DOCSLIB.ORG

Gene Targeting

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Gene Targeting Proc. Nati. Acad. Sci. USA Vol. 91, pp. 8527-8531, August 1994 Genetics Homology-associated nonhomologous recombination in mammalian gene targeting (homologous recombln ao/ tIe rembt /gene n ts/genome evolut/p v ) KEIKO SAKAGAMI*, YASUYUKI TOKINAGA*, HIROSHI YOSHIKURAt, AND ICHIZO KOBAYASHI* *Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Shiroganedai, Tokyo 108, Japan; and tDepartment of Bacteriology, Medical School, University of Tokyo, Tokyo 113, Japan Communicated by Susumu Ohno, May 19, 1994 ABSTRACT Nonhomologous (illegitimate) recombinatlon and I.K., unpublished). Preliminary reports of these findings of DNA underlies many changes In the genome. It Involves no have appeared (7, 8). or little homology between recombining DNAs and has been conidered unrelated with homologous recombination, which requres long homology. In mouse cells, however, we found MATERIALS AND METHODS recombination products whose sequences suggest that homol- Plasmds, Bacteria, and Cells. The donor plasmid (pIK39), ogous Interaction between DNAs caused nondologous re- the target plasmid (pIK32) (Fig. 1), and a neo+ version of combination with another DNA. The intermediates of homol- pIK32 (pIK30) have been described (9). Three recAl strains ogous recombination wereapparently trapped at various stages ofE. coli K-12, DH1, DH5, and DH1OB, usedforpropagation and shunted to nonhomologous recombination. In one product, of plasmids, have been described (9, 10) as was a mouse cell the nonhomologous recombination disrupted gene conversion. line, C127 (3). In another, it took place exactly at the end of long homology Establishment of the Target in the Cells. Target plasmid shared between two DNAs. This finding eplains why gene pIK32 was transferred to C127 by calcium phosphate trans- targeting needs long uninterrpted homology and why mam- fection (3). The cells in a focus were recovered with a mailan homologous recombination Is often nonconservative. penicillin cup and spread. Six colonies were subcloned. We discuss possible consequences and roles of this type of Extrachromosomal DNA from each ofthem was isolated by homology-driven gene destruction nism. the Hirt method and was analyzed by the Southern method. One clone carrying plasmid indistinguishable from pIK30was chosen as the target cell line. Its copy number was estimated Several types of DNA recombination have played important to be 160 per cell by a dot blot method with total DNA roles in genome changes and evolution. These include ho- preparation. mologous recombination, which requires long homology be- Gene Targtig to lid DNA. The cell line (C127 car- tween recombining DNAs, and nonhomologous recombina- rying pIK32) was spread to a cell density of 3-4 x 105 cells tion, which requires no or very short homology. These two per 6-cm (diameter) dish. The donor plasmid (10 jg) was recombination mechanisms have been considered quite sep- transferred by a calcium phosphate method (day 0). In arate. experiment 1, 200 jug ofG418 per ml was added on day 3. Its DNA transfer into cultured mammalian cells provides an concentration was increased to 400 jag/ml on day 6. G418R interesting experimental system for the analysis of recombi- (G418-resistant) colonies were counted and isolated with a nation (1). The major route of homologous recombination penicillin cup on day 13. G418 selection was continued until between transferred DNAs is nonconservative (two parental extrachromosomal DNA was prepared by the Hirt method 2 DNAs producing only one progeny DNA) rather than con- months after transfection. In experiment 2, G418 selection servative (two parental DNAs producing two progeny DNAs) started on day 3 at 200 pg/ml. Colony isolation was on day (2, 3). Their nonhomologous recombination is frequent. Ho- 26. DNA was isolated 3 months after transfection. Circular mologous recombination