Mutant/mutation
The mutant expresses a C-terminal mCherry tagged P28 protein.

For tagging P28 the CRISPR/Cas9 method was used as described for mutant RMgm-1095  with a slight modification of the pYCm vector. In this vector the original hdhfr slectable marker was replaced by the hdhfr-yfcu selectable marker. This allows for killing mutated parasites that still contain remaining episomal plasmids by 5-FC treatment of mice. Removal of the episomal plasmid is essential for subsequent mutations in the same parasite using the CRISPR/Cas9 method (see also mutants RMgm-4069 and RMgm-4070.

Protein (function)
P28 is one of two major surface proteins (P25 and P28) of the plasma membrane of ookinetes. The proteins are conserved between different Plasmodium species. The proteins are characterized by a secretory N-terminal signal sequence followed by three or four epidermal growth factor (EGF) domains and a glycosylphosphatidylinositol (GPI) anchor.

Phenotype
Normal development of blood and mosquito stages indicating an negative effect of tagging on the function of P28 (however also parasites lacking expression of P28 show only slight reduction of ookinete/oocyst development; see mutant RMgm-1)
No expression of P28::mCherry in asexual blood stages and gametocytes.
P28::mCherry expression in female gametes, zygotes, ookinetes and very young (1 day) oocysts. No expression in 4-day oocysts.

Additional information
See mutant RMgm-1095 for a detailed description of the CRISPR/Cas9 method.
Both for tagging P28 and for disruption of CTRP the CRISPR/Cas9 method was used as described for mutant RMgm-1095  with a slight modification of the pYCm vector. In this vector the original hdhfr slectable marker was replaced by the hdhfr-yfcu selectable marker. This allows for killing mutated parasites that still contain remaining episomal plasmids by 5-FC treatment of mice. Removal of the episomal plasmid is essential for subsequent mutations in the same parasite using the CRISPR/Cas9 method.

Other mutants