Generation Of Plasmodium Yoelii Malaria Parasite Carrying Double Fluorescence Reporters In Gametocytes

Malaria is an infections disease caused by Plasmodium infection in mammals and transmitted by Anopheles mosquitoes.Plasmodium parastie has a complex life cycle,alternating between vertebrate and Anopheles,including asexual and sexual stage.The initiation of sexual stage development of Plasmodium is gametocyte,and the gametocyte is the only and essential celluar stage for the transmission of Plasmodium from vertebrate hosts to Anopheles mosquitoes.In the current research of Plasmodium,the development of the sexual stage of malaria parasites are hot spots.However,due to the low proportion of female gametocytes and male gametocytes in the erythrocyte stage(less than 0.1%),single-sex gametocytes are difficult to separate and collect independently,which limits the in-depth development of Plasmodium gametocytic biology.In the Plasmodium falciparum and Plasmodium berghei,the fluorescent protein gene was introduced by transfection to obtain the male and female gametocyte fluorescent reporter strains,and the higher purity male and female gametocytes were isolated by flow cytometry for the relevant research.However,the above transgenes are expression edements of a fluorescent protein gene driven by a promoter inserted into a specific locus in the genome.Wheteher this kind of transgenic method can achieve accurate single-sex male and female gametocyte fluorescent protein expression remains uncertain.In this study,based on the CRSIPR/Cas9 gene modification method,the female gametocyte-specific gene ccp2 and the male gametocyte-specific gene Dhcl were selected in Plasmodium yoelii,the mCherry tag and the GFP tag were endogenously fused,respectively.Immunofluorescence experiments and live cell fluorescence observation confirmed that the ccp2::mCherry strain specifically expresses red fluorescent protein in the female gametocyte;the Dhcl::gfp strain specifically expresses green fluorescent protein in the male gametocyte.Further,the ccp2::mCherry strain and the Dhcl::gfp strain were gene-hybridized by mosquito transmission experiments,and the DFsc7 double-fluorescent reporter strain which expresses specific fluorescence in the female/male gametocyte was successfully obtained.Analysis and comparison of the ability of three fluorescent insect strains to grow and develop,the results showed that the growth of the three tag strains in the vertebrate host(proliferative rate of asexual growth,gametocyte production)and Anopheles vector(oocyst,salivary gland sporozoite)were consistent with the wild type;none of the three tag strains have plasmid residues,and genetic modification of other target sites can be performed on themThe tagged strain specifically expressing the fluorescence in the gametocyte phase is used in the following three aspects:1.Analysis of DNA ploidy cahnges in the process of male gametocyte activation by flow cytometry;2.Analysis of changes in DNA ploidy during fertilization of male and female gametes;3.Collection of high-purity single-sex gametocytes by flow sortingThe three tag strains prepared in this study enriched the cell platform and experimental tools for the biological study of the sexual development stage of Plasmodium yoelii.