Normalization of muscle plasmid uptake by Southern blot: application to SERCA1 promoter analysis.
Direct injection of plasmid DNA into muscle allows the study of promoters in a physiological environment. Because of the variability of reporter gene activity, attempts have been made to normalize activity to muscle plasmid uptake by coinjection of a second "control" plasmid whose reporter gene is constitutively expressed by a viral promoter. The purpose of this study was to evaluate the use of a control plasmid vs. Southern blot to normalize for differences in uptake of plasmids containing promoter fragments of the skeletal muscle-specific sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) gene. Results showed that the correlation of luciferase activity from control vs. SERCA1 plasmids is poor and that normalization by a virally driven control plasmid increased variability of SERCA1 luciferase activity. In several cases, the presence of a control plasmid inhibited SERCA1 reporter expression. When Southern blot analysis was used to normalize for differences in plasmid uptake there was less variability than with coinjection, and correlations between plasmid uptake and SERCA1 luciferase activity were better. Moreover, there were no inhibitory effects of a control plasmid allowing for optimization of injection conditions of the SERCA1 deletion constructs. The use of Southern analysis is suggested to determine whether plasmid uptake is differentially affected by physiological stimuli, muscle types, or plasmid sizes under study.
Mitchell-Felton
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Department of Health Sciences, Boston University, Boston, Massachusetts 02215, USA.
Kandarian
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SC
D000818:Animals; D015139:Blotting, Southern; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques / Q000592:standards*; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D008845:Microinjections; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D010957:Plasmids / Q000493:pharmacokinetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D017931:DNA Primers; D008156:Luciferases; D000252:Calcium-Transporting ATPases