediated regulable target gene expression in vivo.

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.

Adenoviridae↗
PubMed2005

Differential recognition of response elements determines target gene specificity for p53 and p63.

p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63+/+ mouse, it is undetectable in these tissues in the p63-/- mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.

Amino Acid Sequence↗
PubMed1997

Stability of HPRT marker gene expression at different gene-targeted loci: observing and overcoming a position effect.

For sophisticated gene targeting procedures requiring two sequential selective steps to operate efficiently it is essential that the marker genes used are not prone to position effects. The double replacement gene targeting procedure, to produce mice with subtle gene alterations, is based on the use of hypoxanthine phosphoribosyltransferase ( HPRT) minigenes in HPRT-deficient embryonic stem cells. Our standard HPRTminigene, under the control of the mouse phosphoglycerate kinase-1 gene promoter, was stably expressed at five of six target loci examined. At the remaining locus, DNA ligase I (Lig1), expression of this minigene was highly unstable. A different minigene, under the control of the mouse HPRT promoter and embedded in its natural CpG-rich island, overcame this position effect and was stably expressed when targeted to the identical site in the Lig1 locus. The promoter region of the stably expressed minigene remained unmethylated, while the promoter of the unstably expressed minigene rapidly became fully methylated. The difference in the stability of HPRT minigene expression at the same target locus can be explained in the context of the different lengths of their CpG-rich promoter regions with associated transcription factors and a resulting difference in their susceptibility to DNA methylation, rather than by differences in promoter strength.

Animals↗
PubMed2004

Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes.

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. A majority of these mutations are missense mutations in the DNA-binding domain. As a result, the mutated p53 gene encodes a full-length protein incapable of transactivating its target genes. In addition to this loss of function, mutant p53 can have a dominant negative effect over wild-type p53 and/or gain of function activity independently of the wild-type protein. To better understand the nature of the tumorigenic activity of mutant p53, we have investigated the mechanism by which mutant p53 can exert a dominant negative effect. We have established several stable cell lines capable of inducibly expressing a p53 mutant alone, wild-type p53 alone, or both proteins concurrently. In this context, we have used chromatin immunoprecipitation to determine the ability of wild-type p53 to bind to its endogenous target genes in the presence of various p53 mutants. We have found that p53 missense mutants markedly reduce the binding of wild-type p53 to the p53 responsive element in the target genes of p21, MDM2, and PIG3. These findings correlate with the reduced ability of wild-type p53 in inducing these and other endogenous target genes and growth suppression in the presence of mutant p53. We also showed that mutant p53 suppresses the ability of wild-type p53 in inducing cell cycle arrest. This highlights the sensitivity and utility of the dual inducible expression system because in previous studies, p53-mediated cell cycle arrest is not affected by transiently overexpressed p53 mutants. Together, our data showed that mutant p53 exerts its dominant negative activity by abrogating the DNA binding, and subsequently the growth suppression, functions of wild-type p53.

Cell Cycle↗
PubMedDate not supplied

Polyethyleneimine-based immunopolyplex for targeted gene transfer in human lymphoma cell lines.

BACKGROUND: Specific and efficient delivery of genes into targeted cells is a priority objective in non-viral gene therapy. Polyethyleneimine-based polyplexes have been reported to be good non-viral transfection reagents. However, polyplex-mediated DNA delivery occurs through a non-specific mechanism. This article reports the construction of an immunopolyplex, a targeted non-viral vector based on a polyplex backbone, and its application in gene transfer over human lymphoma cell lines. METHODS: Targeting elements (biotin-labeled antibodies), which should recognize a specific element of the target cell membrane and promote nucleic acid entry into the cell, were attached to the polyplex backbone through a bridge protein (streptavidin). Immunopolyplex transfection activity was studied in several hematological cell lines [Jurkat (CD3+/CD19-), Granta 519 (CD3-/ CD19+), and J.RT3-T3.5 (CD3-/CD19-)] using the EGFP gene as a reporter gene and anti-CD3 and anti-CD19 antibodies as targeting elements. Transfection activity was evaluated via green fluorescence per cell and the percentage of positive cells determined by flow cytometry. RESULTS: A significant selectivity of gene delivery was observed, since the anti-CD3 immunopolyplex worked only in Jurkat cells while the anti-CD19 immunopolyplex worked only in the Granta cell line. Moreover, transfection of a CD3+/CD3- cell mixture with anti-CD3 immunopolyplexes showed up to 16-fold more transfection in CD3+ than in CD3- cells. Several non-specific transfection reagents showed poor or no transfection activity. CONCLUSION: It is concluded that immunopolyplex is a good non-viral vector for specific and selective nucleic acid delivery. Immunopolyplex design allows easy replacement of the targeting element (antibody) - the streptavidin-polyplex backbone remaining intact - thereby conferring high versatility.

