DOCSLIB.ORG

Suppressor-Linker Insertional Mutagenesis: Positions Ofviable

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Suppressor-Linker Insertional Mutagenesis: Positions Ofviable Proc. Nati. Acad. Sci. USA Vol. 81, pp. 4149-4153, July 1984 Genetics Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: Positions of viable insertion mutations (virus replication/synthetic DNA/oligonucleotides) LESLIE I. LOBEL AND STEPHEN P. GOFF Department of Biochemistry and Institute for Cancer Research, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032 Communicated by Harold S. Ginsberg, March 20, 1984 ABSTRACT A highly efficient method for the generation thetic oligonucleotide sequence containing a restriction en- of insertion mutations is described. The procedure involves the zyme recognition site is inserted at the position of cleavage use of a 220-base-pair (bp) Ec6RI fragment bearing the SuIIIl in the target DNA. The resulting mutations can be readily suppressor tRNA gene as an insertional mutagen. The plasmid mapped simply by determining the site of cleavage by the DNA to be mutagenized is linearized by a variety of means, restriction enzyme that recognizes the inserted sequence. and the suppressor fragment is ligated into the site of cleavage. We have constructed several libraries of mutants of Mo- Successful insertion mutants can be readily detected in Esche- loney murine leukemia virus (M-MuLV) by the insertion of richia coli carrying lac- amber mutations on MacConkey lac- EcoRI linkers at random positions in a cloned DNA copy of tose plates; virtually 100% of the red colonies contain inser- the viral genome. During the course of these studies, we tions of the fragment. Subsequent removal of the SulIl+ gene found that the efficiency of the process was highly variable; and recyclization leaves a 12-bp insertion if the original cleav- linkers were added poorly to ends formed by some nucleases age was blunt-ended and a 9-bp insertion if the original cleav- and very few linker insertions could be successfully formed. age generated 3-bp cohesive termini. This technique, as well as To circumvent this difficulty, we have developed an im- conventional linker mutagenesis with decamer and dodecamer proved method by which rare insertion events can be readily linkers, was used to generate a large library of insertion muta- detected, obviating the screening of large numbers of clones tions in cloned DNA copies of the genome of Moloney murine by electrophoresis to identify insertions. By this method, leukemia virus. A number of viable mutants were isolated large libraries of mutants, free of wild-type parental DNA, bearing 9-, 10-, and 12-bp insertions in various domains of the can be efficiently constructed. In this report, we describe genome. The map positions of the viable mutations suggest the method and its application to the genetic analysis of the that the viral long terminal repeats and portions of the gag and genome of M-MuLV. We have screened libraries of mutants env genes are quite insensitive to alteration. Although most of and, after transfection of NIH/3T3 cells, have recovered nu- the mutations were stable for many passages, some of the mu- merous clones that are still replication competent. The posi- tants lost the inserted DNA; we presume that the insertion was tions of the mutations in these clones define noncoding and somewhat deleterious in these mutants and that continued pas- coding regions of the viral genome that are dispensible for sage of the virus selected for overgrowth by a revertant. replication or that can tolerate insertions without deleterious effects on the virus. The in vitro mutagenesis of cloned genes has been one of the most powerful tools in the elucidation of the relationship be- MATERIALS AND METHODS tween structure and function of DNA sequences. By intro- Bacteria. Escherichia coli strain HB101 (rec A13,- hsdRl, ducing defined alterations in a sequence and correlating the hsdM-, lac Y1, supE44) was used as the recipient cell for changes with alterations in gene function, it has been possi- most DNA transformations (12). To detect the presence of ble to define such features as transcriptional promoters (1- the SuIII+ suppressor gene, strain CC114 [del(ara,leu)7697, 4), RNA splicing signals (5), RNA polyadenylylation signals lacZ- amYJ4, galU, galK, hsdR-, hsdM', strAr, rifr, argE- (6), ribosome binding sites (7), and protein domains needed am, srl::TnIO, recAl] was used as recipient. This strain, con- for particular enzymatic activities (8, 9). Our power to carry structed and generously provided by C. Manoil, gave high out such analyses has been increased enormously as the transformation efficiencies comparable to that of HB101. techniques used to make the defined alterations have be- Plasmid DNAs. Plasmids