A comparative analysis of constitutive promoters located in adeno-associated viral vectors.

The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin c...

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Autores principales:	Lkhagvasuren Damdindorj, Sivasundaram Karnan, Akinobu Ota, Ekhtear Hossain, Yuko Konishi, Yoshitaka Hosokawa, Hiroyuki Konishi
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/522231ee79154f3eb755a24a41acf241
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spelling	oai:doaj.org-article:522231ee79154f3eb755a24a41acf2412021-11-25T06:02:38ZA comparative analysis of constitutive promoters located in adeno-associated viral vectors.1932-620310.1371/journal.pone.0106472https://doaj.org/article/522231ee79154f3eb755a24a41acf2412014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25170953/?tool=EBIhttps://doaj.org/toc/1932-6203The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.Lkhagvasuren DamdindorjSivasundaram KarnanAkinobu OtaEkhtear HossainYuko KonishiYoshitaka HosokawaHiroyuki KonishiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 8, p e106472 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lkhagvasuren Damdindorj Sivasundaram Karnan Akinobu Ota Ekhtear Hossain Yuko Konishi Yoshitaka Hosokawa Hiroyuki Konishi A comparative analysis of constitutive promoters located in adeno-associated viral vectors.
description	The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.
format	article
author	Lkhagvasuren Damdindorj Sivasundaram Karnan Akinobu Ota Ekhtear Hossain Yuko Konishi Yoshitaka Hosokawa Hiroyuki Konishi
author_facet	Lkhagvasuren Damdindorj Sivasundaram Karnan Akinobu Ota Ekhtear Hossain Yuko Konishi Yoshitaka Hosokawa Hiroyuki Konishi
author_sort	Lkhagvasuren Damdindorj
title	A comparative analysis of constitutive promoters located in adeno-associated viral vectors.
title_short	A comparative analysis of constitutive promoters located in adeno-associated viral vectors.
title_full	A comparative analysis of constitutive promoters located in adeno-associated viral vectors.
title_fullStr	A comparative analysis of constitutive promoters located in adeno-associated viral vectors.
title_full_unstemmed	A comparative analysis of constitutive promoters located in adeno-associated viral vectors.
title_sort	comparative analysis of constitutive promoters located in adeno-associated viral vectors.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/522231ee79154f3eb755a24a41acf241
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A comparative analysis of constitutive promoters located in adeno-associated viral vectors.

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