Additional remarks phenotype | Mutant/mutation
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. This expression cassette has been introduced in the neutral p230p locus of the GIMOp230p line that has the yfcu/hdhfr positive/negative selectable marker integrated into the neutral p230p locus. This line is used to introduce transgenes into the p230p locus using negative selection with 5-FC (see also RMgm-687). The mutant does not contain a drug-selectable marker.
See below for details of using this line to conditional change gene expression by rapamycin induced expression of Cre recombinase (DiCre) resulting in the excision or inversion of the floxed (flanked by loxP) DNA segments in the Plasmodium genome.
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 recognises 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
Evidence for expression of Cre recombinase (DiCre) after rapamycin-treatment of blood stages (in vitro and in vivo) (using the rapamycin-binding FKBP12 and FRB proteins to dimerise the two enzyme halves of Cre).
P. berghei parasites were synchronized by overnight culture in schizont media and a Nycodenz gradient was used to harvest schizonts, as described above. Purified schizonts were intravenously injected into naive mice, where they maintained their synchronicity for 48 h. Rapamycin (Sigma) was dissolved in DMSO to 4 mg ml−1 to generate the stock solution. DiCre test parasites were induced either in vitro and in vivo. For in vitro induction, parasites were removed from the host by cardiac puncture 2 h post invasion (hpi) and cultured in schizont media with 200 mM rapamycin. For in vivo induction, the host animals were injected intraperitoneally with 4 mg kg−1 rapamycin 2 hpi. Parasites were harvested at various time points via tail bleed or cardiac puncture for DNA and flow cytometry analysis.
Additional information
In the paper this transgenic line expressing DiCre has been used to introduce a
second DNA construct that included a module containing modified loxP sites (lox66 and lox71) in a head-to-head orientation flanking a strong constitutive promoter (hsp70) used to express a selection marker. This module was inserted in front of the ap2-g open reading frame (ORF; PBANKA_1437500), interrupting and uncoupling its native promoter. In this position, the unidirectional induced lox recombination event would invert the hsp70 promoter and over-express ap2-g.
Evidence is presented that (conditional) over-expression of ap2-g results in a high conversion rate of asexual blood stages into gametocytes.
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