Synthetic design of strong promoters.
Identifieur interne : 002605 ( Main/Exploration ); précédent : 002604; suivant : 002606Synthetic design of strong promoters.
Auteurs : Michael R. Schlabach [États-Unis] ; Jimmy K. Hu ; Mamie Li ; Stephen J. ElledgeSource :
- Proceedings of the National Academy of Sciences of the United States of America [ 1091-6490 ] ; 2010.
Descripteurs français
- KwdFr :
- Analyse sur microréseau (), Animaux, Cellules HeLa, Cytomegalovirus (génétique), Cytométrie en flux, Facteurs de transcription (métabolisme), Humains, Lignée cellulaire, Oligonucléotides (génétique), Protéines à fluorescence verte (génétique), Régions promotrices (génétique) (génétique), Régulation de l'expression des gènes, Sites de fixation (génétique), Souris, Séquences d'acides nucléiques régulatrices (génétique), Transcription génétique, Éléments activateurs (génétique) (génétique).
- MESH :
- génétique : Cytomegalovirus, Oligonucléotides, Protéines à fluorescence verte, Régions promotrices (génétique), Sites de fixation, Séquences d'acides nucléiques régulatrices, Éléments activateurs (génétique).
- métabolisme : Facteurs de transcription.
- Analyse sur microréseau, Animaux, Cellules HeLa, Cytométrie en flux, Humains, Lignée cellulaire, Régulation de l'expression des gènes, Souris, Transcription génétique.
English descriptors
- KwdEn :
- Animals, Binding Sites (genetics), Cell Line, Cytomegalovirus (genetics), Enhancer Elements, Genetic (genetics), Flow Cytometry, Gene Expression Regulation, Green Fluorescent Proteins (genetics), HeLa Cells, Humans, Mice, Microarray Analysis (methods), Oligonucleotides (genetics), Promoter Regions, Genetic (genetics), Regulatory Sequences, Nucleic Acid (genetics), Transcription Factors (metabolism), Transcription, Genetic.
- MESH :
- chemical , genetics : Green Fluorescent Proteins, Oligonucleotides.
- genetics : Binding Sites, Cytomegalovirus, Enhancer Elements, Genetic, Promoter Regions, Genetic, Regulatory Sequences, Nucleic Acid.
- chemical , metabolism : Transcription Factors.
- methods : Microarray Analysis.
- Animals, Cell Line, Flow Cytometry, Gene Expression Regulation, HeLa Cells, Humans, Mice, Transcription, Genetic.
Abstract
We have taken a synthetic biology approach to the generation and screening of transcription factor binding sites for activity in human cells. All possible 10-mer DNA sequences were printed on microarrays as 100-mers containing 10 repeats of the same sequence in tandem, yielding an oligonucleotide library of 52,429 unique sequences. This library of potential enhancers was introduced into a retroviral vector and screened in multiple cell lines for the ability to activate GFP transcription from a minimal CMV promoter. With this method, we isolated 100 bp synthetic enhancer elements that were as potent at activating transcription as the WT CMV immediate early enhancer. The activity of the recovered elements was strongly dependent on the cell line in which they were recovered. None of the elements were capable of achieving the same levels of transcriptional enhancement across all tested cell lines as the CMV enhancer. A second screen, for enhancers capable of synergizing with the elements from the original screen, yielded compound enhancers that were capable of twofold greater enhancement activity than the CMV enhancer, with higher levels of activity than the original synthetic enhancer across multiple cell lines. These findings suggest that the 10-mer synthetic enhancer space is sufficiently rich to allow the creation of synthetic promoters of all strengths in most, if not all, cell types.