with chromosomal DNA is much DNA plasmids were recovered in E. coli DH1OB by electro- rarer than nonhomologous recombination with it. The fre- poration (Bio-Rad Gene Pulser; 2.5 kV, 25 uF, and 200 fi) by quency of homologous recombination is very dependent on selection for kanamycin resistance. the length of homology and sensitive to minor sequence Sequence Determination and Analysis. Parts ofthe products divergence (4, 5). These properties have been problems in were subcloned in pUC118 and/or pUC119 as described in gene targeting. the figure legends. (For the "donor correction"-type prod- For the analysis of recombination, we have employed a uct, 7881-8246 and 8246-9169 in the coordinate ofthe donor mammalian plasmid shuttle vector derived from bovine pap- were subcloned in pUC118.) DNA sequences were deter- illomavirus type 1 (BPV-1), which replicates in a chromatin- mined with T7 DNA polymerase using the M13 universal like structure in the nuclei of cultured mouse cells (3, 6). We primer (5'-CGACGTTGTAAAACGACGGCCAGT) or the found novel recombination products carrying a nonhomolo- M13 reverse primer (5'-CAGGAAACAGCTATGAC) in a gous joint as well as a homologous joint. Their sequences Pharmacia sequencer. strongly suggest that the process of homologous interaction itself led to the nonhomologous rearrangements. They pro- vide clues to the mechanism of homologous interaction and RESULTS gene targeting in mammalian cells. We detected similar Experimental Design: Gene Targeting to a homology-associated nonhomologous recombination in some Plasmid. Fig. 1A illustrates our recombination substrates. Escherichia coli mutants (K. Kusano, K.S., Y.T., E. Ueda, The target mammalian plasmid consists of BPV-1, an E. coli plasmid and a neomycin phosphotransferase gene (neo) with The publication costs ofthis article were defrayed in part by page charge a deletion. In the first step, we established this target as a payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: BPV, bovine papillomavirus; G418R, G418-resistant. 8527 Downloaded by guest on October 1, 2021 8528 Genetics: Sakagarni et al. Proc. Natl. Acad. Sci. USA 91 (1994) A Substrates B Target correction C Aberrant product '6,z in Hp NT RI RI Donor plasmid 8979 8696 8192 a4 N e~~~~~N q Sal bI 1-,8185 R 1005 7958 1079bp A1 505 A Hr ?889Sa Nr Nr 4433 Bam 4185 Sa Target plasmid _ part of pSV2neo C end deletion _ BP'V- mammalian olasmid neo gene L[] N end deletion S pML2d ( bacterial plasmid ) RI Hp FIG. 1. Experimental system. (A) Substrates. Homologous regions between the donor and the target are drawn as parallel lines. The direction ofthe arrows indicates the direction ofmajor transcription. Restriction enzyme sites are as follows: Bam, BamHI; RI, &oRI; RV, EcoRV; Hin, HindIII; Hp, Hpa I; Sal, SalI; X, Xho I; Na, Nae I; Nr, Nar I. (B) Precise target correction by homologous recombination. The solid line includes joints by homologous recombination. The sequences of the region between deletion a and deletion b and of the 1079-bp long region to the left ofdeletion a in B were shown to be retained. (C) Product with nonhomologous recombination at the end ofhomology. There is gene conversion between the donor and the target at deletion a. Around deletion b, where homology between the donor neo segment (II) and the target neo segment (I) is disrupted, there is nonhomologous recombination (dotted line) between the donor neo DNA (II) and a site outside of the neo homology (III). There is another homologous recombination event outside of the neo homology. Thus the solid line includes two homologous recombination events. From experiment 2. Ps, Pst I site. plasmid in a mouse cell line, taking advantage of the mor- region (II) and the third region (III) took place where the phological transformation. In the second step, we introduced homology between the first region (I) and the second region the donor plasmid, with a neo gene with a different, nonover- (II) ends. This