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Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent

The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimo...

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Autores principales: Vila, Marta, Díaz-Santos, Encarnación, de la, Marta, Rodríguez, Herminia, Vargas, Ángeles, León, Rosa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528124/
https://www.ncbi.nlm.nih.gov/pubmed/23211713
http://dx.doi.org/10.3390/md10122749
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author Vila, Marta
Díaz-Santos, Encarnación
de la, Marta
Rodríguez, Herminia
Vargas, Ángeles
León, Rosa
author_facet Vila, Marta
Díaz-Santos, Encarnación
de la, Marta
Rodríguez, Herminia
Vargas, Ángeles
León, Rosa
author_sort Vila, Marta
collection PubMed
description The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.
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spelling pubmed-35281242012-12-28 Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent Vila, Marta Díaz-Santos, Encarnación de la, Marta Rodríguez, Herminia Vargas, Ángeles León, Rosa Mar Drugs Article The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies. MDPI 2012-12-04 /pmc/articles/PMC3528124/ /pubmed/23211713 http://dx.doi.org/10.3390/md10122749 Text en © 2012 by the authors; licensee MDPI, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0/ This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Vila, Marta
Díaz-Santos, Encarnación
de la, Marta
Rodríguez, Herminia
Vargas, Ángeles
León, Rosa
Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent
title Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent
title_full Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent
title_fullStr Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent
title_full_unstemmed Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent
title_short Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent
title_sort promoter trapping in microalgae using the antibiotic paromomycin as selective agent
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528124/
https://www.ncbi.nlm.nih.gov/pubmed/23211713
http://dx.doi.org/10.3390/md10122749
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