Development of reliable transgenic systems for inducing in vitro-cultured hematopoietic cell proliferation in Cherax quadricarinatus

The absence of a stable and efficient transgenic system for crustacean cells has significantly impeded advancements in gene function studies and the establishment of continuous cell lines. In this study, we successfully developed a functional transgenic system for both two-dimensional (2D)- and three-dimensional (3D)-cultured hematopoietic tissue (HPT) cells of Cherax quadricarinatus , through the modification of crustacean viral promoters and the optimization of transfection methods. To evaluate the effectiveness of this system, the coding sequence of crayfish hematopoietic factor, astakine, was cloned and inserted into an expression vector as a transgene. Subsequent optimization of truncated crustacean viral promoters, along with their strategic combinations, led to the design of a highly efficient expression vector for crayfish cells, driven by the hybrid promoter OpIE2-P2-WSV249 (-300/-1) . Following the optimization of transfection parameters, stable expression of the exogenous gene astakine was successfully achieved in both 2D and 3D cultures of crayfish HPT cells using electroporation and chemical transfection techniques. Notably, chemical transfection resulted in astakine overexpression with an efficiency of approximately 18 %, which was associated with a 7 % enhancement in the proliferation rate of the transgenic cells. Furthermore, these cells exhibited sustained, healthy growth for months. In summary, this study presented a reliable transgenic system for gene functional analysis in shrimp and crayfish, laying the foundation for the development of immortalized cell lines in crustaceans. • Clone CqAst into expression vectors to evaluate the transgenic system. • Novel-built OpIE2-P2-WSV249 (-300/-1) promoter can drive crayfish gene expression. • Transgene achieved in 2D/3D HPT cells via electroporation & chemical transfection. • Chemical transgenic system efficiently overexpressed CqAst in HPT cells. • CqAst overexpression promoted transgenic cell proliferation and long-term growth.