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An Entry/Gateway(® )cloning system for general expression of genes with molecular tags in Drosophila melanogaster

BACKGROUND: Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological l...

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Autores principales: Akbari, Omar S, Oliver, Daniel, Eyer, Katie, Pai, Chi-Yun
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2654426/
https://www.ncbi.nlm.nih.gov/pubmed/19178707
http://dx.doi.org/10.1186/1471-2121-10-8
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author Akbari, Omar S
Oliver, Daniel
Eyer, Katie
Pai, Chi-Yun
author_facet Akbari, Omar S
Oliver, Daniel
Eyer, Katie
Pai, Chi-Yun
author_sort Akbari, Omar S
collection PubMed
description BACKGROUND: Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway(® )vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells. RESULTS: The Entry/Gateway(® )system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway(® )based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei. CONCLUSION: We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system successfully expresses functional fluorescent CP190 fusion proteins. The fluorescent CP190 proteins exist in insulator bodies of various numbers and sizes among cells from multiple living tissues. Furthermore, live imaging of the movements of these fluorescent-tagged proteins suggests that the assembly and disassembly of insulator bodies are normal activities in living cells and may be directed for regulating transcription.
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spelling pubmed-26544262009-03-12 An Entry/Gateway(® )cloning system for general expression of genes with molecular tags in Drosophila melanogaster Akbari, Omar S Oliver, Daniel Eyer, Katie Pai, Chi-Yun BMC Cell Biol Methodology Article BACKGROUND: Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway(® )vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells. RESULTS: The Entry/Gateway(® )system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway(® )based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei. CONCLUSION: We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system successfully expresses functional fluorescent CP190 fusion proteins. The fluorescent CP190 proteins exist in insulator bodies of various numbers and sizes among cells from multiple living tissues. Furthermore, live imaging of the movements of these fluorescent-tagged proteins suggests that the assembly and disassembly of insulator bodies are normal activities in living cells and may be directed for regulating transcription. BioMed Central 2009-01-29 /pmc/articles/PMC2654426/ /pubmed/19178707 http://dx.doi.org/10.1186/1471-2121-10-8 Text en Copyright © 2009 Akbari et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Article
Akbari, Omar S
Oliver, Daniel
Eyer, Katie
Pai, Chi-Yun
An Entry/Gateway(® )cloning system for general expression of genes with molecular tags in Drosophila melanogaster
title An Entry/Gateway(® )cloning system for general expression of genes with molecular tags in Drosophila melanogaster
title_full An Entry/Gateway(® )cloning system for general expression of genes with molecular tags in Drosophila melanogaster
title_fullStr An Entry/Gateway(® )cloning system for general expression of genes with molecular tags in Drosophila melanogaster
title_full_unstemmed An Entry/Gateway(® )cloning system for general expression of genes with molecular tags in Drosophila melanogaster
title_short An Entry/Gateway(® )cloning system for general expression of genes with molecular tags in Drosophila melanogaster
title_sort entry/gateway(® )cloning system for general expression of genes with molecular tags in drosophila melanogaster
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2654426/
https://www.ncbi.nlm.nih.gov/pubmed/19178707
http://dx.doi.org/10.1186/1471-2121-10-8
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