A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum

Lade...
Vorschaubild
Dateien
Serif_2-6ss0tqyo960o9.pdf
Serif_2-6ss0tqyo960o9.pdfGröße: 969.5 KBDownloads: 795
Datum
2017
Herausgeber:innen
Kontakt
ISSN der Zeitschrift
Electronic ISSN
ISBN
Bibliografische Daten
Verlag
Schriftenreihe
Auflagebezeichnung
ArXiv-ID
Internationale Patentnummer
Angaben zur Forschungsförderung
Projekt
Open Access-Veröffentlichung
Open Access Green
Core Facility der Universität Konstanz
Gesperrt bis
Titel in einer weiteren Sprache
Forschungsvorhaben
Organisationseinheiten
Zeitschriftenheft
Publikationstyp
Zeitschriftenartikel
Publikationsstatus
Published
Erschienen in
Zusammenfassung

Reverse genetics techniques are powerful tools for studying gene functions. In the model diatom Phaeodactylum tricornutum, RNAi-mediated knockdown of genes still is the most commonly used reverse genetics technique. Due to the diploidic life cycle missing reproduction in lab cultures, many commonly used techniques to create knockout instead of knockdown lines are not applicable in P. tricornutum. These limitations can be overcome by using genome editing approaches like TALEN (Transcription activator-like effector nucleases), and/or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats), allowing the introduction of targeted mutagenesis events. Both techniques have recently been adapted exemplarily for diatoms, however, no concise guidelines exist yet for routine utilization of these tools and the subsequent characterization of the mutants. We therefore have adapted a cost-effective TALEN generation system previously established for mammalian cells for the use in P. tricornutum, allowing the assembly of TALENs in about two weeks. We further provide protocols for: a) choosing a TALEN target site in order to avoid potentially ineffective and/or off-target prone TALEN constructs, b) efficient transformation of P. tricornutum with both TALEN constructs, utilizing two antibiotics resistance markers, c) effective screening of the transformants. In order to test our system we chose the blue-light dependent transcription factor Aureochrome 1a (PtAureo1a) as a target gene due to the known phenotype of previously characterized P. tricornutum RNAi knockdown strains. Our TALEN approach appears to be highly efficient: targeted mutation events were detected in 50% of all transformants obtained, whereas 21% of the transformants were found to be bi-allelic knockout lines. Furthermore, most TALEN transformed cell lines were found to be genetically homogeneous without the need for re-plating, which greatly facilitates the screening process.

Zusammenfassung in einer weiteren Sprache
Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Phaeodactylum tricornutum, TALEN, PtAUREO1a knockout, Blue light-dependent transcription factor
Konferenz
Rezension
undefined / . - undefined, undefined
Zitieren
ISO 690SERIF, Manuel, Bernard LEPETIT, Kristoffer WEISSERT, Peter G. KROTH, Carolina RÍO BÁRTULOS, 2017. A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum. In: Algal Research. 2017, 23, pp. 186-195. ISSN 2211-9264. Available under: doi: 10.1016/j.algal.2017.02.005
BibTex
@article{Serif2017relia-37721,
  year={2017},
  doi={10.1016/j.algal.2017.02.005},
  title={A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum},
  volume={23},
  issn={2211-9264},
  journal={Algal Research},
  pages={186--195},
  author={Serif, Manuel and Lepetit, Bernard and Weißert, Kristoffer and Kroth, Peter G. and Río Bártulos, Carolina}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/37721">
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2017-02-27T13:07:39Z</dcterms:available>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dc:rights>terms-of-use</dc:rights>
    <dc:language>eng</dc:language>
    <dc:contributor>Río Bártulos, Carolina</dc:contributor>
    <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/37721/1/Serif_2-6ss0tqyo960o9.pdf"/>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dcterms:title>A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum</dcterms:title>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2017-02-27T13:07:39Z</dc:date>
    <dcterms:abstract xml:lang="eng">Reverse genetics techniques are powerful tools for studying gene functions. In the model diatom Phaeodactylum tricornutum, RNAi-mediated knockdown of genes still is the most commonly used reverse genetics technique. Due to the diploidic life cycle missing reproduction in lab cultures, many commonly used techniques to create knockout instead of knockdown lines are not applicable in P. tricornutum. These limitations can be overcome by using genome editing approaches like TALEN (Transcription activator-like effector nucleases), and/or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats), allowing the introduction of targeted mutagenesis events. Both techniques have recently been adapted exemplarily for diatoms, however, no concise guidelines exist yet for routine utilization of these tools and the subsequent characterization of the mutants. We therefore have adapted a cost-effective TALEN generation system previously established for mammalian cells for the use in P. tricornutum, allowing the assembly of TALENs in about two weeks. We further provide protocols for: a) choosing a TALEN target site in order to avoid potentially ineffective and/or off-target prone TALEN constructs, b) efficient transformation of P. tricornutum with both TALEN constructs, utilizing two antibiotics resistance markers, c) effective screening of the transformants. In order to test our system we chose the blue-light dependent transcription factor Aureochrome 1a (PtAureo1a) as a target gene due to the known phenotype of previously characterized P. tricornutum RNAi knockdown strains. Our TALEN approach appears to be highly efficient: targeted mutation events were detected in 50% of all transformants obtained, whereas 21% of the transformants were found to be bi-allelic knockout lines. Furthermore, most TALEN transformed cell lines were found to be genetically homogeneous without the need for re-plating, which greatly facilitates the screening process.</dcterms:abstract>
    <dc:contributor>Serif, Manuel</dc:contributor>
    <dc:contributor>Lepetit, Bernard</dc:contributor>
    <dc:creator>Río Bártulos, Carolina</dc:creator>
    <dcterms:issued>2017</dcterms:issued>
    <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/37721"/>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/52"/>
    <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/>
    <dc:contributor>Kroth, Peter G.</dc:contributor>
    <dc:creator>Serif, Manuel</dc:creator>
    <dc:contributor>Weißert, Kristoffer</dc:contributor>
    <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/37721/1/Serif_2-6ss0tqyo960o9.pdf"/>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/52"/>
    <dc:creator>Weißert, Kristoffer</dc:creator>
    <dc:creator>Lepetit, Bernard</dc:creator>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dc:creator>Kroth, Peter G.</dc:creator>
  </rdf:Description>
</rdf:RDF>
Interner Vermerk
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Kontakt
URL der Originalveröffentl.
Prüfdatum der URL
Prüfungsdatum der Dissertation
Finanzierungsart
Kommentar zur Publikation
Allianzlizenz
Corresponding Authors der Uni Konstanz vorhanden
Internationale Co-Autor:innen
Universitätsbibliographie
Ja
Begutachtet
Diese Publikation teilen