DOCSLIB.ORG

Prospects for Insertional Mutagenesis in Zebrafish

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Prospects for Insertional Mutagenesis in Zebrafish MINIREVIEW From screens to genes: prospects for insertional mutagenesis in zebrafish Alexander F. Schier, 1 Alexandra L. Joyner, Ruth Lehmann, and William S. Talbot I Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, and Department of Cell Biology, New York University Medical Center, New York, New York 10016 USA The systematic, and now classic, genetic screens for mu- general defects (growth retardation or degeneration) and tations affecting the development of Drosophila and were discarded. Complementation analysis of the remain- Caenorhabditis elegans have laid the foundation for the ing mutants indicated that the two screens identified >400 study of developmental mechanisms in these inverte- genetic loci involved in various aspects of development. brates (N/isslein-Volhard and Wieschaus 1980; Horvitz Based on the average allele frequency, estimates indicate and Sternberg 1991). The subsequent molecular isolation that >50% of genes that could be identified by such and characterization of the affected genes has allowed a screens remain to be isolated. Nevertheless, the screens thorough understanding of the mechanisms underlying were efficient in inducing and identifying mutants: 1.3- development. A similar genetic approach has recently 1.4 mutants were identified in the progeny of each F2 been taken in a vertebrate. Large-scale genetic screens in family. These numbers are similar to the efficiency by zebrafish (Danio rerio) have identified >400 genes essen- which embryonic lethal mutations were identified in the tial for the early development of this vertebrate (Driever Drosophila chemical mutagenesis screens. The major lim- et al. 1996; Haffter et al. 1996). Phenotypic analysis of itation of zebrafish compared to Drosophila is, therefore, zebrafish mutants has already provided interesting in- not the rate of chemical mutagenesis but the limited sights into developmental mechanisms, but only a hand- number of fish that can be raised, housed, and screened. ful have been cloned. Because the mutagen ethylni- trosourea (ENU) predominantly causes point mutations, Insertional mutagenesis with pseudotyped retroviruses cloning of the mutations is rather laborious at this stage, disrupts essential genes and depends on positional cloning and candidate gene Insertional mutants provide an extremely useful tool to approaches (for review, see Collins 1995). The recent de- rapidly clone disrupted genes. This approach has been velopment of pseudotyped retroviruses as insertional very successful in C. elegans and Drosophila, using mutagens in zebrafish provides a novel tool to identify transposable elements. For instance, P element-medi- and efficiently clone zebrafish mutations. As described ated mutagenesis in Drosophila has allowed the cloning by Hopkins and co-workers (Gaiano et al. 1996b; Allende of many of the genes originally identified in chemical et al., this issue), retroviral vectors insert into the ze- mutagenesis screens, and has also led to the identifica- brafish genome and can disrupt essential genes. The in- tion and cloning of many more genes in recent years (for tegrated DNA can then be used to isolate affected genes review, see Spradling et al. 1995). Experiments in the rapidly. We discuss the strategies and results of recent mouse introducing transgenes by pronuclear injections, chemical and insertional genetic screens and assess dif- gene trapping, or retroviral integration have also high- ferent approaches to clone essential zebrafish genes. lighted the power of insertional mutagenesis in vertebrates (for review, see Jaenisch 1988; Gossler and Zachgo 1993). Chemical mutagenesis with ENU efficiently induces The retroviral approach has now been applied success- mutations in essential genes fully to disrupt and rapidly clone essential genes in ze- brafish (Allende et al. 1996; Gaiano et al. 1996b). Burns Two large-scale chemical mutagenesis screens were re- et al. (1993) initially developed a pseudotyped retroviral cently performed, using a standard three-generation in- vector that contains a genome based on the Moloney breeding scheme, to identify embryonic and larval mu- murine leukemia virus (MoMLV) and the envelope gly- tants as homozygous diploid animals (Driever et al. coprotein of the vesicular stomatitis virus (VSV). The 1996; Haffter et al. 1996). Germ-line mutations were in- pseudotyped retrovirus was used to infect zebrafish cell duced by ENU in males (GO; see Fig. 1A), transmitted to lines (Burns et al. 1993) and embryos (Lin et al. 1994a), their F1 offspring, expanded by breeding to produce F2 and proviral insertions were initially transmitted at a families, and bred to homozygosity in F3 embryos by low frequency to subsequent generations. More recently interbreeding F2 fish. The two screens led to the identi- higher-titer virus stocks (2 x 10 9 cfu/ml) have been pro- fication of >6000 mutants, two-thirds of wh:ch showed duced, and the efficiency of insertion and germ-line transmission has been improved 100-fold (Gaiano et al. 1Corresponding authors. 