Figure 5(A) Schematic diagram outlining the generation of CRISPR/Cas9-mediated Pogz+/∆ mouse model. A region spanning critical exons 9 and 10 of the murine Pogz gene on chromosome 3 was deleted using dual sgRNA CRISPR. (B) Expected and observed genotypic distribution of offspring of heterozygous Pogz+/∆ crosses. Genotype was determined by PCR at time of weaning (3 weeks). Data are represented as a bar graph showing the mean ± SEM (n=22 individual litters, across 5 different breeding pairs). (C) Expression analysis of Pogz by western blot in mouse embryonic fibroblasts (MEFs) generated from E12.5 embryos with the indicated genotype. NIH3T3 cells were used as a comparison. Gadph was used a loading control. Observed genotypic distribution of offspring of heterozygous Pogz+/∆ crosses at specified embryonic day. Each litter is considered a biological replicate. Data are represented as a bar graph showing the mean ± SEM (n for each embryonic day is specified). (D) (E) The body mass of male wild-type (WT) or Pogz+/∆ mice was monitored weekly for 4 weeks post-weaning. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ). At least 4 mice per genotype was monitored. Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05. (F) The indicated organ mass of male wild-type (WT) or Pogz+/∆ mice was calculated relative to total body mass. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ) (n=7 6 mice per genotype). Significance was determined by unpaired two-tailed t-test. *P<0.05. (G) Representative movement traces of the indicated mice used for quantification in (H) and (I). (H) Quantification of the distance travelled (left panel) and the average speed (right panel) of each mouse (n=6 mice per genotype) in the open field. Data are represented as a bar graph showing the mean ± SEM and each mouse is represented by a a round dot (WT) or a square (Pogz+/∆ ). Significance was determined by unpaired two-tailed t-test. *P<0.005. (I) The percentage of time that each mouse spent in the middle of the open field was quantified and represented as the mean ± SEM, each mouse being represented by a round dot (WT) or a square (Pogz+/∆ ) (n = 6 mice per genotype). Significance was determined by unpaired two-tailed t-test. *P<0.005. (J) Schematic diagram outlining the conditioning/experimental set up quantified in (K) of the contextual fear tests. (K) The percentage of freezing time in the different experimental conditions (context test, left panel; cue (tone) test, right panel) was monitored for each mouse and is represented as the mean ± SEM, each mouse being represented by a round dot (WT) or a square (Pogz+/∆ ) (n=6 mice per genotype). Significance was determined by two-way ANOVA followed by a Sidak's test. *P<0.05. (L) Plasma was isolated from cardiac punctures of wild-type (WT) or Pogz+/∆ mice (8 weeks) and assessed for circulating levels of specified immunoglobulin isotypes. Each mouse is represented by a round dot (WT) or a square (Pogz+/∆ ) (n=6 mice per genotype). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.005.