used in this work include p8.2 come increasingly sophisticated (10). (13), a complete permuted copy of the M-MuLV genome in- The use of these techniques is sometimes limited by our serted in the HindIII site of pBR322; pACYC177 (14), a vec- ability to choose likely target regions for mutagenesis. When tor containing no EcoRI sites; pMOV9 (15), containing an there is no information defining which parts of a gene are integrated proviral copy of the M-MuLV genome; pBR328 essential for a particular function, then narrowly targeted in (16), a vector with a single Pvu II site; and piVX (17), a plas- vitro mutagenesis is not helpful in the localization of that mid carrying the SuIII+ gene. function. In such cases, it is necessary to generate a large Enzymatic Reactions. Synthetic oligomers (New England library of mutants with defined alterations scattered Biolabs) were phosphorylated with T4 polynucleotide kinase throughout the cloned DNA and to screen these mutants for (Bethesda Research Laboratories) at a concentration of 1 ,ug a desired phenotype. One of the best ways to construct such per 10 A.l in buffer (50 mM Tris HCl, pH 7.4/10 mM a library is by "linker insertion" mutagenesis (11): a circular MgCI2/10 mM dithiothreitol) containing 1 mM ATP (5 Ci/ target DNA is linearized with a nuclease, and a short syn- mmol; 1 Ci = 37 GBq) for 1 hr at 370C. One-tenth of the preparation (0.1 pug) was ligated to DNA (1-5 ,ug) in the same The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: M-MuLV, Moloney murine leukemia virus; kb, kilo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. base(s); bp, base pair(s). 4149 Downloaded by guest on September 27, 2021 4150 Genetics: Lobel and Goff Proc. NatL. Acad. Sci. USA 81 (1984) buffer overnight at 15'C before purification by agarose gel DNAs were linearized by treatment with restriction enzymes electrophoresis. Restriction enzymes were used according to Hae III or Alu I. These enzymes produce blunt ends and can the specifications of the' manufacturer (New England Bio- make cleavages at a large number of sites in the M- labs). Partial cleavages were carried out at 250C for various MuLV genome: a total of 64 and 36 sites, respectively, are times (1-60 min). Typically, 40 gg of DNA in 200 1.l of the present in the 8.8-kb provirus. After limited digestion, link- recommended buffer was digested with 1 unit of enzyme, ers were added with T4 DNA ligase, and the full-length lin- and aliquots were removed for analysis. Optimal times for ear molecules were isolated by agarose gel electrophoresis. formation of full-length linear DNAs were usually 20-40 The mixture was digested with EcoRI, circles were reformed min. Cohesive ends of DNA fragments were filled in with the by ligation, and clones were prepared from bacterial trans- Klenow fragment (18) of DNA polymerase I (New England formants. With this technique, our best experiments pro- Biolabs) in buffer (50 mM Tris HCl, pH 7.4/10 mM duced populations in which -50% of the DNAs were found MgCl2/10 mM dithiothreitol) containing the four deoxyri- to contain novel EcoRI sites. In some cases, however, only bonucleotides (1 mM each). Ends were blunted before addi- 10% contained an EcoRI site. Furthermore, independently tion of linkers by treatment with S1 nuclease (19) (Boeh- isolated inserts at a given site sometimes generated different ringer Mannheim) in buffer (300 mM NaCl/30 mM sodium phenotypes, suggesting that sometimes insertions of differ- acetate, pH 4.6/4 mM ZnCl2) at 20'C for 20 min. Reactions ent lengths had occurred. with terminal transferase (20, 21) obtained from Bethesda The positions of a large number of these insertion muta- Research Laboratories were carried out with -0.5 ,ug of tions were determined by digestion with pairs of restriction DNA in 100 ,ul of buffer (200 mM potassium cacodylate, pH enzymes (EcoRI and HindIII, or EcoRI and Sal I) followed 7.2/1. mM mercaptoethanol/1 mM CoC12/50 ,uM dGTP or by agarose gel electrophoresis. Once the approximate map dCTP) for 1-4 min at 25°C. DNAs to be joined were mixed, position was determined from these digests, the exact site of heated to 70°C for 5 min, and then cooled to 40°C for 4 hr insertion was mapped by measuring the distance from the before transformation of bacteria. linker to a nearby restriction site on acrylamide gels. When DNA sequence analysis was as described (22). two Hae III or Alu I sites were close together, DNA se- Bacterial Transformations. Transformation of strain quence analysis was used to determine the insertion site. CC114 was carried out according to standard protocols, Mutations were assigned numbers according to the base po- treating the cells with buffer (50 mM CaCl2/10 mM Tris HCl, sition of the insertion starting at the left edge of the left long pH 7.4) to induce competence.
