Figure 4.

Characterization of TgGT1_280480. (A) Scheme showing the strategy used for TgGT1_280480 gene knockout. CRISPR/Cas9 gene knockout strategy was done in the RH strain. Genomic region (green) and recombination fragment, protospacer (blue), and selection marker (orange; DHFR) are shown. (B) Top panel, insertion of the drug cassette disrupts the TgGT1_280480 gene. PCR product using the primers indicated in the cartoon in A produce a product of 5 kbp validating the insertion in Δtg280480 (fragment is marked in the scheme in A) while a fragment of 1.9 kbp is obtained from the PCR reaction of the parental strain. Bottom panel, PCR positive control from parental and Δtg280480. (C) qPCR analysis of total RNA harvested from Δtg280480 mutant strain and parental strain RH. Left, expression level obtained with primers downstream of the drug cassette insertion site. Right, mRNA levels using primers upstream and downstream of the drug cassette insertion site. (D) Plaque assays showing growth of plaques formed by Δtg280480 mutant and RH parasites. Plaque size measurements and statistic analysis n = 3, P<0.05. (E) Changes in cytosolic Ca²⁺ levels of Fura-2 AM loaded tachyzoites [Δtg280480 mutants and RH). Parasites were in suspension as described in Material and Methods and Ca²⁺ (2 mM) was added at the time indicated. The bar graph to the right shows the quantification and statistical analysis from three biological experiments, each in duplicate. (F) Changes in cytosolic Ca²⁺ levels of Fura-2 AM loaded tachyzoites [Δtg280480 mutants and RH). 1 μM Thapsigargin and 2 mM Calcium (Ca²⁺) were added at the times indicated. The bar graph to the right shows the quantification and statistical analysis of three biological experiments, each in duplicate. P<0.05.