Successful overexpression of <i>AGGF1</i> mRNA and protein in skeletal muscle by gene transfer using a plasmid-based delivery system.

<p><b>A)</b> Real time PCR analysis for expression of human <i>AGGF1</i> mRNA derived from an <i>AGGF1</i> expression plasmid injected into the gastrocnemius muscle in a mouse ischemic hindlimb model. The RT-PCR primers are specific to human <i>AGGF1</i> and not able to amplify the endogenous mouse <i>AGGF1</i> mRNA. Time points are in days after the injection of plasmid DNA. Injection of the empty vector DNA served as negative control. The data are shown as the amount of plasmid-derived human <i>AGGF1</i> mRNA normalized to control <i>GAPDH</i> levels. To avoid the contamination of plasmid DNA, RNA samples were treated with DNase. In addition, RT-PCR analysis with RNA samples without reverse transcription did not yield any product. <b>B</b>) Expression of AGGF1 protein in gastrocnemius muscles by Western blot analysis at time points of 7, 14, 28 days after plasmid DNA was injected. The anti-AGGF1 antibody recognizes both human AGGF1 derived from the injected plasmid and endogenous mouse AGGF1 protein. <b>C</b>) The images from Western blot analysis in <b>B</b>) were scanned, quantified, and plotted. The intensity of AGGF1 signal was normalized to the signal of control β-actin. <b>D</b>) Immunohistochemical analysis of the sections of ischemic hindlimb muscle from mice injected with an empty vector (Control) and with an <i>AGGF1</i> expression plasmid (AGGF1) with an anti-AGGF1 antibody seven days later by injection of plasmid DNA. *, <i>P</i><0.05.