Load more

Recommended publications
  • Controlled Insertional Mutagenesis Using a LINE-1 (Orfeus) Gene-Trap
    Controlled insertional mutagenesis using a LINE-1 PNAS PLUS (ORFeus) gene-trap mouse model Kathryn A. O’Donnella,b,1,2, Wenfeng Ana,b,3, Christina T. Schruma,b, Sarah J. Wheelanc, and Jef D. Boekea,b,c,1 Departments of aMolecular Biology and Genetics and cOncology, and bHigh Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Edited* by Fred H. Gage, The Salk Institute for Biological Studies, San Diego, CA, and approved May 30, 2013 (received for review February 7, 2013) A codon-optimized mouse LINE-1 element, ORFeus, exhibits dra- treated with doxycycline. We observed high levels of retro- matically higher retrotransposition frequencies compared with transposition in tissues from double-transgenic mice but not its native long interspersed element 1 counterpart. To establish in control littermates, and the amount of retrotransposition a retrotransposon-mediated mouse model with regulatable and increases with increased doxycycline dose. Induction of the tet- potent mutagenic capabilities, we generated a tetracycline (tet)- ORFeus element with high doses of doxycycline during embryo- regulated ORFeus element harboring a gene-trap cassette. Here, genesis led to a reduced number of double-transgenic mice, we show that mice expressing tet-ORFeus broadly exhibit robust likely due to a significant burden of mutations and embryonic retrotransposition in somatic tissues when treated with doxycy- lethality in these animals. Unexpectedly, a significant percentage cline. Consistent with a significant mutagenic burden, we observed of double-transgenic agouti mice developed white spots, suggest- a reduced number of double transgenic animals when treated with ing that somatic mutations occurred at different times in de- high-level doxycycline during embryogenesis.
    [Show full text]
  • Transposon Insertion Mutagenesis in Mice for Modeling Human Cancers: Critical Insights Gained and New Opportunities
    International Journal of Molecular Sciences Review Transposon Insertion Mutagenesis in Mice for Modeling Human Cancers: Critical Insights Gained and New Opportunities Pauline J. Beckmann 1 and David A. Largaespada 1,2,3,4,* 1 Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] 2 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA 3 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA 4 Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA * Correspondence: [email protected]; Tel.: +1-612-626-4979; Fax: +1-612-624-3869 Received: 3 January 2020; Accepted: 3 February 2020; Published: 10 February 2020 Abstract: Transposon mutagenesis has been used to model many types of human cancer in mice, leading to the discovery of novel cancer genes and insights into the mechanism of tumorigenesis. For this review, we identified over twenty types of human cancer that have been modeled in the mouse using Sleeping Beauty and piggyBac transposon insertion mutagenesis. We examine several specific biological insights that have been gained and describe opportunities for continued research. Specifically, we review studies with a focus on understanding metastasis, therapy resistance, and tumor cell of origin. Additionally, we propose further uses of transposon-based models to identify rarely mutated driver genes across many cancers, understand additional mechanisms of drug resistance and metastasis, and define personalized therapies for cancer patients with obesity as a comorbidity. Keywords: animal modeling; cancer; transposon screen 1. Transposon Basics Until the mid of 1900’s, DNA was widely considered to be a highly stable, orderly macromolecule neatly organized into chromosomes.