1996a). In a pilot screen, -1/70 of insertions have now GENES & DEVELOPMENT 10:3077-3080 1996 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/96 $5.00 3077 Schier et al. Figure 1 Possible strategies forinsertional GO Injection of pseudotyped ~r ~ retrovlrus into GO embryos mutagenesis in zebrafish. (A) An F3 screen- leads to mature GO fish with ing protocol similar to that previously used mosaic integration of retroviral for insertional mutagenesis except that F1 fish from a GO intercross can carry one or more transgenes fish carrying multiple insertions are identi- /1\ fied by Southern blot and used to make the I:1 F2 generation. The large-scale chemical screens employed a similar breeding scheme (see text for details). 'a' would then correspond to a mutation in a gene leading A. Identification of F1 individuals with B. Identification of F1 male and C. Production of gynogenetlc three or more insertions (Fish 1) by female with same insertion (Fish 2 F2 embryos from FI females to an embryonic or larval phenotype. (B, C) Southem blot, Breeding of two FI fish and 5) by Southern blot. Breeding (e.g. Fish 4); no Southern blot. with three or more different insertions results in F2 homozygotes In the case of gynogenetic F2 screening protocols. (B) Animals ho- generates F2 families with 6 or more hapioids, half of F2 embryos different insertions have a particular insertion mozygous for an insertion (b) can be ob- tained in the F2 generation if male and fe- male F1 animals carrying the same trans- l gene can be identified by Southern blot. (C) A screen of gynogenetic F2 animals would Each particular insertion is carded by eliminate the need to identify transgenic half of F2 individuals, An intercross of F2 siblings heterozygous for a given Screen for phenotypes fish, because only the maternal genome insertion ("a") results in F3 homozygotes contributes to gynogenotes. Half of a clutch of F2 haploids will inherit any transgene (c) Screen for phenotypes present in a F1 female. Gynogenetic dip- loids (not shown), which do not display the epigenetic abnormalities associated with haploids, can also be used; in this case, the fraction of homozygous progeny will vary (from 5-50%), depending on the distance Screen for phenotypes between the transgene and its centromere. been found to produce embryonic or larval phenotypes present insertional mutagenesis protocol is >20-fold less (Allende et al. 1996; Gaiano et al. 1996b). efficient in inducing lethal mutations as compared to To obtain transgenic fish, blastula embryos were in- chemical mutagenesis (Allende et al. 1996; Gaiano et al. jected with -20,000 infectious particles, some of which 1996b). Using F2 families that carry two insertions, one infect germ cells (see Fig. 1). Retroviral insertions were in about 35 F2 families would show an embryonic or transmitted to F1 offspring by breeding GO founder fish larval phenotype, compared to virtually every F2 family and Southern blot analysis was performed to determine in chemical mutagenesis screens. At this rate for in- which F1 animals carried retroviral insertions. About sertional mutagenesis, it is unlikely that all muta- 30% of F1 embryos derived from a given GO fish were tions identified in the chemical screens will be isolated. found to be transgenic, and, on average, 11 different in- However, variations on the breeding and screening sertions were present in the mosaic germline of each GO schemes, and potential advances in retroviral vectors founder fish (Gaiano et al. 1996a). F2 families were raised could make insertional mutagenesis more widely applic- and F2 fish heterozygous for the same insertion were able. intercrossed. The resulting F3 progeny were screened for Some benefit could be derived by increasing the num- mutant phenotypes. In a small-scale screen, Gaiano et al. ber of retroviral insertions in each F2 family (Fig. 1A). (1996b) analyzed 217 retroviral insertions and found When two founders are crossed to each other, -15% of three lethal mutations correlating with insertions. In to- F1 animals harbor three or more insertions. By breeding tal, four insertional mutations have now been identified, F1 fish with several insertions, F2 families could be and three have been cloned using the retroviral insertion to raised that contain six or more different insertions and isolate flanking genomic DNA by inverse PCR. The cloned these animals can be identified by Southern analysis of flanking DNA was sequenced, and disrupted transcription
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]