Antigens, CD19↗
PubMed2004

Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link.

Saccharomyces cerevisiae served as a model fungal system to examine functional genomics of oxidative stress responses and reactions to test antioxidant compounds. Twenty-two strains of S. cerevisiae, including a broad spectrum of singular gene deletion mutants, were exposed to hydrogen peroxide (H2O2) to examine phenotypic response to oxidative stress. Responses of particular mutants treated with gallic, tannic or caffeic acids, or methyl gallate, during H2O2 exposure, indicated that these compounds alleviated oxidative stress. These compounds are also potent inhibitors of aflatoxin biosynthesis in Aspergillus flavus. To gain further insights into a potential link between oxidative stress and aflatoxin biosynthesis, 43 orthologs of S. cerevisiae genes involved in gene regulation, signal transduction (e.g., SHO1, HOG1, etc.) and antioxidation (e.g., CTT1, CTA1, etc.) were identified in an A. flavus expressed sequence tag library. A successful exemplary functional complementation of an antioxidative stress gene from A. flavus, mitochondrial superoxide dismutase (sodA), in a sod2Delta yeast mutant further supported the potential of S. cerevisiae deletion mutants to serve as a model system to study A. flavus. Use of this system to further examine functional genomics of oxidative stress in aflatoxigenesis and reduction of aflatoxin biosynthesis by antioxidants is discussed.

Aflatoxins↗
PubMed2000

Gene targeted agents: new opportunities for rational drug development.

In addition to traditional drug development methods designed to modulate the activity of protein targets, knowledge of disease gene DNA sequences provides an opportunity for the highly rational design of therapeutic agents that act at the DNA level through sequence-specific interactions. Among the ligands capable of binding DNA in a precise, sequence-specific manner are oligonucleotides, peptide nucleic acids and polyamides. Various strategies employing these agents to either transiently or permanently alter gene expression have been investigated over the past decade. During the past two to three years, important steps have been taken to illustrate the therapeutic potential of these ligands. Triple-helix (triplex) forming oligonucleotides have been particularly effective DNA-targeting agents with a wide range of applications, including the positive and negative transcriptional regulation of target genes, as well as the controlled delivery of site-specific mutations. This review will focus upon recent advances involving the use of sequence-specific DNA-binding ligands to modify gene expression and/or structure, with particular emphasis on the use of triplex-forming oligonucleotides in these roles.

Animals↗
PubMed2001

A sensitive and rapid assay for homologous recombination in mosquito cells: impact of vector topology and implications for gene targeting.

BACKGROUND: Recent progress in insect transgenesis has been dramatic but existing transposon-based approaches are constrained by position effects and potential instability. Gene targeting would bring a number of benefits, however progress requires a better understanding of the mechanisms involved. Much can be learned in vitro since extrachromosomal recombination occurs at high frequency, facilitating the study of multiple events and the impact of structural changes among the recombining molecules. We have investigated homologous recombination in mosquito cells through restoration of luciferase activity from deleted substrates. The implications of this work for the construction of insect gene targeting vectors are discussed. RESULTS: We show that linear targeting vectors are significantly more efficient than circular ones and that recombination is stimulated by introducing double-strand breaks into, or near, the region of homology. Single-strand annealing represents a very efficient pathway but may not be feasible for targeting unbroken chromosomes. Using circular plasmids to mimic chromosomal targets, one-sided invasion appears to be the predominant pathway for homologous recombination. Non-homologous end joining reactions also occur and may be utilised in gene targeting if double-strand breaks are first introduced into the target site. CONCLUSIONS: We describe a rapid, sensitive assay for extrachromosomal homologous recombination in mosquito cells. Variations in substrate topology suggest that single-strand annealing and one-sided invasion represent the predominant pathways, although non-homologous end joining reactions also occur. One-sided invasion of circular chromosomal mimics by linear vectors might therefore be used in vitro to investigate the design and efficiency of gene targeting strategies.

Animals↗
PubMed2006

Genetic modification of sheep by nuclear transfer with gene-targeted somatic cells.

