Figure 5. Generation of an AMA1 conditional knockout.

Figure 5

Generation of an AMA1 conditional knockout. A) Wild type T. gondii expressing the tetracycline-sensitive transactivator (designated “AMA1” parasites) were transfected with a plasmid containing AMA1 under control of the regulatable promoter. This creates parasites (“AMA1/AMA1-myc”) that express both endogenous and regulatable AMA1 in the absence of anhydrotetracycline (Atc), and only endogenous AMA1 after incubation with Atc. Expression of endogenous and regulatable AMA1 can be visualized by Western blotting (top panel); the epitope tag on the regulatable copies results in an electrophoretic mobility shift. The endogenous AMA1 locus was subsequently interrupted by targeted gene disruption, creating parasites (“Δama1/AMA1-myc”) whose sole source of AMA1 can be regulated by Atc. The relative AMA1 expression levels, as determined by quantitative Western blotting, are shown below each lane. B) Host cell invasion by the AMA1, AMA1/AMA1-myc, and Δama1/AMA1-myc parasites, each grown with or without Atc, was measured using the Laser Scanning Cytometer-based invasion assay. Parasites expressing just 10% of wild type levels of AMA1 (Δama1/AMA-myc minus Atc) have wild type levels of invasion. However, parasites expressing undetectable (<0.5%) levels of AMA1 (Δama1/AMA-myc plus Atc) are significantly defective in invasion. The LSC-based assay allows for a large sample size (tens of thousands of parasites) to be attained. Adapted from ref. 8.

From: Current and Emerging Approaches to Studying Invasion in Apicomplexan Parasites

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