    [Show full text]
  • Retroviral Insertional Mutagenesis: Past, Present and Future
    Oncogene (2005) 24, 7656–7672 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Retroviral insertional mutagenesis: past, present and future AG Uren1,2, J Kool1,2, A Berns*,1 and M van Lohuizen*,1 1Division of Molecular Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands Retroviral insertion mutagenesis screens in mice are site of the provirus. Consequently, these viruses have powerful tools for efficient identification of oncogenic been widely used in genetic screens for mutations mutations in an in vivo setting. Many oncogenes identified involved in the onset of tumorigenesis in various model in these screens have also been shown to play a causal role organisms. in the development of human cancers. Sequencing and annotation of the mouse genome, along with recent improvements in insertion site cloning has greatly Transforming retroviruses as tools for genetic screening facilitated identification of oncogenic events in retro- virus-induced tumours. In this review, we discuss the Oncogenic retroviruses can be divided into two classes: features of retroviral insertion mutagenesis screens, acute and slow transforming viruses. Acute transform- covering the mechanisms by which retroviral insertions ing retroviruses induce polyclonal tumours within 2 to 3 mutate cellular genes, the practical aspects of insertion weeks after infection of the host. Transformation by site cloning, the identification and analysis of common these viruses is mediated by the expression of viral insertion sites, and finally we address the potential for use oncogenes such as v-Abl in the Abelson Murine of somatic insertional mutagens in the study of non- leukaemia Virus (reviewed in Shore et al., 2002), and haematopoietic and nonmammary tumour types.
    [Show full text]
  • A Review on the Population Genomics of Transposable Elements
    G C A T T A C G G C A T genes Review On the Population Dynamics of Junk: A Review on the Population Genomics of Transposable Elements Yann Bourgeois and Stéphane Boissinot * New York University Abu Dhabi, P.O. 129188 Saadiyat Island, Abu Dhabi, UAE; [email protected] * Correspondence: [email protected] Received: 4 April 2019; Accepted: 21 May 2019; Published: 30 May 2019 Abstract: Transposable elements (TEs) play an important role in shaping genomic organization and structure, and may cause dramatic changes in phenotypes. Despite the genetic load they may impose on their host and their importance in microevolutionary processes such as adaptation and speciation, the number of population genetics studies focused on TEs has been rather limited so far compared to single nucleotide polymorphisms (SNPs). Here, we review the current knowledge about the dynamics of transposable elements at recent evolutionary time scales, and discuss the mechanisms that condition their abundance and frequency. We first discuss non-adaptive mechanisms such as purifying selection and the variable rates of transposition and elimination, and then focus on positive and balancing selection, to finally conclude on the potential role of TEs in causing genomic incompatibilities and eventually speciation. We also suggest possible ways to better model TEs dynamics in a population genomics context by incorporating recent advances in TEs into the rich information provided by SNPs about the demography, selection, and intrinsic properties of genomes. Keywords: transposable elements; population genetics; selection; drift; coevolution 1. Introduction Transposable elements (TEs) are repetitive DNA sequences that are ubiquitous in the living world and have the ability to replicate and multiply within genomes.
    [Show full text]
  • Meeting Report: the Role of the Mobilome in Cancer Daniel Ardeljan1,2, Martin S
    Published OnlineFirst July 18, 2016; DOI: 10.1158/0008-5472.CAN-15-3421 Cancer Meeting Report Research Meeting Report: The Role of the Mobilome in Cancer Daniel Ardeljan1,2, Martin S. Taylor3, Kathleen H. Burns1,4, Jef D. Boeke5, Michael Graham Espey6, Elisa C. Woodhouse6, and Thomas Kevin Howcroft6 Abstract Approximately half of the human genome consists of repetitive in developmental and pathologic contexts, including many sequence attributed to the activities of mobile DNAs, including types of cancers. However, we have limited knowledge of the DNA transposons, RNA transposons, and endogenous retro- extent and consequences of L1 expression in premalignancies viruses. Of these, only long interspersed elements (LINE-1 or and cancer. Participants in this NIH strategic workshop consid- L1) and sequences copied by LINE-1 remain mobile in our species ered key questions to enhance our understanding of mechan- today. Although cells restrict L1 activity by both transcriptional isms and roles the mobilome may play in cancer biology. and posttranscriptional mechanisms, L1 derepression occurs Cancer Res; 76(15); 1–4. Ó2016 AACR. Introduction demonstration of retrotransposition in cultured HeLa cells (2). A somatically acquired L1 insertion was shown to disrupt the Mobile DNAs, including DNA transposons, RNA transposons, adenomatous polyposis coli (APC) tumor suppressor gene in a and endogenous retroviruses, are highly abundant sequences that case of colorectal cancer (3). Nearly a decade of advances in DNA make up a major portion of eukaryotic genomes. Although sequencing have both underscored the importance of L1 activity human genomes no longer possess active DNA transposons, in causing heritable variation through retrotransposition in the which integrate via a "cut-and-paste" mechanism, they are instead germline and also demonstrated that widespread somatic retro- enriched in active retrotransposons that integrate via RNA inter- transposition occurs in many cancers.
