Mutant/mutation
In the mutant the endogenous P. berghei csp gene has been replaced by the P. falciparum csp gene. This has been performed by the GIMO method of transfection. The P. falciparum csp gene is under control of the 5'- and 3'-UTR regions of the P. berghei csp gene. The mutant does not contain a drug-selectable marker. The mutant also expresses mCherry and luciferase under control of constitutive promoters.

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
Normal development throughout the complete life cycle showing complementation of P. berghei CSP by P. falciparum CSP

Additional information
Generation of line 3079cl1:
In the first step we deleted the Pb csp CDS and replaced it with the positive-negative selectable marker, to create a Pb csp deletion GIMO line (PbANKA-CS GIMO; see RMgm-4681). In order to do this, we generated a construct (pL2153) that is based on the standard GIMO DNA construct pL0034. This construct contains the positive-negative (hdhfr::yfcu) selection marker (SM) cassette, and was used to insert both the Pbcsp 5’ and 3’ gene targeting regions (TR), encompassing the full length promoter and transcription terminator sequences, respectively, and was transfected into the parental parasites (1868cl1) using standard methods of transfection. Selection and cloning resulted in line 3065cl1 (see RMgm-4681).

In the second step we replaced the positive-negative SM in the PbANKA-CSP GIMO genome with the Pfcsp CDS by GIMO transfection to create the P. berghei chimeric CSP replacement line. This was achieved by modifying the construct used in the first step (pL1929); specifically, the hdfhr::yfcu SM cassette was removed and replaced with Pfcsp CDS sequence, generating plasmid pL1972. The Pfcsp CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL1972 construct was sequenced to ensure there were no mutations in the Pfcsp CDS. The constructs was linearized using ApaI and NotI restriction enzymes outside of the 5’ and 3’ TRs before transfection. The construct was used to transfect parasites of the PbANKA-CSP GIMO line (line 2151cl1) using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice. Negative selection results in selection of chimeric parasites where the hdhfr::yfcu SM in the csp locus of PbANKA-CSP GIMO line is replaced by the CDS of Pfcsp. Selected chimeric parasites were cloned by the method of limiting dilution (resulting in line 3079cl1). Correct integration of the constructs into the genome of chimeric parasites was analysed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes. This method creates chimeric ‘gene replacement’ P. berghei parasites that lack the Pbcsp CDS but express PfCSP ( under the control of the Pbcsp regulatory sequences. 

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