Fig. 2

Identification and quantification of exogenous DNA uptake by sperm using conventional PCR and absolute real-time PCR. Total DNA was extracted from sperm from experimental groups treated with pgRNA-Cas9 after removing unbound or non-internalized plasmids. A Conventional PCR and gel electrophoresis were performed to confirm plasmid internalization into sperm. Gel electrophoresis detected fragments of 250 bp between the U6 promoter and the guide region. L: 50 bp DNA ladder; L1-L4: 0, 0.75, 1, and 2 mM MBCD + pgRNA-Cas9 groups, respectively; L5: DMSO/gRNA-Cas9 group; L6: negative control group (HTF + 0.4%BSA); and L7: NTC. B The diagram illustrates the method employed to determine the copy number of internalized plasmid per sperm. C Quantification of plasmid copy numbers internalized per sperm cell using absolute real-time PCR. The data are shown as mean ± SEM of two independent experiments performed in triplicate (n = 2). Differences between means with a p < 0.05 are indicated by different small letters. A one-way ANOVA statistical test was used to compare the groups, and a Games-Howell Post Hoc test was performed for pairwise comparisons. NTC: No template control