DOCSLIB.ORG

Insertional Mutagenesis of Drosophila Heterochromatin with Single P Elements

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read 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. screen for local transposition of the PZ element in the male Strains containing single P element insertions will greatly germ line (18). facilitate the structural and functional analysis of this poorly Genetic Analysis of Heterochromatic Insertions. General understood genomic component. information concerning single P element insertion screens was provided previously (14, 20). Briefly, the initial screen Like those ofmost eukaryotes, the chromosomes ofDrosoph- was carried out as follows (Fig. 1). F1 males of genotype ila melanogaster are divided into large, conspicuous euchro- X(PZ)/ Y; Sb A2-3/ry containing an X-linked PZ insertion and matic and heterochromatic domains (see Fig. 1) distinguished the A2-3 transposase source were generated to activate the by divergent structural and functional properties (1). Drosoph- element. Individual F1 males were crossed to C(1)RM/O; ila euchromatin accounts for approximately 70%o of the ge- ry/ry females in vials. F2 C(1)RM/ Y; ry/ry females carried a nome, and it can be subdivided by the detailed banding pattern Y chromosome to suppress position effect and were virgins in polytene chromosomes into approximately 5000 regions because their X/O male siblings were sterile due to the lack averaging 25 kb in length (2). In contrast, metaphase chromo- of a Y chromosome. A single F2 female bearing a new PZ some banding has resolved Drosophila heterochromatin into insertion as indicated by ry+ (i.e., wild type) eye color was about 60 regions averaging 1000 kb (3-5). collected from each vial and mated to X'Y/O; ry/ry males. Drosophila heterochromatin houses diverse genetic func- Candidate lines bearing heterochromatic insertions were tions, despite an abundance of satellite and repetitive DNAs identified by the failure of more than the expected 50% of F3 (6). More than 20 vital genes have been identified within progeny females to express ry+. Other lines were discarded autosomal heterochromatin (7, 8) and six Y chromosome and the sites of the insertions in the candidate lines were fertility factors are required exclusively during spermatogen- mapped to individual chromosomes and balanced (see be- esis (4, 9). Other heterochromatic regions interact with low). The screens involving Y95-2 were similar to the initial specific euchromatic genes (10, 11), while rearrangements screen except as follows. Transposition was activated in thatjuxtapose heterochromatin and euchromatin cause char- X/Y95-2; Sb A2-3/ry males or C(1)RM/Y95-2; Sb A2-3/TM6, acteristic local disruptions in gene expression known as ry females. Because insertions on neither the A2-3 nor the position-effect variegation (12, 13). Technical difficulties in TM6, ry chromosome were desired, no third chromosome analyzing heterochromatic regions rich in repetitive DNA insertions were retained from the screen in which the Y95-2 have limited our ability to systematically analyze these element was mobilized in females. phenomena. Insertions were balanced by scoring the meiotic segrega- Insertional mutagenesis with single P elements (14) has the tion of ry+ expression in those progeny where it was ex- potential to greatly facilitate studies of heterochromatin and pressed. In cases where expression was too weak, an extra its genes. However, previous attempts to insert genetically Y chromosome or PCR assays were used to determine marked P elements into heterochromatin have had only segregation. Southern blots were carried out on lines derived limited success. Insertions onto Dp1187, a well-defined from Y95-2 and bearing Y-linked insertions to verify that new 1300-kb minichromosome (15), were obtained only within a bands flanking the ends of the PZ element were present. small block of subtelomeric heterochromatin, not within a These tests revealed that some candidate strains derived from much larger region of centromeric heterochromatin (16-18). Y95-2 arose by internal Y chromosome rearrangement rather It remained uncertain whether genetically marked P elements than by transposition; these were not studied further. are simply unable to insert into centromeric heterochromatin Strains bearing lethal heterochromatic PZ insertions were or ifsuch insertions were not detected due to position effects. tested for genetic complementation with strains bearing de- We now report that single P element insertions can be ficiencies that uncover all the known heterochromatic vital genes. The strains used for CH(2)5 were as follows: Dfi2L)C' and the strains used for all The publication costs ofthis article were defrayed in part by page charge (21), Dft2L)ltxlO, Dft2L)lth2l (22); payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: DAPI, 4',6-diamidino-2-phenylindole. 