Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system.
Identifieur interne : 000716 ( Main/Corpus ); précédent : 000715; suivant : 000717Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system.
Auteurs : Kerry Ann Lutz ; Zora Svab ; Pal MaligaSource :
- Nature protocols [ 1750-2799 ] ; 2006.
English descriptors
- KwdEn :
- Attachment Sites, Microbiological (genetics), Genetic Engineering (methods), Genetic Markers (MeSH), Integrases (genetics), Integrases (metabolism), Plant Leaves (genetics), Plant Leaves (radiation effects), Plant Proteins (genetics), Plant Proteins (metabolism), Plants, Genetically Modified (MeSH), Recombination, Genetic (genetics), Tobacco (genetics).
- MESH :
- chemical , genetics : Integrases, Plant Proteins.
- chemical , metabolism : Integrases, Plant Proteins.
- chemical : Genetic Markers.
- genetics : Attachment Sites, Microbiological, Plant Leaves, Recombination, Genetic, Tobacco.
- methods : Genetic Engineering.
- radiation effects : Plant Leaves.
- Plants, Genetically Modified.
Abstract
Incorporation of a selectable marker gene in the plastid genome is essential to uniformly alter the thousands of genome copies in a tobacco cell. When transformation is accomplished, however, the marker gene becomes undesirable. Here we describe plastid transformation vectors, the method of plastid transformation using tobacco leaves and alternative protocols for marker gene excision with the P1 bacteriophage Cre-loxP site-specific recombination system. Plastid vectors carry a marker gene flanked with directly oriented loxP sites and a gene of interest, which are introduced into plastids by the biolistic process. The transforming DNA integrates into the plastid genome by homologous recombination via plastid targeting sequences. Marker gene excision is accomplished by a plastid-targeted Cre protein expressed from a nuclear gene. Expression may be from an integrated gene introduced by Agrobacterium transformation (Transformation Protocol), by pollination (Pollination Protocol) or from a transient, non-integrated T-DNA (Transient Protocol). Transplastomic plants are obtained in about 3 months, yielding seed after 2 months. The time required to remove the plastid marker and nuclear genes and to obtain seed takes 10-16 months, depending on which protocol is used.
DOI: 10.1038/nprot.2006.118
PubMed: 17406323
Links to Exploration step
pubmed:17406323Le document en format XML
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When transformation is accomplished, however, the marker gene becomes undesirable. Here we describe plastid transformation vectors, the method of plastid transformation using tobacco leaves and alternative protocols for marker gene excision with the P1 bacteriophage Cre-loxP site-specific recombination system. Plastid vectors carry a marker gene flanked with directly oriented loxP sites and a gene of interest, which are introduced into plastids by the biolistic process. The transforming DNA integrates into the plastid genome by homologous recombination via plastid targeting sequences. Marker gene excision is accomplished by a plastid-targeted Cre protein expressed from a nuclear gene. Expression may be from an integrated gene introduced by Agrobacterium transformation (Transformation Protocol), by pollination (Pollination Protocol) or from a transient, non-integrated T-DNA (Transient Protocol). Transplastomic plants are obtained in about 3 months, yielding seed after 2 months. The time required to remove the plastid marker and nuclear genes and to obtain seed takes 10-16 months, depending on which protocol is used.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">17406323</PMID><DateCompleted><Year>2007</Year><Month>09</Month><Day>21</Day></DateCompleted><DateRevised><Year>2008</Year><Month>03</Month><Day>24</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Electronic">1750-2799</ISSN><JournalIssue CitedMedium="Internet"><Volume>1</Volume><Issue>2</Issue><PubDate><Year>2006</Year></PubDate></JournalIssue><Title>Nature protocols</Title><ISOAbbreviation>Nat Protoc</ISOAbbreviation></Journal><ArticleTitle>Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system.</ArticleTitle><Pagination><MedlinePgn>900-10</MedlinePgn></Pagination><Abstract><AbstractText>Incorporation of a selectable marker gene in the plastid genome is essential to uniformly alter the thousands of genome copies in a tobacco cell. When transformation is accomplished, however, the marker gene becomes undesirable. Here we describe plastid transformation vectors, the method of plastid transformation using tobacco leaves and alternative protocols for marker gene excision with the P1 bacteriophage Cre-loxP site-specific recombination system. Plastid vectors carry a marker gene flanked with directly oriented loxP sites and a gene of interest, which are introduced into plastids by the biolistic process. The transforming DNA integrates into the plastid genome by homologous recombination via plastid targeting sequences. Marker gene excision is accomplished by a plastid-targeted Cre protein expressed from a nuclear gene. Expression may be from an integrated gene introduced by Agrobacterium transformation (Transformation Protocol), by pollination (Pollination Protocol) or from a transient, non-integrated T-DNA (Transient Protocol). Transplastomic plants are obtained in about 3 months, yielding seed after 2 months. The time required to remove the plastid marker and nuclear genes and to obtain seed takes 10-16 months, depending on which protocol is used.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Lutz</LastName><ForeName>Kerry Ann</ForeName><Initials>KA</Initials><AffiliationInfo><Affiliation>Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, New Jersey 08854, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Svab</LastName><ForeName>Zora</ForeName><Initials>Z</Initials></Author><Author ValidYN="Y"><LastName>Maliga</LastName><ForeName>Pal</ForeName><Initials>P</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Nat Protoc</MedlineTA><NlmUniqueID>101284307</NlmUniqueID><ISSNLinking>1750-2799</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005819">Genetic Markers</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D010940">Plant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.7.7.-</RegistryNumber><NameOfSubstance UI="C045073">Cre recombinase</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.7.7.-</RegistryNumber><NameOfSubstance UI="D019426">Integrases</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001287" MajorTopicYN="N">Attachment Sites, Microbiological</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005818" MajorTopicYN="N">Genetic Engineering</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005819" MajorTopicYN="N">Genetic Markers</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D019426" MajorTopicYN="N">Integrases</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D018515" MajorTopicYN="N">Plant Leaves</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000528" MajorTopicYN="N">radiation effects</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010940" MajorTopicYN="N">Plant Proteins</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D030821" MajorTopicYN="N">Plants, Genetically Modified</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D011995" MajorTopicYN="N">Recombination, Genetic</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014026" MajorTopicYN="N">Tobacco</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2007</Year><Month>4</Month><Day>5</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2007</Year><Month>9</Month><Day>22</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2007</Year><Month>4</Month><Day>5</Day><Hour>9</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">17406323</ArticleId><ArticleId IdType="pii">nprot.2006.118</ArticleId><ArticleId IdType="doi">10.1038/nprot.2006.118</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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