A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells.
Identifieur interne : 001070 ( PubMed/Curation ); précédent : 001069; suivant : 001071A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells.
Auteurs : Karim Benabdellah [Espagne] ; Alejandra Gutierrez-Guerrero [Espagne] ; Marién Cobo [Espagne] ; Pilar Mu Oz [Espagne] ; Francisco Martín [Espagne]Source :
- PloS one [ 1932-6203 ] ; 2014.
Descripteurs français
- KwdFr :
- Animaux, Cellules souches pluripotentes (cytologie), Cellules souches pluripotentes (métabolisme), Différenciation cellulaire (génétique), Données de séquences moléculaires, Expression des gènes, Extinction de l'expression des gènes, Globines bêta (génétique), Humains, Hématopoïèse, Lentivirus (génétique), Lignée cellulaire, Ordre des gènes, Poulets, Région de contrôle de locus, Régulation de l'expression des gènes, Séquence nucléotidique, Transgènes, Vecteurs génétiques (génétique), Éléments isolateurs.
- MESH :
- cytologie : Cellules souches pluripotentes.
- génétique : Différenciation cellulaire, Globines bêta, Lentivirus, Vecteurs génétiques.
- métabolisme : Cellules souches pluripotentes.
- Animaux, Données de séquences moléculaires, Expression des gènes, Extinction de l'expression des gènes, Humains, Hématopoïèse, Lignée cellulaire, Ordre des gènes, Poulets, Région de contrôle de locus, Régulation de l'expression des gènes, Séquence nucléotidique, Transgènes, Éléments isolateurs.
English descriptors
- KwdEn :
- Animals, Base Sequence, Cell Differentiation (genetics), Cell Line, Chickens, Gene Expression, Gene Expression Regulation, Gene Order, Gene Silencing, Genetic Vectors (genetics), Hematopoiesis, Humans, Insulator Elements, Lentivirus (genetics), Locus Control Region, Molecular Sequence Data, Pluripotent Stem Cells (cytology), Pluripotent Stem Cells (metabolism), Transgenes, beta-Globins (genetics).
- MESH :
- chemical , genetics : beta-Globins.
- cytology : Pluripotent Stem Cells.
- genetics : Cell Differentiation, Genetic Vectors, Lentivirus.
- metabolism : Pluripotent Stem Cells.
- Animals, Base Sequence, Cell Line, Chickens, Gene Expression, Gene Expression Regulation, Gene Order, Gene Silencing, Hematopoiesis, Humans, Insulator Elements, Locus Control Region, Molecular Sequence Data, Transgenes.
Abstract
Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3' LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells.
DOI: 10.1371/journal.pone.0084268
PubMed: 24400083
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Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada, Spain.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada</wicri:regionArea></affiliation></author><author><name sortKey="Gutierrez Guerrero, Alejandra" sort="Gutierrez Guerrero, Alejandra" uniqKey="Gutierrez Guerrero A" first="Alejandra" last="Gutierrez-Guerrero">Alejandra Gutierrez-Guerrero</name><affiliation wicri:level="1"><nlm:affiliation>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada, Spain.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada</wicri:regionArea></affiliation></author><author><name sortKey="Cobo, Marien" sort="Cobo, Marien" uniqKey="Cobo M" first="Marién" last="Cobo">Marién Cobo</name><affiliation wicri:level="1"><nlm:affiliation>Human DNA Variability Department, GENYO - 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Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada, Spain.</nlm:affiliation><country xml:lang="fr">Espagne</country><wicri:regionArea>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada</wicri:regionArea></affiliation></author></analytic><series><title level="j">PloS one</title><idno type="eISSN">1932-6203</idno><imprint><date when="2014" type="published">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Cell Differentiation (genetics)</term><term>Cell Line</term><term>Chickens</term><term>Gene Expression</term><term>Gene Expression Regulation</term><term>Gene Order</term><term>Gene Silencing</term><term>Genetic Vectors (genetics)</term><term>Hematopoiesis</term><term>Humans</term><term>Insulator Elements</term><term>Lentivirus (genetics)</term><term>Locus Control Region</term><term>Molecular Sequence Data</term><term>Pluripotent Stem Cells (cytology)</term><term>Pluripotent Stem Cells (metabolism)</term><term>Transgenes</term><term>beta-Globins (genetics)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term><term>Cellules souches pluripotentes (cytologie)</term><term>Cellules souches pluripotentes (métabolisme)</term><term>Différenciation cellulaire (génétique)</term><term>Données de séquences moléculaires</term><term>Expression des gènes</term><term>Extinction de l'expression des gènes</term><term>Globines bêta (génétique)</term><term>Humains</term><term>Hématopoïèse</term><term>Lentivirus (génétique)</term><term>Lignée