3539 Downloaded by guest on October 2, 2021 3540 Genetics: Zhang and Spradling Proc. Natl. Acad Sci. USA 91 (1994) element insertions recovered previously in subtelomeric (15) 1\ i. or chromosome 4 (27, 28) heterochromatin all displayed position-effect variegation. Insertions lying deeper within heterochromatin might have been missed if marker gene expression had been completely suppressed. Such insertions i -XA hE I might be recovered under conditions where heterochromatic position effects were strongly suppressed by the presence of an extra Y chromosome. Furthermore, insertions subject to ./ t s 1 *a..f; ) ^ / :: )E strong position effect should be easily identified, since I ': marker gene expression from these sites would be lost or V Y, / greatly reduced in a normal genetic background. V X To test this approach we carried out a single P element mutagenesis screen in which new transpositions of an V P. A X-linked P element marked with the rosy+ (by+) eye color gene (hereinafter called "PZ") were identified in flies bearing FiG. 1. Single P element insertional mutagenesis of heterochro- an extra Y chromosome (Fig. 1B; for further details, see matin. (A) Schematic drawings of the Drosophila melanogaster Materials andMethods). When flies from 4% individual lines chromosomes illustrate the location and size of heterochromatin bearing new independent PZ transpositions were crossed to (stippled boxes) and euchromatin (open boxes). Centromeres are remove the extra Y chromosome, 10 strains displayed a ry- represented as small circles. The positions ofthe starting P elements X(PZ) and Y95-2 are shown. (B) Crossing scheme for generating eye color (Table 1). The relatively high frequency of these Y-dependent insertions. For details and genetic nomenclature see "Y-dependent" insertions (10/4% = 2%6) compared with the Materials and Methods. rate of heterochromatic insertions in a previous study (24/ 7825 = 0.3%) (16) encouraged further studies. CH(3) strains were as follows: Df(3L)2-30, Df(3L)9-56, A Genetically Silent P Elment Insertion Within Y Chromo- Dft3L)2-66, D~ft3L)1-166, Dff3L)8A-80, Dff3L)6-61, Dff3L)3- some Heteroebromatin. We next characterized line 95-2, 52, Df(3L)1-16, Dff3LR)10-26, Df(3LR)6B-29, Dff3R)10-65, generated during previous experiments to investigate local P and Dfl3R)4-7S (8). Complementation was observed in all element transposition (18). In addition to a euchromatic crosses except as noted in the text. insertion, 9S-2 males were found by Southern blotting to Reversion tests by PZ excision were carried out essentially contain a second PZ insertion on their Y chromosome that as described (14). One hundred independent lines that failed was separated into a derivative line called Y95-2. Fig. 2 shows to express ry+ were generated from each strain and tested for that a single band complementary to the P element probe was viability in combination with the starting insertion. (A few of observed in strain Y95-2 DNA from males but not females as these derivatives still retained the original element but were expected for a Y-linked insertion. Surprisingly, the Y95-2 phenotypically ry- due to position effect.) In the case of insertion appeared to be structurally normal but failed to strains CH(2)S and CH(3)7, >50% of ry derivatives were viable when heterozygous with the starting chromosome; the express ry+ as judged by eye color, even in the presence of other lines produced no viable derivatives. an extra Y chromosome to suppress position-effect variega- DNA Probes.
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]
  • 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]
  • An Insertional Mutagenesis Approach to Dictyostelium Cell Death
    Cell Death and Differentiation (1998) 5, 416 ± 425 1998 Stockton Press All rights reserved 13509047/98 $12.00 http://www.stockton-press.co.uk/cdd An insertional mutagenesis approach to Dictyostelium cell death Sophie Cornillon1, Robert A. Olie1 and Pierre Golstein1,2 unicellular organism under favorable conditions but becomes multicellular by aggregation upon starvation (Raper, 1984). 1 Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Aggregation is followed with cell differentiation and morpho- Marseille cedex 9, France genesis, leading within 24 h to the formation of a 1 ± 2 mm 2 Corresponding author: Pierre Golstein, tel: 33 (4) 91 26 94 68; fax: 33 (4) 91 26 high fungus-like structure called sorocarp. The sorocarp 94 30; e-mail: [email protected] comprises two main populations of cells: viable spores in a Received 14.10.97; revised 18.11.97; accepted 28.11.97 bolus, and vacuolated cells tightly compacted in a stalk. Stalk Edited by G. Melino cells have been shown to be non-viable, as they do not re- grow in culture medium (Whittingham and Raper, 1960). Stalk cells thus may be considered an example of developmental Abstract PCD. Dictyostelium discoideum is an attractive model to study Programmed cell death (PCD) in Dictyostelium shows a PCD. Differentiation is simple, ultimately leading to only two pattern of ordered degeneration similar to that observed in main types of cells (viable spore and dead stalk cells). The higher eukaryotes but somewhat different from the most main inducers of differentiation are known, including in studied form of PCD, i.e.
    [Show full text]