product carried a nonhomologous joint near lapping deletion, into this cell line. Homologous recombina- deletion b, where the homology between the two interacting tion between the two mutant neo genes should restore a neo DNAs (I and II) is interrupted (Fig. 1C). The two functional neo gene and should make the cell resistant to drug homologous DNAs (I and II) experienced a gene conversion G418. The donor plasmid introduced to the cells harboring event, at deletion a, without flanking crossing-over. The the target plasmid produced G418R colonies at a frequency nonhomologousjoint lay only 3 bp away from the point where "5% of that obtained with its neo+ version (Table 1). The the homology ends (I and II) (Fig. 2A). The nonhomologous production ofrecombinant G418R colonies required the pres- joint itselfwas typical in that it involved only 4-bp homology. ence of both target DNA and donor DNA (Table 1). In the Two more independent transformants had exactly the same third step, we recovered these neo+ plasmid molecules in a sequence. This strongly suggests that the nonhomologous recA strain of E. coli by kanamycin selection. joint was formed in the mouse cells rather than in E. coli. The structure of the recovered plasmids was analyzed by This specific configuration immediately suggests its own restriction mapping and sequencing. One species showed origin. The end of the homology-dependent interaction be- precise correction of the mutation of the target by homolo- tween the two DNA segments (I and II) produced some gous recombination (Fig. 1B). A second neo+ species showed unusual structure, like a single-stranded region or a strand precise correction of the donor mutation (data not shown). break, which caused the nonhomologous recombination with We found, however, that neo+ products carrying at least one a third DNA (Ill) (Fig. 2B). The homology-dependent inter- nonhomologous joint constituted the majority (Table 2). action could be strand exchange (Fig. 2C) or protein- Nonhomologous Recombination at the End of Long Homol- mediated alignment and/or protection. ogy. The sequence of the first type of this group strongly Partial Gene Conversion.
Load more

Recommended publications
  • Gene Targeting in Plants: 25 Years Later HOLGER PUCHTA* and FRIEDRICH FAUSER
    Int. J. Dev. Biol. 57: 629-637 (2013) doi: 10.1387/ijdb.130194hp www.intjdevbiol.com Gene targeting in plants: 25 years later HOLGER PUCHTA* and FRIEDRICH FAUSER Botanical Institute II, Karlsruhe Institute of Technology, Karlsruhe, Germany ABSTRACT Only five years after the initiation of transgenic research in plants, gene targeting (GT) was achieved for the first time in tobacco. Unfortunately, the frequency of targeted integration via homologous recombination (HR) was so low in comparison to random integration that GT could not be established as a feasible technique in higher plants. It took another 25 years and great effort to develop the knowledge and tools necessary to overcome this challenge, at least for some plant species. In some cases, the overexpression of proteins involved in HR or the use of negative select- able markers improved GT to a certain extent. An effective solution to this problem was developed in 1996, when a sequence-specific endonuclease was used to induce a double-strand break (DSB) at the target locus. Thus, GT frequencies were enhanced dramatically. Thereafter, the main limitation was the absence of tools needed to induce DSBs at specific sites in the genome. Such tools became available with the development of zinc finger nucleases (ZFNs), and a breakthrough was achieved in 2005 when ZFNs were used to target a marker gene in tobacco. Subsequently, endogenous loci were targeted in maize, tobacco and Arabidopsis. Recently, our toolbox for genetic engineering has expanded with the addition of more types of site-specific endonucleases, meganucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas system.
