Baculovirus and plasmid vector-mediated transgenic experiments in the embryonic cell cultures developed from the freshwater crustacean Daphnia magna

Abstract

Abstract Cell culture represents an indispensable tool for investigating fundamental biological processes. Nevertheless, technical challenges such as low cell yield, sub-optimal cell differentiation, and inadequate attachment to the growth substrate have restricted the application of this tool in many studies. Here, we introduce an easy protocol for the preparation of primary cell cultures from Daphnia magna embryos, offering a versatile approach to address cell biological questions in conjunction with the robust in vivo model of D. magna. The development of transgenic cells is an emerging interdisciplinary field that can be used for the fundamental understanding of normal and pathological responses of cells and the improvement of tissue functionality. The application of this technology to primary cells is still in its infancy but promises to accelerate research. In this work, embryonic cell culture is developed from D. magna; and is used to standardize viral (BacIe1-GFP) and plasmid vector (pCS-EF1α1-DSRed2)-mediated transgenic experiments. The standardized conditions methodology for developing embryonic cell culture, Cellfectin-mediated transfection and baculovirus-mediated transduction methods envisage strengthening the crustacean cell line research and bringing forth the Daphnia cell culture system as a 'model' in vitro system for crustaceans. Additionally, the simplicity and flexibility of the methodology described are expected to lead to widespread use in many biological research areas, including their wide application to ecotoxicological and epigenetic studies which are currently limited to in vivo studies. This is the first report on the optimization of cell culture medium for freshwater crustaceans and the use of baculovirus for transduction studies in D. magna embryonic cell culture.