FIG 2.

Amplification of transgenic parasites in immunocompromised mice. (A) Amplification strategy for obtaining tagged INS1 parasites. Approximately 5 × 10⁷ sporozoites were cotransfected with 50 μg tagging plasmid and 30 μg CRISPR/Cas9 plasmid per cuvette. Each Ifngr1^−/− mouse was gavaged with 200 μl 8% sodium bicarbonate solution 5 min before being infected by gavaged with 2.5 × 10⁷ transfected sporozoites in 100 μl of DPBS. A second round of selection was conducted in NSG mice. Each mouse was gavaged with fecal slurry containing 2 × 10⁴ oocysts obtained at 13 dpi from the first round of selection. All mice received 16 g/liter paromomycin drinking water from the first day postinfection (dpi) for the duration of the experiment. (B) Diagram of the INS1-3HA-tagged locus in stable transgenic parasites. C. parvum was cotransfected with INS1-3HA-Nluc-P2A-neo tagging plasmid and CRISPR/Cas9 plasmid containing an INS1 sgRNA specific to the INS1 locus. (C) Relative luminescence per milligram of feces from transgenic C. parvum oocysts. Each data point represents a single pellet, and each connecting line represents an individual infected NSG mouse from round two amplification of transfected parasites. (D) The number of oocysts per milligram of feces was measured by qPCR. Each data point represents a single pellet, and each connecting line represents an individual NSG mouse from round two of amplification of transfected parasites. (E) PCR analysis of INS1-3HA oocysts amplified in mice from round two. WT, wild type. HA, INS1-3HA transgenic parasites. The product 5′ Ins is specific for the 5′ CRISPR targeting site of INS1-3HA. The product 3′ Ins is specific for the 3′ CRISPR targeting site of INS1-3HA. Control, product is specific to the INS3 locus. Primers are defined in Table S1. (F) Diagram of the INS1-GFP-tagged locus in stable transgenic parasites. C. parvum was cotransfected with the INS1-GFP-Nluc-P2A-neo tagging plasmid and CRISPR/Cas9 plasmid containing an INS1 gRNA specific to the INS1 locus. (G) Relative luminescence per milligram of feces from transgenic C. parvum oocysts. Each data point represents a single pellet, and each connecting line represents an individual NSG mouse infected with INS1-GFP parasites from round two. (H) The number of oocysts per milligram of feces was measured by qPCR. Each data point represents a single pellet, and each connecting line represents an individual NSG mouse infected with INS1-GFP parasites from round two. (I) PCR analysis of INS1-GFP oocysts amplified in mice from round two. WT, wild type. GFP, INS1-GFP transgenic parasites. The product 5′ Ins is specific for the 5′ CRISPR targeting site of INS1-GFP. The product 3′ Ins is specific for the 3′ CRISPR targeting site of INS1-GFP. Control, product is specific to the INS3 locus. Primers are defined in Table S1.