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Mar. Drugs 2012, 10(12), 2749-2765; doi:10.3390/md10122749
Article

Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent

1
, 1, 1
, 2
, 2 and 1,*
Received: 11 September 2012; in revised form: 12 October 2012 / Accepted: 15 November 2012 / Published: 4 December 2012
(This article belongs to the Special Issue Marine Algae)
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Abstract: 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.
Keywords: APHVIII; Chlamydomonas reinhardtii; microalgae transformation; paromomycin; promoter trapping APHVIII; Chlamydomonas reinhardtii; microalgae transformation; paromomycin; promoter trapping
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Vila, M.; Díaz-Santos, E.; de la Vega, M.; Rodríguez, H.; Vargas, Á.; León, R. Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent. Mar. Drugs 2012, 10, 2749-2765.

AMA Style

Vila M, Díaz-Santos E, de la Vega M, Rodríguez H, Vargas Á, León R. Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent. Marine Drugs. 2012; 10(12):2749-2765.

Chicago/Turabian Style

Vila, Marta; Díaz-Santos, Encarnación; de la Vega, Marta; Rodríguez, Herminia; Vargas, Ángeles; León, Rosa. 2012. "Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent." Mar. Drugs 10, no. 12: 2749-2765.


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