    [Show full text]
  • Insertional Mutagenesis of Drosophila Heterochromatin with Single P Elements
    Proc. Natl. Acad. Sci. USA Vol. 91, pp. 3539-3543, April 1994 Genetics Insertional mutagenesis of Drosophila heterochromatin with single P elements PING ZHANG AND ALLAN C. SPRADLING Howard Hughes Medical Institute Research Laboratories, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210 Contributed by Allan C. Spradling, December 28, 1993 ABSTRACT Insertional mutagenesis with transposable P efficiently generated throughout diverse regions of hetero- elements has greatly facilitated the identification and analysis chromatin by suppressing position effects genetically. of genes located throughout the 70% of the Drosophila mela- nogaster genome classified as euchromatin. In contrast, genet- ically marked P elements have only rarely been shown to MATERIALS AND METHODS transpose into heterochromatin. By carrying out single P Drosophila Stocks. Flies were grown on standard corn element insertional mutagenesis under conditions where posi- meal/agar media (1), at 220C. Unless stated otherwise, strains tion-effect variegation was suppressed, we efficiently generated and mutations are as described in ref. 19. The same starting strains containing insertions at diverse sites within centromeric strain used previously (16) was employed for the screen in and Y-chromosome heterochromatin. The tendency of P ele- Fig. 1. It contains an X-linked insertion ofthe PZ transposon, ments to transpose locally was shown to operate within het- which carries the rosy+ (ry+) eye color gene. The isolation of erochromatin, and it further enhanced the recovery of hetero- 24 lines containing PZ insertions on the Y chromosome was chromatic insertions. Three of the insertions disrupted vital reported previously (16). The 95-2 strain was identified in a genes known to be present at low density in heterochromatin.
    [Show full text]
  • Hepatitis B Virus-Related Insertional Mutagenesis Implicates SERCA1 Gene in the Control of Apoptosis
    Oncogene (2000) 19, 2877 ± 2886 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Hepatitis B virus-related insertional mutagenesis implicates SERCA1 gene in the control of apoptosis Mounia Chami1,7, Devrim Gozuacik1,7, Kenichi Saigo1,2,7, Thierry Capiod3, Pierre Falson4, Herve Lecoeur5, Tetsuro Urashima2, Jack Beckmann6, Marie-Lyse Gougeon5, Michel Claret3, Marc le Maire4, Christian Bre chot1 and Patrizia Paterlini-Bre chot*,1 1U-370 INSERM, Necker Institute, 75015 Paris, France; 2Second Department of Surgery, Chiba University, Chiba 263-8852, Japan; 3U-442 INSERM, Universite Paris Sud, 91405 Orsay, France; 4URA CNRS 2096, CEA Saclay, 91191 Gif sur Yvette, France; 5Pasteur Institute, 75015 Paris, France; 6Centre National GeÂnotypage, 91057 Evry, France We have used the Hepatitis B Virus DNA genome as a and Yun, 1998) and NF-kB signaling (Chirillo et al., probe to identify genes clonally mutated in vivo,in 1996). It has also been suggested that it directly human liver cancers. In a tumor, HBV-DNA was found interacts with cellular proteins controlling cell growth to be integrated into the gene encoding Sarco/Endoplas- (p53 (Truant et al., 1995; Wang et al., 1994a)), DNA mic Reticulum Calcium ATPase (SERCA), which pumps repair (ERCC (Wang et al., 1994b) and UVDDB (Lee calcium, an important intracellular messenger for cell et al., 1995; Sitterlin et al., 1997)), senescence (Sun et viability and growth, from the cytosol to the endoplasmic al., 1998), NFkB activity (Weil et al., 1999), basal reticulum. The HBV X gene promoter cis-activates transcription (RBP5 (Cheong et al., 1995) and RMP chimeric HBV X/SERCA1 transcripts, with splicing of (Dorjsuren et al., 1998)), signal transduction (ATF/ SERCA1 exon 11, encoding C-terminally truncated CREB (Williams and Andrisani, 1995)) and with SERCA1 proteins.