For many years the lack of germline competent embryonic stem cell lines in livestock meant that the targeted modification of endogenous genes was not possible in these species. The demonstration that livestock could be cloned by nuclear transfer from cultured somatic cells has now provided an alternative route to accomplish gene targeting. This chapter describes protocols for culturing primary sheep fibroblasts, introducing and selecting targeted modifications into them and then using these modified cells in nuclear transfer experiments.

Animals↗
PubMed2004

Gene targeting in yeast is initiated by two independent strand invasions.

To study the mechanism of gene targeting, we examined heteroduplex DNA (hDNA) formation during targeting of two separate chromosomal locations in Saccharomyces cerevisiae. We examined both replacement of the entire gene with a heterologous selectable marker and correction of a single base pair insertion mutation by gene targeting, and in all cases our results were consistent with separate strand invasion/resolution at the two ends of the targeting fragment as the dominant mechanism in wild-type cells. A small subset of transformants was consistent with assimilation of a single strand of targeting DNA encompassing both flanking homology regions and the marker into hDNA. hDNA formation during correction of a point mutation by targeted integration was conspicuously altered in a mismatch repair-deficient background and was consistent with single-strand invasion/assimilation without mismatch correction, confirming that gene targeting by this pathway is actively impeded in wild-type yeast. Finally, inversion of one targeted locus and mutation of an active origin of DNA replication at the other locus affected hDNA formation significantly, suggesting that formation of productive interactions between the targeting DNA and the targeted site in the chromosome is sensitive to local DNA dynamics.

Carrier Proteins↗
PubMed1999

Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis.

Gene targeting, defined as the introduction of site-specific modifications into the genome by homologous recombination, has revolutionarized the field of mouse genetics and allowed the analysis of diverse aspects of gene function in vivo. It is now possible to engineer specific genetic alterations ranging from subtle mutations to chromosomal rearrangements and more recently, even tissue-specific inducible gene targeting with temporo-spatial control has become feasible. This review tries to recapitulate what we have learned in this extremely rapidly expanding field during the past decade. Diverse aspects of the technique will be discussed starting from basic construct design to the analysis of complex phenotypes, including recent advances on inducible expression system. Many examples from different areas of biomedical research are given to illustrate the purpose and limitations of the employed experimental approaches.

Animals↗
PubMedDate not supplied

FAD mutant PS-1 gene-targeted mice: increased A beta 42 and A beta deposition without APP overproduction.

To investigate the consequences of mutant presenilin-1 (PS-1) expression under the control of the normal PS-1 gene, a gene-targeted mouse bearing the FAD mutation P264L was made. Gene-targeted models are distinct from transgenic models because the mutant gene is expressed at normal levels, in the absence of the wild-type protein. PS-1(P264L/P264L) mice had normal expression of PS-1 mRNA, but levels of the N- and C-terminal protein fragments of PS-1 were reduced while levels of the holoprotein were increased. When crossed into Tg(HuAPP695.K670N/M671L)2576 mice, the PS-1(P264L) mutation accelerated the onset of amyloid (Abeta) deposition in a gene-dosage dependent manner. Tg2576/PS-1(P264L/P264L) mice also had Abeta deposition that was widely distributed throughout the brain and spinal cord. APP(NLh/NLh)/PS-1(P264L/P264L) double gene-targeted mice had elevated levels of Abeta42, sufficient to cause Abeta deposition beginning at 6 months of age. Abeta deposition increased linearly over time in APP(NLh/NLh)/PS-1(P264L/P264L) mice, whereas the increase in Tg2576 mice was exponential. The APP(NLh/NLh)/PS-1(P264L/P264L) double gene-targeted mouse represents an animal model that exhibits Abeta deposition without overexpression of APP.

Alzheimer Disease↗
PubMed1998

Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments.

