Figure 1.

Changes in Gene and Protein Expression after i.m. Administration of Reprogramming pDNA in Mouse GA

(A) BALB/c mice were i.m. injected into the GA with 50 μg pOKSM or pGFP in 50 μL 0.9% saline or with 50 μL saline alone. (B–D) GAs were dissected 2, 4, and 8 days after injection with pOKSM or saline, and real-time qRT-PCR was performed to determine changes in gene expression of (B) reprogramming factors and GFP transgene encoded in pOKSM, (C) endogenous pluripotency markers, and (D) genes involved in myogenesis. Gene expression levels were normalized to day 2 for Oct3/4 and GFP mRNA. **p < 0.01 and *p < 0.05 indicate statistically significant differences between day 2 and day 4 post-injection (p.i.), assessed by one-way ANOVA; n.a. indicates no amplification of the target. Expression levels of other transcripts were normalized to saline-injected controls and *p < 0.05 indicates statistically significant differences in gene expression between pOKSM- and saline-injected groups, assessed by one-way ANOVA or Welch ANOVA. Data are presented as 2ˆ^-ΔΔCt ± propagated error, n = 3. (E) 10-μm-thick GA sections, obtained 2 or 4 days after saline, pOKSM, or pGFP injection, were stained with anti-NANOG and anti-GFP antibodies. Images were taken with a slide scanner at 20× magnification; scale bars represent 50 μm. (F) Number of GFP⁺ cell clusters per GA. *p < 0.05 and **p < 0.01 indicate statistically significant differences in the number of GFP⁺ clusters compared to saline-injected controls, and ꞩ for p < 0.05 indicates statistically significant differences between 2 and 4 days after pOKSM injection, assessed by one-way ANOVA and Tukey's post-hoc test (n = 2 GAs, 3 whole sections/muscle). (G) Number of NANOG⁺GFP⁺ cell clusters per GA. ***p < 0.001 indicates statistically significant differences in the number of NANOG⁺GFP⁺ clusters between pOKSM-injected muscles (2 days p.i.) and the rest of the groups, assessed by one-way ANOVA and Tukey’s test (n = 2 GAs, 3 whole sections/muscle).