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Matthew H. Porteus - One of the best experts on this subject based on the ideXlab platform.
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Mammalian Gene Targeting with designed zinc finger nucleases
Molecular therapy : the journal of the American Society of Gene Therapy, 2005Co-Authors: Matthew H. PorteusAbstract:Gene Targeting by homologous recombination is a powerful method to manipulate the genome precisely and could be exploited to correct Genetic defects. Zinc finger nucleases are designed proteins that fuse a zinc finger DNA binding domain to the nuclease domain from the FokI restriction endonuclease. Zinc finger nucleases were Generated that stimulated Gene Targeting from half-site sequences from the human beta-globin Gene and the human common gamma-chain Gene. Zinc finger nucleases were also Generated that stimulated Gene Targeting at full sites from the green fluorescent protein Gene and the human CD8alpha Gene. This work built on the prior zinc finger design work of others and in Targeting these four Genes had a 100% success rate at designing nucleases to the consensus half-site 5'-GNNGNNGNN-3' and the consensus full site 5'-NNCNNCNNCNNNNNNGNNGNNGNN-3', suggesting that zinc finger nucleases can be empirically designed to stimulate Gene Targeting in a large portion of the mammalian genome.
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Gene Targeting using zinc finger nucleases
Nature biotechnology, 2005Co-Authors: Matthew H. Porteus, Dana CarrollAbstract:The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for basic and applied research. Gene Targeting is a process in which a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to manipulate the genome. In the past, the application of Gene Targeting to mammalian cells has been limited by its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of Gene Targeting by introducing DNA double-strand breaks in target Genes, which then stimulate the cell's endogenous homologous recombination machinery. Recent results have shown that ZFNs can be used to create Targeting frequencies of up to 20% in a human disease-causing Gene. Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability.
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Efficient Gene Targeting mediated by adeno-associated virus and DNA double-strand breaks.
Molecular and cellular biology, 2003Co-Authors: Matthew H. Porteus, Toni Cathomen, Matthew D. Weitzman, David BaltimoreAbstract:Gene Targeting is the in situ manipulation of the sequence of an endogenous Gene by the introduction of homologous exogenous DNA. Presently, the rate of Gene Targeting is too low for it to be broadly used in mammalian somatic cell Genetics or to cure Genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate Gene Targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and Gene Targeting with rAAV. Both random integration and spontaneous Gene Targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target Gene can stimulate Gene Targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated Gene Targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated Gene Targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute Gene Targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a Gene Targeting substrate and a second vector expresses the nuclease that creates a DSB in the target Gene. The frequencies of Gene Targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of Gene Targeting.
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Chimeric Nucleases Stimulate Gene Targeting in Human Cells
Science (New York N.Y.), 2003Co-Authors: Matthew H. Porteus, David BaltimoreAbstract:Correction of Gene defects in human somatic cells by Targeting as has been used in murine embryonic stem cells (1, 2) has been precluded by the low spontaneous rate of Gene Targeting (3). However, creation of a DNA double-stranded break (DSB) in the genomic target (DSB-GT) can stimulate homologous recombination by over 1000-fold (4). We can rapidly and quantitatively measure Gene Targeting by correcting a mutation in a green fluorescent protein (GFP) Gene that has been stably integrated into the genome (5) (fig. S1). With an optimized GFP Gene Targeting system, the introduction of a DSB by I–Sce I (Sce) stimulated GT >40,000-fold and the absolute rate of Gene Targeting reached 3 to 5% (fig. S2). Such a system, however, depends on the prior introduction of a Sce binding site into the target Gene and cannot be used for endogenous Genes. Chimeric nucleases (CNs) have the potential to create sequence-specific DSBs (6). CNs—fusions between zinc finger binding DNA binding domains and the endonuclease domain of Fok I—can sitespecifically cleave naked DNA in vitro (6), extrachromosomal DNA in Xenopus oocytes (7), and chromosomal DNA in Drosophila (8). CNs work as dimers, and their efficiency depends on the spacing and orientation of the zinc finger binding sites with respect to the length of the amino acid linker between the DNA binding and endonuclease domains (7, 9). QQR is an artificial zinc finger DNA binding domain that recognizes the sequence 5′-GGGGAAGAA-3′ with nanomolar affinity (10). We modified QQR chimeric nucleases (QQR-CNs) (7, 9) (Fig. 1A) and tested whether they stimulated Gene Targeting (Fig. 1B). The background rate of Gene Targeting was 0.71 events per million transfected cells (fig. S1C). QQRL18-CN stimulated Gene Targeting 17-fold on target QQR6 and 260-fold on target QQR8 (Fig. 1B). QQRL0-CN did not stimulate Gene Targeting on target QQR8, but it was as efficient as Sce in stimulating Gene Targeting by over 2000-fold on target QQR6 (Fig. 1B). QQRL18-CN showed some preference for an 8–base pair (bp) spacing between binding sites whereas QQRL0-CN preferred 6-bp spacing. Thus, removing the linker between the zinc spacfinger and the nuclease domains increased the activity and specificity of the fusion protein in mammalian cells. As controls, we showed that the CNs did not stimulate Gene Targeting if (i) they lacked a nuclear localization signal, (ii) there was a single binding site rather than an inverted repeat binding site in the target, and (iii) the cognate binding site was changed. Thus, homodimers of CNs are potent stimulators of Gene Targeting in human somatic cells.