DOI: 10.1073/pnas.0914803107
PubMed: 20133776
Affiliations:
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Schlabach</name><affiliation wicri:level="2"><nlm:affiliation>Department of Genetics, Division of Genetics, Howard Hughes Medical Institute, Brigham and Women's Hospital, Harvard University Medical School, Boston, MA 02115, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Genetics, Division of Genetics, Howard Hughes Medical Institute, Brigham and Women's Hospital, Harvard University Medical School, Boston, MA 02115</wicri:regionArea><placeName><region type="state">Massachusetts</region></placeName></affiliation></author><author><name sortKey="Hu, Jimmy K" sort="Hu, Jimmy K" uniqKey="Hu J" first="Jimmy K" last="Hu">Jimmy K. Hu</name></author><author><name sortKey="Li, Mamie" sort="Li, Mamie" uniqKey="Li M" first="Mamie" last="Li">Mamie Li</name></author><author><name sortKey="Elledge, Stephen J" sort="Elledge, Stephen J" uniqKey="Elledge S" first="Stephen J" last="Elledge">Stephen J. Elledge</name></author></analytic><series><title level="j">Proceedings of the National Academy of Sciences of the United States of America</title><idno type="eISSN">1091-6490</idno><imprint><date when="2010" type="published">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Binding Sites (genetics)</term><term>Cell Line</term><term>Cytomegalovirus (genetics)</term><term>Enhancer Elements, Genetic (genetics)</term><term>Flow Cytometry</term><term>Gene Expression Regulation</term><term>Green Fluorescent Proteins (genetics)</term><term>HeLa Cells</term><term>Humans</term><term>Mice</term><term>Microarray Analysis (methods)</term><term>Oligonucleotides (genetics)</term><term>Promoter Regions, Genetic (genetics)</term><term>Regulatory Sequences, Nucleic Acid (genetics)</term><term>Transcription Factors (metabolism)</term><term>Transcription, Genetic</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Analyse sur microréseau ()</term><term>Animaux</term><term>Cellules HeLa</term><term>Cytomegalovirus (génétique)</term><term>Cytométrie en flux</term><term>Facteurs de transcription (métabolisme)</term><term>Humains</term><term>Lignée cellulaire</term><term>Oligonucléotides (génétique)</term><term>Protéines à fluorescence verte (génétique)</term><term>Régions promotrices (génétique) (génétique)</term><term>Régulation de l'expression des gènes</term><term>Sites de fixation (génétique)</term><term>Souris</term><term>Séquences d'acides nucléiques régulatrices (génétique)</term><term>Transcription génétique</term><term>Éléments activateurs (génétique) (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Green Fluorescent Proteins</term><term>Oligonucleotides</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Binding Sites</term><term>Cytomegalovirus</term><term>Enhancer Elements, Genetic</term><term>Promoter Regions, Genetic</term><term>Regulatory Sequences, Nucleic Acid</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Cytomegalovirus</term><term>Oligonucléotides</term><term>Protéines à fluorescence verte</term><term>Régions promotrices (génétique)</term><term>Sites de fixation</term><term>Séquences d'acides nucléiques régulatrices</term><term>Éléments activateurs (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Transcription Factors</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Microarray Analysis</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Facteurs de transcription</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Cell Line</term><term>Flow Cytometry</term><term>Gene Expression Regulation</term><term>HeLa Cells</term><term>Humans</term><term>Mice</term><term>Transcription, Genetic</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Analyse sur microréseau</term><term>Animaux</term><term>Cellules HeLa</term><term>Cytométrie en flux</term><term>Humains</term><term>Lignée cellulaire</term><term>Régulation de l'expression des gènes</term><term>Souris</term><term>Transcription génétique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">We have taken a synthetic biology approach to the generation and screening of transcription factor binding sites for activity in human cells. All possible 10-mer DNA sequences were printed on microarrays as 100-mers containing 10 repeats of the same sequence in tandem, yielding an oligonucleotide library of 52,429 unique sequences. This library of potential enhancers was introduced into a retroviral vector and screened in multiple cell lines for the ability to activate GFP transcription from a minimal CMV promoter. With this method, we isolated 100 bp synthetic enhancer elements that were as potent at activating transcription as the WT CMV immediate early enhancer. The activity of the recovered elements was strongly dependent on the cell line in which they were recovered. None of the elements were capable of achieving the same levels of transcriptional enhancement across all tested cell lines as the CMV enhancer. A second screen, for enhancers capable of synergizing with the elements from the original screen, yielded compound enhancers that were capable of twofold greater enhancement activity than the CMV enhancer, with higher levels of activity than the original synthetic enhancer across multiple cell lines. These findings suggest that the 10-mer synthetic enhancer space is sufficiently rich to allow the creation of synthetic promoters of all strengths in most, if not all, cell types.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Massachusetts</li></region></list><tree><noCountry><name sortKey="Elledge, Stephen J" sort="Elledge, Stephen J" uniqKey="Elledge S" first="Stephen J" last="Elledge">Stephen J. Elledge</name><name sortKey="Hu, Jimmy K" sort="Hu, Jimmy K" uniqKey="Hu J" first="Jimmy K" last="Hu">Jimmy K. Hu</name><name sortKey="Li, Mamie" sort="Li, Mamie" uniqKey="Li M" first="Mamie" last="Li">Mamie Li</name></noCountry><country name="États-Unis"><region name="Massachusetts"><name sortKey="Schlabach, Michael R" sort="Schlabach, Michael R" uniqKey="Schlabach M" first="Michael R" last="Schlabach">Michael R. Schlabach</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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