Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of cdpk1. The mutant expresses the yeast FlpL recombinase under the control of the trap promoter  and contains a FRTed ORF (open reading frame) of the cdpk1 locus. 

This mutant has been generated by replacement of the endogenous cdpk1 gene by an cdpk1 gene with a FRTed ORF in the mutant RMgm-747 that expresses FlpL. This mutant expresses the yeast FlpL recombinase under the control of the promoter of trap and does not contain a selectable marker. The flpl gene is integrated into the silent 230p locus by double cross-over integration.

The cdpk1 ORF is removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-269, RMgm-747). Removal of the FRTed ORF of cdpk1  has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase in sporozoites that resulted in the excision of the ORF of cdpk1 which was flanked by FRT sequences.

Protein (function)
In malaria parasites, intracellular Ca²⁺ signals are translated into stage-specific cellular responses by members of a family of Ca2+-dependent protein kinases (CDPKs), which combine an N-terminal kinase domain with a C-terminal, calmodulin-like regulatory domain in the same polypeptide. By analysis of a promoter-swap mutant that expresses CDPK1 only in asexual blood stages and not in gametocytes and ookinetes it has been shown that CDPK1 plays an essential role in ookinete formation (see RMgm-773; RMgm-966).

Phenotype
The Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence CDPK1 expression specifically in liver stages. Removal of the FRTed ORF of cdpk1  has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase in sporozoites that resulted in the excision of the FRTed ORF of cdpk1.

The efficiency of 'slincing' CDPK1 expression (i.e. the efficiency of excision of the cdpk1 gene) has (only) been analysed by PCR analysis of parasites recovered after mosquito transmission.

Equal numbers of sporozoites were recovered from salivary glands of CDPK1 cKO-infected and FlpL/TRAP-infected mosquitoes, demonstrating that CDPK1 is not required for parasite invasion of salivary glands. To determine if CDPK1 plays a role in hepatocyte invasion, CDPK1 cKO sporozoites were used to infect the human hepatoma cell line, HepG2. FlpL/TRAP sporozoites were used as controls in this and subsequent experiments. There was no significant difference in the number of liver stages formed by CDPK1 cKO or control sporozoites. These results demonstrate that CDPK1 is not essential for the parasite’s invasion of hepatocytes or subsequent intrahepatic development. To study CDPK1’s role in parasite egress from infected HepG2 cells, the number of extracellular merosomes released in CDPK1 cKO and control infected HepG2 cultures was determined. There was no significant difference in the number of merosomes present in both. These results indicate that CDPK1 is not essential for parasite development in and egress from hepatocytes.

Additional information
See also RMgm-518 and RMgm-966 for unsuccessful and successful attempts to select for mutants that lack expression of CDPk1

Other mutants
RMgm-518: unsuccessful attempts to disrupt cdpk1
RMgm-966: A mutant lacking expression of CDPK! (successful disruption of cdpk1!)
RMgm-773: A promoter exchange mutant that expresses CDPK1 under control of the the asexual blood stage specific clag promoter
RMgm-772: A mutant expressing CDPK1-GFP from the endogenous cdpk1