David W. Russell - One of the best experts on this subject based on the ideXlab platform.
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The effects of polymorphisms on human Gene Targeting
Nucleic acids research, 2013Co-Authors: David R. Deyle, Gaoying Ren, David W. RussellAbstract:DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce Gene Targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human Gene Targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific Targeting or to improve unfavorable vector designs that introduce deletions.
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Chromosomal position effects on AAV-mediated Gene Targeting.
Nucleic acids research, 2010Co-Authors: Anda M. Cornea, David W. RussellAbstract:The effects of chromosomal position and neighboring genomic elements on Gene Targeting in human cells remain largely unexplored. To study these, we used a shuttle vector system in which murine leukemia virus (MLV)-based proviral targets present at different chromosomal locations and containing mutations in the neomycin phosphotransferase (neo) Gene were corrected by adeno-associated virus (AAV)-mediated Gene Targeting. Sixteen identical target loci present in HT-1080 human sarcoma cells were all successfully corrected by Gene Targeting. The Gene Targeting frequencies varied by as much as 10-fold, and there was a clear bias for correction of one of the targets in clones containing two target sites. The Targeting frequency at each site was correlated to the proximity and density of various genomic elements, and we found a significant association of higher Targeting frequencies at loci near a subset of dinucleotide microsatellite repeats (r = -0.55, P < 0.05), in particular GT repeats (r = -0.87, P < 0.0001). Additionally, there was a correlation between meiotic recombination rates and Targeting frequencies at the target loci (r = 0.52, P < 0.05). There was no correlation between surrounding chromosomal transcription units and Targeting frequencies. Our results indicate that certain chromosomal positions are preferred sites for Gene Targeting in human cells.
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Gene Targeting with viral vectors.
Molecular therapy : the journal of the American Society of Gene Therapy, 2005Co-Authors: Paul C. Hendrie, David W. RussellAbstract:Genetic manipulation of cells for scientific and therapeutic goals can be achieved by both Gene-addition and Gene-Targeting methods. Gene Targeting precisely alters a Gene in its natural chromosome location, providing distinct advantages over Gene-addition approaches. Classic Gene-Targeting delivery systems (microinjection, electroporation, or calcium phosphate transfection) have led to major scientific advances, but are too inefficient in their current state to be used for some applications, including Gene therapy. This review describes the development of Gene-Targeting vectors based on three types of viruses (retrovirus, adenovirus, and adeno-associated virus) and discusses the design, possible mechanisms of action, and applications of Gene-Targeting vectors based on adeno-associated virus.
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Gene Targeting by adeno-associated virus vectors is cell-cycle dependent.
Human gene therapy, 2005Co-Authors: Grant D. Trobridge, Roli K. Hirata, David W. RussellAbstract:Adeno-associated virus (AAV) vectors can be used to introduce site-specific mutations into homologous chromosomal sequences. There are many potential applications of this technique, but the process of AAV-mediated Gene Targeting and factors that influence Targeting efficiency are not completely understood. We investigated the dependence of AAV-mediated Gene Targeting on the host cell-cycle status. The frequency of Gene Targeting by AAV vectors was compared in dividing and serum-arrested normal human fibroblast cultures. Gene Targeting occurred in arrested fibroblast cultures at 0.15 to 1.1% the frequency of dividing cultures, and only took place in cells that had undergone DNA synthesis. Gene Targeting was also reduced when DNA synthesis was inhibited by hydroxyurea.
