Figure 5

Total and episomal transgene DNA quantification. (A) Real-time PCR quantification of total GFP transgenes after muscle electrotransfer. GFP expression vector containing the indicated MAR element were introduced in mice muscles by electrotransfer. At the indicated day after the electrotransfer, total DNA extracts were prepared from the muscles. 120 ng of total DNA was used for qPCR assays. GFP copy numbers were normalised to those of the cellular GAPDH gene, and they are represented as the average number of GFP gene copies per diploid myofiber nuclei. (B) Muscles of mice were collected 1 or 5 day after the electrotransfer of GFP-expression plasmids containing or not a MAR element, as indicated. Total DNA was extracted and processed to quantify the GFP transgene and GAPDH cellular gene by quantitative PCR (total GFP DNA). Alternatively, the DNA preparations were treated with the PS-DNase exonuclease to degrade all transgenes occurring as chromosomally integrated linear DNA molecules (Episomal GFP DNA). GFP DNA amounts were normalized to those of GAPDH prior to PS-DNase treatment, and they are expressed as the average number of copies per diploid myofiber nucleus, after substraction of the background non-specific values obtained for not injected muscles.