Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors.
Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical diff...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2010-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC2844420?pdf=render |
| _version_ | 1828373446036291584 |
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| author | Munehiro Uemura Youko Niwa Naoki Kakazu Noritaka Adachi Kazuo Kinoshita |
| author_facet | Munehiro Uemura Youko Niwa Naoki Kakazu Noritaka Adachi Kazuo Kinoshita |
| author_sort | Munehiro Uemura |
| collection | DOAJ |
| description | Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations. |
| first_indexed | 2024-04-14T07:17:53Z |
| format | Article |
| id | doaj.art-5e275374c8c3493081c6f64bae15e696 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T07:17:53Z |
| publishDate | 2010-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-5e275374c8c3493081c6f64bae15e6962022-12-22T02:06:15ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-01-0153e984610.1371/journal.pone.0009846Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors.Munehiro UemuraYouko NiwaNaoki KakazuNoritaka AdachiKazuo KinoshitaFeasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.http://europepmc.org/articles/PMC2844420?pdf=render |
| spellingShingle | Munehiro Uemura Youko Niwa Naoki Kakazu Noritaka Adachi Kazuo Kinoshita Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors. PLoS ONE |
| title | Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors. |
| title_full | Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors. |
| title_fullStr | Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors. |
| title_full_unstemmed | Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors. |
| title_short | Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors. |
| title_sort | chromosomal manipulation by site specific recombinases and fluorescent protein based vectors |
| url | http://europepmc.org/articles/PMC2844420?pdf=render |
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