Generation of Slc29a3 ^−/− and Tlr7 ^−/− mice. (A and I) Genomic configuration of Slc29a3 and Tlr7 genes showing 20mer gRNA target sites to introduce a mutation into exon 2 of Slc29a3 and exon 5 of Tlr7. The PAM sequence is highlighted by the red box. (B) Genomic PCR with the primer set (Fw and Rv) shown in A reveals an insertional mutation in the targeted allele of Slc29a3. (C) Direct sequencing of the gRNA target site of Slc29a3. The inserted sequence containing the stop codon is shown by the blue box. (D–G) NF-κB reporter assay using HEK293T cells transfected with TLR7 and human Unc93B1. Transfected cells were left unstimulated or stimulated with indicated nucleoside ligands (100 μM) with or without polyU (5 μg/ml). The results are represented as mean values ± SD of triplicates. (H) IL-12 p40 production by BM-Mphs left unstimulated or stimulated with a combination of polyU (1 μg/ml) and the indicated nucleoside (100 μM). The results are represented by mean values ± SD of triplicates. (J) Sequence data of the gRNA target site on the Tlr7 allele showing a 4-bp deletion in the fifth exon of Tlr7 (blue). (K) FACS analyses show the lack of TLR7 protein in splenic B cells, splenic monocytes, and BM-derived pDCs in Slc29a3^‒/‒Tlr7^‒/‒ mice. Red and gray histograms represent intracellular staining with and without anti-TLR7 mAb, respectively. The data shown in D–H and K are representative of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.