    [Show full text]
  • Tn7-Based Genome-Wide Random Insertional Mutagenesis of Candida
    Methods Tn7-Based Genome-Wide Random Insertional Mutagenesis of Candida glabrata Irene Castan˜o, Rupinder Kaur, Shihjung Pan, Robert Cregg, Alejandro De Las Pen˜as, Nini Guo, Matthew C.Biery, Nancy L.Craig, and Brendan P.Cormack 1 Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA We describe and characterize a method for insertional mutagenesis of the yeast pathogen Candida glabrata using the bacterial transposon Tn7.Tn7 was used to mutagenize a C. glabrata genomic fosmid library. Pools of random Tn7 insertions in individual fosmids were recovered by transformation into Escherichia coli. Subsequently, these were introduced by recombination into the C. glabrata genome. We found that C. glabrata genomic fragments carrying a Tn7 insertion could integrate into the genome by nonhomologous recombination, by single crossover (generating a duplication of the insertionally mutagenized locus), and by double crossover, yielding an allele replacement. We were able to generate a highly representative set of ∼104 allele replacements in C. glabrata, and an initial characterization of these shows that a wide diversity of genes were targeted in the mutagenesis. Because the identity of disrupted genes for any mutant of interest can be rapidly identified, this method should be of general utility in functional genomic characterization of this important yeast pathogen. In addition, the method might be broadly applicable to mutational analysis of other organisms. Candida species, primarily Candida albicans and Candida gla- pects of virulence can be isolated and characterized. An effi- brata, are important human pathogens, responsible for 7% of cient genetic analysis depends on a method of random inser- all hospital-acquired blood stream infections (Schaberg et al.
    [Show full text]
  • Observed Incidence of Tumorigenesis in Long-Term Rodent Studies of Raav Vectors
    Gene Therapy (2001) 8, 1343–1346 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt BRIEF COMMUNICATION Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors A Donsante1, C Vogler2, N Muzyczka3, JM Crawford4, J Barker5, T Flotte3, M Campbell-Thompson4, T Daly1,6 and MS Sands1 1Department of Internal Medicine, and 2Department of Pathology, St Louis University School of Medicine, St Louis, MO; 3Powell Gene Therapy Center, 4Department of Pathology, University of Florida, Gainesville, FL; and 5Jackson Laboratory, Bar Harbor, ME, USA Gene therapy using recombinant adeno-associated virus normal mice overexpressing human GUSB for the presence vectors (rAAV) is generally considered safe. During the of tumors and increased hepatocyte replication. The results course of a study designed to determine the long-term effi- of these studies do not indicate that MPSVII mice or mice cacy of rAAV-mediated gene therapy initiated in newborn overexpressing human GUSB are susceptible to tumor for- mice with the lysosomal storage disease, mucopolysacchar- mation; however, the number of animals examined is too idosis type VII (MPSVII), a significant incidence of hepato- small to draw definitive conclusions. Results from quantitat- cellular carcinomas and angiosarcomas was discovered. A ive PCR performed on the tumor samples suggest that the hepatocellular carcinoma was first detected in a 35-week- tumors are probably not caused by an insertional old mouse and by 72 weeks of age, three out of five rAAV- mutagenesis event followed by the clonal expansion of a treated MPSVII mice had similar lesions. These types of transformed cell.