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design and packaging of adeno associated virus Gene Targeting vectors
Journal of Virology, 2000Co-Authors: Roli K. Hirata, David W. RussellAbstract:Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) Gene addition by chromosomal integration or episomal transGene expression or (ii) Gene Targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of mutations in chromosomal Genes with high fidelity and specificity. In this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter Genes into normal human cells and subsequently corrected these mutations with AAV Gene Targeting vectors. We find that increasing the length of homology between the AAV vector and the target locus improves Gene correction rates, as does positioning the mutation to be corrected in the center of the AAV vector genome. AAV-mediated Gene Targeting increases with time and multiplicity of infection, similar to AAV-mediated Gene addition. However, in contrast to Gene addition, genotoxic stress did not affect Gene Targeting rates, suggesting that different cellular factors are involved. In the course of these studies, we found that (i) vector genomes less than half of wild-type size could be packaged as monomers or dimers and (ii) packaged dimers consist of inverted repeats with covalently closed hairpins at either end. These studies should prove helpful in designing AAV Gene Targeting vectors for basic research or Gene therapy.
Didier G. Schaefer - One of the best experts on this subject based on the ideXlab platform.
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Gene Targeting in Physcomitrella patens
Current opinion in plant biology, 2001Co-Authors: Didier G. SchaeferAbstract:Gene-Targeting efficiency in the land plant Physcomitrella patens (Bryophyta) can only be compared with that observed in Saccharomyces cerevisiae. Sequencing programs and microbiological molecular Genetic approaches are now being developed to unravel the precise function of plant Genes. Physcomitrella patens, as the new 'green yeast', might well become a major tool for functional genomic studies of multicellular eukaryotes.
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Efficient Gene Targeting in the moss Physcomitrella patens.
The Plant journal : for cell and molecular biology, 1997Co-Authors: Didier G. Schaefer, Jean-pierre ZrydAbstract:The moss Physcomitrella patens is used as a Genetic model system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. Transformation experiments designed to target three single-copy genomic loci were performed to determine the efficiency of Gene Targeting in this plant. Mean transformation rates were 10-fold higher with the Targeting vectors and molecular evidence for the integration of exogenous DNA into each targeted locus by homologous recombination is provided. The efficiency of Gene Targeting determined in these experiments is above 90%, which is in the range of that observed in yeast and several orders of magnitude higher than previous reports of Gene Targeting in plants. Thus, Gene knock-out and allele replacement approaches are directly accessible to study plant development in the moss Physcomitrella patens. Moreover, efficient Gene Targeting has so far only been observed in lower eukaryotes such as protozoa, yeasts and filamentous fungi, and, as shown here the first example from the plant kingdom is a haplobiontic moss. This suggests a possible correlation between efficient Gene Targeting and haplophase in eukaryotes.
Dana Carroll - One of the best experts on this subject based on the ideXlab platform.
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Gene Targeting using zinc finger nucleases
Nature biotechnology, 2005Co-Authors: Matthew H. Porteus, Dana CarrollAbstract:The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for basic and applied research. Gene Targeting is a process in which a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to manipulate the genome. In the past, the application of Gene Targeting to mammalian cells has been limited by its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of Gene Targeting by introducing DNA double-strand breaks in target Genes, which then stimulate the cell's endogenous homologous recombination machinery. Recent results have shown that ZFNs can be used to create Targeting frequencies of up to 20% in a human disease-causing Gene. Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability.
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enhancing Gene Targeting with designed zinc finger nucleases
Science, 2003Co-Authors: Marina Bibikova, Kelly J Beumer, Jonathan K Trautman, Dana CarrollAbstract:Gene Targeting—the process of Gene replacement by homologous recombination—is a very useful but typically inefficient technique ( [1][1] ). A General method for improving the efficiency of Gene Targeting would be valuable in many circumstances, as would the extension of this Genetic tool to a
Ralf Kühn - One of the best experts on this subject based on the ideXlab platform.
