Extended Data Fig. 8: Generation and additional characterization of Api5-knockout mice.
From: The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death
a, Schematic strategy for the generation and genotyping of Api5 knockout mouse. The CRISPR-Cas9 gene targeting mixture containing sgRNAs #1 and #2 targeting exon 1 of Api5 and Cas9 mRNA were injected into zygotes generated from Atg16L1f/f females impregnated by Atg16L1ΔIEC males. The 23 resulting chimeras were screened through a PAGE-based genotyping approach4 in which amplicons generated using the indicated primers and tail DNA from chimeras and wildtype mice as templates were annealed. Heteroduplexes signifying mismatches between the wildtype sequence and the disrupted locus were used to identify 12 candidate knockout mice. Three candidates were selected for further backcrossing and breeding, one of which successfully produced reproductively viable offspring. b, Representative genotyping gel images. Middle gel shows byproducts from first PCR reaction and annealing process using primers from (a). Since Api5+/+ and Api5−/− mice cannot be distinguished by this approach, their PCR products were denatured and annealed with wild-type B6 tail DNA in a second reaction shown in the right gel, which yields multiple bands in the presence of sequence mismatches in Api5−/− and Api5+/− mice but not Api5+/+ mice. c, Sequencing of the Api5 locus from Api5 mutant mice identified a dinucleotide AT insertion after the third codon that causes a frameshift leading to an early stop codon at amino acid position 30. d, Expected and observed number of offspring mice with indicated genotypes from Atg16L1 flox/flox villinCre+ Api5+/− and Atg16L1 flox/flox villinCre− Api5+/− breeders. e, Proportion of the indicated IEL subpopulations of the small intestine from Api5+/+and Api5+/− mice. n = 3 (Api5+/+) and 3 (Api5+/−). f, g, Representative H&E images (f) and quantification (g) of Paneth cells per organoid from Atg16L1ΔIEC (ΔIEC) mice co-cultured with ~5 x 104 γδ or αβ IELs harvested from Api5+/+ or Api5+/− mice. n = 30 per condition. h, i, Representative images (h) and viability (i) of small intestinal organoids from ΔIEC mice. Scale bar 50 μm. In organoid experiments, intestinal crypts were harvested from 3 mice per genotype, and the viability assay was performed in triplicate. An ANOVA with Tukey’s multiple-comparison test in (g, i). Data points in (e) are individual mice, data points in (g) represent individual organoids, and data points in (i) represent organoid viability in each well. Bars represent means ± SEM. At least two independent experiments were performed.