cellulaire</term><term>Ordre des gènes</term><term>Poulets</term><term>Région de contrôle de locus</term><term>Régulation de l'expression des gènes</term><term>Séquence nucléotidique</term><term>Transgènes</term><term>Vecteurs génétiques (génétique)</term><term>Éléments isolateurs</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>beta-Globins</term></keywords><keywords scheme="MESH" qualifier="cytologie" xml:lang="fr"><term>Cellules souches pluripotentes</term></keywords><keywords scheme="MESH" qualifier="cytology" xml:lang="en"><term>Pluripotent Stem Cells</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Cell Differentiation</term><term>Genetic Vectors</term><term>Lentivirus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Différenciation cellulaire</term><term>Globines bêta</term><term>Lentivirus</term><term>Vecteurs génétiques</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Pluripotent Stem Cells</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Cellules souches pluripotentes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Cell Line</term><term>Chickens</term><term>Gene Expression</term><term>Gene Expression Regulation</term><term>Gene Order</term><term>Gene Silencing</term><term>Hematopoiesis</term><term>Humans</term><term>Insulator Elements</term><term>Locus Control Region</term><term>Molecular Sequence Data</term><term>Transgenes</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Données de séquences moléculaires</term><term>Expression des gènes</term><term>Extinction de l'expression des gènes</term><term>Humains</term><term>Hématopoïèse</term><term>Lignée cellulaire</term><term>Ordre des gènes</term><term>Poulets</term><term>Région de contrôle de locus</term><term>Régulation de l'expression des gènes</term><term>Séquence nucléotidique</term><term>Transgènes</term><term>Éléments isolateurs</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3' LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells. </div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">24400083</PMID><DateCompleted><Year>2014</Year><Month>09</Month><Day>10</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>13</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Electronic">1932-6203</ISSN><JournalIssue CitedMedium="Internet"><Volume>9</Volume><Issue>1</Issue><PubDate><Year>2014</Year></PubDate></JournalIssue><Title>PloS one</Title><ISOAbbreviation>PLoS ONE</ISOAbbreviation></Journal><ArticleTitle>A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells.</ArticleTitle><Pagination><MedlinePgn>e84268</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1371/journal.pone.0084268</ELocationID><Abstract><AbstractText>Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3' LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells. </AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Benabdellah</LastName><ForeName>Karim</ForeName><Initials>K</Initials><AffiliationInfo><Affiliation>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gutierrez-Guerrero</LastName><ForeName>Alejandra</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Cobo</LastName><ForeName>Marién</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Muñoz</LastName><ForeName>Pilar</ForeName><Initials>P</Initials><AffiliationInfo><Affiliation>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Martín</LastName><ForeName>Francisco</ForeName><Initials>F</Initials><AffiliationInfo><Affiliation>Human DNA Variability Department, GENYO - Centre for Genomic and Oncological Research (Pfizer/University of Granada/Andalusian Regional Government), PTS Granada, Granada, Spain.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2014</Year><Month>01</Month><Day>06</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>PLoS One</MedlineTA><NlmUniqueID>101285081</NlmUniqueID><ISSNLinking>1932-6203</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D055544">beta-Globins</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D001483" MajorTopicYN="N">Base Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002454" 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MajorTopicYN="N">Hematopoiesis</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D038101" MajorTopicYN="Y">Insulator Elements</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D016086" MajorTopicYN="N">Lentivirus</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D019901" MajorTopicYN="Y">Locus Control Region</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008969" MajorTopicYN="N">Molecular Sequence Data</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D039904" MajorTopicYN="N">Pluripotent Stem Cells</DescriptorName><QualifierName UI="Q000166" MajorTopicYN="N">cytology</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D019076" 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