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Cre/loxP recombination system and Gene Targeting.
Methods in molecular biology (Clifton N.J.), 2002Co-Authors: Ralf Kühn, Raul M. TorresAbstract:Embryonic stem (ES) cell technology has clearly established itself as a powerful technique for the examination of Gene function in vivo. The vast majority of Gene-Targeting experiments to date have been designed simply to inactivate the function of the Gene of interest by the targeted insertion of a selectable marker into the ES genome. Homologous recombinant ES cells are used without further modification for the Generation of mice that bear a permanently modified allele in all cells from the onset of development. In contrast to this “conventional” Gene-Targeting strategy, in recent years, the use of the Cre/loxP recombination system in conjunction with Gene Targeting has greatly expanded the versatility and avenues with which biologic questions can be addressed in the mouse. In addition to the Generation of subtle mutations, this system allows for a number of other genotypic options in ES cells or mice by strategically incorporating Cre recombinase recognition (loxP) sites into the genome and the subsequent expression of recombinase in vitro or in vivo. In particular, when Cre is expressed in mice harboring a loxP-containing target Gene, the desired Gene modification can be restricted to certain cell types or developmental stages of the mouse (conditional Gene Targeting) depending on the tissue specificity and timing of recombinase expression. There is no definitive rule to decide whether, for a particular experiment, conventional or conditional Gene Targeting is more appropriate since this depends on the specific biologic question and the peculiarities of the Gene studied.
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cre loxp recombination system and Gene Targeting
Methods of Molecular Biology, 2002Co-Authors: Ralf Kühn, Raul M. TorresAbstract:Embryonic stem (ES) cell technology has clearly established itself as a powerful technique for the examination of Gene function in vivo. The vast majority of Gene-Targeting experiments to date have been designed simply to inactivate the function of the Gene of interest by the targeted insertion of a selectable marker into the ES genome. Homologous recombinant ES cells are used without further modification for the Generation of mice that bear a permanently modified allele in all cells from the onset of development. In contrast to this “conventional” Gene-Targeting strategy, in recent years, the use of the Cre/loxP recombination system in conjunction with Gene Targeting has greatly expanded the versatility and avenues with which biologic questions can be addressed in the mouse. In addition to the Generation of subtle mutations, this system allows for a number of other genotypic options in ES cells or mice by strategically incorporating Cre recombinase recognition (loxP) sites into the genome and the subsequent expression of recombinase in vitro or in vivo. In particular, when Cre is expressed in mice harboring a loxP-containing target Gene, the desired Gene modification can be restricted to certain cell types or developmental stages of the mouse (conditional Gene Targeting) depending on the tissue specificity and timing of recombinase expression. There is no definitive rule to decide whether, for a particular experiment, conventional or conditional Gene Targeting is more appropriate since this depends on the specific biologic question and the peculiarities of the Gene studied.
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Laboratory Protocols for Conditional Gene Targeting
1997Co-Authors: Raul M. Torres, Ralf KühnAbstract:Section 1: The Cre-loxP recombination system and Gene Targeting in ES cells 1. Gene Targeting strategies 2. Cre-loxP recombination system 3. Gene Targeting vectors - consideration and use of loxP 4. Nonselectablae modifications and removal of selection marker Genes 5. Large deletions 6. Gene replacement 7. Targeted integrations 9. Conditional Gene modification 10. LoxP-containing transGenes 11. Conditional Gene Targeting Section 2: Working with ES cells 12. ES cell transfection overview 13. Embryonic feeder cells 14. ES cells - handling and use 15. ES cell culture and transfection 16. ES clone picking 17. Identification of homologous recombinants 18. Freezing ES clones 19. Thawing ES clones 20. Genomic DNA preparation 21. Cre-mediated neomycin deletion in ES cells 22. Generation of mice - preparation 23. Mouse breeding for ES cell injection or aggregation 24. Injection needles and holders 25. Blastocyst preparation/injection 26. Embryo aggregation 27. Blastocyst transfer Appendices References Selected references for further reading
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Advances in Gene Targeting methods
Current opinion in immunology, 1997Co-Authors: Ralf Kühn, Frieder SchwenkAbstract: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.