G9a crispr cas9 ko h and g9a hdr plasmids

Custom designed siRNA oligos against Cdg7_FLc_1000 and a scrambled siRNA were synthesized by Integrated DNA Technologies (Coralville, Iowa, USA) and transfected into cells with Lipofectamine RNAimax (Invitrogen, USA). The Cdg7_FLc_1000 expression plasmid was generated by reverse transcription-PCR amplification of Cdg7_FLc_1000 cDNA, using RNA from C. parvum sporozoites (Iowa strain) and cloned into the pcDNA3.1(+) vector in accordance with the manufacturer’s protocol (Invitrogen). Full-Cdg7_FLc_1000 was transfected into cells with lipofectamine 2000 (Invitrogen), a pcDNA3.1(+) empty vector was transfected as a control. The primer sequences for Cdg7_FLc_1000 plasmid generation are listed as following: forward (NheI), 5′-CGGCTAGCAGTTTTTACATTTTGTATCTCAGTT-3′ and reverse (KpnI), 5′-GGGGTACCTGAGCGAAATTAGAGTAGTCTGA-3′. Stable HCT-8-G9a^−/− cells were generated through transfection of cells with the G9a-CRISPR/Cas9 KO^(h) and G9a-HDR plasmids (Santa Cruz, USA). HCT-8 cells stably expressing the empty vector were selected for controls.

C. parvum oocysts used in this study were the Iowa isolate purchased from Bunch Grass Farm. Oocysts were firstly treated with 20% sodium hypochlorite at 4°C for 20 min, and then washed twice with Phosphate Buffered Saline (PBS) and RPMI-1640. Viable oocysts were resuspended in RPMI-1640, and respectively used to infect human carcinoma intestinal epithelial HCT-8 cells (ATCC) and non-carcinoma small intestinal epithelial FHs 74 Int cells (INT) (ATCC) with a ratio of oocysts to host cells at 5:1 to 10:1 in the serum free mediums to establish in vitro infection models. Stable HCT-8-G9a^−/− cells were generated through transfection of cells with the G9a-CRISPR/Cas9 KO^(h) and G9a-HDR plasmids (Santa Cruz), as previously reported (Ming et al., 2017 ). Cells were washed with PBS to remove oocyst walls, oocysts and free sporozoites, and changed with complete culture medium at 4 h post infection.