Mutant/mutation
In the mutant the Pyaph gene (acylated pleckstrin-homology domain-containing protein, putative) of P. yoelii with a mutated PKAc gene that has  loxP sequences inserted in intron1 and in the 3'UTR. The aph gene is C-terminally-tagged with cmyc

In addition, the mutant contains a diCre expression cassette containing the two genes Cre59-FKBP and Cre60-FRB placed on either side of the bidirectional eef1a promoter (see the parent line RMgm-5083 for details about this transgene expression cassette). 

Protein (function)

According to a model largely generated by characterization of T. gondii MIC2 (a homolog of Plasmodium TRAP proteins) and AMA1, diacylglycerol is produced during a signaling cascade involving phospholipase C. The diacylglycerol is further converted to phosphatidic acid (PA) by a diacylglycerol kinase on the inner leaflet of the plasma membrane, which is then recognized by acylated pleckstrin homology (pH) domain-containing protein (APH) on the cytosolic face of the microneme membrane. The binding of APH to PA facilitates juxtaposition of these two membranes, to release the contents of micronemes.

The rapamycin-inducible Cre recombinase (DiCre) established for use in P. falciparum uses the rapamycin-binding FKBP12 and FRB proteins to dimerise the two enzyme halves. In the DiCre system, Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin‐binding protein (either FKBP12 or FRB, the rapamycin‐binding domain of the FKBP12‐rapamycin‐associated protein mTOR). Rapamycin‐induced heterodimerization of the two components restores recombinase activity (rapamycin‐mediated dimerization of two distinct, enzymatically inactive polypeptides approximately corresponding to the individual domains of Cre (residues Thr19–Asn59, called Cre59, and Asn60‐343, called Cre60) each fused to a different rapamycin‐binding protein (FKBP12 and FRB respectively) results in the reconstitution of Cre recombinase activity). This site–specific recombinase recognizes short, 34 bp sequences called loxP sites and catalyzes the excision or inversion of the floxed (flanked by loxP) DNA segment.

Phenotype
From the abstract: 'We found that APH deletion' (i.e. knockdown after RAP treatment) resulted in a reduction in parasite asexual growth and erythrocyte invasion, with some parasites retaining the ability to invade and grow without APH. APH deletion impaired the secretion of microneme proteins, MTRAP and AMA1, and upon contact with erythrocytes the secretion of MTRAP, but not AMA1, was observed.'

Additional information
About rapamycin (RAP) treatment: ' RAP (Sigma-Aldrich) was dissolved with dimethyl sulfoxide (DMSO) at 4 mg/mL and stored at 􀀀 20 ◦C. RAP (4 mg/kg) or an equivalent volume of DMSO were intraperitoneally (i.p.) injected into mice infected with PyAPH-iKO parasites when the parasitemias reached 20–30%, and parasites were collected 24 h later. Schizonts were enriched by density gradients using Nycodenz (1.077 g/mL) and incubated in vitro at 15 ◦C for 3 h for maturation. Matured schizonts (107) were then intravenously (i.v.) injected to BALB/c mice and RAP (4 mg/kg) or DMSO were immediately administrated (i.p.). Blood was collected at 3, 12, 24, 36, 60, and 84 h after parasite inoculation and thin blood smears and gDNA were prepared. Blood smears were stained with Giemsa's solution and parasitemias were determined.' 

Other mutants