We also created null mice with BALB/c background from TCR mRNA and lacked -GalCer/CD1d-restricted iNKT cells. absence of a biased TCR repertoire. Concerning iNKT cell functions, these animals have thus far been employed to investigate the role of iNKT cells in allergen-induced pulmonary inflammation12 and -GalCer-mediated suppression of tumor metastases13. Additional studies are needed to reassess iNKT cell functions and those of type 2 NKT cells with these novel locus mutation WS 3 Two gene-targeted single lead RNAs (called Traj18_sgRNA1 and Traj18_sgRNA2) (Supplemental Fig.?1a) were designed to target the gene segment. We first validated whether the sgRNAs could WS 3 identify and cleave the target sequence using an system, as explained previously15. In brief, the targeted genome segment of the locus (Supplemental Fig.?1b), including sgRNA target sequence, was inserted between the split-EGFP (enhanced green fluorescent protein) fragments that share 400?bp of DNA sequence, under control of the CAG promoter (pCAG-EGxnFP-target) and used as a reporter plasmid. We co-transfected pCAG-EGxnFP-target and pCAG-T3-hCas9-pA with or without pU6-sgRNA (Supplemental Fig.?1c) into HEK293T cells and the levels of reconstituted EGFP expression were evaluated by fluorescence microscopy (Supplemental Fig.?1d) and circulation cytometry (Supplemental Fig.?1e) 48 hrs after transfection. Both Traj18_sgRNA1 and Traj18_sgRNA2 worked effectively, as revealed by EGFP expression in approximately 40% of the transfected cells. Generation of mice with a partial deletion of the gene segment by CRISPR/Cas9 technology Following validation of sgRNAs in HEK293T cells, we proceeded to generate gene-targeted mutant mice by zygote injection. sgRNA and hCas9 mRNA were placed under the phage T3 promoter followed by transcription using T3 RNA polymerase (Supplemental Fig.?2a) and injected into the pronuclei of fertilized eggs of B6 mice. Pups derived from these fertilized eggs were genotyped by sequence analysis. Eight out of 11 mice from your Traj18_sgRNA1 (Supplemental Fig.?2b, Supplemental Table?1) and 10 out of 17 mice from your Traj18_sgRNA2 (Supplemental Fig.?2c, Supplemental Table?1) contained a partial deletion in the locus. We selected three founder mice and established four new strains with a mutant mice We compared the TCR repertoire diversity in sorted pre-selection double-positive (DP) thymocytes (TCRlow CD4+ CD8+ CD69?) from (encoded V14) that contains iNKT-TCR, or (encoded V2), the most frequently used TCR in T cells, by using a specific forward primer for each V encoding sequence and a reverse primer for the sequence encoding the TCR constant region (C). The products were purified and subjected to next-generation sequencing analysis. All four gene segments as WT B6 mice, except for (Fig.?1a). Selective deficiency in was confirmed in usage in or PCR products were prepared and subjected to next-generation sequencing analysis. The graphs show percentages of productive gene segment rearrangements. Data represents mean??SD of three biologically indie samples per group. (b) gene segment usage in or transcripts analyzed by next-generation sequencing. (c) Frequencies of iNKT cells (TCR+, -GalCer/CD1d dimer+) in WS 3 total thymocytes isolated from WT B6, in the present mouse strain. Open in a separate window Physique 2 male mice were fed with a HFD or a normal chow diet (ND) starting from 8 weeks of age. For WT B6 and mice gained less excess weight than WT B6 mice, whereas there was no significant difference in the weight gain between mice (Fig.?3a,b). Open in a separate window Physique 3 Impact of iNKT cell-deficiency on metabolic parameters. (a) Curve of relative body weight (BWdn/BWd0??100%) of WT B6 and gene segments upstream of allele might have caused inadvertent alterations in TCR gene transcription and rearrangement7. This impaired TCR repertoire diversity might have resulted in the loss of some unique T cell subsets, raising issues about experimental results obtained with this mouse strain. To avoid the Sirt7 unintended effects caused by the gene segments, we and other groups12C14 tried to generate null mice with an undisturbed TCR repertoire. Two groups12,13 explained deletion mice produced around the C57BL/6 background in which was deleted along with the gene segment by traditional homologous recombination in C57BL/6 ES cells with Cre/loxP and/or FLP/FRT method. Zhang exon resulting in lack of iNKT cells even in the presence of transcript, highlighted the importance of the CDR3 sequence of iNKT-TCR in the acknowledgement of -GalCer/CD1d. Here we employed another genome editing CRISPR/Cas9 technology to generate four strains of null mice with C57BL/6 background. We also produced null mice with BALB/c background from TCR mRNA and lacked -GalCer/CD1d-restricted iNKT cells. Even though minor populace of -GalCer/CD1d-restricted cells with a.