Artículo
Promoter trapping in microalgae using the antibiotic paromomycin as selective agent
Autor/es | Vila Spinola, Marta
Díaz Santos, Encarnación Vega Naranjo, Marta de La Rodríguez Martínez, Herminia Vargas Muñoz, María de los Ángeles León, Rosa |
Departamento | Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular |
Fecha de publicación | 2012 |
Fecha de depósito | 2018-08-06 |
Publicado en |
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Resumen | 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 ... 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. |
Agencias financiadoras | Junta de Andalucía Junta de Andalucía |
Identificador del proyecto | P09-CVI-5053
BIO-214 |
Cita | Vila Spinola, M., Díaz Santos, E., Vega Naranjo, M.d.L., Rodríguez Martínez, H., Vargas Muñoz, M.d.l.Á. y León, R. (2012). Promoter trapping in microalgae using the antibiotic paromomycin as selective agent. Marine Drugs, 10, 2749-2765. |
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marinedrugs-10-02749.pdf | 710.6Kb | [PDF] | Ver/ | |