Simple Transgene Overexpression using Scallop Hemocyte Culture Platform Enables Functional Genetic Research in Molluscs.

Despite being evolutionarily and commercially important, molluscs have been a traditionally challenging group to study, due to their difficulty in maintenance under lab conditions and the lack of a genetic toolkit. Previously, we showed that transgene expression can be attained in molluscan cells with reporter genes under a molluscan virus promoter sequence. Following up, we developed a simple, efficient and rapid transgene expression platform using primary hemocyte culture of Farrer's scallop Chlamys farreri, a marine bivalve mollusc. The protocol consists of two steps: collection and seeding of hemocytes and incubation for 1 to 4 days with DNA-reagent mixture. We evaluated seven transfection reagents for three bivalve species and found that X-tremeGENE 360 was highly efficient for DNA transfection, particularly for C. farreri. Subsequently, C. farreri hemocyte culture and transfection conditions were examined, such as culture medium, size and form of DNA, and the mixing ratio of DNA and transfection reagent. Using this protocol, we visualized the subcellular localization of four bivalve oncogenes, Cf-Mdm2-like, Cf-c-Myc-like, Cf-Mortalin-like, and Cf-Ras-like, tagged with EGFP. Our hemocyte platform provides an easy entry to study cellular and molecular biology of molluscs and can be readily adapted for advanced methods such as live imaging and DNA-protein interaction assays, making the study of molluscs more accessible to the scientific community.