1. Generation of GAMA knockout (KO) by CRISPR-CAS9.
Generation of GAMA knockout (KO) by CRISPR-CAS9. (A) Schematic representation of CRISPR-CAS9-facilitated double homologous replacement of TgGAMA with a chloramphenicol acetyltransferase (CAT) selection cassette. Prom, promoter; sgRNA, single guide RNA. (B) Western blot analysis of parental, Δgama mutant, and Δgama/GAMA lysates. (C) Immunofluorescence confirmation of Δgama and Δgama/GAMA with rabbit anti-GAMA antibody. (D) Red-green invasion assay with HFF host cells. Data are the average ± the standard error of the mean of at least three independent experiments. *, P < 0.05; n.s., not significant (Student’s t test). (E) Δgama mutant tachyzoites are modestly attenuated in virulence; Δgama/GAMA tachyzoites show virulence kinetics similar to those of parental Δku80:Δhxg tachyzoites. Mice were infected subcutaneously with 200 tachyzoites. The data represent infection of 20 mice each with Δku80:Δhxg and Δgama mutant tachyzoites and infection of 10 mice with Δgama/GAMA tachyzoites. Concomitant plaque assays were performed to corroborate the numbers of viable tachyzoites injected. **, P < (Gehan-Breslow-Wilcoxon and Mantel-Cox tests). The values beside the blot in panel B are molecular sizes in kilodaltons.
My-Hang Huynh, and Vern B. Carruthers mSphere 2016; doi: /mSphere