    [Show full text]
  • Cancer Gene Discovery: Exploiting Insertional Mutagenesis
    Published OnlineFirst August 8, 2013; DOI: 10.1158/1541-7786.MCR-13-0244 Molecular Cancer Review Research Cancer Gene Discovery: Exploiting Insertional Mutagenesis Marco Ranzani1, Stefano Annunziato1, David J. Adams2, and Eugenio Montini1 Abstract Insertional mutagenesis has been used as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. Retroviruses have been used for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using retrovirus-based approaches. Most recently, lentiviral vectors have appeared on the scene for use in cancer gene screens. Lentiviral vectors are replication-defective integrating vectors that have the advantage of being able to infect nondividing cells, in a wide range of cell types and tissues. In this review, we describe the various insertional mutagens focusing on their advantages/limitations, and we discuss the new and promising tools that will improve the insertional mutagenesis screens of the future.
    [Show full text]
  • Insertional Mutagenesis Approaches and Their Use in Rice for Functional Genomics
    plants Review Insertional Mutagenesis Approaches and Their Use in Rice for Functional Genomics 1, , 2, 1, 1 1 Hasthi Ram y *, Praveen Soni y, Prafull Salvi y , Nishu Gandass , Ankita Sharma , Amandeep Kaur 1 and Tilak Raj Sharma 1,* 1 Department of Agri-Biotechnology, National Agri-Food Biotechnology Institute (NABI), Sector-81, SAS Nagar, Mohali 140306, India 2 Department of Botany, Rajasthan University, Jaipur 302004, India * Correspondence: [email protected] (H.R.); [email protected] (T.R.S.) Equal Contributions. y Received: 12 July 2019; Accepted: 7 August 2019; Published: 29 August 2019 Abstract: Insertional mutagenesis is an indispensable tool for engendering a mutant population using exogenous DNA as the mutagen. The advancement in the next-generation sequencing platform has allowed for faster screening and analysis of generated mutated populations. Rice is a major staple crop for more than half of the world’s population; however, the functions of most of the genes in its genome are yet to be analyzed. Various mutant populations represent extremely valuable resources in order to achieve this goal. Here, we have reviewed different insertional mutagenesis approaches that have been used in rice, and have discussed their principles, strengths, and limitations. Comparisons between transfer DNA (T-DNA), transposons, and entrapment tagging approaches have highlighted their utilization in functional genomics studies in rice. We have also summarised different forward and reverse genetics approaches used for screening of insertional mutant populations. Furthermore, we have compiled information from several efforts made using insertional mutagenesis approaches in rice. The information presented here would serve as a database for rice insertional mutagenesis populations.
    [Show full text]
  • The Evolution of Gene Therapy in the Treatment of Metabolic Liver Diseases
    G C A T T A C G G C A T genes Review The Evolution of Gene Therapy in the Treatment of Metabolic Liver Diseases Carlos G. Moscoso 1,* and Clifford J. Steer 1,2,* 1 Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota Medical School, Minneapolis, MN 55455, USA 2 Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455, USA * Correspondence: [email protected] (C.G.M.); [email protected] (C.J.S.); Tel.: +1-612-625-8999 (C.G.M. & C.J.S.); Fax: +1-612-625-5620 (C.G.M. & C.J.S.) Received: 18 May 2020; Accepted: 6 August 2020; Published: 10 August 2020 Abstract: Monogenic metabolic disorders of hepatic origin number in the hundreds, and for many, liver transplantation remains the only cure. Liver-targeted gene therapy is an attractive treatment modality for many of these conditions, and there have been significant advances at both the preclinical and clinical stages. Viral vectors, including retroviruses, lentiviruses, adenovirus-based vectors, adeno-associated viruses and simian virus 40, have differing safety, efficacy and immunogenic profiles, and several of these have been used in clinical trials with variable success. In this review, we profile viral vectors and non-viral vectors, together with various payloads, including emerging therapies based on RNA, that are entering clinical trials. Genome editing technologies are explored, from earlier to more recent novel approaches that are more efficient, specific and safe in reaching their target sites. The various curative approaches for the multitude of monogenic hepatic metabolic disorders currently at the clinical development stage portend a favorable outlook for this class of genetic disorders.
    [Show full text]