Puromycin selection for stable transfectants of the oyster-infecting parasite Perkinsus marinus

Perkinsus marinus is a marine protozoan parasite that infects natural and farmed oysters, attracting attention from researchers in both fisheries and evolutionary biology. The functions of almost all cellular components and organelles are, however, poorly understood even though a draft genome sequence of P. marinus is publicly available. One of the major obstacles for a functional study of the parasite is limited experimental means for genetic manipulation: a transfection method was established in 2008, and the first drug selection system with bleomycin was reported in 2016. We here introduce the second drug-selectable marker for selection of P. marinus transfectants. The parasite growth is efficiently inhibited by puromycin (IC50 = 4.96 mu g/mL), and transfection of its resistance gene, puromycin-N-acetyl-transferase (pac), confers resistance to the drug on the parasite. Stable transfectants can be obtained within 2 months by treating with puromycin at 100 mu g/mL. Furthermore, combining puromycin and bleomycin treatment can select transfectants co-expressing two marker genes. This dualtransfection method raises the possibility of using co-localization to identify the cellular localization of novel proteins in P. marinus, thereby contributing to the understanding of cellular functions and pathogenesis.