MOESM7 of Chimeric Plasmodium falciparum parasites expressing Plasmodium vivax circumsporozoite protein fail to produce salivary gland sporozoites
2018-08-09T05:00:00Z (GMT) by
Additional file 7. Generation and genotyping a P. falciparum mutant line lacking expression of CSP (PfΔcsp). A. The PfΔcsp line was generated using CRISPR/Cas9 methodology. The pfcsp gene was replaced by insertion of a mCherry expression cassette (mCherry under control of the of the constitutive gapdh promoter) using donor-DNA plasmids pLf0086. A schematic representation of the pfcsp locus before and after insertion of the construct showing the location of the restriction sites (A: AvaII), sizes (in bp) of restriction fragments (red for Southern blot analysis), location of primers (p), PCR amplicons and sizes of the fragments (in black) used to analyse correct disruption of the pfcsp and insertion of the mCherry cassette (B, C). HR1, HR2: pfcsp homology (targeting) regions. The figure is not shown to scale. Primer sequences can be found in Additional file 8. B. Diagnostic PCR confirming correct integration of the mCherry cassette into the PfCSP locus. 5′ integration PCR (lane 2; primers p15/p19); pfcsp open reading frame (lane 3; primers p17/p18); P. falciparum sequestrin as a control gene (lane 1; primers p22/p23); mCherry gene (lane 4; primers P20/P21) of cloned parasites of PfΔcsp (cl3) and WT. C. Southern blot analysis of AvaII/XhoI restricted DNA of WT and PfΔcsp parasites confirms the specific integration of the mCherry cassette into the pfcsp gene locus. DNA was hybridized with a probe targeting the homology region 2 (upper panels; HR2; primers p3/p4; see (A) of pfcsp. The hybridization pattern observed with the HR2 probe identified the expected different-sized DNA fragments in WT and pf-pvcsp parasites (2057 bp and 4105 bp). In addition to show absence of donor-DNA plasmid and presence of single cross-over events DNA was hybridized with a probe for the ampicillin gene (lower panels; intermediate donor-DNA plasmid pLf0040 digested with AatII and PvuI).