    [Show full text]
  • CRISPR-Cas9-Mediated Efficient Directed Mutagenesis and RAD51
    CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella patens Cécile Collonnier, Aline Epert, Kostlend Mara, François Maclot, Anouchka Guyon-Debast, Florence Charlot, Charles White, Didier Schaefer, Fabien Nogué To cite this version: Cécile Collonnier, Aline Epert, Kostlend Mara, François Maclot, Anouchka Guyon-Debast, et al.. CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51- independent gene targeting in the moss Physcomitrella patens. Plant Biotechnology Journal, Wiley, 2017, 15 (1), pp.122 - 131. 10.1111/pbi.12596. inserm-01904879 HAL Id: inserm-01904879 https://www.hal.inserm.fr/inserm-01904879 Submitted on 25 Oct 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Plant Biotechnology Journal (2017) 15, pp. 122–131 doi: 10.1111/pbi.12596 CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella
    [Show full text]
  • ES Cell Targeting Handbook
    TABLE OF CONTENTS Overview of ES Core Facility Introduction Generation of Gene-Targeted ES Cells Karyotyping of Positive ES Clones ES Cell Request Form General Information for the Generation of Targeted Cells Principles of Gene Targeting Requirements for the Design of Targeting Constructs Screening Assay for the Identification of Targeted ES Clones Overview of ES Cell Culture ES Cell Factors Affecting Successful Chimera Production FAQ Overview of ES Core Facility Our Mission The ES Core Facility (ECF) was founded by the NINDS Core Center Grant and was established to benefit the contributors of this proposal. The mission of ECF is to effectively produce ES cell lines with a high probability of germline transmission. Core Service Services provided by the Core for a typical project include: • Provide guidance on the design of targeting construct • Generate targeted ES cell lines for the production of chimeric mice • Karyotyping ES cells to be micro-injected into blastocysts Consultation is available from ECF directors and staff members on the entire procedures of generating gene knock-out mice. Application for Service Prior to the initiation of a project, a brief meeting is generally required between the investigator and ECF facility staff resulting in a mutually acceptable research strategy. This strategy will outline specifics of the project including knockout strategy, KO construct design, screening assays, and other procedural issues relevant to the generation of targeted ES cells. In addition, a completed service application form, signed by the principal investigator and approved by the Core Director, will also be required. The Core Director will prioritize the service requests according to the difficulty of the project and work load.
    [Show full text]
  • Highly Efficient Transient Gene Expression and Gene Targeting in Primate Embryonic Stem Cells with Helper-Dependent Adenoviral Vectors
    Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors Keiichiro Suzuki*, Kaoru Mitsui*, Emi Aizawa*, Kouichi Hasegawa†‡, Eihachiro Kawase§, Toshiyuki Yamagishi¶, Yoshihiko Shimizuʈ, Hirofumi Suemori†, Norio Nakatsuji§**, and Kohnosuke Mitani*†† *Division of Gene Therapy, Research Center for Genomic Medicine, ¶Department of Anatomy and ʈDepartment of Pathology, Saitama Medical University, Hidaka, Saitama 350-1241, Japan; and †Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center and §Department of Development and Differentiation, Institute for Frontier Medical Sciences, and **Institute for Integrated Cell-Material Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan Communicated by C. Thomas Caskey, University of TexasϪHouston Health Science Center, Houston, TX, July 23, 2008 (received for review April 18, 2008) Human embryonic stem (hES) cells are regarded as a potentially gene expression in Ϸ100% of cells have not been established (9, unlimited source of cellular materials for regenerative medicine. 10). Furthermore, unlike mES cells, in which gene targeting via For biological studies and clinical applications using primate ES HR has been used routinely with great success, there are few cells, the development of a general strategy to obtain efficient studies using gene targeting in hES cells (11–17). Gene targeting gene delivery and genetic manipulation, especially gene targeting study in nonhuman primate ES cells has not yet been reported. via homologous recombination (HR), would be of paramount Although three investigative groups have reported that HPRT1 importance. However, unlike mouse ES (mES) cells, efficient strat- gene targeting was achieved in hES cells by using electroporation egies for transient gene delivery and HR in hES cells have not been (11, 12, 17), the frequencies of HR were extremely low at Ϸ1 ϫ established.
