Mutant/mutation
The mutant contains a DiCre expression cassette. The Dicre gene (the N-terminal Cre 59 (residues Thr19-Asn59) and C-terminal Cre 60 (Asn60-Asp343) portions of the Cre fused at their N-terminus to FKBP12 and FRB, respectively) is under control of the constitutive hsp70 promoter. The DiCre expression cassette is introduced into the silent p230p locus. In addition it expresses mCherry and does not contain a drug-selectable marker (see below).

We first generated a parasite line that stably and constitutively expresses both components of the Cre enzyme. We assembled a construct encoding the N-terminal Cre 59 (residues Thr19-Asn59) and C-terminal Cre 60 (Asn60-Asp343) portions of the Cre fused at their N-terminus to FKBP12 and FRB, respectively. The two components were placed under control of the constitutive bidirectional promoter eEF1alpha, and followed by the 3’ untranslated region (UTR) from PfCAM and PbHSP70, respectively. In addition, the construct contained a mCherry cassette under the control of an inactive truncated fragment of HSP70 promoter, and a TgDHFR/TS pyrimethamine resistance cassette. The TgDHFR/TS cassette was flanked by two LoxP sites, to allow Cre-mediated excision and production of drug selectable marker-free parasites). A sequence corresponding to the 3’ UTR of PbDHFR was included at the end of the construct, for homologous recombination at the modified p230p locus of GFP-expressing P. berghei ANKA parasites (PbGFP). 
The resulting parasite population was exposed to a single dose of rapamycin that was administered orally to mice. This treatment resulted in excision of the TgDHFR/TS cassette, as demonstrated by PCR genotyping (Fig 1B), and retention of a single LoxP site. Cloning by limiting dilution resulted in the final selectable-marker free mCherry-expressing PbDiCre parasite line.

Protein (function)
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 catalyses the excision or inversion of the floxed (flanked by loxP) DNA segment.
Different systems have been developed to introduce DiCre into P. falciparum, including integration of DiCre cassettes into the chromosome and expression of the genes from an episome. Excision levels vary greatly between the systems, ranging from 50–98%. In these studies, parasite cultures were treated with 100 nM rapamycin for a minimum of four hours and high levels of excision of floxed DNA sequence were reported. However, both rapamycin and the carrier, DMSO, can negatively affect parasite growth at higher concentrations. 

Phenotype
From the Abstract: 
'We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle'

Additional information
DiCre recombinase mediated excision of targeted DNA sequences in vivo was achieved by administration of 200 μg Rapamycin (1mg/ml stock, Rapamune, Pfizer) to mice by oral gavage, 24 hours prior to transmission to mosquitoes. In order to achieve excision in the mosquito stages, 10 μg rapamycin (1 mg/ml stock solution in DMSO, Sigma-Aldrich) was added to 10 ml 10% sucrose solution and used to feed mosquitoes. The rapamycin dose was refreshed every alternate day along with the sucrose solution.

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