The Pax-5 gene codes for the transcription factor BSAP which is essential for the progression of adult B lymphopoiesis beyond an early progenitor (pre-BI) cell stage. Although several genes have been proposed to be regulated by BSAP, CD19 is to date the only target gene which has been genetically confirmed to depend on this transcription factor for its expression. We have now taken advantage of cultured pre-BI cells of wild-type and Pax-5 mutant bone marrow to screen a large panel of B lymphoid genes for additional BSAP target genes. Four differentially expressed genes were shown to be under the direct control of BSAP, as their expression was rapidly regulated in Pax-5-deficient pre-BI cells by a hormone-inducible BSAP-estrogen receptor fusion protein. The genes coding for the B-cell receptor component Ig-alpha (mb-1) and the transcription factors N-myc and LEF-1 are positively regulated by BSAP, while the gene coding for the cell surface protein PD-1 is efficiently repressed. Distinct regulatory mechanisms of BSAP were revealed by reconstituting Pax-5-deficient pre-BI cells with full-length BSAP or a truncated form containing only the paired domain. IL-7 signalling was able to efficiently induce the N-myc gene only in the presence of full-length BSAP, while complete restoration of CD19 synthesis was critically dependent on the BSAP protein concentration. In contrast, the expression of the mb-1 and LEF-1 genes was already reconstituted by the paired domain polypeptide lacking any transactivation function, suggesting that the DNA-binding domain of BSAP is sufficient to recruit other transcription factors to the regulatory regions of these two genes. In conclusion, these loss- and gain-of-function experiments demonstrate that BSAP regulates four newly identified target genes as a transcriptional activator, repressor or docking protein depending on the specific regulatory sequence context.

Animals↗
PubMed1997

Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission.

The ability to contribute to the germ line is the most important experimental feature of embryonic stem (ES) cells. Using ES cells, it is possible to introduce targeted mutations into any gene and to derive the corresponding mutant mice. A common problem with this technology is that the ES cells often lack or have only a low efficiency of germ line transmission. To address this issue, we examined the relationship between the growth rate and karyotype of ES cells, and their ability to contribute to the germ line. We found that chromosomal abnormalities occurred rather frequently in ES cells. Cells having an abnormal number of chromosomes, in particular trisomy 8, were found in three independently derived ES cell lines, and this abnormality conferred a selective growth advantage on these cells. Selection of abnormal cells led to depletion and eventual loss of normal ES cells during consecutive passages. In comparison with parental ES cells, ES cells with trisomy 8 contributed rarely to the germ line. This realization allowed us to select, based upon ES cell clone morphology, those clones with the highest probability of contributing to the germ line. This insight is of practical value for any given gene targeting experiment as it permits optimization of the rate of success without having to rely on more elaborate tests such as karyotyping individual clones prior to blastocyst injection.

Animals↗
PubMed1999

T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis.

The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used.

Agrobacterium tumefaciens↗
PubMed2002

Gene targeting technology and advances in the pathophysiology of inflammation.

Gene targeting ('knock-out') technology is now widely used in the basic science of all disciplines of pathology and particularly auto-immune and inflammatory disease. Gene targeting is the wilful introduction of precise mutations into the genome of an animal, usually a mouse, affecting the function of a single gene or genes. The phenotyping of knockout mice provides whole animal data on the functions of individual genes in pathophysiological settings, and frequently provides the basis for novel therapeutic strategies. 'Knock-ins', the Cre-LoxP system and conditional knockouts are important new advances. This paper serves as an introduction to the methodology and draws on examples of major advances in inflammation research provided by the targeting of cytokines, in particular the tumour necrosis factor family of ligands.

Animals↗
PubMed2005

Thyroid hormone-regulated target genes have distinct patterns of coactivator recruitment and histone acetylation.

Thyroid hormone receptors (TRs) are ligand-regulated transcription factors that bind to thyroid hormone response elements of target genes. Upon ligand binding, they recruit coactivator complexes that increase histone acetylation and recruit RNA polymerase II (Pol II) to activate transcription. Recent studies suggest that nuclear receptors and coactivators may have temporal recruitment patterns on hormone response elements, yet little is known about the nature of the patterns at multiple endogenous target genes. We thus performed chromatin immunoprecipitation assays to investigate coactivator recruitment and histone acetylation patterns on the thyroid hormone response elements of four endogenous target genes (GH, sarcoplasmic endoplasmic reticulum calcium-adenosine triphosphatase, phosphoenolpyruvate carboxykinase, and cholesterol 7alpha-hydroxylase) in a rat pituitary cell line that expresses TRs. We found that TRbeta, several associated coactivators (steroid receptor coactivator-1, glucocorticoid receptor interacting protein-1, and TR-associated protein 220), and RNA Pol II were rapidly recruited to thyroid hormone response elements as early as 15 min after T3 addition. When the four target genes were compared, we observed differences in the types and temporal patterns of recruited coactivators and histone acetylation. Interestingly, the temporal pattern of RNA Pol II was similar for three genes studied. Our findings suggest that thyroid hormone-regulated target genes may have distinct patterns of coactivator recruitment and histone acetylation that may enable highly specific regulation.

Acetylation↗