The inventors have generated multiple gene knockout strains of T. gondii, listed below.
RH∆hx∆ku80 TIR1 (RH∆ku80∆hxgprt/pTub 5’UTR-OsTir1-3xFlag-DHFR 3’UTR-CAT):
The inventors used the CRISPR/Cas9 system for genome editing, comined with the AID degradation system to generate the Toxoplasma gondii strain. It was used to study plasma membrane association and PKG function in Toxoplasma gondii.
DiCre KB1 (RH∆ku80∆hxgprt/DiCre):
To generate a DiCre recipient strain expressing both subunits in the same genomic locus, p5RT70DiCre-HX was transfected into RH hxgprt− (RH hxgprt−/diCre, referred to here as RH DiCre). Expression of Cre recombinase subunits was confirmed by western blot analysis with antibodies to FKBP12 and FRB.
The ku80∷diCre recipient strain was generated by replacing HX with diCre in the ku80∷HX strain by homologous recombination (ku80∷HX∷diCre, referred to here as ku80∷diCre). The 5′ UTR–DiCre–ku80 3′ UTR cassette was transfected into ku80∷HX strain, and subsequently ku80∷diCre parasites were selected using 6-thioxanthine to remove HX. Integration of diCre into the ku80 locus was confirmed by analytical PCR on genomic DNA using ku80∷HX fw (1) and ku80 rv (1′) primer pair to check for the presence of hx in the ku80 locus.
Pru ∆ku80 SL1 (Pru∆ku80∆hxgprt clone SL1):
The inventors used electroporation to transfect the parental Pru (Pru∆ku80∆hxgprt) T. gondii strain. All transfected plasmids were linearized 5' of the 5' target DNA flank prior to transfection using unique restriction enzyme sites designed into the targeting plasmids. Forward selections to integrate the pmini-HXGPRT selectable marker were performed in mycophenolic acid and xanthine. Negative selections to excise HXGPRT were performed in 6-thioxanthine. Negative selections using the cytosine deaminase (CD) selectable marker were performed in 5-fluorocytosine. Negative selections to delete UPRT were performed in 5-fluorodeoxyuridine (FUDR). After transfection, parasites were allowed to replicate for 24 h without selection to allow replication and ramp up homologous recombination, and then selections were launched and continuously maintained through verification steps of cloned isolates
ME49 TIR1 (ME49∆ku80::Luciferase∆hxgprt/pTub 5’UTR-OsTir1-3xFlag-DHFR 3’UTR-CAT)
ME49∆hx∆ku80 SL1 (ME49∆ku80::Luciferase∆hxgprt clone SL1)