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Promoter trapping in microalgae using the antibiotic paromomycin as selective agent.

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.

SUBMITTER: Vila M

PROVIDER: S-EPMC3528124 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Promoter trapping in microalgae using the antibiotic paromomycin as selective agent.

Vila Marta M Díaz-Santos Encarnación E de la Vega Marta M Rodríguez Herminia H Vargas Angeles A León Rosa R

Marine drugs 20121204 12

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 prom ...[more]

PMID: 23211713

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Project description:BackgroundPhotodynamic therapy (PDT) using chloroaluminium phthalocyanine (ClAlPc) and paromomycin sulfate (PM) can be effective against New World Leishmania species involved in cutaneous leishmaniasis (CL). The aim of this study is to assay the skin permeation and the antileishmanial effects of a nanoemulsion (NE) containing both ClAlPc and PM in experimental CL by Leishmania (Viannia) braziliensis.Material and methodsCremophor ELP/castor oil-based NEs were prepared by a low-energy method and characterized for their physicochemical parameters. The NEs were used to deliver both ClAlPc and PM to leishmania cells. The in vitro toxicity of NEs were tested in vitro against L. (V.) braziliensis and THP-1 cells. The in vivo toxicity was assessed in non-infected BALB/c mice. Ex-vivo permeation and retention studies using healthy mice skin were also conducted. Finally, the in vivo activity of NE-PM+ClAlPc after PDT was tested in BALB/c mice infected with parasites.ResultsNEs are colloidally stable with average droplet diameter of 30 nm, polydispersity index (PDI) below 0.2, and zeta potential near zero. Both promastigotes and intracellular amastigotes treated with NE-PM, NE-ClAlPc and NE-PM+ClAlPc were inhibited at >50%, >95%, >88%, respectively, after PDT with a phototoxic index (PI) >1.2. No skin ClAlPc permeation was observed. In contrast, PM skin permeation was 80-fold higher using PM-loaded NE formulation in comparison to aqueous PM solution. Topical treatment with NE formulations showed no signs of local toxicity or genotoxicity. In addition, concentrations of PM between 27.3 - 292.5 μM/25 mg of tissue were detected in different organs. In vivo, the NE-PM+ClAlPc treatment did not reduce skin lesions.ConclusionThe Cremophor ELP/castor oil NE formulation increases the permeation of PM through the skin and can be used to co-deliver PM plus ClAlPc for combined PDT protocols. However, the lack of efficacy in the in vivo model evidences that the therapeutical scheme has to be improved.

Dataset Information

Promoter trapping in microalgae using the antibiotic paromomycin as selective agent.

Publications

Promoter trapping in microalgae using the antibiotic paromomycin as selective agent.

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