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Published on: June 28, 2018
Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus
Hirokazu Sakamoto1, Kiyoshi Kita2, Motomichi Matsuzaki1
1Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Abstract:
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 100μg/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.
Insights
Researchers developed a new drug selection system for Perkinsus parasites, enabling easier genetic manipulation. This breakthrough aids in understanding and controlling infections in commercially important shellfish like clams and oysters.
Area of Science:
- Marine biology
- Parasitology
- Molecular biology
Background:
- Perkinsus species are significant unicellular marine parasites impacting valuable mollusk fisheries.
- Understanding Perkinsus biology is crucial for developing effective infection control strategies.
- Limited genetic manipulation tools hinder molecular studies of these parasites, necessitating manual cell isolation.
Purpose of the Study:
- To establish a novel drug selection system for genetic manipulation of Perkinsus parasites.
- To overcome the limitations of manual cell isolation for creating transfected cell lines.
- To facilitate functional analyses of Perkinsus parasites.
Main Methods:
- Introduction of a drug selection system using the antibiotic bleomycin.
- Development of a vector containing the Sh-ble resistance gene.
- Testing the sensitivity of Perkinsus marinus to bleomycin and the efficacy of the Sh-ble gene for selection.
Main Results:
- Perkinsus marinus demonstrated sensitivity to bleomycin, with 100μg/ml completely inhibiting proliferation.
- Co-expression of the Sh-ble gene allowed for specific selection of transfected Perkinsus cells in the presence of bleomycin.
- The developed system simplifies the establishment of transfected Perkinsus cell lines.
Conclusions:
- A bleomycin-based drug selection system has been successfully established for Perkinsus parasites.
- This system significantly advances the capability for genetic manipulation and functional studies in Perkinsus.
- The new method is expected to accelerate research into Perkinsus biology and disease management in mollusks.
