Organoid-based system and reporter for the study of sexual reproduction.

Many advances have been made recently in our understanding of 's asexual cycle and sexual differentiation. However, the process of fertilization, which is required for transmission of infectious oocysts, is not well understood. Typical cancer cell-based culture only allows robust exploration of asexual cycle and sexual differentiation of . To facilitate exploration of sexual reproduction in we developed an organoid-based culture system that supports full life cycle and a novel fertilization reporter. Organoid-derived monolayers (ODMs) supported fertilization and oocyst production and maintained the infection for up to 3 weeks. ODM-derived oocysts were infectious . Fertilization was confirmed by successfully mating two strains of and with a novel fertilization switch reporter. The fertilization switch reporter utilizes a DiCre system in which cre fragments are expressed under the control of sexual stage promoters, resulting in a rapamycin-inducible switch in fluorescent protein expression from mCherry to mNeonGreen after fertilization that is spatially and temporally controlled. This results in mCherry-positive parasites in the first generation and offspring that express mNeonGreen. validation of the fertilization switch reporter demonstrated the precision and efficiency of the fertilization switch reporter and confirmed excision of the mCherry gene sequence only after rapamycin treatment. The start of a second generation of parasites was also shown in the ODMs and rarely in HCT8s. Use of this reporter in ODMs can help investigate the life cycle post-sexual differentiation in a physiologically relevant system.IMPORTANCEOrganoid-derived monolayers enable the study of previously inaccessible aspects of 's biology. This system overcomes the disadvantages of previous organoid-based methods for culture. It is faster and simpler than previously described systems, uses defined differentiation media to increase reproducibility, enables real-time observation, supports parasite fertilization and oocyst production, and provides a physiologically relevant tissue culture system to facilitate cell biology studies. The organoid-derived monolayer system could facilitate the study of host-pathogen interactions, -host specificity, or innate or cellular immune responses to infection stimulated in the intestinal epithelium. The fertilization switch reporter could be used to test factors or drugs that may have potential to interfere with sexual reproduction. Organoid-based cell cultures in combination with the fertilization switch reporter could increase our understanding of sexual reproduction in leading to vital information for the development of sexual reproduction inhibitors or vaccines that could shorten disease duration and prevent transmission.