Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus

- Perkinsus marinus is sensitive to bleomycin (IC50 = 1.94 μg/ml).
- Cells transfected with pMOE-mCherry-ble gain bleomycin resistance.
- 100 μg/ml bleomycin completely blocks the growth of untransfected parasites.

Perkinsus species are notorious unicellular marine parasites that infect commercially important mollusk species including clams and oysters. Recent accumulation of molecular information will greatly facilitate the understanding of Perkinsus biology and development of strategies to control infection. However, the limited availability of methods for genetic manipulation has hindered molecular-based studies of the parasites. In particular, the lack of a drug selection system requires manual isolation of fluorescent cells under a microscope to establish transfected cell lines. Here, we introduce a drug selection system using a glycopeptide antibiotic, bleomycin, and a vector containing the resistance gene Sh-ble. Perkinsus marinus is sensitive to bleomycin, and 100 μg/ml of this drug completely blocks its proliferation. Concomitant expression of Sh-ble enables us to specifically select transfected cells in the presence of the drug. We believe that this system provides new opportunities for functional analyses of this parasite.

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