    [Show full text]
  • Gene Targeting in Plants
    The EMBO Journal vol.7 no.13 pp.4021 -4026, 1988 Gene targeting in plants Jerzy Paszkowski, Markus Baur, Although integration of transforming DNA into the Augustyn Bogucki and Ingo Potrykus homologous chromosomal DNA occurs efficiently in yeast, other fungi and Dictyostelium discoideum (Hinnen et al., Friedrich Miescher Institut, PO Box 2543, CH4002 Basel, 1978; De Lozanne et al., 1987; Miller et al., 1987), Switzerland non-homologous ('illegitimate') integration of foreign DNA Present address: Swiss Federal Institute of Technology, Institute of into the genome of higher eukaryotic cells makes it difficult Plant Sciences, ETH-Zentrum, Universitatstrasse 2, CH-8092 Zurich, to assay the frequency of gene targeting to a desired locus. Switzerland Only recently has the application of selection (Lin et al., Communicated by J.-D.Rochaix 1985; Thomas et al., 1986; Doetschman et al., 1987; Song et al., 1987; Thomas and Capecchi, 1987) and screening Although the generation of transgenic plants is now (Smithies et al., 1985) systems allowed comparison of the routine, the integration of foreign genetic information has frequencies of homologous and illegitimate integration of so far been at random sites in the genome. We now transformed DNA in cultured mammalian cells. In plants present evidence for directed integration into a predicted the high transformation frequencies reported for DNA location in the host plant genome. Protoplasts of molecules with no homology to the plant genome (Shillito transgenic tobacco (Nicotiana tabacum) plants carrying et al., 1985; Negrutiu et al., 1987) indicate efficient copies of a partial, non-functional drug-resistance gene illegitimate integration. Therefore, the detection of gene in the nuclear DNA were used as recipients for DNA targeting through homologous DNA recombination requires molecules containing the missing part of the gene.
    [Show full text]
  • E Cient Targeted Gene Insertion in Maize Using Agrobacterium
    Ecient Targeted Gene Insertion in Maize using Agrobacterium-Mediated Delivery Sergei Svitashev ( [email protected] ) Corteva Agriscience https://orcid.org/0000-0003-1667-7430 Dave Peterson Corteva Agriscience Pierjuigi Barone Corteva Agriscience https://orcid.org/0000-0002-2579-0913 Brian Lenderts Corteva Agriscience Chris Schwartz Corteva Agriscience Lanie Feigenbutz Corteva Agriscience Grace St. Clair Corteva Agriscience Spencer Jones Department of Molecular Engineering, Corteva Agriscience™ Agriculture Division of DowDuPont™ Article Keywords: Genome Editing, Gene Knock-outs, Nucleotide Replacement, Vector Design, Trait Product Development Posted Date: December 22nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-99643/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on May 2nd, 2021. See the published version at https://doi.org/10.1111/pbi.13613. Page 1/17 Abstract CRISPR-Cas is a powerful DNA double strand break technology with wide-ranging applications in plant genome modication. However, the eciency of genome editing depends on various factors including plant genetic transformation processes and types of modications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knock-outs in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-dened genomic location remain highly challenging. Here we report an ecient, heritable, selectable marker-free site-specic gene insertion in maize using Agrobacterium-mediated delivery. Advancements in maize transformation and new vector design enabled targeted insertion with frequencies as high as 8–10%.
    [Show full text]
  • Concentrating Mechanisms and Development of Molecular Tools for Chlamydomonas Reinhardtii
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses and Dissertations in Biochemistry Biochemistry, Department of 12-2013 Transcriptomic Analyses of the CO2-Concentrating Mechanisms and Development of Molecular Tools for Chlamydomonas reinhardtii Andrew J. Brueggeman University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/biochemdiss Part of the Biochemistry Commons, and the Molecular Biology Commons Brueggeman, Andrew J., "Transcriptomic Analyses of the CO2-Concentrating Mechanisms and Development of Molecular Tools for Chlamydomonas reinhardtii" (2013). Theses and Dissertations in Biochemistry. 13. https://digitalcommons.unl.edu/biochemdiss/13 This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses and Dissertations in Biochemistry by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. TRANSCRIPTOMIC ANALYSES OF THE CO2-CONCENTRATING MECHANISM AND DEVELOPMENT OF MOLECULAR TOOLS FOR CHLAMYDOMONAS REINHARDTII by Andrew J Brueggeman A Dissertation Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Biochemistry Under the Supervision of Professor Donald P. Weeks Lincoln, Nebraska December, 2013 TRANSCRIPTOMIC ANALYSES OF THE CO2-CONCENTRATING MECHANISM AND DEVELOPMENT OF MOLECULAR TOOLS FOR CHLAMYDOMONAS REINHARDTII Andrew J Brueggeman, Ph.D. University of Nebraska, 2013 Advisor: Donald P. Weeks Microalgae, such as Chlamydomonas reinhardtii, account for a large percentage of photosynthesis that occurs on the planet. Many algae possess a Carbon- Concentrating Mechanism, or CCM, that actively transports inorganic carbon (Ci) into the cell to create artificially high internal levels of CO2, enhancing their rate of carbon fixation.
