Fig 1.
Knockout of the Il2rg and Rag2 genes.
(A) Schematic diagram of the gene knockout strategy and targeted sequences of the mutant F0 progenies. Blocks indicate exon regions. Guide RNAs (gRNAs) targeted to exon 2 of Il2rg and exon 3 of Rag2 are highlighted in yellow; the protospacer adjacent motif (PAM) sequences are highlighted in pink. Targeted sequences of the F0 progenies are listed for comparison with the wild-type sequences. Short red lines indicate deletions and blue characters indicate insertions. Progenies that simultaneously carry mutations in both Il2rg and Rag2 are indicated with red boxes. (B) Schematic representation of the method used for CRISPR-based knockout experiments in rat embryos. (C) RT-PCR analyses of Il2rg and Rag2 from single-knockout (sKO) and double-knockout (dKO) rats (15-week-old) were conducted. Gapdh was used as the control. Here, images of different gels were partially cropped to facilitate display as a single figure. (D) Body weights of F344/Jcl wild-type, Il2rg-sKO, and Il2rg/Rag2-dKO rats were measured weekly, beginning at the fifth week postpartum. The graphs show the body weights of male (left) and female (right) rats. For both sKO and dKO rats, six male and six female rats were assessed. The reference weight data of the F344/Jcl wild-type rats were provided by CLEA Japan, Inc. (Tokyo, Japan) (n = 20). sKO and dKO rats were sometimes significantly heavier than the wild-type rats, but no growth retardation was observed. Data are presented as means with standard deviations. Multiple comparisons of each group (sKO or dKO) versus F344/Jcl were conducted using Dunnett’s test. Asterisks or “ns” represent adjusted p-values: ns, 0.12; *, 0.033; **, 0.002; ***, <0.001.
Fig 2.
Knockout of Il2rg and Rag2 caused deficient development of lymphocytes.
(A) Images of thymus specimens from F344/Jcl wild-type, double heterozygote, Il2rg-single-knockout (sKO), and Il2rg/Rag2-double-knockout (dKO) rats. (B) Images of spleen specimens from 4-week-old double heterozygote (top) and Il2rg-sKO (bottom) rats. (C) Serum IgM, IgG, and IgA levels in F344/Jcl wild-type, double heterozygote, Il2rg-sKO, and Il2rg/Rag2-dKO rats, measured by ELISA. Multiple comparisons of each group (sKO, dKO, or he/he; double heterozygote) versus F344/Jcl were conducted using Dunnett’s test. Asterisks or “ns” represent adjusted p-values: ns, 0.1234; *, 0.0332; **, 0.0021; ***, <0.0002; ****, <0.0001.
Fig 3.
Analysis of hematological parameters.
(A) Numbers of white blood cells (WBCs) and red blood cells (RBCs) in wild-type (WT), single-knockout (sKO), and double-knockout (dKO) rats. (B–D) Numbers of lymphocytes (LYM), monocytes (MON), granulocytes (GRA), and their ratios relative to all blood cells. The threshold of statistical significance was set at p = 0.0167 (Bonferroni correction of p = 0.05/3). WT, n = 20; sKO, n = 13; dKO, n = 20.
Fig 4.
Flow cytometry analysis of cell populations in peripheral blood.
(A) Analyses of SSC and CD3. Dot plots show the distributions of the cells after lymphoid gating. The SSC low/CD3+ T cell group is indicated in the box as SSCloCD3+. (B) Analyses of CD4 and CD8. Dot plots show the distributions of CD3+ cells. (C) Analyses of CD45RA and CD161a. Cells from the SSCloCD3− gate were divided into groups. CD3−/CD45RA+ B cells and CD3−/CD161a+ NK cells are shown in the boxes.
Table 1.
Comparison of biochemical parameters among F344/Jcl, sKO, and dKO rats.
Table 2.
Microbiological monitoring results.