Figure 2: Analysis of genomic mutations in mice.
From: Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs
(A) PCR amplicons of target sites in murine FVII were subjected to T7E I assays. The amplicons from mutant mice (a) and from mutant mice mixed with wild type (WT) amplicons (b, 1:1) were digested to identify biallelic mutations. Amplicons from WT mice served as negative controls (c). (B) Amplicons of off-target site (OT3–2) from FVII mutant mouse were subjected to T7E I digestion (a). The amplicons were mixed with WT amplicons(1:1) and digested to identify biallelic mutations (b). Amplicons from WT mice were served as negative controls. The target and off-target sites were subsequently confirmed by either PCR-sequencing or TA cloning-sequencing. (C) Detection of plasma prothrombin time (PT) in FVII mutant mice. a, b values with the different numbers within the same table column were indicative of significant differences (P < 0.05). Plasma from four WT mice served as controls. (D) Western blot analysis of FVII expression in plasma of mutant mice. Total plasma proteins were separated by SDS-PAGE, and FVII expression was detected by Western blot. The samples H78, H69, H68, and O68 showed a decreased FVII expression to an extent lower than its 1/2 WT level. FVII proteins in Western blot were indicated out by arrow. (E) Founder mice (H78, H69, H68, and O68) were breed by mating with wild-type mice, and the rates of germline transmission were recorded. a values within the same column showed no significant differences (P > 0.05).