    [Show full text]
  • Improved CRISPR Genome Editing Using Small Highly Active and Specific Engineered RNA-Guided Nucleases
    ARTICLE https://doi.org/10.1038/s41467-021-24454-5 OPEN Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases Moritz J. Schmidt1,4, Ashish Gupta1,4, Christien Bednarski1,4, Stefanie Gehrig-Giannini1, Florian Richter1, Christian Pitzler1, Michael Gamalinda1, Christina Galonska1, Ryo Takeuchi2, Kui Wang2, Caroline Reiss2, Kerstin Dehne1, Michael J. Lukason3, Akiko Noma2, Cindy Park-Windhol2, Mariacarmela Allocca2, Albena Kantardzhieva2, Shailendra Sane2, Karolina Kosakowska2, Brian Cafferty2, Jan Tebbe1, Sarah J. Spencer3, ✉ Scott Munzer2, Christopher J. Cheng2, Abraham Scaria2, Andrew M. Scharenberg2, André Cohnen 1,5 & 1234567890():,; ✉ Wayne M. Coco 1,5 Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a “GG” dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases. 1 Bayer AG, Leverkusen, Germany. 2 Casebia Therapeutics LLC, Cambridge, MA, USA.
    [Show full text]
  • Two Mind Genes in Physcomitrella Patens Are Functionally Redundant
    © 2005 The Japan Mendel Society Cytologia 70(1): 87–92, 2005 Two minD Genes in Physcomitrella patens Are Functionally Redundant Ryuuichi D. Itoh1,*, Katsuaki Takechi2, Akihiro Hayashida2, Shin-ichiro Katsura1 and Hiroyoshi Takano2 1 Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, Okinawa 903–0213, Japan 2 Graduate School of Science and Technology, Kumamoto University, Kumamoto 860–8555, Japan Received January 18, 2005; accepted January 26, 2005 Summary We investigated two genes that encode a homolog of the bacterial cell division site de- terminant MinD, PpMinD1 and PpMinD2, which were previously identified in the moss Physcomitrella patens. Southern analysis suggested that the P. patens genome does not contain minD genes other than PpMinD1 and PpMinD2. Molecular phylogenetic analysis and the results of a tran- sient expression assay using MinD : green fluorescent protein (GFP) fusions implied that PpMinD1 and PpMinD2 are cyanobacterium-derived, closely related genes encoding chloroplast-targeted MinD proteins. Disruption of the genomic PpMinD1 or PpMinD2 locus had no detectable effects on plastid division. This implies that there is functional redundancy in the two minD genes in P. patens. Key words Chloroplast division, gene targeting, knockout, minD, moss, Physcomitrella patens. The genes essential for plastid division have been identified in Arabidopsis thaliana, and most encode proteins that are homologous to bacterial cell division proteins such as FtsZ, ARC11/MinD, MinE, ARTEMIS, ARC6/Ftn2 (for review, see Osteryoung and Nunnari 2003), and GC1/SulA (Maple et al. 2004, Raynaud et al. 2004). The involvement of prokaryote-type cell division factors in plastid division is quite reasonable in view of the endosymbiotic theory, which holds that plastids arose from cyanobacteria engulfed by endocytosis.
