Gene Disruption of Plasmodium falciparum p52 Results in Attenuation of Malaria Liver Stage Development in Cultured Primary Human Hepatocytes (original) (raw)
Figure 1
Generation of P. falciparum parasites lacking expression of P52.
(A) Illustration of the DNA construct (m144) used for the targeted gene disruption of p52 and the _p52_-genomic locus before and after integration. Shown are the p52 gene and target sequence (amplified using 1624 & 1625), the paralog of p52, p36, and the T. gondii dhfr/ts selection cassette. In addition, primer pairs and restriction sites for diagnostic PCR and Southern analysis are shown (see B and C). hrp – histidine rich protein. (B) Southern analysis of _BstN_I/_SnaB_I digested genomic DNA of Wt and Δp52 demonstrates correct disruption of p52. DNA was hybridized with a p52 specific probe detecting a 3.3 kb fragment in Wt, a 2.2 kb fragment for intact plasmid and the expected fragments of 1.3 kb and a 4.2 kb band (see A) in the two Δp52 clones (Δp52-1 and Δp52 -2). (C) PCR analysis of genomic DNA of Wt and Δp52 clones and the plasmid DNA (construct) demonstrates correct disruption of p52. Genomic DNA from Wt and Δp52 asexual parasites and sporozoites was used as template for the PCR reactions. The Wt specific PCR was performed using primers 1638 and 1676 amplifying a 2.1 kb fragment. PCR primer pairs 1638 and L430, specific for integration of the DNA construct (see A) amplify a 2.0 kb fragment. Primer pairs 190 and 191 amplifying a 1.8 kb fragment from T. gondii dhfr/ts were used as a control.