Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene.
Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvemen...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3466256?pdf=render |
| _version_ | 1830487356658941952 |
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| author | Sivasundaram Karnan Yuko Konishi Akinobu Ota Miyuki Takahashi Lkhagvasuren Damdindorj Yoshitaka Hosokawa Hiroyuki Konishi |
| author_facet | Sivasundaram Karnan Yuko Konishi Akinobu Ota Miyuki Takahashi Lkhagvasuren Damdindorj Yoshitaka Hosokawa Hiroyuki Konishi |
| author_sort | Sivasundaram Karnan |
| collection | DOAJ |
| description | Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines. |
| first_indexed | 2024-12-21T18:59:41Z |
| format | Article |
| id | doaj.art-ad78a4692be04eacb9f746cc45783c23 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T18:59:41Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-ad78a4692be04eacb9f746cc45783c232022-12-21T18:53:32ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01710e4738910.1371/journal.pone.0047389Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene.Sivasundaram KarnanYuko KonishiAkinobu OtaMiyuki TakahashiLkhagvasuren DamdindorjYoshitaka HosokawaHiroyuki KonishiGene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines.http://europepmc.org/articles/PMC3466256?pdf=render |
| spellingShingle | Sivasundaram Karnan Yuko Konishi Akinobu Ota Miyuki Takahashi Lkhagvasuren Damdindorj Yoshitaka Hosokawa Hiroyuki Konishi Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene. PLoS ONE |
| title | Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene. |
| title_full | Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene. |
| title_fullStr | Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene. |
| title_full_unstemmed | Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene. |
| title_short | Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene. |
| title_sort | simple monitoring of gene targeting efficiency in human somatic cell lines using the piga gene |
| url | http://europepmc.org/articles/PMC3466256?pdf=render |
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