    [Show full text]
  • Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Caltech Authors MOLECULAR AND CELLULAR BIOLOGY, May 2003, p. 3558–3565 Vol. 23, No. 10 0270-7306/03/$08.00ϩ0 DOI: 10.1128/MCB.23.10.3558–3565.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved. Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand Breaks Matthew H. Porteus,1* Toni Cathomen,2 Matthew D. Weitzman,2 and David Baltimore1 California Institute of Technology, Pasadena, California 91125,1 and Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California 920372 Received 18 November 2002/Returned for modification 6 January 2003/Accepted 3 March 2003 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.
    [Show full text]
  • Overview of Gene Targeting by Homologous Recombination 4.29.10
    Overview of Gene Targeting by Homologous UNIT 4.29 Recombination The analysis of mutant organisms and cell tion of the mutation present in the targeting lines has been important in determining the construct into the target gene. Once identified, function of specific proteins. Until recently, mutant ES cell clones can be microinjected into mutants were produced by mutagenesis fol- a normal blastocyst in order to produce a lowed by selection for a particular phenotypic chimeric mouse. Because many ES cell lines change. Recent technological advances in gene retain the ability to differentiate into every cell targeting by homologous recombination in type present in the mouse, the chimera can have mammalian systems enable the production of tissues, including the germ line, with contribu- mutants in any desired gene (Mansour, 1990; tion from both the normal blastocyst and the Robertson, 1991; Zimmer, 1992). This technol- mutant ES cells. Breeding germ-line chimeras ogy can be used to produce mutant mouse yields animals that are heterozygous for the strains and mutant cell lines. Because most mutation introduced into the ES cell, and that mammalian cells are diploid, they contain two can be interbred to produce homozygous mu- copies, or alleles, of each gene encoded on an tant mice. autosomal (nonsex) chromosome. In most Homologous recombination can also be cases, both alleles must be inactivated to pro- used to produce homozygous mutant cell lines. duce a discernible phenotypic change in a mu- Previously, inactivation of both alleles of a gene tant. The conversion from heterozygosity to required two rounds of homologous recombi- homozygosity is accomplished by breeding in nation and selection (te Riele et al., 1990; Cruz the case of mouse strains and by direct selection et al., 1991; Mortensen et al., 1991).
    [Show full text]
  • Efficient Gene Targeting by Homologous Recombination in Rice
    RESEARCH ARTICLE Efficient gene targeting by homologous recombination in rice Rie Terada1, Hiroko Urawa1, Yoshishige Inagaki1,2, Kazuo Tsugane1, and Shigeru Iida1* Published online: 9 September 2002, doi:10.1038/nbt737 Modification of genes through homologous recombination, termed gene targeting, is the most direct method to characterize gene function. In higher plants, however, the method is far from a common practice. Here we describe an efficient and reproducible procedure with a strong positive/negative selection for gene targeting in rice, which feeds more than half of the world’s population and is an important model plant. About 1% of select- ed calli and their regenerated fertile plants were heterozygous at the targeted locus, and only one copy of the selective marker used was found at the targeted site in their genomes. The procedure’s applicability to other genes will make it feasible to obtain various gene-targeted lines of rice. Rice (Oryza sativa L.) is an important staple food for more than half of genomic sequence is well characterized3,4,6. As a model gene to be http://www.nature.com/naturebiotechnology the world’s population and is a model plant for other cereal species1–3. targeted, we chose the gene Waxy, which encodes granule-bound With the elucidation of the entire genome sequence of rice3–6, it is starch synthase, a key enzyme in amylose synthesis (Fig. 1)16,17.We becoming clear that a large proportion of predicted rice genes have no selected Waxy because its mutations affect the quality and quanti- recognizable homologs in Arabidopsis, in which <10% of putative ty of rice grain and because the associated phenotype in pollen genes have been studied experimentally7.
    [Show full text]