(Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for "Dermo" disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR4 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes and . In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of PmMOE[MOE1]:GFP and 25.0 × 10 cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062965 | PMC |
http://dx.doi.org/10.3389/fbioe.2021.623278 | DOI Listing |
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