Two base pair deletion in IL2 receptor γ gene in NOD/SCID mice induces a highly severe immunodeficiency

摘要

The generation of NIG(NSIG) mice by IVF and CRISPR/Cas9 system. a Schematic diagram of sgRNA targeting the mouse IL2Rg gene loci. The exon 1–8 region of the mouse IL2Rg gene is shown. The exon 6 sequence (upper case) are shown with 2 sgRNA sequences (labeled in red), and the PAM domain sequence NGG in yellow. b Schematic illustration of IVF and microinjection. Female NOD/SCID mice are super-ovulated with PMSG and hCG, followed by oocyte retrieval. Sperm is collected from male NOD/SCID mice. The oocytes and sperm are incubated to generate fertilized eggs and embryos, which are then microinjected with sgRNA and Cas9 protein. The injected embryos are transferred into pseudopregnant surrogate mothers (foster mothers). c Founder mice were genotyped by T7E1 assay after PCR amplification, and the T7E1 products were electrophoresed in 2.4% agarose gel. Mice were labeled #1–13, and M: marker, WT: wild type (negative control). WT allele is represented by a single band at 633 bp, while the heterozygous alleles (red labeling) are obtained as two bands at 453 bp and 633 bp. d Sequence analysis of the mutated IL2Rg alleles. The wild type sequence of exon 6 is shown on top. The sgRNA targeting sites are shown in bold letters. The mutant alleles of each mouse are labeled with the mouse ID number. The deleted sequences are marked in dash. e IL2Rg mRNA expression level results of F4 homo mutant mice. The mRNA levels of IL2Rg were determined by qRT-PCR analysis from mice tail tissue. Mice were labeled NOD.CB17/Prkdcscid/JKrb as NOD/SCID (positive control) and (−2 bp)−/− (F4 homo mutant) mouse as NIG(NSIG)